18.1 Plasma protein diagnostics

Lothar Thomas

The term plasma proteins refers to the proteins of the blood plasma circulating between the blood and the interstitial fluid compartments. Specimens are serum, plasma, urine, cerebrospinal fluid, saliva, amnion fluid, aspirated fluids (ascites, pleural exudate) and feces. Under physiological conditions, the distribution of these proteins is at a steady state between the fluid compartments /1/. The term also includes other proteins such as immunoglobulins, enzymes, and blood clotting factors.

Functionally, plasma proteins are classified into:

• Transport proteins; they bind and transport hardly water-soluble substances; albumin, for example, is an important transport protein
• Acute phase proteins; they are associated with inflammation; the C-reactive protein, for example, is an important acute phase protein
• Proteins of the immune defense systems, for example immunoglobulins and complement factors
• Proteins shed within the scope of cell membrane changes, for example the soluble transferrin receptor
• Factors and inhibitors of plasmatic coagulation
• Oncofetal proteins; they are produced by tumors or physiologically produced during the fetal period (e.g., α-fetoprotein).

Diagnostically important plasma proteins are listed in Tab. 18.1-1 – Diagnostically significant plasma proteins.

18.1.1 Plasma protein synthesis and distribution

The serum concentration of plasma proteins depends on synthesis, catabolism, distribution in intracellular and extracellular fluid compartments, loss into “third space” (ascites, pleural effusion) or to the outside (proteinuria) /1/. Plasma proteins are determined in cases of:

• Normo-, hypo- or hyperproteinemia defined by determination of total protein in serum
• Dysproteinemia, a disturbance in the plasma protein composition. In this case, serum protein electrophoresis shows an atypical protein pattern
• Special clinical questions as single protein (e.g., haptoglobin in presumed hemolytic anemia).

18.1.2 Plasma protein synthesis

Whereas immunoglobulins (Ig) are produced by the plasma cells, the non-Ig proteins are primarily synthesized in the hepatocytes, with the following exceptions: β₂-microglobulin and the transferrin receptor, which are shed by cells as surface proteins, as well as complement factor D, a protease involved in the activation of the alternative complement pathway and produced by adipocytes.

Like lysosomal proteins and cell membrane proteins, plasma proteins are produced by the polyribosomes of the rough endoplasmic reticulum. The general mechanism for the synthesis of proteins is the same in all body cells. The synthesis of proteins includes the following steps:

Transcription 

DNA in the cell nucleus is the carrier of the genetic information, and a copy is produced in the form of RNA by RNA polymerase /2/. After the intervening sequences (or introns) are turned off, the messenger RNA (mRNA) is produced. This mRNA transfers the information regarding which protein will be synthesized from the cell nucleus to the cytoplasm. The regulation of transcription is achieved through regulatory proteins (transcription factors) that bind to specific DNA sequences of the gene to be regulated, and exert either a positive or negative effect on protein synthesis. For example, if an acute phase reaction occurs, the pro inflammatory cytokines will bind to the hepatocyte plasma membranes. This signal induces to the activation of the nuclear proteins that bind to the corresponding DNA sequences in the nucleus, thus leading to an amplified transcription of acute phase proteins such as C-reactive protein.

Translation

The mRNA base sequence is translated into a defined amino acid sequence. Translation takes place on the ribosomes of the endoplasmic reticulum, to which the mRNA becomes attached.

Protein synthesis

Plasmaprotein synthesis takes place in the ribosomes of the endoplasmic reticulum. The ribosomes are composed of ribonucleic acids and proteins that are produced by the nucleolus. Most proteins acquire two additional peptides on the N-terminal end during synthesis, a hydrophobic peptide and a pro peptide. The hydrophobic peptide is composed of 15–30 amino acids, and serves as a pilot peptide to guide the protein through the membrane of the endoplasmic reticulum in its lumen. The pilot peptide is immediately cleaved after the protein is discharged through the lumen of the endoplasmic reticulum. The folding of the peptide into its final tertiary structure takes place via the formation of disulfide bridges in the endoplasmic reticulum. Certain proteins are transported from the lumen of the endoplasmic reticulum into the lumen of the Golgi apparatus for secretion. The pro peptide remains on the protein while it matures in the vesicles of the Golgi apparatus or is stored in the secretory granules. The pro peptide is cleaved shortly before the release of the protein from these organelles /3/.

Co translational or post translational protein modification

Protein modifications start as the protein passes through the lumen and the cisterns of the endoplasmic reticulum and Golgi apparatus. The majority of the plasma proteins are glycoproteins and some are lipoproteins.

Other co translational or post translational modifications

Oxidation of C atoms 3 and 4 which results in the formation of thioester binding, phosphorylation of, for example, α₂HS-glycoprotein and binding of phospholipids.

18.1.3 Glycoproteins

The glycosylation of proteins is important for the folding into a tertiary structure, and for their functioning and degradation /4/. There are two types of protein glycosylation, N-glycosylation and O-glycosylation. They are classified into:

• N-glycosylated type: most proteins are glycosylated by forming a covalent N-bond between the amino acid asparagine and a carbohydrate residue composed mainly of mannose. This step begins in the lumen of the endoplasmic reticulum. In a subsequent process that occurs over multiple stages, a transformation of the carbohydrate chains takes place wherein the terminal position is often bound to N-acetyl neuraminic acid, while galactose is linked at the preterminal position.
• O-glycosylated type: some plasma proteins contain sugar residues that are bound via the OH-groups of the amino acids serine and threonine. O-glycosylation takes place in the Golgi apparatus.

Microheterogeneity of glycoproteins

All glycoproteins exhibit heterogeneity, since they are derived from a population with various oligosaccharide sequences (glycoforms). The variation comes from the different numbers of carbohydrate chains, the varying chain length and the differing end substitutions that are borne by the amino acid skeleton. As a result, each glycoprotein can occur in forms having different net electric charges. This is manifested in the electrophoresis as micro heterogeneity. Thus, for example, acidic α₁-glycoprotein exhibits seven bands with isoelectric focusing at acidic pH. Differences in the glycosylation of IgG have an effect on immunoregulation /5/.

18.1.4 Lipoproteins

Lipoproteins are proteins that are coupled covalently with lipids (e.g., in the cell membranes). Thus, a fatty acid can bind to an N-terminal glycine as an amide, or to cysteine as a thioester. Plasma lipoproteins are non covalent aggregates of lipids and proteins.

18.1.5 Release of proteins into the plasma

Following post translational modification, the secretory plasma proteins are stored in vesicles that are separate from the Golgi apparatus. These vesicles fuse with the plasma membrane of the cell, and the proteins are released into the extravascular compartment via exocytosis. The pro peptide plays an important role in this process.

18.1.6 Plasma protein distribution

Plasma proteins are continuously redistributed in both directions between the vascular and interstitial compartments. This takes place via diffusion through the capillary walls, pinocytotic transport through the capillary endothelium or over the intercellular connections of the tissue cells /6/. The distribution depends on molecular weight. The higher the molecular weight, the greater will be the intravascular proportion of that protein (Tab. 18.1-2 – Distribution of plasma proteins between the intravascular and extravascular compartments). Large proteins, such as fibrinogen, α₂-macroglobulin and IgM remain predominantly in the extravascular compartment, and will only enter circulation to a limited extent via the intercellular connections between the endothelial cells or through pinocytosis. However, the reverse flow from the tissue into the blood vessels occurs through the lymph vessels. Glomerular filtered plasma proteins are absorbed by the cells of the proximal tubule through pinocytosis, and are degraded in these tubule cells.

The quantity of plasma proteins transferred from the intravascular to the extravascular compartment is organ-dependent; the levels are high in the liver, but quite low in the brain so that the protein concentration in the cerebrospinal fluid is about 300-fold lower than in the plasma. In the case of systemic disorders, however, the vascular permeability can change drastically and can lead to the development of exudations in the form of effusions or edema.

18.1.7 Plasma protein catabolism

Plasma protein degradation occurs in all somatic cells (see also Section 18.2.7 – Pathophysiology). The released amino acids are used by the cells for resynthesis of proteins; only essential amino acids must be taken in with food. Desialation plays an important role in the breakdown of glycoproteins. The intactness of the carbohydrate side chains protects the protein from degradation /7/. Removal of sialic acids and reduction of carbohydrates by membrane-bound or circulating enzymes favors pinocytosis and intracellular degradation by lysosomal enzymes /8/.

Organs and tissues that are significantly involved in the degradation of proteins include:

• The hepatocytes; they are easily reached by plasma proteins since the liver sinusoids lack basement membranes and endothelial cells have marked intracellular fenestrations
• The kidneys; following glomerular filtration, low molecular weight proteins are taken up by the brush border of tubular cells by pinocytosis and subsequently degraded by lysosomal enzymes
• The endothelial cells of capillaries; although these cells display only minimal capacity for pinocytosis, their catabolic potential is high because of the large size of the capillary bed.

The degradation rate of plasma proteins varies and is described by their half-life (albumin 19 days, ceruloplasmin 4 days, transferrin 8 days, IgG 23 days, IgA 5 days and IgM 5 days).

Degradation of senescent proteins is non-enzymatic by way of post translational modification based on various reactions /9/:

• Glycation; the most common reaction is the binding of glucose or other reducing substances to proteins. The carbonyl groups of the sugars and the amino groups of the protein form a Schiff base that quickly forms a stable keto amine through molecular rearrangement (Amadori product). In further reactions involving oxidation, the Amadori product is converted into an advanced glycation end product (AGE). Glycated hemoglobin is a typical Amadori product (see Fig. 3.6-1 – Glycation of N-terminal valine of hemoglobin with glucose and subsequent Amadori rearrangement).
• Direct oxidation by reactive oxygen species forming advanced oxidation protein products (AOPPs). The main AOOPs include methionine sulfoxide from oxidation of methionine and 3-nitro tyrosine from nitration of tyrosine.
• Carbamylation from the binding of isocyanic acid to amino acids, especially to ε-NH₂ groups of lysine residues. The isocyanides form due to spontaneous dissociation of urea or reaction of thiocyanate catalyzed by myeloperoxidase in the presence of H₂O₂.

The organism tries to maintain the total protein concentration in the intravascular compartment at a constant level within certain limits. For instance, in infectious diseases, the elevated level of acute phase proteins and immunoglobulins is compensated for by a reduction in the concentration of negative acute phase proteins (albumin, transthyretin, transferrin, apolipoproteins). In multiple myeloma, the monoclonal increase in immunoglobulins is compensated for a long time by suppression of polyclonal immunoglobulin production.

18.1.8 Modifications in plasma protein synthesis

The daily physiological turnover of plasma proteins is about 25 g and depends on the amino acid pool available for protein synthesis. This pool, in turn, is dependent on numerous variables.

Reduced plasma protein synthesis

Genetically induced; inflammation, hepatopathies (liver cirrhosis, acute hepatitis), nutritional protein deficiency, hypothyroidism, malabsorption syndrome, alcoholism, lymphoma, metastasized carcinoma.

Increased plasma protein synthesis

Inflammation, fever, hyperthyroidism, hyper cortisolism, increased release of growth hormone, protein-losing syndrome, iron deficiency, stimulation of the immune systems and clonal increase in the number of immunoglobulin-producing plasma cells (multiple myeloma).

Genetic impacts on plasma protein synthesis

Impacts may present clinically in the form of protein deficiency, protein increase or protein dysfunction. For instance, a protein may:

• Not be synthesized (e.g., hereditary IgA deficiency)
• Be synthesized with a structural defect and thus not be released from the cell (e.g., hereditary α₁-antitrypsin deficiency)
• Be secreted as a structurally similar but functionally inactive variant (e.g., C1-esterase inhibitor in hereditary angioneurotic edema).

18.1.9 Diagnostic significance of plasma proteins

One-time determination

In certain clinical concerns, the determination of the concentration or activity of particular plasma proteins provides important help in the diagnosis of diseases, for example:

• CRP if systemic inflammation is suspected
• Haptoglobin in presumed intravascular hemolysis
• α₁-antitrypsin in pulmonary emphysema.

In addition, the finding of a normal plasma protein concentration is also of important differential diagnostic value in order to rule out certain diseases.

Plasma protein profile

The plasma protein profile allows differential diagnostic conclusions.

Monitoring

During the course of the disease (acute symptoms) or during therapy, the plasma protein concentration and its changes during the disease course may provide conclusions as to the activity, severity and possible complications of the condition. The time in point when normalization is achieved has important prognostic implications.

18.1.10 Indication

Important indications for plasma protein determination are listed in Tab. 18.1-3 – Important indications for plasma protein determination.

18.1.11 Specimen

Because of the high intravascular proportion of plasma proteins in relation to the interstitial fluid, a concentrating effect may occur before or during blood collection, thus simulating falsely elevated protein concentrations. This is the case, for example, if the patient does not sit for at least 15 min. prior to blood collection and venous occlusion during blood collection lasts for more than 3 min. The sample should be analyzed the same day. If this is not possible, it is better to store the sample at 4 °C than at –20 °C if deep-freezing at –70 °C is not feasible. Storage at 20 °C is associated with a measurable decline in protein concentration after 36 hours that affects many proteins.

Immunonephelometric determination in serum and in lithium heparin plasma yields comparable results /10/.

18.1.12 Method of determination

Principle

Immunochemical methods are used to determine plasma proteins. For the determination of an unknown plasma protein concentration a constant amount of specific antibodies is employed. Refer to Section 52.1-4 – Principles of immunochemical methods. The critical factors which determine whether the immunocomplexes which are formed can be detected in a soluble form or a precipitable form include besides the plasma protein concentration to be determined, the ratio of the plasma protein to the antibody concentration (curve according to Heidelberger and Kendall, refer to Fig. 52.1-5 – Precipitin curve. In antibody excess, soluble immune complexes are formed. The measurement of the immune complex concentration is made nephelometrically or turbidimetrically.

In immunonephelometry, light from a helium neon laser is directed through the cuvette and is scattered by the immune complexes; this scattered light is then focused on a detector by means of a lens system. The electrical signal of the detector is proportional to the light scattering intensity. The concentration of the plasma protein concentration can be determined from the light scattering signal with the help of a calibration curve. In the case of kinetic-nephelometric plasma protein determination, changes in the scattered light are measured at short intervals, while the endpoint method allows the reaction to occur for a defined period of time (e.g., 15 or 30 min.). By adding further plasma protein or antibodies, it is possible to check whether the measurement occurred on the slope of the Heidelberger and Kendall curve (see Fig. 52.1-5 – Precipitin curve). This is the case if the addition of sample (plasma protein) results in an increase of the measurement signal or if the addition of antibodies produces no change in the measurement signal.

In immunoturbidimetry soluble immune complexes are formed by the addition of the plasma protein to antibodies and a buffer containing accelerator that allows kinetic measurement according to the fixed time principle. The increase of absorption at 334 or 340 nm is measured within a defined time period.

Calibrators for the plasma protein assays are standardized using the BCR/IFCC/CAP RPPHS reference material, also know as CRM 470.

The detection limit for the immunonephelometric and immunoturbidimetric tests, which is about 10 mg/L, can be increased by a factor of 10 to 100 if the specific antibodies are bound to latex particles (latex-enhanced assays).

18.1.13 Quality assurance

Since the introduction of the reference preparation BCR/IFCC/CAP RPPHS, also known as CRM 470 (now ERM-DA470), which contains reference values for the plasma proteins listed in Tab. 18.1-4 – Plasma proteins of the ERM reference materials, the accuracy of plasma protein determinations has improved /11/. ERM-DA472/IFCC has been certified for CRP. The successor reference preparation of ERM-DA470, referred to as ERM-DA470k/IFCC, was reproduced in the same quality and spiked with CRP and β₂-microglobulin /12/.

References

1. Ritchie RF, Navolotskaia O, eds. Serum proteins in clinical medicine, Vol I. Scarborough: Foundation for Blood Research, 1996.

2. Dreyfuss G, Hentze M, Lamond AI. From transcript to protein. Cell 1996; 85: 963–72.

3. Hong W. Protein transport from the endoplasmatic reticulum to the Golgi apparatus. J Cell Science 1998; 111: 2831–9.

4. Lee YC. Biochemistry of carbohydrate-protein interaction. Faseb J 1992; 6: 3193–200.

5. Hounsell EF, Davies MJ. Role of protein glycosylation in immune regulation. Ann Rheumat Dis 1993; 52: S22-S29.

6. Davis A. Plasma protein synthesis and distribution. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine, Vol II. Scarborough: Foundation for Blood Research, 1999.

7. Ciechanover A, Gonen H, Elias S, Mayer A. Degradation of proteins by the ubiquitin-mediated proteolytic pathway. The New Biologist 1990; 2: 227–34.

8. Lord JM, Davey J, Frigerio R, Roberts LM. Endoplasmatic reticulum-associated protein degradation. Cell & Developmental Biology 2000; 11: 159–64.

9. Jaisson S, Gillery P. Evaluation of nonenzymatic posttranslational modification – derived products as biomarkers of molecular aging of proteins. Clin Chem 2010; 56: 1401–12.

10. Develter M, Blanckaert N, Komarek A, Bossuyt X. Can heparin plasma be used instead of serum for nephelometric analysis of serum proteins. Clin Chem 2006; 52: 1609–10.

11. Merlini G, Blirup-Jensen S, Johnson MA, Sheldon J, Zegers I. Standardizing plasma protein measurements worldwide: a challenging enterprise. Clin Chem Lab Med 2010; 48: 1567–75.

12. Zegers I, Keller T, Schreiber W, Sheldon J, Albertini R, Blirup-Jensen S, et al. Characterization of the new serum protein reference material ERM-DA470k/IFCC: value assignment by immunoassay. Clin Chem 2010; 56: 1880–8.

13. Ritzmann SE. Serum protein abnormalities. Boston: Little, Brown, 1984.

14. Shenkin A, Cederblad G, Isaksson EM. Laboratory assessment of protein-energy status. Clin Chim Acta 1996; 253: S5–S59.

15. Ellervik C, Tybjaerk-Hansen A, Nordestgaard BG. Total mortality by transferrin saturation levels: two general population studies and a metaanalysis. Clin Chem 2011; 57: 459–66.

16. Ranasinghe RNK, Biswas M, Vincent RP. Prealbumin: The clinical utility and analytical methodologies. Ann Clin Biochem 2022; 59 (1): 7–14.

17. Bernstein LH, Leukhardt-Fairfield CJ, Pleban W, Rudolph R. Usefulness of data on albumin and prealbumin concentration in nutritional support. Clin Chem 1989; 35 (2): 271–4.

18. Fang F, Daya N, Coresh J, Christenson RH, Selvin E. Glycated albumin for the diagnosis of diabetes in US adults. Clin Chem 2022: 68 (3): 413–21.

18.2 Total protein (TP)

Lothar Thomas

The determination of TP is based on the following premises:

• Each individual protein reacts the same way in the method of determination as any of many other proteins in the serum, plasma or body fluids
• All proteins are pure polypeptide chains with approximately 16% of their mass consisting of nitrogen
• The proteins are compared to bovine serum albumin since the latter is used as a calibrator in the method of determination.

18.2.1 Indication

Presence of the following symptoms, conditions or diseases: inflammation, proteinuria, edema, polyuria, chronic renal disease, chronic liver disease, chronic diarrhea, malignant tumor, increased susceptibility to infections, bone pain, rheumatic symptoms of undeterminable localization, lymphomas, external and internal hemorrhages, pregnancy, pre- and postoperative state, monoclonal gammopathy, shock, burns, patients requiring intensive medical care, investigation of an acute decrease in hemoglobin.

18.2.2 Method of determination

The Biuret method has proven useful for quantitative determination of serum TP. Determination of TP in urine, cerebrospinal fluid (CSF) and other body fluids is performed using various methods. The most reliable method is the Biuret reaction after acid precipitation of proteins. For CSF and urine, dye-binding methods, especially the Coomassie method, and light scattering techniques are employed.

Biuret method

Principle: the Biuret method is dependent on the presence of peptide bonds in the proteins. If a protein solution is treated with Cu(II) ions in a weakly alkaline solution, a colored chelate is formed between the Cu(II) ion and the carboxyl oxygen and amide nitrogen of the peptide bond. A requirement for this reaction to take place is the presence of at least two peptide bonds (tripeptide). Amino acids and dipeptides do not react. The intensity of the resulting violet color varies in a linear fashion with the number of peptide bonds and hence with the protein concentration over a broad range. The Biuret reagent contains copper sulfate, sodium potassium tartrate, potassium iodide and sodium hydroxide. The Cu(II) ions are kept in solution as a tartrate complex at alkaline pH while potassium iodide prevents auto reduction of Cu(II).

The method is not standardized, and the molarities of the Biuret reagent components have been modified in many ways, but a candidate reference method is described /1/. Bovine serum albumin is recommended for calibrating the TP determination. If determined manually, 1 part serum is added to 50 parts Biuret reagent, and after a 30 min. incubation at room temperature the absorption of the test sample and that of the standard are measured against Biuret reagent at 546 nm.

Coomassie method

Principle: the textile dye Coomassie Brillant Blue G 250 (CBB-G250) is in its leukoform in slightly acidic solution, and has an absorption maximum at 465 nm. CBB-G250 reacts rapidly with proteins forming a protein-dye complex which causes shift of the absorption maximum to 595 nm /2/. At low levels, the absorption is approximately linear with the protein concentration. This method is acceptable for TP determinations in urine and cerebrospinal fluid.

Turbidimetric method

Principle: in specimens that are low in protein such as urine and cerebrospinal fluid are denatured by addition of trichloroacetic acid. The denatured proteins scatter short-wavelength light. The measurable light scattering signal is proportional to the TP concentration over a certain concentration range. Light scattering methods are used for TP determination in cerebrospinal fluid /3/ and urine specimens /4/.

18.2.3 Specimen

Serum, plasma (heparin), urine, CSF, aspirated fluid: 1 mL

18.2.4 Reference interval

Refer to references /5, 6/ and Tab. 18.2-1 – Reference intervals for total protein.

18.2.5 Clinical significance

This section is limited to the clinical interpretation of TP levels in serum and plasma. For an assessment of

• TP in urine, refer to Section 12.9 – Urinary proteins
• TP in CSF refer to Chapter 46 – Laboratory diagnosis of neurological diseases
• TP in extravascular fluids refer to Chapter 47 – Extravascular fluids.

Deviations of serum TP from the reference interval indicate the presence of dysproteinemia or are a sign of hypoproteinemia or hyperproteinemia based on disorders in the water and electrolyte balance.

From a differential diagnostic point of view, the conditions can be distinguished by additional performance of serum protein electrophoresis and determination of hematocrit.

Electrophoretically, dysproteinemia is associated with a quantitative shift in the pattern of the protein fractions, while hematocrit is unchanged.

Both dehydration and hyper hydration lead to uniform increases or reductions in serum protein concentrations, while no shift in protein bands occurs during serum protein electrophoresis. However, the hematocrit is abnormal.

18.2.5.1 Hypoproteinemia

Hypoproteinemia is mostly due to a reduction in albumin, and is less frequently due to a decrease in antibody synthesis (Tab. 18.2-2 – Diseases and conditions that may cause hypoproteinemia). Clinical symptoms of pronounced hypoproteinemia include the development of edema and effusions in body cavities. Hypoproteinemia may be due to:

• Disturbance in protein synthesis
• Protein malnourishment
• Protein malabsorption
• Protein-losing syndrome
• Dilution hypoproteinemia.

18.2.5.2 Hyperproteinemia

Hyperproteinemia is rarer than hypoproteinemia because in the event of an increase in globulins, a regulatory reduction in albumin ensues. Therefore, in hyperglobulinemia, the total protein concentration remains within the reference interval for a long period of time. Hyperproteinemia above 80 g/L is found in about 3.5% of clinical patient samples.

Only pronounced, monoclonal gammopathies, severe chronic-inflammatory disorders and certain auto immunological processes (e.g., autoimmune hepatitis) cause hyperproteinemia (Tab. 18.2-3 – Diseases and conditions that may cause hyperproteinemia).

18.2.6 Comments and problems

Blood collection

The blood should be collected with the patient in a supine position since blood samples obtained in an upright position lead to the measurement of up to 10% higher TP concentration. The discrepancy is even higher in patients who tend to develop edema. After more than 3 min. of venous occlusion during blood collection, the protein concentration may increase by up to 10%. Blood sampling after vigorous muscle activity may lead to a rise in TP concentration by up to 12%.

Serum or plasma can be used for TP determination. Due to fibrinogen, average TP concentration in plasma is higher than in serum (i.e., by 2.5 g/L in blood donors, by 3.6 g/L in outpatients, by 4.6 g/L in in-patients and by 6.6 g/L in in-patients with a CRP > 50 mg/L) /8/. The patient does not need to be in a fasting state for the blood collection.

Method of determination

The Biuret method is used employing many variations of the Biuret reagent. These can be roughly divided into two groups /9/:

a) Low NaOH concentration (0.1–0.2 mol/L) and high CuSO₄ concentration (10–30 mmol/L).

b) High NaOH concentration (0.5–0.8 mol/L) and low CuSO₄ concentration (4–6 mmol/L). The Biuret reagents of these groups have a low reagent blank value, while the determination is only linear up to a protein concentration of 1.4 g/L as a rule.

The Biuret method reacts with some amino acids, dipeptides and other substances forming a 5-membered or 6-membered ring complex with Cu. These complexes have a higher absorption maximum (blue) than peptides and proteins (pink) /10/.

Interfering factors

Infusion solutions: protein-containing infusion solutions such as oxypolygelatin as well as polypeptides that are derived from degraded gelatin and cross-linked via urea bridges are also detected in the reaction to a varying extent, depending on the Biuret reagent used. Polyglucose such as dextran and sugar solutions such as glucose, mannitol, sorbitol and fructose lead to color intensification and simulate elevated protein levels; dextran, in addition, causes turbidity. Hydroxyethyl starch and synthetic materials such as polyvinylpyrrolidone do not enter into the reaction /9/.

Other interfering substances: ammonium salts (e.g., contained in enzyme preparations) may simulate falsely low levels as a result of protein precipitation. Tris(hydroxymethyl)-aminomethane leads to falsely elevated TP concentrations since it reacts with the Biuret reagent to yield a protein-like color reaction.

Hemolysis: 0.8 g of Hb/L resemble a 2% protein increase /11/. If bovine serum albumin is used as the standard, each mg of Hb mimics approximately 2 mg of protein since the globin enters into the Biuret reaction.

Lipemia: markedly lipemic serum samples simulate elevated TP concentrations by causing turbidity of the reagent mixture and must be cleared prior to determination.

Bilirubin: serum concentrations > 5 mg/dL (85 μmol/L) cause falsely increased TP levels in the Biuret reaction unless a sample blank (with the exception of copper sulfate) is measured.

X-ray contrast media: depending on their composition, X-ray contrast media can cause falsely elevated TP concentrations.

Coomassie method and light scattering technology: while the Biuret reaction has about the same percental absorption coefficient for all proteins, this does not apply to the Coomassie method /11/. Using the light scattering technology, globulins are determined too low in comparison to albumin /12/.

Stability

In a closed container at room temperature up to 1 week, at 4 °C up to 1 month, in a deep-frozen state > 1 year.

18.2.7 Pathophysiology

Plasma TP is composed of > 100 structurally known proteins; the biological function is known in detail for about 50 proteins. Albumin, α₁-, α₂- and β-globulins are synthesized by the parenchymal cells of the liver, while the proteins of the γ-globulin fraction (i.e., the immunoglobulins) are synthesized by plasma cells. The half-life of proteins ranges from a few hours (e.g., acute phase proteins) to as long as 3 weeks (e.g., IgG and albumin).

The liver has a significant functional reserve capacity for protein synthesis (3-fold for the synthesis of albumin, 6-fold for the synthesis of fibrinogen), and translation and transcription are usually not disturbed with the onset of liver disease /13/.

Glucagon and malnutrition (due to a lack in nutrients and/or poor dietary habits), especially a deficiency in the amino acid tryptophan, exert an inhibitory effect on the synthesis of proteins.

Glucocorticoids, growth hormone, insulin and thyroid hormones have a stimulatory effect on the hepatic synthesis of proteins.

Hepatic protein degradation takes place after endocytic uptake of proteins by the hepatocytes. This involves, for example, binding of glycoproteins to an asialoglycoprotein receptor on the hepatocyte membrane after removal of the N-acetylneuraminic acid (NANA) that is located in a terminal position at the carbohydrate side chains. The glycoproteins are subsequently internalized by pinocytosis.

Protein degradation involves two pathways:

• In the lysosomes mediated by the action of peptidases and proteases under acid pH conditions
• In the cytosol, by proteolytic enzymes under neutral pH conditions. The proteins to be degraded are bound to the protein ubiquitin that is present in cytosol, and thus targeted proteins are broken down by proteases first into peptides and ultimately into amino acids /14/.

Decreases in hepatic protein degradation can result from:

• A reduced number of asialoglycoprotein receptors on the hepatocyte. This condition has been described, for example, in liver cirrhosis. As a result of this, plasma concentration of asialylated glycoproteins is elevated.
• Increased sialylation (covalent binding of NANA) of proteins and, thus, a delay in desialylation and a reduced uptake by the hepatocytes. Increased sialylation has been described for GGT in acute alcoholic liver injury and for ALP in primary biliary cirrhosis.

The degradation of nonsialylated albumin is still largely unknown.

The physiological importance of plasma proteins lies in the maintenance of the colloid osmotic pressure as well as in their function as a vehicle for lipids, metabolic products, hormones and minerals; several proteins display enzymatic activity.

Many abnormal processes in the organism cause an alteration in the protein composition of the plasma (dysproteinemia) but often do not lead to protein concentrations outside the reference interval.

Plasma volume-induced changes of TP concentration, as seen after infusions or severe diarrhea, may be recognized by a synchronous pattern of hematocrit and TP concentration.

Absolute changes in total protein in plasma are due to either a decrease in albumin or an increase or decrease in immunoglobulins. Proteins that physiologically migrate in α₁-, α₂- and β-globulin fraction during electrophoresis are rarely subject to changes that result in marked hypoproteinemia or hyperproteinemia. An absolute increase in albumin does not occur.

References

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9. Kleine TO. Interferenz von Infusionslösungen mit der Biuret-Reaktion in einem vollmechanisierten und einem manuellen System. Med Welt 1979; 30: 102–7.

10. Hortin GL, Meilinger B. Cross reactivity of amino acids and other compounds in the Biuret reaction: Interference with urinary peptide measurements. Clin Chem 2005; 51: 1411–9.

11. Sonntag O. Störung klinisch-chemischer Untersuchungen durch Hämolyse. J Clin Chem Clin Biochem 1986; 24: 127–39.

12. Nishi H, Kestner J, Elin RJ. Four methods for determining total protein compared by using purified protein fractions from human serum. Clin Chem 1985; 31: 95–8.

13. Gerok W. Protein- und Aminosäurestoffwechsel bei Leberkrankheiten. In: Seidel D, Lang H (eds). Funktion und Funktionsdiagnostik der Leber. Heidelberg: Springer, 1985: 65–97.

14. Ciecanover A, Gonen H, Elias S, Mayer A. Degradation of proteins by the ubiquitin-mediated proteolytic pathway. The New Biologist 1990; 2: 227–34.

18.3 Serum protein electrophoresis

Lothar Thomas

Serum protein electrophoresis (SPE) is performed to diagnose dysproteinemias. The separation of serum proteins takes place at alkaline pH as a function of the protein’s net charge, isoelectric point and molecular weight. For medical diagnostic purposes, proteins are separated:

• Either on a cellulose acetate medium into the six classic factions transthyretin, albumin, α₁-, α₂-, β- and γ-globulins in healthy individuals
• Or on agarose gel medium, where specimens from healthy individuals exhibit 8–11 fractions
• Using capillary zone electrophoresis, where up to eight protein fractions can be separated in healthy individuals.

18.3.1 Indication

Diagnosis and disease monitoring in patients with

• Monoclonal gammopathies
• Acute and chronic inflammatory response
• Protein-losing syndromes (kidney, gastrointestinal tract, skin, exudates and transudates).

Pathological results in basic laboratory investigations, for example:

• Elevated erythrocyte sedimentation rate
• Proteinuria
• Increased or decreased serum total protein concentration.

18.3.2 Method of determination

Zone electrophoresis on cellulose acetate medium /1, 2/

Principle: the fractionation of serum proteins on certain media, especially cellulose acetate is a function of the voltage, electroendosmosis, pH of the separation buffer and the pK value of the individual protein. The placement of the sample on the supporting medium relative to the cathode and anode will affect and determine the degree of separation and resolution of the individual proteins. A multi-sample applicator is used to apply the sample, which is most often placed near the cathode. The separation of the proteins takes place at constant voltage (200 to 250 V) with a separation time of approximately 20 min. in a separation buffer of pH 8.2–8.6. The serum proteins migrate anodically and separate into bands (zones) comprising the fractions albumin, α₁-, α₂-, β- and γ-globulins. The bands are stained with protein dyes (Ponceau Red S, Amido Black 10B); any non specifically adsorbed dye on the supporting medium is washed out in de colorizing baths. After staining, proteins are identifiable by a characteristic pattern of colored bands which appear on the electrophoretogram, and by the relative intensities of these bands. The quantitative evaluation of the electrophoretogram is performed by densitometric scanning after rendering the cellulose acetate support transparent.

Automatic print out of the densitometer provides following results:

• A scan that indicates the optical density of individual bands (Fig. 18.3-1 – Zone electrophoresis on cellulose acetate sheet)
• The percentages representing the proportions of the individual band relative to the optical density of the entire electrophoretogram
• The protein concentration of the individual band in g/L relative to the total protein concentration in the sample.

Agarose gel electrophoresis /3/

Principle: serum protein separation using agarose as supporting medium is comparable with electrophoresis on cellulose acetate medium, except that a separation time of 30–60 min. is necessary. The higher electroendosmosis, provides a better separation of serum proteins (Fig. 18.3-2 – Separation of serum proteins on agarose gel electrophoresis).

Agarose gel electrophoresis is widely used as a basic method for the separation of proteins, which can be further supplemented by other techniques that demonstrate the proteins more sensitively (e.g., by immunochemical techniques such as immunoelectrophoresis or immunofixation electrophoresis or by enzymatic methods for the measurement of isoenzymes).

Capillary zone electrophoresis (CZE) /4/

Principle: the separation of proteins is carried out in liquid medium in a narrow-bore capillary (20–200 μm), to which a high-voltage potential is applied. In this system, the electroendosmotic separation effect on the individual proteins is greater than their electrophoretic mobility. The separation takes place in the direction of the cathode. There, the proteins are quantified through UV measurement of the peptide bonds. With the help of a data processing program, a graph comparable to that from zone electrophoresis is produced for the following 8 fractions: pre albumin, albumin, α₁ acidic glycoprotein, α₂-globulin, hemopexin, transferrin, complement, and γ-globulin.

18.3.3 Specimen

Serum: 1 mL

18.3.4 Reference interval

Refer to Tab. 18.3-1 – Reference intervals of serum protein electrophoresis.

18.3.5 Clinical significance

The use of serum protein electrophoresis represents an effective way to diagnose dysproteinemia.

18.3.5.1 Dysproteinemia

Dysproteinemias are quantitative or qualitative changes in the protein composition of serum and are closely related to numerous diseases. In the electrophoretogram, dysproteinemias are linked to a disease state and are mainly recognizable when albumin or protein groups are affected exhibiting an increase, or decrease of protein fractions or an extra band . Examples of such proteins or protein groups are albumin, acute phase proteins, the transthyretin-transferrin group and immunoglobulins.

Albumin

The proportion of albumin decreases in any condition associated with an absolute rise in globulins (α, β, γ); thus, total protein usually remains within the reference interval.

Acute phase proteins

Acute phase proteins migrate in the α₁- and α₂-globulin fraction and are elevated by 50–300% in acute inflammatory conditions but are decreased in acute hepatitis, chronic active liver disease and protein-losing syndrome.

Transthyretin-transferrin group

Pre albumin, also known as transthyretin, migrates in front of the albumin band; usually, 50–70% of the transthyretin are bound in a complex with the retinol-binding protein. Both proteins are decreased in nutritional protein deficiency or in general energy deficiency conditions due to fasting states or in patients under intensive care.

Transferrin migrates in the β-globulin fraction, is elevated in iron deficiency and decreased in a protein and energy deficiency state and in the presence of any acute and chronic inflammatory condition. This also applies to anemia in chronic disease.

Members of the transthyretin-transferrin protein group react to all cases of acute and chronic inflammation with a decrease and are termed as the negative acute phase proteins.

Immunoglobulins

These proteins have antibody function and represent the γ-globulin but in part also the β-globulin fraction. Increases in immunoglobulins are referred to as gammopathies.

Polyclonal gammopathies

In the electrophoretogram these gammopathies cause a broad-based γ-globulin band and are due to a disease that activates the humoral immune defense.

Monoclonal gammopathies

The monoclonals form a narrow-based M-spike within the globulin band. The M-spike is caused by the excessive production of an immunoglobulin or an immunoglobulin fragment by a single plasma cell clone. Clinically, this presents mainly as multiple myeloma or as Waldenström’s macroglobulinemia. The M-spike is localized in the γ– globulin band or β-globulin band, and is referred to as M-spike (M = monoclonal = myeloma = M-formation of the gradient with the albumin fraction in the electrophoretogram).

Oligoclonal gammopathies

Selective increases of one or several Ig classes or Ig subclasses but of both light chain types. The Ig class or Ig subclass has limited heterogeneity of antibodies. The γ-globulin zone displays one or mostly several bands (sawtooth pattern).

Isolated protein changes

With the exception of albumin, α₁-antitrypsin and IgG, they are not detectable by cellulose acetate electrophoresis but better detectable by capillary zone electrophoresis.

Extragradient

There may be some bands that deviate from the usual electrophoretogram. These appear in SPE with a frequency of approximately 0.7%, and are either supernumerary, and thus situated atypically between the normal bands, or directly superimposed over another band. They become recognizable when their protein concentration exceeds 2 g/L. They result either from the extreme increased production of a plasma protein, or from a technical error while the SPE is being carried out.

18.3.5.2 Clinical association with the SPE

The SPE does not allow a diagnosis to be directly established; however, based on the presence of dysproteinemia and the pattern of the electrophoretogram (constellation type), the following interpretations are possible:

• Allocation of certain diseases or groups of diseases to characteristic types of pattern
• Evaluation of disease activity
• Monitoring of disease course.

Because of the quantitative determination of individual plasma proteins, SPE has lost a lot of its value and is/was used:

• Originally, to diagnose reaction patterns of diseases (Tab. 18.3-2 – Reaction patterns in serum protein electrophoresis)
• In the diagnosis of monoclonal spikes (Tab. 18.3-3 – Dysproteinemias with M-spikes and extra gradients).

Typical graphs of dysproteinemias are shown in Fig. 18.3-3 – Scans of serum electrophoretograms.

18.3.6 Comments and problems

Specimen

Serum should be used since fibrinogen in plasma leads to the formation of an extra gradient within the β-globulin region /8/.

Reference interval

SPE is not standardized. Cellulose acetate electrophoresis with Ponceau red staining and CZE have generally comparable reference intervals.

Capillary zone electrophoresis (CZE)

CZE has the following advantages over cellulose acetate electrophoresis and agarose gel electrophoresis:

• Markedly lower imprecision /4, 6/
• CZE and immunonephelometric assays yield very similar results in the quantitative determination of albumin /4/.

α₁-antitrypsin (AAT) deficiency: CZE should not be used for screening for AAT deficiency if the result is based on the lower reference interval value (Tab. 18.3-1 – Reference intervals of serum protein electrophoresis). In a study /9/, however, 86% of the ZZ phenotypes and 29% of the MZ phenotypes were detected at a cutoff ≤ 0.21 g/L (see also Section 18.5 – α₁-antitrypsin (AAT)).

Hemolytic serum: causes a small peak in the anodic part of the γ-globulin band.

Monoclonal gammopathies: CZE is more sensitive in the detection of monoclonal gammopathies than cellulose acetate electrophoresis (sensitivity 74%) and agarose gel electrophoresis (sensitivity 86%). However, diagnostic sensitivity is only 95% compared to immunofixation electrophoresis /10/.

Problems with the detection of proteins by CZE /11/:

• Low concentration of monoclonal IgA (total IgA below 3.2 g/L). The monoclonal IgA overlooked by CZE migrates in the β-globulin zone and is hidden under the C3 or transferrin band
• Low concentration of monoclonal IgM (total IgM below 2.1 g/L)
• Free light chains in serum
• Monoclonal IgD
• Monoclonal Ig with a high isoelectric point, that migrate in the cathodic part of the γ-globulin zone are readily detectable in agarose gel electrophoresis, but may be not detectable in CZE.

Specificity of SPE in monoclonal gammopathies

Monoclonal gammopathies are detected in serum protein electrophoresis by the presence of M-gradient or hypogammaglobulinemia. However, extra bands hiding monoclonal immunoprotein can infrequently occur. The laboratory must verify such electropherograms by extending the analysis order (reflex testing) and additionally perform immunofixation electrophoresis. In a study /12/, reflex testing had to be performed in 13.2% of 5992 SPEs. When doing this, numerous monoclonal gammopathies were detected that would have been missed had the assessment of the M-gradient been the sole criterion for monoclonal gammopathy (Tab. 18.3-4 – Reflex testing upon abnormalities in serum protein electrophoresis).

Interference of monoclonal antibody therapies with SPE

Therapeutically used monoclonal antibodies represent both chimeric human – mouse immunoglobulins such as rituximab (Rituxan), siltuximab, infliximab (Remicade), cetuximab (Erbitux) and humanized antibodies such as trastuzumab (Herceptin), vevacizumab (Avastin), adalimumab (Humira). Under therapy, these IgG Kappa monoclonal antibodies reach a concentration of approximately 100 mg/L and higher. They can be detected by immunofixation electrophoresis and CZE and, in SPE, migrate in the middle of the γ-globulin zone, whereas rituximab and trastuzumab migrate in the cathodic portion of the γ-globulin zone /13/.

The therapeutic monoclonal antibodies become undetectable in patients approximately 3 months after the cessation of therapy (5 half-lives).

Stability

Serum stored in a closed container at room temperature is stable for 1 day and at 4 °C for up to 1 week.

Quality assurance

In the laboratory, control sera are comparable to human sera for measurement controls of the day-to-day precision and the accuracy.

References

1. Kohne J. Zur Technik der Zelluloseazetat-Elektrophorese. Ärztl Lab 1964; 10: 269–78.

2. Thomas L (ed). Serumeiweiss-Elektrophorese. München: Urban und Schwarzenberg, 1981; 1–49.

3. Bienvenu J, Graziani MS, Arpin F, et al. Multicenter evaluation of the Paragon CZE 2000 capillary zone electrophoresis system for serum protein electrophoresis and monoclonal component typing. Clin Chem 1998; 44: 599–605.

4. Jeppsson JO, Laurell CB, Franzen B. Agarose gel electrophoresis. Clin Chem 1979; 25: 629–38.

5. Grabner W, Bergner D, Wermuth G. Mikrozonenelektrophoresen auf Membranfolien. Ärztl Lab 1970; 16: 193–201.

6. Lichtinghagen R, Pietsch D, Brand K. Evaluation of an automated capillary electrophoresis system for serum protein electrophoresis with the determination of gender-specific reference values. Clin Lab 2010; 56: 119–26.

7. Wuhrmann F, Wunderly C (eds). Die Bluteiweisskörper des Menschen. Basel: Schwabe, 1957: 159–78.

8. Tate J, Caldwell G, Daly J, Gillis D, Jenkins M, Jovanovic S, et al. Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand. Ann Clin Biochem 2012; 49: 242–56.

9. Slev PR, Williams BG, Harville TO, Ashwood ER, Bornhorst JA. Efficacy of the detection of α₁-antitrypsin Z deficiency variant by routine serum protein electrophoresis. Am J Clin Pathol 2008; 130: 568–72.

10. Bossuyt X, Bogaerts A, Schiettekate G, Blanckaert N. Detection and classification of paraproteins by capillary immunofixation/substraction. Clin Chem 1998; 44: 760–4.

11. Bossuyt X, Marien G. False-negative results in detection of monoclonal proteins by capillary zone electrophoresis. Clin Chem 2001; 47; 1477–9.

12. Katzmann JA, Stankowski-Drengler TJ, Kyle RA, Lockington KS, Snyder MR, Lust JA, et al. Specificity of serum and urine protein electrophoresis for the diagnosis of monoclonal gammopathies. Clin Chem 2010; 56: 1899–1900.

13. Mc Cudden CR, Voorhees PM, Hainsworth SA, Whinna HC, Chapman JF, Hammett-Stabler CA, et al. Interference of monoclonal antibody therapies with serum protein electrophoresis. Clin Chem 2010; 56: 1897–8.

18.4 Albumin

Lothar Thomas

Albumin is the most important binding and transport protein of the organism. Its physiological functions include:

• Maintenance of colloidal osmotic pressure in the vessels
• Binding and transport of metabolites, metal ions, bilirubin, free fatty acids, phospholipids, amino acids, hormones (steroid hormones, thyroid hormones) and drugs
• To serve as an amino acid pool for the tissues through albumin hydrolysis
• Major antioxidant in plasma
• Binding and removal of substances produced during cell regeneration.

Determination of albumin is clinically relevant:

• In serum for diagnostic investigation of dysproteinemia
• In urine for early diagnosis of kidney damage, especially in diabetes mellitus and hypertension (see Section 12.9 – Urinary proteins)
• In cerebrospinal fluid for detecting blood-brain barrier dysfunction (see Chapter 46 – Laboratory diagnosis of neurological diseases).

This following section is limited to a description of the significance of albumin in serum.

18.4.1 Indication

• Protein loss (nephrotic syndrome, burns, exudative enteropathy)
• Diagnostic investigation of edematous state
• Prognosis in elderly, hospitalized patients as well as mortality in patients with poly trauma and patients under intensive care
• Index of nutritional state in developing countries.

18.4.2 Method of determination

Immunonephelometric or immunoturbidimetric assays, bromocresol green or bromocresol purple method /1/.

18.4.3 Specimen

Serum, heparin anticoagulated blood: 1 mL

18.4.4 Reference interval

Refer to Ref. /2/ and Tab. 18.4-1 – Reference interval for albumin in serum.

18.4.5 Clinical significance

Only a decrease in the serum albumin concentration is of clinical significance. Hyper albuminemia based on an absolute increase in the albumin level does not occur.

18.4.5.1 Hypoalbuminemia

Hypoalbuminemia may be caused by /3/:

• Reduced synthesis (e.g., as seen in liver dysfunction) or in protein deficient diets
• Expansion of the extravascular compartments (e.g., as in capillary leakage, sepsis or shock)
• Losses into “third space” (e.g., in the case of edema, ascites or pleural effusion)
• Losses to the exterior (e.g., as observed in nephrotic syndrome, burns or exudative enteropathy)
• Acute phase response (albumin synthesis is down regulated in favor of the acute phase proteins); albumin is a negative acute phase protein
• Pregnancy because the plasma volume increases by 40%
• Congenital defect of albumin synthesis.

The concentration of albumin in serum is also viewed as a global, rough indicator of the health and nutritional status of an individual. This especially applies to the elderly and to chronically ill individuals. This does not come as a surprise because albumin responds by a decrease in concentration in numerous clinical disorders (Tab. 18.4-2 – Diseases and conditions associated with hypoalbuminemia). In large epidemiological studies, albumin was associated with many health-related variables. Although the results of these studies may be partially inconsistent, they do indicate that sociodemographic, lifestyle-related and illness-related factors correlate with hypoalbuminemia /4/.

18.4.6 Comments and problems

In comparison to immunoturbidimetric and immunonephelometric assays, the bromocresol green method yields higher concentrations, but approximately 10% lower concentrations in lithium heparin plasma than in serum /15/.

If blood is not collected with the patient in a supine position or having been sitting for at least 15 minutes, a 5–10% increase in the albumin concentration should be anticipated because of hemoconcentration.

18.4.7 Pathophysiology

Albumin has a molecular weight of 66.3 kDa, is synthesized in the liver and is the only plasma protein of that size with no carbohydrate chain. Daily synthesis rate ranges from 150–250 mg/kg of body weight; this requires about 12–20% of the hepatic protein synthesis capacity /16, 17/.

The functional domains of albumin are shown in Fig. 18.4-1 – Albumin structure and functional domains. The N-terminal part of albumin is the binding site for divalent forms of transition metals such as iron, cobalt, nickel and copper. The N-terminus consists of an aspartate-alanine-histidine sequence and is unstable. In the presence of hypoxemia, for example in acute myocardial infarction, free radicals are produced and acidosis develops. Under these conditions, albumin is modified (ischemia-modified albumin, IMA) and the bound transition metals are released /18/. The concentration of IMA is determined based on its binding capacity for cobalt in the Albumin Cobalt Binding Test. IMA binds less cobalt than normal albumin does.

Besides binding metals, albumin transports fatty acids, acts as an antioxidant and has detoxifying capacity. These properties are due to the thiol residue of cysteine 34 (Fig. 18.4-1 – Albumin structure and functional domains). In patients with stroke, hepatic failure or spontaneous bacterial peritonitis, albumin therapy leads to improved clinical condition. It is assumed that this effect is due to the anti oxidative and detoxifying properties of albumin /7/.

Synthesis of albumin is decreased by:

• Increase in the oncotic pressure in extracellular fluid of the liver
• Reduced availability of amino acids
• IL-6-induced stimulation of synthesis of acute phase proteins.

Thyroxine, glucocorticoids and anabolic steroids exert a stimulatory effect on the synthesis of albumin.

The exchangeable albumin pool ranges from 3.5–5.0 g per kg of body weight corresponding to 250–350 g in an individual weighing 70 kg. Approximately 35–40% of this quantity are located in the extravascular space with the largest portion of it in skin and muscles. The liver itself stores only approximately 0.3 g. Albumin that is synthesized by hepatocytes reaches the circulation via the hepatic vein. Approximately 10-fold the quantity of albumin synthesized daily travels from the intravascular to the interstitial space and returns again via lymph vessels.

In comparison to an upright position, plasma albumin concentration declines by about 15% after a supine position has been assumed for at least 30 min.

The half-life of albumin is 19 days. Approximately 0.1 g are lost daily via diffusion into the gastrointestinal tract and 15 mg through the kidneys. Catabolism of albumin, like that of other plasma proteins, occurs in many tissues, especially the capillary endothelial cells, and is the result of continuous pinocytosis. Catabolism is decreased in hypoalbuminemia, although fractional degradation is normal. The plasma albumin concentration mostly depends on defects in distribution and much less on defects in synthesis. If food is withheld, albumin concentration declines below the lower reference interval value after one week at the earliest. In nutritional protein deficiency, the extent of edema correlates only weakly with albumin concentration.

More pronounced albumin losses to the exterior (e.g., as seen in patients with nephrotic syndrome) lead to an increased synthetic rate. Since synthesis of albumin is linked to synthesis of cholinesterase, the activity of this enzyme is increased in serum in patients with albumin loss. Absolute albumin increases in serum do not occur. Elevated levels are almost always due to pseudo hyperalbuminemia (e.g., as in exsiccosis).

Many drugs bind to albumin. Hypoalbuminemia can, therefore, be associated with an increase in the free, pharmacologically active portion of a drug. Drugs that are strongly bound to albumin are, for example, phenytoin and valproic acid. In these patients, hypoalbuminemia can lead to an increased pharmacological effect despite constant dosage of the drug. The binding capacity of albumin for therapeutic drugs may also be altered. For instance, in the presence of renal insufficiency, albumin has a decreased binding capacity for phenytoin and salicylic acid.

Genetic structural variants of albumin are detected in serum protein electrophoresis or are incidental findings (e.g., during the determination of thyroid hormones). For instance, in familial dysalbuminemic hyperthyroidism, total T4 is elevated in the face of normal FT4. The underlying cause is an abnormal albumin with increased binding capacity for T4.

References

1. Hill PG. The measurement of albumin in serum and plasma. Ann Clin Biochem 1985; 22: 565–78.

2. Johnson AM, Guder WG. Albumin. In: Ritchie RF, Novolotskaia O (eds). Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996; 600-1–600-10.

3. Whicher J, Spence C. When is serum albumin worth measuring? Ann Clin Biochem 1987; 24: 572–80.

4. Reuben DB, Moore AA, Damesyn M, Keeler E, Harrison GG, Greendale GA. Correlates of hypoalbuminemia in community-dwelling older persons. Am J Clin Nutr 1997; 66: 38–45.

5. Whittacker PG, Lind T. The intravascular mass of albumin during human pregnancy: a serial study in normal and diabetic women. Br J Obstet Gynecol 1993; 100: 587–92.

6. Rothschild MA, Oratz M, Schreiber SS. Albumin synthesis. N Engl J Med 1972; 286: 816–21.

7. Jalan R, Schnurr K, Mookerjee RP, Sen S, Cheshire L, Hodges S, et al. Alterations in the functional capacity of albumin in patients with decompensated cirrhosis is associated with increased mortality. Hepatology 2009; 50: 555–64.

8. Dramaix M, Hennart P, Brasseur D, et al. Serum albumin concentration, arm circumference, and edema and subsequent risk of dying in children in central Africa. BMJ 1993; 307: 710–3.

9. Rady MY, Ryan T, Starr NY. Perioperative determinants of morbidity and mortality in elderly patients undergoing cardiac surgery. Crit Care Med 2001; 29: S163– S172.

10. Yuki RL, Bar-Or D, Harris L, Shapiro H, Winkler JV. Low albumin level in the emergency department: a potential independent predictor of delayed mortality in blunt trauma. J Emergency Med 2003; 25: 1–6.

11. Feldman JG, Gange SJ, Bacchetti P, Cohen M, Young M, Squires KE, et al. Serum albumin is a powerful predictor of survival among HIV-1-infected women. J Acquired Immune Deficiency Syndromes 2003; 33: 66–73.

12. Gamsjäger T, Brenner L, Sitzwohl C, Weinstabl C. Half-lives of albumin and cholinesterase in critically ill patients. Clin Chem Lab Med 2008; 40: 1140–2.

13. Campagnoli M, Hansson P, Dolcini L, Caridi G, Gagnino M, Candiano G, et al. Analbuminemia in a Swedish male is caused by the Kayseri mutation (c228–229delAT). Clin Chim Acta 2008; 396: 89–92.

14. Galliano M, Campagnoli M, Rossi A, Wirsing von König CH, Lyon AW, Cefle K, et al. Molecular diagnosis of analbuminemia: A novel mutation identified in two American and two Turkish Families. Clin Chem 2002; 48: 844–9.

15. Meng QH, Krahn J. Lithium heparinised blood-collection tubes give fasely low albumin results with an automated bromcresol green method in haemodialysis patients. Clin Chem Lab Med 2008; 46: 396–400.

16. Peters jr T. Serum albumin. In: Putman FW (ed). The plasma proteins. Structure, function, and genetic control, Vol I 2nd ed. New York: Academic Press, 1975: 133–81.

17. Johnson AM, Guder WG. Albumin. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine, Vol I. Scarborough: Foundation for Blood Research, 1996: 600-1–600-12.

18. Collison PO, Gaze DC. Ischaemia-modified albumin: clinical utility and pitfalls in measurement. J Clin Pathol 2008; 61: 1025–8.

18.5 α₁-antitrypsin (AAT)

Lothar Thomas

AAT belongs to the family of serine protease inhibitors, also referred to as serpins. These inhibitors form irreversible complexes with, and thus inactivate, serine proteases such as elastase, chymotrypsin, trypsin and thrombin. AAT is, therefore, also referred to as α₁-proteinase inhibitor (α₁-Pi). It is encoded by the gene SERPINA1. Approximately 120 genetic variants of AAT are known. AAT deficiency is usually diagnosed following the diagnosis of chronic obstructive pulmonary disease (COPD), liver disease or, in some countries, within the scope of newborn screening if familial AAT deficiency is known to exist.

For clinical purposes allelic variants of AAT have been classified in 3 main categories / /1/, 2/:

• Normal (referred to M; M1, M2, M3, M4): the normal variants are characterized by plasma levels in the general population
• Deficient variants: these variants are characterized by missense mutations e.g., the common Pi*Z (Glu342 Lys) and Pi*S (Glu264Val) mutations. Included are the variants with small deletions (M_malton, M_heerlen, Q0_amersfoort, M_würzburg) with decreased but still detectable serum concentrations of AAT
• Null variants (Q0) with no detectable AAT serum concentration. Null variants result from nonsense or frameshift mutations leading to premature stop codons.

Contrary to AAT deficiency, elevated AAT has no clinical relevance. However, causes of elevated AAT must be taken into consideration when assessing AAT deficiency.

18.5.1 Indication

The American Thorax Society/European Respiratory Society (ATS/ERS) /2/ recommend the examination for AAT deficiency in:

• Patients with COPD, asthma, unexplainable liver disease and necrotizing panniculitis
• Individuals with persistent air flow restriction
• Siblings of individuals with known AAT deficiency.

18.5.2 Method of determination

Three strategies are followed for diagnosing AAT deficiency:

• Determination of serum AAT concentration. If the concentration is below a certain cutoff value, the phenotype of the AAT deficiency is determined.
• Phenotyping of AAT for determining the isoform pattern. A disadvantage of this approach is that Pi*null alleles cannot be identified because no proteins are synthesized in this variant.
• Genotyping of SERPINA1 [Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1].

Instead of classifying the AAT type, some laboratories immediately perform AAT genotyping if the AAT concentration is lower than a specific cutoff value.

AAT concentration

Serum protein electrophoresis: among the proteins of the α₁-globulin fraction, AAT is the protein that is predominantly stainable by protein dyes. In homozygous AAT deficiency, the α₁-globulin fraction may be strongly diminished or absent. A normal α₁-globulin band does not rule out AAT deficiency. See also Section 18.3.6 – Comments and problems.

Radial immunodiffusion, immunonephelometry, immunoturbidimetry: For principle, see Section 52.1.5 – Direct antigen or antibody assays.

Proteinase inhibitor (Pi) capacity

Principle: depending on the AAT concentration in the patient’s sample, addition of patient serum to a trypsin-catalyzed reaction inhibits the activity of a defined quantity of trypsin that was previously added /4/. In the test reaction, residual active trypsin releases p-nitroaniline from the added substrate (benzyl-arginine-p-nitroanilide or tosyl-glycyl-lysine-4-nitro anilide acetate); the absorption increase of this reaction product is spectrophotometrically measured at 405 nm. The Pi capacity is primarily determined under therapeutic aspects.

Determination of the AAT phenotype

The AAT phenotype of a patient is determined using isoelectric focusing (IEF) in polyacrylamide gel with a pH gradient of 3.5–5.0. The variants (isoforms) of AAT present in band patterns based on their migration in a pH gradient. Z0 (null), ZZ and SZ, which are the essential of 100 Pi variants with pathogenic significance, can be easily differentiated /4/. The patterns show multiple bands of different mobility, reflecting different AAT glycosylation. Interpretation of the results is influenced by artifacts such as age of the sample and storage conditions.

Classification of the AAT genotype

Genomic DNA is extracted from EDTA whole blood, and PCR is performed followed by melting point analysis. Primers are used for genes containing the Z allele and the S allele /5/. Most commercial tests for molecular identification detect the common AAT variants Pi*S and Pi*Z. However, more than 30 variants also associated with AAT deficiency are not detected.

18.5.3 Specimen

• AAT concentration (serum): 1 mL
• Pi capacity (citrate plasma): 2 mL
• DNA analysis (EDTA blood): 5 mL

18.5.4 Reference interval

* Values are 5th and 95th percentiles. Conversion: mg/L × 19.6 = μmol/L

18.5.5 Clinical significance

AAT is primarily synthesized by the hepatocytes, but also by monocytes, alveolar macrophages and granulocytes. The serum concentration depends on the genotype.

18.5.5.1 Increase in AAT

During acute phase response, AAT concentration usually increases to a maximum of 3-fold. Levels > 5 g/L are measured in patients with squamous cell cancer and adenocarcinoma. With the exception of tuberculosis, levels of such magnitude do not occur in any other pulmonary disease /7/. A 1–2-fold increase in concentration can also occur during pregnancy and when taking oral contraceptives.

18.5.5.2 AAT deficiency

AAT alleles are autosomal co dominantly inherited. More than 100 genetic variants have been described. Pi*M with six subtypes M 1–M 6 is the normal allele and is present in more than 90% of the normal population. The normal subtypes are distinguished by different amino acids due to the substitution of individual bases in the DNA. The PiMM phenotype is associated with a normal concentration and inhibitor capacity of AAT.

Clinical symptoms of AAT deficiency involve the lungs, liver and skin. The following symptoms suggest the presence of AAT deficiency:

• Unexplainable COPD
• Viral hepatitis-marker-negative or non-alcohol-induced liver disease
• Necrotizing panniculitis.

Fig. 18.5-1 – Algorithm for diagnosing AAT deficiency shows an algorithm for diagnosing AAT deficiency /13/.

The majority of patients with decreased AAT concentration and COPD or AAT-deficiency-induced liver disease are either homozygous for PiZ*, compound heterozygous for S and Z alleles (PiSZ) or have a null allele.

Approximately 3.4 million people worldwide (1 in 1500 to 1 in 10,000, depending on the population group) have severe AAT deficiency, and 116 million are carriers of a Pi*Z allele or Pi*S allele, with the highest prevalence found in Europe /8/.

The following distinction is made:

• Individuals with severe AAT deficiency due to homozygosity (PiZZ) or compound heterozygosity (PiSZ) or null alleles. These individuals have an increased risk of COPD in their 3rd to 5th decade of life and of chronic liver disease at a later age.
• Individuals with mild to moderate AAT deficiency have a low risk of COPD. This refers to genotypes that are heterozygous for AAT deficiency-related alleles such as PiMZ (odds ratio 2.31 compared to PiMM) or the Pi*S allele /9/. The latter is more common in many European populations than the Pi*Z allele. Characteristics of selected AAT variants are listed in Tab. 18.5-1 – AAT deficiency and related diseases.

In many patients, AAT deficiency is not detected and the mean interval between initial symptoms and diagnosis is 8 years. Patients are 46 years of age on average at the time of diagnosis and have already consulted a doctor at least three times because of their symptoms /8/. In all, only about 5% of AAT deficiency-related cases are detected since many patients are asymptomatic and only 1% of the patients with COPD have AAT deficiency /1/.

18.5.5.3 Assessment of AAT concentration

The determination of the AAT concentration within the scope of screenings is an important criterion for the detection of AAT deficiency. The lower reference interval value should not be used as a cutoff for the screening because the protein concentrations of PiMM types and deficiency-related types overlap. Severe AAT deficiency is generally ruled out in concentrations above 1.0 g/L /8/, but types with mild AAT deficiency, who also have a risk of COPD, may be missed. For instance, in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) /9/, 95% of the AAT variants, including all clinically relevant ones, were detected at an AAT concentration ≤ 1.13 g/L. Classification of the patients in subgroups based on the AAT concentration led to the genotype distribution presented in Tab. 18.5-2 – AAT concentration and SERPINA1 gene variants.

The prevalence of the ZZ phenotype is 1 in 1639 newborns according to a Swedish study /10/ and 1 in 5097 in a study /11/ performed in the USA. The decrease in AAT concentration in these individuals is below 35% of the mean of PiMM types, corresponding to below 0.70 g/L. This also applies to phenotypes SZ, Szero and Zerozero.

Approximately 5–7% of the population have a deficiency-related or silent allele (MZ, M0) that is linked to a mild to moderate decrease, corresponding to an AAT concentration < 1.0 g/L (Tab. 18.5-3 – Clinical manifestations of AAT deficiency).

Liver disease should not be ruled out in the differential diagnosis of neonatal cholestasis associated with normal AAT concentrations. This is due to rare heterozygous variants. Therefore, it is recommended in neonatal cholestasis and suspected AAT deficiency to perform electrophoretic AAT phenotyping or genotyping /12/.

In carriers of a deficiency allele, the reason for an AAT concentration that still falls within the reference interval may be the acute phase function of the protein in inflammation. Such cases can be discriminated by the simultaneously elevated C-reactive protein.

In AAT concentrations < 1.0 g/L, phenotyping is performed using IFE, whereas up to 22% of the carriers of deficiency-related alleles (M_heerlen, Q0_amersfoort, M_würzburg, Q0_soest) are diagnosed by genotyping /14/.

Substitution therapy in patients with AAT deficiency is performed by using highly purified AAT. Treatment consists of a once-weekly administration of 60 mg/kg of body weight. This dosage is sufficient to maintain a mean serum AAT concentration of 0.3 g/L /15/. For further information refer to Ref. /26/.

18.5.6 Comments and problems

Blood collection

Anticoagulants such as buffered citrate, potassium oxalate, sodium fluoride or EDTA lead to falsely low AAT levels when determining both the concentration and the trypsin inhibitory capacity; heparin, on the other hand, does not interference in either method /21/.

Method of determination

The genotyping of individuals with AAT deficiency using commercial assays only detects those with Pi*Z and Pi*S alleles, but not those with null alleles or other rare variants.

Reference interval

When determining the AAT concentration cutoff value, below which further genotyping and phenotyping are to be performed, care must be taken to take the reagent manufacturer into account because there still are marked differences between the individual manufacturers. In the literature, the cutoff values range between ≤ 1.1 and ≤ 1.0 g/L /8, 9/.

18.5.7 Pathophysiology

AAT is a glycoprotein with a molecular weight of 51 kDa and occurs in approximately equal concentrations in plasma and in interstitial fluid. Its synthetic sites, besides hepatocytes, include alveolar macrophages and monocytes. The daily synthetic rate is 34 mg/kg of body weight and the half-life is 6–7 days.

AAT belongs to the serpin family, including /22/:

• Antithrombin which causes the inactivation of released proteases of the coagulation system, such as thrombin and FXa
• The C1 esterase inhibitor which controls the activation of the complement system
• The plasmin activator (PA) inhibitor (PAI). The PAI inhibits the PA, which converts plasminogen into plasmin and thus activates fibrinolysis.

The AAT, also known as α₁-proteinase inhibitor, inhibits the serine proteases trypsin, chymotrypsin as well as pancreatic and especially polymorphonuclear-granulocytic elastase. Elastase cleaves connective tissue structures such as collagen and elastin. This step is necessary for the formation of pus and the liquefaction of an inflammatory site. However, in tissue surrounding the inflammatory site, AAT limits the elastase effect in order to prevent tissue damage from becoming too widespread.

The serpins differ from other proteinase inhibitor families by the complex manner of protease inhibition associated with a drastic change in structure (Fig. 18.5-2 – Serine protease inactivation by α₁-antitrypsin (AAT)). For instance, mutations in the gene SERPINA1 lead to a change in protein conformation so that the protein can still be synthesized but no longer secreted in the endoplasmic reticulum. This results in a conformation disorder including the precipitation of protein aggregates in the hepatocyte and hepatocellular degeneration as well as AAT deficiency /22/.

AAT exerts its effect mainly on epithelial and serous surfaces. Development of pulmonary emphysema in patients with AAT deficiency is thought to be caused by an imbalance between elastase and AAT. Imbalance is due to an activation of granulocytes by the platelet-activating factor, which is released by macrophages and granulocytes; as a mediator of inflammation, this platelet-activating factor stimulates granulocytes to release peroxidases and elastase.

A significant part of the mutants in the gene SERPINA1 has no influence on protein expression and function. However, several alleles encode an AAT that either exists in the circulation at decreased concentration or is dysfunctional.

Based on their pathomechanism, disease-associated alleles are classified in /13/:

• Deficiency alleles such as Pi*Z. The Glu342Lys mutation changes the protein structure of the AAT protein. This results in an aggregation and polymerization of the molecule in the endoplasmic reticulum of the hepatocytes including the precipitation and formation of hepatocellular inclusion bodies.
• PiS-protein allele. The protein of this Glu264Val mutation polymerizes but is secreted and relatively quickly eliminated from the blood; as a result, serum concentration is only 60% of the normal value.
• Null alleles; they either produce no transcript or garbled or unstable AAT causing degradation already prior to secretion from the cell /23/.
• Dysfunctional alleles encode an AAT that does not bind to elastase but to other proteins such as antithrombin.

Refer also to Tab. 18.5-4 – Characteristics of selected α₁-antitrypsin variants.

Individuals with the PiSS phenotype always produce sufficient AAT so that severe AAT deficiency does not manifest and clinical symptoms manifest but rarely.

Carriers of a Pi*Z allele are characterized by decreased AAT concentration and decreased inhibitor capacity regarding polymorphonuclear-granulocytic elastase. This results in unrestricted elastase activity and development of COPD. The Z variant of AAT has an unstable conformation leading to the polymerization of several AAT molecules and the formation of hepatocellular inclusion bodies.

Hence, liver disease in carriers of Pi*Z alleles is not based on AAT deficiency but on abnormal AAT polymerization. The β chain of the AAT molecule in the rough endoplasmic reticulum has an altered conformation causing the entire molecule to become unstable. As a result, the peptide chain of the second AAT molecule is able to incorporate into the structure of the first molecule causing the formation of a dimer (Fig. 18.5-3 – Polymerization of two AAT molecules). The entire process can multiply, leading to the formation of polymers that can no longer be released from the endoplasmic reticulum and precipitate as a result. The resulting inclusion bodies are subject to degradation. The development of hepatocellular necrosis is thought to depend on the balance between AAT aggregation and inclusion body degradation. The fact that aggregate formation increases with increasing temperature is an explanation why newborns with the ZZ phenotype with fever more often develop liver disease than those without fever.

References

1. American Thoracic Society/European Respiratory Society Statement. Standards for the diagnosis and management of alpha₁-antitrypsin deficiency. Am J Respir Crit Care Med 2003; 168: 818–900.

2. Balderacci AM, Barzon V, Ottaviani S, Corino A, Zorzetto M, Wencker M, et al. Comparison of different algorithms in laboratory diagnosis of alpha₁-antitrypsin deficiency. Clin Chem Lab Med 2021; 59 (8): 1384–91.

3. Witt J, Tritscher W. α₁-antitrypsin: Referenzwerte im Serum und Plasma mit Benzoyl-D, L-arginin-p-nitroanilid, einem chromogenen Substrat. J Clin Chem Clin Biochem 1982; 20: 587–91.

4. Jeppsson JO, Franzen B. Typing of genetic variants of alpha 1-antitrypsin by electrofocusing. Clin Chem 1982; 28: 219–25.

5. Braun A, Meyer P, Cleve H, Roscher AA. Rapid and simple diagnosis of the two common α₁-proteinase inhibitor deficiency alleles PiZ and PiS by DNA analysis. Eur J Clin Chem Clin Biochem 1996; 34: 761–4.

6. Luisetti M, Seersholm N. α₁-Antitrypsin deficiency. 1. Epidemiology of α₁-antitypsin deficiency. Thorax 2004; 59: 164–9.

7. Silverman EK, Sandhaus RA. Alpha₁-antitrypsin deficiency. N Engl J Med 2009; 360: 2749–57.

8. Kalsheker NA. α₁-antitrypsin deficiency: best clinical practice. J Clin Pathol 2009; 62: 865–9.

9. Zorzetto M, Russi E, Senn O, Imboden M, Ferrarotti I, Tinelli C, et al. SERPINA1 gene variants in individuals from the general population with reduced α₁-antitrypsin concentrations. Clin Chem 2008; 54: 1331–8.

10. Sveger T. Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants. N Engl J Med 1976; 294: 1316–21.

11. US Consensus Bureau. 2000 Consensus of population and Housing; summary population and housing characteristics, PHC 1-1, Washington, DC: US Consensus Bureau, 2002.

12. Lang T, Mühlbauer M, Strobelt M, Weidinger S, Hadorn HB. Alpha-1 antitrypsin deficiency in children: liver disease is not reflected by low serum levels of alpha-1 antitrypsin – a study on 48 pediatric patients. Eur J Med Res 2005; 10: 509–14.

13. Snyder MR, Katzmann JR, Butz ML, Yang P, Dawson DB, Halling KC, et al. Diagnosis of α₁-antitrypsin deficiency: an algorithm of quantification, genotyping, and phenotyping. Clin Chem 2006; 52: 2236–42.

14. Prins J, van der Meijden BB, Kraaijenhagen RJ, Wielders JPM. Inherited chronic obstructive pulmonary disease: new selective-sequencing workup for α₁-antitrypsin deficiency identifies 2 previously unidentified null alleles. Clin Chem 2008; 54: 101–7.

15. McElvaney NG, Crystal RG. Therapy of α₁-AT deficiency. In: Crystal RG (ed). Alpha 1-antitrypsin deficiency. New York: Marcel Dekker, 1996: 319–31.

16. Agusti A, Hogg JC. Update in the pathogenesis of chronic obstructive pulmonary disease. N Engl J Med 2019; 381: 1248–56.

17. Tanash HA, Nilsson PM, Nilsson JA, et al. Clinical course and prognosis of never smokers with severe alpha₁-antitrypsin deficiency. Thorax 2008; 63: 1091–5.

18. Larsson C. Natural history and life expectancy in severe alpha1-antitrypsin deficiency PiZ. Acta Med Scand 1978; 204: 345–51.

19. McBean J, Sable A, Maude J, Robinson-Bostom L. α₁-antitrypsin deficiency panniculitis. Cutis 2003; 71: 205–9.

20. Bernspang E, Carlson J, Piitulainen E. The liver in 30-year-old individuals with alpha₁-antitrypsin deficiency. Scand J Gastroenterol 2009; 44: 1349–55.

21. Berninger RW, Teixeira F. α₁-antitrypsin: the effect of anticoagulants on the trypsin inhibitor capacity, concentration and phenotype. J Clin Chem Clin Biochem 1985; 23: 277–81.

22. Carrell RW, Lomas DA. Alpha1-antitrypsin deficiency – a model for conformational diseases. N Engl J Med 2002; 346: 45–53.

23. Lee JH, Brantly M. Molecular mechanisms of alpha1-antitrypsin null alleles. Respiratory Medicine 2000; 94 Suppl C: S7–S11.

24. Lomas DA. Loop-sheet polymerization: the mechanism of alpha₁-antitrypsin deficiency. Respiratory Medicine 2000; 94, suppl C: S3–S6.

25. Stoller JK, Aboussouan L. Alpha₁-antitrypsin deficiency. Lancet 2005; 365: 2225–36.

26. Chapman KR, Chrostowska-Wynimko J, Koczulla AR, Ferrarotti I, McElvaney NG. Alpha I antitrypsin to treat lung disease in alpha I antitrypsin deficiency: recent developments and clinical implications. Int J of COPD 2018; 13: 419–32.

18.6 Alcohol biomarkers

Lothar Thomas

Laboratory tests to detect light drinking and alcohol abuse offer the opportunity to objectively verify the information about alcohol consumption provided by a person or patient. A differentiation is made between /1/:

• Direct biomarkers; these markers are created when ethanol is metabolized or reacts with substances in the body
• Indirect biomarkers, which undergo typical changes in response to acute or chronic alcohol consumption e.g., increase in gamma glutamyl transferase or increase of mean cell volume (MCV) of red cells.

An overview is provided of the alcohol markers ethanol, ethyl glucuronide, carbohydrate deficient transferrin, and some indirect biomarkers.

18.6.1 Ethanol

Alcohol refers to ethanol which may carry synonyms of ethyl alcohol. The molecular weight of ethyl alcohol (chemical formula: CH₃CH₂OH) is 46 Da and the density is 0.79. If another alcohol is consumed, it must be specifically identified and the medical effect differently assessed /2/.

Note the following Tables and Figures:

• Tab. 18.6-1 – Effects of alcohol consumption
• Tab. 18.6-2 – What is a standard drink?
• Tab. 18.6-3 – Acute metabolic changes following alcohol consumption
• Fig. 18.6-1 – Metabolism of ethanol.

18.6.2 Ethyl glucuronide (EtG)

Anaerobic metabolim of ethanol generates direct markers of alcohol like ethyl glucuronide (EtG). This compound is a product of the conjugation reaction catalyzed by UDP-glucuronosyltransferase (Fig. 18.6-2 – Structure of ethyl glucuronide). EtG is nonvolantile, water-soluble, stable, and detectable long after complete elimination of alcohol. EtG is concentrated by almost 200 times in urine as compared to serum; the window of detection in urine is up to 90 h. This makes EtG a preferred marker for the detection of alcohol consumption in medical and forensic diagnostics /3/.

18.6.3 Carbohydrate-deficient transferrin

The structure of transferrin (Tf), the Fe³⁺-transporting protein, usually consists of two branched carbohydrate chains with a sialic acid residue at the end of each of the four branches. Tetrasialo transferrin, for example, is intact Tf. Long-term continuous consumption of more than 60–80 g of pure alcohol per day causes defective Tf glycosilation. This results in an increase in Tf isoforms lacking one or two N glycans (e.g., disialo Tf, monosialo Tf and asialo Tf) (Fig. 18.6-3 – Structure of transferrin and disialotransferrin). These isoforms are correlated with chronic alcohol abuse and are collectively referred to as carbohydrate-deficient transferrin (CDT). The quantitative determination of CDT is used for diagnosing chronic alcohol abuse. CDT is currently the best indirect biomarker in this context because it yields the lowest rate of false-positive results compared to other laboratory assays /4/.

18.6.4 Indication

Based on the window between the last drink and the collection of the sample different biomarkers can show a positive result /2, 5/:

• Breath test: up to 10–12 h after the last drink
• Alcohol in blood or urine: up to 10–12 h after the last drink
• Ethyl glucuronide in blood: up to 20 h after the last drink
• Ethyl glucuronide in urine: up to 24 h after small quantities of alcohol, up to 130 h after excessive consumption
• Carbohydrate deficient transferrin: chronic excessive drinking.

18.6.5 Method of determination

Alcohol in blood, serum, and plasma

Principle of alcohol dehydogenase (ADH) method: in the forward direction ethanol is oxidized in the presence of NADP to aldehyde and NAPH₂. The reaction is catalyzed by the enzyme ADH (EC 1.1.1.2).

Ethyl glucuronide in urine

High performance liquid chromatography coupled to tandem mass spectrometry with deuterated internal standards /6/.

Immunoassay: Refer to Ref. /7/.

CDT immunoassay in serum/plasma

Principle: the CDT immunoassay is based on the application of a monoclonal antibody that is specifically directed against CDT isoforms lacking one or several N-glycans /8/. The Tf concentration is determined simultaneously in a second immunoassay. Polystyrene particles coated with a monoclonal anti-CDT antibody are agglutinated by CDT-coated polystyrene particles. The CDT in the sample inhibits the reaction between the antibody coated and CTD-coated particles in a dose-dependent manner. The extent of agglutination depends on the CDT concentration in the sample. The increase in light scattering measured by nephelometry is recorded and %CDT is calculated based on simultaneously determined Tf.

18.6.6 Specimen

Serum: 1 mL

Urine: 5 mL

18.6.7 Reference interval

Threshold to differentiate between patients with and without alcohol dependence:

• Ethanol in serum/plasma: 5 mg/L. Threshold for recent alcohol intake; end of drinking not longer than 1 day ago /2/.
• Ethyl glucuronide in urine: 100–200 μg/L. Any drinking the night before should be detectable the following morning with these cutoffs /9/
• CDT in serum/plasma: relative threshold (immunoassay): ≤ 2.5% /8/.

18.6.8 Clinical significance

Alcohol biomarkers are ordered to objectively evaluate alcohol consumption, excessive alcohol abuse, and to monitor alcohol abstinence. The selection of the biomarker is dependent on the amount of consumed alcohol and the width of the time window between the last alcohol consumption and collection of the sample to be analyzed.

The strategies for the evaluation of alcohol consumption are classified into direct and indirect methods /10/.

• Direct methods encourage self-reporting of patients (Alcohol Use Disorder Identification Test or AUDIT).
• Indirect methods comprise clinical tests, indirect questionnaires and laboratory investigations.

Laboratory investigations can raise the suspicion of alcohol abuse but must always be considered in combination with medical history, clinical examination and direct evaluation /11/.

• Diseases induced by chronic alcohol abuse are shown in Tab. 18.6-4 – Diseases induced by chronic alcohol abuse
• Typical laboratory markers of alcoholism are shown in Tab. 18.6-5 – Laboratory investigations of alcohol consumption.

18.6.8.1 Definitions

Alcohol consumption is defined according to the Dietary Guidelines for Americans 2015–2020 of the US Department of Health and Human Services and US Department of Agriculture:

• Moderate drinking is up to 1 standard drink per day for women and up to 2 drinks per day for men
• A heavy drinking day is defined as over 3 standard drinks for women and over 4 standard drinks for men
• Binge drinking is a pattern of drinking that brings blood alcohol concentration levels to 0.08 g/dL (17.4 mmol/L). This typically occurs after 4 standard drinks for women and 5 drinks for men in about 2 hours.
• Heavy alcohol use is binge drinking on 5 or more days in the past month.
• Low risk drinking for developing alcohol use disorders is no more than 3 drinks on any single day and no more than 7 drinks per week (for women) and no more than 4 drinks on any single day and no more than 14 drinks per week (for men).
• Alcohol use disorder is a chronic relapsing brain disease characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences.

For definition of standard drink refer to Tab. 18.6-2 – What is a standard drink?

18.6.8.2 Ethanol: marker of acute alcohol consumption

Measures of blood alcohol such as breath test, alcohol determination in blood or urine can only detect alcohol use during the preceding 12 hours, making them suitable for detection current intoxication only /12/.

18.6.8.3 Ethyl glucuronide: marker of short term alcohol consumption

Ethyl glucuronide (EtG) can be detected in blood within 45 min. after alcohol consumption and the time window in serum is by up to 8 h longer compared to ethanol. In urine EtG can be proven for up to about 24 h even after consumption of small quantities. After excessive consumption the window of detection is up to 130 hours /13/.

Results of a dose ranging alcohol challenge study have shown the following results /9/:

• Any drinking the night before are detectable the following morning with EtG cutoffs of 100 or 200 μg/L. Twenty-four hours after drinking, sensitivity is poor for light drinking, but good for heavier consumption.
• At 48 hours, sensitivity is low following 6 drinks or less.
• Increasing the cutoff to 500 μg/L leads to substantially reduced sensitivity.

The results of a study using ethyl glucuronide in urine to detect light and heavy drinkers presented the following results /14/:

• The 100 μg/L cutoff detected > 76% of light drinking for two days, and 66% at 5 days
• The 100 μg/L cutoff detected 84% (1day) to 79% (5 days) of heavy drinking
• The 200 μg/L cutoff detected > 55% of light drinking and > 66% of heavy drinking across 5 days
• The 500 μg/L cutoff identified 68% of light drinking and 78% of heavy drinking for 1 day with detection of light (2–5 days < 58%) and heavy drinking (5 days < 71%) decreasing thereafter.

EtG in urine is a suitable marker both for alcohol withdrawal or quit drinking programs and for abstinence checks prior to proposed liver transplantation or inclusion in the waiting list /1/.

18.6.8.4 Carbohydrate-deficient transferrin: marker of chronic excessive alcohol intake

Carbohydrate-deficient transferrin (CDT) is the most specific biomarker of chronic alcohol abuse. Increased amounts of CDT appear with high prevalence in serum if alcohol abuse of 50–80 g of alcohol per day are consumed on at least 7 consecutive days. The %CDT decreases to within the reference interval after abstinence with a half-life of approximately 14 days /15/. The %CDT varies only slightly in unchanged alcohol consumption. During alcohol withdrawal, the decrease in %CDT in relation to the initial value is conclusive in longitudinal assessment /16/.

The diagnostic sensitivity of %CDT for detection of chronic alcohol abuse is 30–50% for women and 50–70% for men /15/.

The diagnostic specificity of %CDT is > 90% and, thus, markedly higher than that of GGT. Hence, %CDT is currently the best biomarker for excluding chronic alcohol abuse /15/.

Gender dependence

It has been found that CDT concentration (U/L, mg/L) is gender-related, while %CDT is not /16/.

Pregnant women tend to have increased CDT concentrations. However, these increases are small and, therefore, irrelevant in the clinical assessment of %CDT /17/.

Advantages of %CDT determination

No correlations have been found between the serum concentrations of CDT and Tf /15/. The proportion of CDT in Tf (%CDT) is recommended as an assessment criterion for compensating changes in Tf concentration due to electrolyte/water imbalance or an increase/decrease in Tf concentration. %CDT has the following advantages over concentration data:

• Fewer false-positive findings (increased diagnostic specificity) in individuals with normal alcohol consumption and elevated Tf concentration (e.g., in the presence of iron deficiency) /18/.
• Fewer false-negative findings (increased diagnostic sensitivity) in individuals with chronic alcohol abuse and low Tf concentration due to acute or chronic infections, hemochromatosis and tumor diseases /15/.

GGT and %CDT

No correlations have been found between GGT activity and %CDT. The combined determination of CDT and GGT makes sense due to the high diagnostic sensitivity of GGT and the good diagnostic specificity of CDT.

%CDT and MCV

No correlation has been found between %CDT and MCV. The combined determination of %CDT and MCV does not make sense because both have good diagnostic specificity /15/.

18.6.9 Comments and problems

Blood collection

Venous blood collection without specific preparation of the patient; CDT is not influenced by food intake or time of day. Daily intraindividual CDT fluctuation is 8%.

Method of determination

In chromatographic and electrophoretic methods, the TF phenotype D leads to falsely high %CDT and the TF phenotype B leads to falsely low %CDT values. This is not the case in the homogeneous CDT immunoassay. The latter assay is also not interfered by the serum of patients with hepatic disease.

Influence factors

CDT is largely unaffected by drugs. Disulfiram, an aldehyde dehydrogenase inhibitor used for the treatment of alcohol dependence, does not affect CDT. False-positive %CDT findings are listed in Tab. 18.6-6 – Diseases and conditions that may lead to false-positive CDT findings.

EtG can be detected in samples at low levels and can be positive after exposure to alcohol from non-beverage sources or incidental exposure, which lead to false positives. Some healthcare workers, who are exposed to alcohol containing hand washes repeatedly throughout the day, might be positive if tested shortly thereafter. Using a cutoff of 200 μg/l might reduce the risk of such false positives /9/.

Stability

CDT in serum is stable for 30 hours at room temperature, for up to 7 days at 4 °C, and for several months to years at –22 °C; CDT increases by 25% after 3 days at room temperature. CDT is not affected by repeated freezing and defrosting. When stored at 4 °C in airtight test tubes, EtG concentration remains relatively constant.

18.6.10 Pathophysiology

Alcohol

The biochemical effects of alcohol are represented in Fig. 18.6-4 – Biochemical effects of ethanol.

Alcohol content in drinks: some notes /19/

• The amount of alcohol contained in drinks is expressed most commonly as percent by volume (% v). The (% v) multiplied with the density of ethanol gives the percentage of the drink in grams (Tab. 18.6-7, equation 1)
• The total amount of alcohol consumed in grams is calculated according to Tab. 18.6-7, equation 2
• The concentration of blood alcohol is calculated from the amount of alcohol consumed taking into account the reduction weight. Explanation: muscles and brain take up more alcohol in relation to bone and adipose tissue. The body weight must be corrected by the reduction factor 0.7 for calculating the relation of the amount of alcohol to the body weight (body weight × 0.7 = reduction weight). The reduction weight multiplied by the blood alcohol concentration (‰) expresses the amount of consumed alcohol (Tab. 18.6-7, equation 3).
• Within a few hours following alcohol consumption, the blood alcohol level can be used to calculate the amount of alcohol consumed (Tab. 18.6-7, equation 4)
• The serum alcohol concentration (mmol/L) is converted into blood alcohol concentration (‰) according to Tab. 18.6-7, equations 5.

Ethyl glucuronide (EtG)

None oxidative metabolism of ethanol generates compounds, which are called direct markers of alcohol. This group includes EtG, ethyl sulfate and phosphatidyl ethanol. EtG is formed by glucuronidation following exposure to ethanol. The molecular mass is 222 g/mol, the molecular formula is C₈H₁₄O₇, and the elimination half-life is 2–3 hours. The concentration of serum EtG is a function of two opposing influences; dose of alcohol consumed and time window elapsed between consumption and sample collection. The concentration in urine is dependent on diuresis. The intake of alcohol contained in large volumes of water results in a steep decline of EtG level in urine. Therefore a minimum requirement on the urine creatinine concentration is > 20 mg/dL (1.77 mmol/L).

Carbohydrate-deficient transferrin (CDT)

Tf is a glycoprotein consisting of a single polypeptide chain containing 679 amino acid residues and carrying two N-linked carbohydrate chains at position 413 and 611 (Fig. 18.6-3 – Structure of transferrin (top) and disialotransferrin).

There are three known causes of Tf heterogeneity:

• Variation of the amino acid structure of the polypeptide chain due to genetic polymorphism. Using starch gel electrophoresis, the wild type TfC can be differentiated from the more quickly migrating type TfB and the slower cathodic type TfD. All genetic variants have normal iron binding capacity.
• The iron content. Among the four isoforms, diferric Tf binds iron in the N-terminal and C-terminal domains, monoferric Tf binds iron in the N-terminal and C-terminal domains and apo-Tf has no bound iron ions. The N-terminal domain has dominant iron binding capacity.
• The carbohydrate chains. Each of the two oligosaccharide chains in the N-terminal domain structurally varies in branching, with mostly bi- or tri-antennary structure. Each antenna is terminated by sialic acid.

The following CDT isoforms are distinguished: asialo Tf (< 0.5%), monosialo Tf (< 0.9%), disialo Tf (< 2.5%), trisialo Tf (4.5–9.0%), tetrasialo Tf (64–80%), pentasialo Tf (12–18%), hexasialo Tf (1.0–3.0%) and heptasialo Tf (< 1.5%). The isoelectric point of the isoforms depends on the number of sialic acid residues.

The alcohol-induced increase in CDT concentration is due to ethanol-induced and/or acetaldehyde-induced defective synthesis of the N-carbohydrate chains of Tf. For instance, lower activities of galactosyl transferase and N-acetyl glucosaminyl transferase are measured in the serum of alcoholics.

References

1. Andresen-Streichert H, Müller A, Glahn A, Skopp G, Sterneck M. Alcohol biomarkers in clinical and forensic contexts. Dtsch Arztebl Int 2018; 115: 309–15.

2. NCCLS. Blood alcohol testing in the clinical laboratory. Proposed Guideline. NCCLS Vol 8 No. 10; 1988.

3. Shukla L, Sharma P, Genesha S, Ghadigaonkar D, Thomas E, Kandasamy A, et al. Value of ethyl glucuronide and ethylsulfate in serum as biomarkers of alcohol consumption. Indian J Psycol Med 2017; 39: 481–7.

4. Maenhout TM, Uytterhoeven M, Lecocq E, De Buyzere ML, Delanghe JR. Immunonephelometric carbohydrate-deficient transferrin results and transferrin variants. Clin Chem 2013; 59: 997–1000.

5. Lowe JM, Mc Donell MG, Leickly E, Angelo FA, Villadarga R, McPherson S, , et al. Determining ethyl glucuronide cutoffs when detecting self-reported alcohol use in addiction treatment patients. Alcohol Clin Exp Res 2015; 39: 905–10.

6. Weimann W, Schaefer P, Thierauf A, Schreiber A, Wurst FM. Confimatory analysis of etylglucuronide in urine by liquid-chromatography/electrospray ionization/tandem mass spectrometry according to forensic guidelines. J Amer Mass Spectrometry 2004; 15: 188–93.

7. McDonell MG, Srebnik D, Angelo F, Sugar AM, Howell D, Rainey C, et al. Evaluation of ethyl glucuronide immunoassay urinanalysis in five alcohol dependent outpatients. Am J Addict 2011; 20: 482–5.

8. Delanghe JR, Helander A, Wielders JPM, Pegelharing JM, Roth HJ, Schellenbeg F, et al. Development and multicenter evaluation of the N latex CDT direct immunonephelometric assay for serum carbohydrate-deficient transferrin. Clin Chem 2007; 53: 1115–21.

9. Jatlow PI, Agro A, Wu R, Nadim H, Toll BA, Ralevski E, et al. Ethylglucuronide and ethyl sulfate assays in the clinical trial, interpretation and limitations: results of a dose ranging alcohol challenge study and two clinical trials. Alcohol Clin Exp Res 2014; 38: 2056–65.

10. Sharpe PC. Biochemical detection and monitoring of alcohol abuse and abstinence. Ann Clin Biochem 2001; 38: 652–64.

11 Topic A, Djukic M. Diagnostic characteristics and application of alcohol biomarkers. Clin Lab 2013; 59: 233–45.

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26. Sharpe PC. Biochemical detection and monitoring of alcohol abuse and abstinence. Ann Clin Biochem 2001; 38: 652–64.

18.7 Ceruloplasmin: Wilson’s disease and other copper disorders

Lothar Thomas

Ceruloplasmin (Cp) is the copper (Cu)-carrying protein in the circulation. A Cp molecule binds 6 Cu atoms; 90% of the Cu in plasma are present in the form of Cp-Cu complexes functioning as the source of Cu supply to peripheral organs such as the brain and kidneys.

Cp is synthesized in the liver. The hepatocytes secrete Cp as copper (Cu)-carrying protein into the circulation. Cp is an acute phase protein and, in serum protein electrophoresis, migrates in the α₂-globulin band.

Moreover, the functions of Cp include:

• Ferroxidase activity (regulation of the oxidative status of iron and other metal ions); for instance, Cp oxidizes Fe²⁺ to Fe³⁺
• Anti oxidative effect (influence of the redox state of plasma) due to the prevention of metal-ion-catalyzed oxidation of lipids in the cell membrane
• Oxidation of nitrogen monoxide (NO^.) and influence on NO^. homeostasis (see Section 19.2 – Oxidative stress).

The serum Cp concentration is diagnostically relevant because a low level suggests the presence of Wilson’s disease and Menkes disease. For diagnosing Wilson’s disease, Cp should be assessed in association with the urinary Cu excretion. See Section 10.4 – Copper.

18.7.1 Indication

• Exclusion of Wilson’s disease in all children with suspected autoimmune hepatitis or non-alcoholic fatty liver disease (NAFLD). Wilson’s disease is to be suspected in children above 5 years of age who have sonographically detectable fatty liver or any form of acute liver failure /1, 2/.
• Hepatitis-marker-negative liver disease in childhood or adolescence (suspected Wilson’s disease)
• Patients with extra-pyramidal, cerebellar or cerebral symptoms of subacute or chronic nature. The most common initial symptoms are difficulties in speaking and swallowing (suspected Wilson’s disease)
• Neurodegenerative symptoms and signs of connective tissue disease in infants and small children (suspected Menkes disease).

18.7.2 Method of determination

Immunoassay and immunonephelometric or immunoturbidimetric assays.

18.7.3 Specimen

Serum: 1 mL

18.7.4 Reference interval

Refer to References /3, 4/ and Tab. 18.7-1 – Reference intervals for ceruloplasmin.

18.7.5 Clinical significance

Wilson’s disease is only the decrease in serum Cp concentration that is clinically relevant. However, when assessing the Cp concentration, diseases and conditions must be taken into consideration that may lead to an increase in Cp and mask a decrease in concentration.

18.7.5.1 Increase in Cp concentration

Elevated Cp levels in serum up to 3-fold the upper reference interval value may occur as part of the acute phase response in inflammation, especially during bacterial infections. Serum protein electrophoresis shows an increase in the α₂-globulin fraction.

Depending on the dose, intake of oral contraceptives or estrogens during menopause leads to a 20–30% increase in serum Cp concentration. Cp can be elevated as high as three-fold during pregnancy.

18.7.5.2 Decrease in Cp concentration

Diseases that may be associated with a decrease in the serum Cp concentration include Wilson’s disease, nutritional copper deficiency, Menkes disease and other neurological copper disorders /5/.

18.7.5.3 Wilson’s disease (WD)

WD may present as liver disease, a neurologic disorder,.a psychiatric illness or a combination of these disorders. Clinically evident WD is relentlessly progressive and ultimatively fatal, if untreated /6/. WD is an autosomal recessive condition, results in a Cu deposition in the hepatocytes, brain, iris and kidney. The excretion of Cu into the bile is delayed and the incorporation of Cu in Cp is impaired. Most patients present with predominantly hepatic or predominantly neurological Cu disorders. WD is a monogenic, autosomal recessively inherited condition. The causative gene ATP7B encodes a Cu-transporting P-type ATPase /7/. The latter can be associated with symptomatic or asymptomatic involvement of the liver. WD can manifest at any age, although usually before the age of 50 years older age does not rule it out /6/.

Liver disease of WD

In children hepatic WD ranges from mild liver disease and fatty liver disease to cirrhosis. Infrequently, WD manifests as acute liver failure, the first manifestation of WD.

Clinical findings: severe coagulopathy, hepatic encephalopathy, acute intravascular hemolysis.

Laboratory findings: the liver enzyme pattern indicates moderate elevations of ALT, AST, and ALP and progression to renal failure. A ratio of increase of ALP/total bilirubin of < 4 and a ratio of AST/ALT > 2.2 was 100% sensitive and specific for diagnosing WD.

Neurologic disorders of WD

The disorders involve either increased movement with tremor and dystonia or decreased movement, resembling parkinsonian rigidity /6/.

Psychiatric disorders of WD

The symptoms are highly variable, although depression is common /6/.

Laboratory findings

• Serum ceruloplasmin (Cp): Concentrations of 11 to 14 mg/dL are reported although levels < 5 mg/dL strongly supports WD. However serum Cp is not adequate for WD.
• 24 hours copper excretion in urine > 40 mg
• Kayser-Fleischer rings by means of slit-lamp examination
• Hepatic parenchymal copper level of 250 μg/g dry weight
• ATP7B mutations: More than 500 ATP7B mutations have been described. Most are missense mutations, small deletions/insertions in the coding region, or splice junction mutations /8/. Among Caucasians in Europe and North America, the point mutation H1069Q is the most common ATP7B mutation and 50–80% of WD patients carry at least one H1069Q allele.

Firm genotype-phenotype associations for other more common ATP7B mutations do not reveal a clear correlation. In a study /9/, mutations in both chromosomes were diagnosed in 57% of the patients with WD. Approximately 15% of the patients showed no mutation; this was attributed to undetected mutations in the promoter region and exons that had not been analyzed.

There is an association between the presence of the ATP7B H1069Q mutation, the incidence of neurological symptoms and age.

For instance, patients with WD homozygous and heterozygous for the H1069Q mutation or without the H1069Q mutation had /11/:

• Neurological symptoms in 63%, 43% and 15% of the cases, respectively
• A mean age of 20.9, 15.9 and 12.6 years.

In another study /9/, patients with WD presenting with neurological symptoms were 20.2 ± 10.8 years of age, and those with hepatic symptoms were 15.5 ± 9.6 years of age. Diagnosing of the disease took 44.4 months in the patients with neurological symptoms and 14.4 months in the patients with hepatic symptoms.

Prevalence of Wilson’s disease

Data of a study /12/ suggested an unexpectedly high rate (approximately 1 in 40) of ATP7B heterozygote mutation carriers, predicting a 1 : 7.000 prevalence for WD in the United Kingdom population.

Diagnostic approaches of WD

Evidence of WD is based on the correct assignment of clinical symptoms of hepatitis, neurological symptoms of disease, and on laboratory investigations. In a retrospective study /10/, the prevalence of diagnostic parameters in patients with WD are shown in Tab. 18.7-3 – Prevalence of findings in Wilson’s disease patients. Patients with cholestatic liver disease were found at the time of diagnosis in 173 patients with WD at a mean age of 17 years with predominantly hepatic symptoms and at a mean age of 24 years with predominantly neurological symptoms.

The spectrum of laboratory investigations according to the recommendations of the American Association for the Study of Liver Diseases (AASLD) is presented in Tab. 18.7-4 – Diagnosis of Wilson’s disease according to the AASLD practice guidelines.

A diagnostic algorithm /1/ is shown in Fig. 18.7-1 – Diagnostic procedure for Wilson’s disease in patients with unexplainable liver disease.

A laboratory test by itself does not provide evidence of, or rules out, Wilson’s disease. In genetic diagnostics of mutations, total sequencing does not cover the entire gene. Moreover, the determination of the hepatic Cu concentration leaves doubt in the case of ambiguously elevated levels /2/.

A scoring system for WD diagnosis developed at the international WD meeting in Leipzig 2001 is presented in Tab. 18.7-5 – Scoring system for Wilson’s disease.

Wilson’s disease and other disorders associated with a decrease in Cp level are shown in:

• Tab. 18.7-6 – Diseases that may be associated with Cp deficiency.
• Tab. 18.7-7 – Findings in patients with liver disease and acute liver failure.

18.7.6 Comments and problems

Method of determination

Cp is a labile protein and is easily fragmented in serum or during determination. Immunonephelometry and immunoturbidimetry are less affected by this problem than radial immunodiffusion /3/. Serum and urinary Cu concentrations should be determined using the atomic absorption method.

Stability

Storage at 4 °C if determination is performed within 3–4 days. Longer-term storage in a deep-frozen state, only /3/.

18.7.7 Pathophysiology

The populations of industrial countries have a daily dietary intake of 5 mg Cu via the upper gastrointestinal tract. Since many components of the diet are rich in Cu, there is practically no Cu deficiency. Cu is taken up by the liver via the portal circulation within 4 hours and 6–8% Cu bound to Cp appear in the plasma within 24 hours. See also Section 10.4 – Copper.

Cp is a glycoprotein with a molecular weight of 132 kDa and has a carbohydrate content of about 9%. The Cp molecule binds 6 Cu atoms.

Cp is synthesized in the hepatocyte as apoCp. The Cu atoms are incorporated post translationally, followed by the binding of the carbohydrate side chains.

Incorporation of Cu atoms into apoCp occurs intracellularly by P1-type ATPase (ATP7B). The Cu atoms in CP are mostly in the Cu(II) state. The entrance of Cu atoms via the CTR1 receptor into the cell is not the rate-limiting step for uptake of Cu from Cp. It seems highly likely that a reductase is needed to provide Cu(I) for uptake /14/.

In comparison to Cp with a half-life of 4 days, apoCp has an intra- and extracellular half-life of only a few hours.

The body’s Cu homeostasis is critical because the liver is the only organ to eliminate enterally absorbed Cu. This is achieved via excretion into the bile. Normally all of the excess Cu intake is later excreted so that the body’s Cu pool remains constant /8/. Cu is not subject to enterohepatic circulation because it is excreted into the bile as a non-absorbable complex.

Physiological functions of Cp include:

• Regulation of transport, availability and redox potential of iron (Fe) as a result of its ferroxidase activity. For instance, if functional iron is required for erythropoiesis, Fe³⁺ is released from ferritin via reduction to Fe²⁺. However, since the transport protein transferrin can only bind Fe³⁺, Fe²⁺ is immediately oxidized to Fe³⁺ by Cp. Furthermore, all Fe²⁺ resorbed by the intestinal mucosa is oxidized to Fe³⁺ by hephaestin or Cp before binding to transferrin.
• Prevention of metal-ion-catalyzed per oxidation of membrane lipids. This per oxidation is thought to be a causative cofactor of many disorders such as atherosclerosis or neurotoxicity. Cp reacts either directly with the super oxide ion radical (O₂^–.), or oxidizes Fe²⁺ or Cu²⁺ and thus prevents their catalytic effect for lipid per oxidation and the damage of cellular structures /3/.

Paradoxically, Cp changes from antioxidant to oxidant in the absence of divalent cations or under acid pH conditions. For instance, it is thought to play a significant role in LDL oxidation induced by monocytes/macrophages. Thus, under monocyte/macrophage stimulation, increasingly synthesized Cp may promote atherosclerosis.

The hepatocytes play an essential role in the body’s Cu metabolism. Following binding of the Cu to the intracellular Cu transport protein ATP7B, the hepatocyte controls Cu absorption via ATP7B by mediating:

• Binding of Cu to Cp
• Cu excretion into the bile after the Cp is saturated with Cu.

In WD, the cellular Cu balance is disturbed by a mutation-related dysfunction of the Cu transporter ATP7B. This results in intracellular accumulation of Cu. If all metallothionein in the cytoplasm and Cp are saturated, the free Cu has a toxic effect. This toxic effect leads to changes in the mitochondrial structure, defective DNA synthesis, modified protein synthesis and decrease in glutathione. All processes in combination may result in hepatocellular necrosis and Cu release. As these processes usually proceed slowly in most patients, the disease remains clinically inapparent in many cases.

In WD, the liver can produce apo-Cp, but Cu is not incorporated into the Cp molecule and accumulates primarily in the hepatocytes and secondarily in other tissues. In general, no clinical manifestations are observed prior to 5 years of age. They usually develop around the age of 15.

The following stages are distinguishable during the course of Wilson’s disease /11/:

• Cu accumulation; Cu accumulates diffusely in the cytosol of hepatocytes. Cu content of the liver is increased, while aminotransferases are usually normal or only slightly elevated. Elevated levels are only coincidentally detected in clinically asymptomatic patients.
• Cu redistribution; if a critical threshold of Cu accumulation has been reached in the cytosol of the hepatocytes, Cu is redistributed into the lysosomes. During this process, Cu is also released into plasma. In most patients, redistribution occurs slowly and, hence, the patients remain clinically inapparent. In some of them, redistribution takes place rapidly and a lot of Cu is released into plasma. Chronic active hepatitis occurs and may progress to liver failure. Hepatitis or liver failure is caused by intravascular hemolysis due to release of large quantities of Cu into plasma as well as toxicity-related hepatocellular necrosis. From a differential diagnostic point of view, it is important to note that in liver failure due to Wilson’s disease ALT, in relation to bilirubin, is only minimally elevated, the AST/ALT ratio is > 2, and ALP tends to be reduced /13/.
• Development of liver fibrosis and Cu accumulation in extrahepatic organs.

In patients, in whom Cu redistribution in the hepatocyte is accompanied by a clinically inapparent course, histological findings early on include morphological nuclear changes of periportal hepatocytes and steatosis similar to that seen in alcoholic fatty liver. Subsequently, liver fibrosis develops with progression to liver cirrhosis. Among the extrahepatic changes, neurological alterations predominate. The Kayser-Fleischer corneal ring is an ocular manifestation of the disease. Because of splenomegaly, some of the patients have leukopenia and thrombocytopenia.

References

1. Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease: an update. AASLD practice guidelines. Hepatology 2008; 47: 2089–2111.

2. Dockter G. Morbus Wilson – hepatische Manifestationen. Klin Pädiatr 2009; 221: 407–8.

3. Johnson AM. Ceruloplasmin. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996: 13.01–1–8.

4. Dati F, Schumann G, Thomas L, Aguzzi F, Baudner H, Bienvenu O, et al. Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 plasma proteins in serum based on the standardization against IFCC/BCR/ CAP reference material (CRM 470). Eur J Clin Chem Clin Biochem 1996; 34: 517–20.

5. Bandmann O, Weiss KH, Kaler SG. Wilson’s disease and other neurological disorders. Lancet Neurol 2015; 14: 103–13.

6. Roberts EA, Schilsky ML. Current and emerging issues in Wilson's disease. N Engl J Med 2023; 389 (10): 922–33.

7. Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 1993; 5: 327–37.

8. www.wilsondisease.med.ualberta.ca/database.asp

9. Merle U, Weiss KH, Eisenbach C, Tuma S, Ferenci P, Stremmel W. Truncating mutations in the Wilson disease gene ATP7B are associated with very low serun ceruloplasmin oxidase activity and an early onset of Wilson disease. BMC Gastroenterol 2010; 10: 8.

10. Merle U, Schaefer M, Ferenci P, Sremmel W. Clinical presentation, diagnosis and long-term outcome of Wilsons disease: a cohort study. Gut 2007; 56: 115–20.

11. Stapelbroek JM, Bollen CW, van Amstel JK, van Erpecum KJ, van Hattum J, van den Berg LH, et al. The H1069Q-Mutation in ATP7b is associated with late and neurologic presentation of Wilson’s disease: results of a metaanalysis. J Hepatol 2004; 41: 758–63.

12. Coffey AJ, Durkie M, Hague S, McLay K, Emmerson J, Lo C, et al. A genetic study of Wilson’s disease in the United Kingdom. Brain 2013; 136: (Pt 5): 1476–87.

13. Degenhardt S, Blomhard G, Hefter H, et al. Hämolytische Krise mit Leberversagen als Erstmanifestation des M. Wilson. Dtsch Med Wschr 1994; 119: L 1421–6.

14. Ramos D, Mar D, Ishida M, Vargas R, Gaite M, Montgemery A, Linder MC. Mechanism of coppper uptake from blood plasma ceruloplasmin by mammalian cells. Plos One 2016. doi: 10.1371/journal.pone.0149516.

15. Tapper EP, Rahni DO, Arnaout R, Lai M. The overuse of serum ceruloplasmin measurement. Am J Med 2013; 126: 926.e1–926.e5.

16. da Costa CM, Baldwin D, Portmann B, Lolin Y, Mowat AP, Mierli-Vergani G. Value of urinary copper excretion after penicillamine challenge in the diagnosis of Wilson’s disease. Hepatology 1992; 15: 609–15.

17. Roberts EA, Cox DW. Wilson’s disease. Baillières Clin Gastroenerol 1998; 12: 237–56.

18. Stremmel W, Meyerrose KW, Niederau C, Hefter H, Kreuzpainter G, Strohmeyer G. Wilsons disease: clinical presentation, treatment and survival. Ann Int Med 1991; 115: 720–6.

19. Huster D, Weizenegger M, Kress S, Mössner J, Caca K. Rapid detection of mutations in Wilson disease gene ATP7B by DNA strip technology. Clin Chem Lab Med 2004; 42: 507–10.

20. Twomey PJ, Viljoen A, House IM, Reynolds TM, Wierzbicki AS. Copper: caeruloplasmin ratio. J Clin Pathol 2007; 60: 441–2.

21. Das SK, Ray K. Wilson’s disease: un update. Nature Clin Practice Neurology 2006; 2: 482–93.

22. Steindl P, Ferenci P, Dienes HP, Grimm G, Pabinger I Madl C, et al. Wilson’s disease in patients presenting with liver disease: a diagnostic challenge. Gastroenterology 1997; 113: 212–8.

18.8 Haptoglobin and hemopexin: hemolytic disease

Lothar Thomas

Extracellular hemoglobin (Hb) and cell-free heme are toxic breakdown products of hemolyzed erythrocytes. The liver synthesizes the scavenger proteins haptoglobin (Hp) and hemopexin (Hx) which bind extracellular Hb and heme respectively.

Hp binds Hb to form high-molecular weight Hp-Hb complex. In this complexed form, Hb is sequestered within the vasculature and the passage of Hb outside the vessels is prevented. Plasma clearance of Hp-Hb complexes occurs via binding to the CD163 receptor, which is expressed by hepatic and splenic macrophages /1/.

Binding of heme to Hx blocks the pre-oxidant and pro-inflammatory effects of cell-free heme and facilitates receptor-mediated uptake of heme by the liver and spleen /2/.

18.8.1 Indication

Haptoglobin

Diagnosis and monitoring of hemolytic diseases.

Hemopexin

Genetic and non-genetic diseases, e.g., sepsis, sickle cell disease, hemolytic uremic syndrome, hemolytic conditions during stages of malaria and dengue /3/.

18.8.2 Method of determination

Quantitative determination

Immunoassay and immunonephelometric or immunoturbidimetric measurement. For principle, see Section 18.1 – Plasma protein diagnostics.

Haptoglobin sub typing

Polyacrylamide gel electrophoresis, isoelectric focusing.

18.8.3 Specimen

Serum: 1 mL

18.8.4 Reference interval

Refer to References /4, 5, 6/ and Tab. 18.8-1 – Reference intervals for haptoglobin and hemopexin.

18.8.5 Clinical significance

Both a decrease and an increase in Hp concentrations as well as the Hp phenotype may be clinically significant. Decreased concentrations indicate in vivo hemolysis. Increased serum concentrations are mostly due to the acute phase reaction of Hp and are found to occur in conjunction with inflammation, infections and autoimmunopathy. Certain Hp phenotypes are risk factors of systemic diseases.

In serum, Hx is much less often altered than Hp and when such an alteration occurs, Hx is almost exclusively affected by a decrease, as seen in severe hemolytic anemia. As a rule, Hx is determined if Hp has declined to non measurable levels.

Elevated Hx levels are rare and are only of clinical relevance in patients with melanoma.

18.8.5.1 Clinical significance of haptoglobin

Hemolytic reaction

In any hemolytic case, the serum Hp concentration depends on the Hp type of the individual and the concentration of lysed erythrocytes. The allele frequencies (HP1 and HP2) differ significantly worldwide /7/. For instance, the HP1 frequency ranges from 7% in parts of India to 70% in parts of West Africa. The HP1 frequency is between 31% and 40% in Europe, between 20% and 40% in Asians and, in North America, is 41% in Caucasians, 52% in Afro-Americans and 31% in Asians and Orientals.

The distribution of the Hp subtypes in Southern Germany, for example, is 14% for Hp 1-1, 48% for Hp 2-1 and 38% for Hp 2-2 /8/.

Since Hp phenotypes are associated with different serum Hp concentrations, the reference interval for Hp is very broad. Without electrophoretic phenotyping and application of a phenotype-specific reference interval, it is therefore impossible to

• Detect mild chronic hemolysis
• Estimate the severity of a hemolytic reaction merely based on the Hp concentration.

Normal Hp concentrations are to be expected despite an underlying hemolytic disease if a concomitant inflammatory disease leads to increased synthesis of Hp as an acute phase protein. This is the case if the C-reactive protein (CRP) is elevated. Therefore, the CRP should be determined besides Hp for diagnosing a hemolytic reaction (Tab. 18.8-2 – Assessment of haptoglobin in combination with C-reactive protein).

Investigations on the diagnosis and monitoring of hemolytic disease are shown in Tab. 18.8-3 – Investigations on the diagnosis and monitoring of hemolytic anemias.

The classification of hemolytic anemias is presented in Tab. 18.8-4 – Classification of hemolytic anemias.

The behavior of Hp in hemolysis and diseases associated with various Hp haplotypes is shown in Tab. 18.8-5 – Haptoglobin in hemolysis and diseases associated with various haptoglobin haplotypes.

Further diseases and conditions associated with decreased Hp

• Acute and chronic liver diseases
• Malabsorption syndrome
• Congenital Hp decrease or deficiency (e.g., as seen in 30% of black Nigerians and in 1/1,000 Caucasians).

Diseases and conditions associated with elevated Hp concentration

• Acute phase response (e.g., acute and chronic active inflammations, acute tissue necrosis, malignant tumors)
• Intrahepatic and extrahepatic cholestasis, Hodgkin’s disease, nephrotic syndrome, rheumatoid arthritis, iron deficiency anemia
• New synthesis of unknown etiology, such as multiple myeloma, amyloidosis /9/.

18.8.5.2 Clinical significance of hemopexin

Decreased serum Hx concentrations are measurable if the Hp concentration has declined to non measurable levels because of pronounced hemolysis. Heme containing molecules are only bound if their concentration exceeds 6 mg/L.

Even in the presence of pronounced hemolysis, Hx is never completely undetectable in serum /10/. Hx and Hp function at different levels (i.e., initially Hp is reduced, then Hx). Causes of a divergent behavior pattern between Hp and Hx are listed in Tab. 18.8-6 – Causes of a divergent behavior pattern between Hp and Hx.

The Hx concentration in newborns is about 20% of that in adults; Hx concentration is not a criterion for detecting hemolytic reactions during the neonatal period /11/.

Hx is better suited than Hp for assessing the extent of hemolysis. The following facts are in favor of Hx:

• Hp is too sensitive, depends on the Hp phenotype and already displays markedly reduced levels in the presence of mild to moderate hemolysis
• As an acute phase protein, Hp increases in the presence of inflammatory processes and thus may mask a hemolytic reaction
• Hx is still measurable even in massive hemolysis.

Reductions in Hx without an underlying hemolytic reaction

• Chronic liver disease
• Porphyria cutanea tarda
• Malabsorption syndrome.

Diseases and conditions associated with elevated Hx

Rapidly growing melanomas cause an increase in Hx that correlates with tumor growth. In the event of successful treatment, levels return to within the reference interval /12/.

18.8.5.3 Heme induced oxidative modification of hemopexin

Inflammation accompanies hemolysis and injury and is associated with increased oxidative stress driven in part via heme-mediated events. The function and concentration of many proteins is impaired by oxidative modifications from reactive oxygen species (ROS). Heme-Hx complexes resist oxidative damage. Heme binding by Hx protects against heme toxicity in hemolytic diseases and conditions, sepsis and sickle cell disease. This protection is sustained by heme-hemopexin complexes in biological fluids that resist oxidative damage during the heme-driven inflammation.

Apo-hemopexin is vulnerable to inactivation by reactive nitrogen species (RNS) and ROS that covalently modify amino acids. It is supposed that during inflammation apo-hemopexin is nitrated and oxidized in niches of the body containing activated RNS- and ROS-generating immune and endothelial cells, potentially impairing Hx protective extracellular antioxidant function. The reason is tyrosine nitration in the heme binding site of pro-hemopexin/hemopexin /3/.

18.8.6 Comments and problems

Method of determination

In radial immunodiffusion, Hp 2-1 and Hp 2-2 have slower diffusion rates than Hp 1-1 since they are more markedly polymerized.

Reference interval

Hp concentration increases continuously after birth up to the age of about 40 years. Women have higher concentrations than men.

18.8.7 Pathophysiology

Haptoglobin

The HP gene exists in two allele types, HP1 and HP2 so that there are three Hp phenotypes: Hp 1-1, Hp 2-1 and Hp 2-2. These phenotypes are differently distributed worldwide, presumably due to genetic drift and natural selection /13/.

Hp is a glycoprotein composed of four polypeptide chains, two light α-chains and two heavy β-chains. The α-chain is genetically polymorphic; the synthesis is encoded by the two above-mentioned alleles. Little is known about the polymorphism of the β-chain. The structure of the Hp molecule is shown in Fig. 18.8-1 – Structure of haptoglobin.

Hp binds oxyhemoglobin, methemoglobin, isolated hemoglobin α-chains, α/β dimers and heme-free hemoglobin H but not deoxygenated hemoglobin, heme, hemoglobin H, isolated hemoglobin β-chains or myoglobin. Its physiological function is to prevent renal losses of hemoglobin and, thus, a loss in iron; this is based on the fact that unlike Hb the Hp-Hb complex, on account of its high molecular weight, is not glomerularly filtered /14/. Hp is primarily expressed by the liver, but also produced by the lungs, kidneys, spleen, thymus and heart.

Any reduction in the erythrocyte life span leads to an increase in hemolysis. The localization of lysis depends on both the extent of the hemolytic process and the mechanism by which each erythrocyte is damaged /15/. Depending on the phenotype, 1.5 mg of Hp bind approximately 1 mg of Hb in plasma. Clearance of the Hp-Hb complex by the reticuloendothelial system is approximately 15 mg per 100 mL of plasma per hour. If more Hb occurs intravascularly in relation to the quantity of Hp produced by the liver, Hp concentration declines as a measurable sign of hemolysis. In liver parenchymal damage with reduced synthesis capacity, a decrease in Hp occurs early on. If inflammatory processes are simultaneously present, Hp reacts as an acute phase protein and, despite a mild hemolytic process, increased Hp consumption is compensated by an increase in the synthesis rate; hence, the serum concentration remains within the reference interval.

Elimination of free hemoglobin by Hp

Intravascular hemolysis changes Hb from being an intracellular O₂/CO₂ transporter to a highly toxic substance in plasma. The pathologic effect of free Hb arises /13/:

• From the hem iron, which can react with endogenous H₂O₂ to produce free radicals, which in turn may cause oxidative tissue injury, especially in the kidney
• From the potency of hemoglobin to be a scavenger of NO, a signaling molecule that functions as a regulator of smooth muscle relaxation, endothelial adhesion molecule expression, and platelet activation and aggregation. The reaction of hemoglobin with NO is irreversible, leading to the production of nitrate and methemoglobin. NO scavenging limits the bioavailability of NO and thereby impairs NO homeostasis.

The effects of free hemoglobin are neutralized by binding to Hp. The hemoglobin-Hp complex is directed to the CD163 receptor expressing macrophages, which internalize the complex (Fig. 18.8-2 – Utilization of iron from the haptoglobin/hemoglobin complex). In the macrophage, the globin portion of the hemoglobin is degraded in the lysosomes, while heme is degraded by hem oxygenase-1 to Fe²⁺, CO and biliverdin. Fe²⁺ induces the synthesis of apoferritin, which binds Fe²⁺. Thus, Fe²⁺ is prevented from forming reactive oxygen species via the Fenton reaction.

In excessive hemolysis, the Hp-CD163-mediated scavenging mechanism is exhausted and fHb and heme are present in plasma. Both are important contributors to disease states associated with hemolysis which is characterized by free hemoglobin, heme induced inflammation and toxic cell damage. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) impaire antioxidant functions. For instance, 50% of patients with sickle cell disease have endothelial dysfunction due to the constant presence of fHb and heme.

In inflammatory conditions (extra corporeal circulation, sickle cell anemia) the synthesis of Hp and CD163 is up regulated by interleukin-6 to increase the fHb removal capacity. Hp does not bind to CD163. FHb binds with low affinity to CD163, provided that fHb was not oxidized by H₂O₂ /16/. Hp-bound Hb cannot be oxidized because it is protected. In contrast to the high-affinity Ca²⁺-dependent binding of the Hp-Hb complex to CD163, the low-affinity binding of non-oxidized fHb is a possibility for Hb removal if fHb concentrations are high.

The Hp phenotypes differ in their free hemoglobin removal functionality, also regarding CD163 interaction. The protective capacity of the phenotype Hp 1-1 against free hemoglobin-mediated oxidation is thought to be better than that of Hp 2-2. This might explain the different diseases associated with the Hp haplotypes.

Elimination of free hemoglobin by Hx and albumin

If Hp binding capacity has been exhausted, free hemoglobin occurs in plasma. Free hemoglobin is either oxidized to methemoglobin, followed by its dissociation into heme and globin, or oxidation of heme follows after dissociation. In both cases, hematin derivatives with Fe³⁺ are present. Hx binds these hematin derivatives with high affinity and albumin does with low affinity (Fig. 18.8-3 – Intravascular hemoglobin and hematin transport in Hp excess and Hp deficiency). Albumin is not involved until large quantities of hemoglobin are released; in this case, as methemalbumin, it gives the serum a coffee-brown color. Like Hx, albumin transports hematin derivatives to the reticuloendothelial system. If, during degradation of erythrocytes, heme is directly cleaved from hemoglobin due to enzymatic action Hx is mainly consumed while Hp may remains unaffected (e.g., as observed in hemorrhagic pancreatitis).

Oxidation of hemoglobin to methemoglobin and further degradation is achieved by the reticuloendothelial system. Detection of methemoglobin in plasma is always a sign of massive hemolysis.

Free Hb in plasma is glomerularly filtered at a clearance rate of 5 mL/min. In the proximal tubule, Hb is reabsorbed and heme iron released from hemoglobin is stored in the form of ferritin and hemosiderin, followed by its re utilization. If tubular epithelia are overloaded due to pronounced hemolysis, these cells degenerate, are sequestered and appear in urine as hemosiderin-carrying epithelial cells that are stainable with the Prussian blue reaction. Hemosiderinuria is, therefore, considered to be a valuable indicator of severe acute and chronic intravascular hemolysis.

Hemoglobinuria occurs when tubular reabsorption capacity is exhausted because of a massive hemolysis.

Hemopexin

Hx has a molecular weight of 80 kDa and, like Hp, is synthesized in the liver. Whereas Hb is exclusively bound by Hp, heme and heme derivatives are only bound by Hx and albumin. Hx has a high affinity toward heme and removes these substances from the plasma after cleavage of fHb into a heme and a globin portion. It only responds in the presence of pronounced hemolysis (i.e., if free Hp is no longer available). Hx is not an acute phase protein. Hx deficiency develops in patients with sepsis that is life-threatening.

Several diseases in which Hx plasma levels increase are associated with inflammation, increased activity of nitric oxide synthase, and oxidative stress. Thus Hx is expected to be exposed to high levels of oxidative species. Because proteins can be inactivated by oxidative modifications from exposure to ROS and RNS it is supposed that the increased levels of modified Hx might be a physiological response to damage, in which case Hx is functional, i.e., capable of binding to heme or to Hx receptors. The protective extracellular antioxidant function of Hx is impaired during heme-driven inflammation because apo-Hx is is vulnerable to inactivation by RNS that cause tyrosine nitration in the peptide YYCFQGNQFLR in the heme-binding site of Hx /3/.

References

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2. Tolosano E, Fagoonee S, Morello N, Vinci F, Fiorito V. Heme scavening and the other facets of hemopexin. Antioxid Redox Signal 2010; 12: 305–20.

3. Hahl P, Hunt R, Bjes ES, Skaff A, Keightley A, Smith A. Identification of oxidative modifications of hemopexin and their predicted physiological relevance. J Biol Chem 2017; 292: 13658–71.

4. Jeppsson JO. Haptoglobin. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research 1996: 7.04-1–6.

5. Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 1996; 42: 1589–1600.

6. Meiers HG, Lissner R, Mawlawi H, Brüster H. Hämopexinspiegel bei Männern und Frauen in verschiedenen Lebensaltern. Klin Wschr 1974; 52: 453–7.

7. Carter K, Worwood M. Haptoglobin: a review of the major allele frequencies worldwide and their associations with diseases. Int Jnl Lab Hem 2007; 29: 92–110.

8. Braun HJ. Eigenschaften und Funktion menschlicher Serumproteine bei intravasaler Hämolyse. Dtsch Med Wschr 1971; 96: 595–601.

9. Aeschlimann A, Mall TH, Rieder HP. Hyperhaptoglobinämie bei Plasmozytom und Amyloidose. Dtsch Med Wschr 1985; 110: 1128–9.

10. Braun HJ. Eigenschaften, Funktion und Serumkonzentration des menschlichen Hämopexins. Klin Wschr 1971; 49: 445–51.

11. Lundh B, Oski FA, Gardner FH. Plasma hemopexin and haptoglobin in hemolytic diseases of the newborn. Acta Paediat Scand 1970; 59: 121–6.

12. Manuel Y, Defontaine MC, Bourgoin JJ, Dargent M, Sonneck JM. Serum hemopexin levels in patients with malignant melanoma. Clin Chim Acta 1971; 31: 485–6.

13. Nielsen MJ, Moestrup SK. Receptor targeting of hemoglobin mediated by haptoglobins: roles beyond heme scavenging. Blood 2009; 114: 764–71.

14. Marchand A, Galen RS, van Lente F. The predictive value of serum haptoglobin in hemolytic disease. JAMA 1980; 243: 1909–11.

15. Zvi B, Levy AP. Haptoglobin phenotypes, which one is better and when? Clin Lab 2006; 52: 29–35.

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19. Garby L, Noyes WD. Studies on hemoglobin metabolism. 1. Kinetic properties of the plasma hemoglobin pool in normal man. J Clin Invest 1959; 38: 1479–83.

20. Shih AWY, McFarlane A, Verhovsek M. Haptoglobin testing in hemolysis: Measurement and interpretation. Am J Hematol 2014; 89: 443–7.

21. Nakagawa T, Muramoto Y, Hori M, Mihara S, Marubayashi T, Nakagawa K. A preliminary investigation of the association between haptoglobin polymorphism, serum ferritin concentration and fatty liver disease. Clin Chim Acta 2008; 398: 34–8.

22. Levy AP, Hochberg I, Jablonski K, Resnick HE, Lee ET, Best L, et al. Haptoglobin phenotype is an independent risk factor for cardiovascular disease in individuals with diabetes: The Strong Heart Study. J Am Coll Cardiol 2002; 40: 1984–90.

23. Graw JA, Mayeur C, Rosales I, Liu Y, Sabbisetti VS, Riley FE, et al. Haptoglobin or hemopexin therapy prevents acute adverse effects of resuscitation after prolonged storage of red cells. Circulation 2016; 134; 945–60.

24. Hulikova A, Kramer H, Khan H, Swietach P. Detection of intravascular hemolysis in newborn infants using carbonic anhydrase I immunoreactivity. JALM 2020; September: 921–34.

18.9 Immunoglobulins (Ig)

Lothar Thomas

Immunocompetent individuals have an acquired immune system that is divided into the following two functional mutually cooperative but developmentally independent units:

• Thymus (T) lymphocyte system; it represents a functionally heterogeneous group of cells concerned with immune regulation and antigen elimination. The T lymphocyte system is primarily responsible for cellular immunity.
• Bursa or bone marrow (B) lymphocyte system; B lymphocytes differentiate into plasma cells which synthesize and secrete immunoglobulins (Ig) after an antigenic stimulus.

Ig represent a heterogeneous group of proteins with antibody functions (i.e., they are capable of binding antigens). Their synthesis is the adaptive response to the antigen structure that will be bound. The structure of the antibody binding site is synthesized to correspond with the configuration of the antigen with which the antibody will react.

Ig have the following effector functions:

• Formation of immune complexes with antigens that are amenable to phagocytosis
• Binding to the membrane receptors of immune cells in order to activate them
• Reaction with plasma proteins (e.g., with complement components, and activation of these proteins in order to eliminate the antigen).

18.9.1 Structure and function of Ig

Ig have a common basic structure, consisting of two identical heavy (H) chains and two identical light (L) chains (Fig. 18.9-1 – T form of Ig molecule in free solution and V form during antigen binding). The chains are joined together by disulfide bonds. The amino terminal ends of the light and heavy chains of the Ig molecule form the variable region. The variable regions of the Ig differ in their amino acid sequences, since these are responsible for the specificity towards the antigen determinants. Within the variable regions, there are areas of lesser and greater variability in the amino acid composition; these latter are also referred to as the hyper variable regions /1, 2, 3/.

The heavy chains of the Ig molecule consist of a variable and a constant region. Each of the two polypeptide chains is comprised of a series of globular regions also referred to as domains, with substantial amino acid homology.

N-terminal domains of H and L chains contain variable amino acid sequences (V region) determining the antigen specificity. The N-terminal amino acids of the V region form a functional pocket into which the epitope of the antigen fits. Surfaces of the antigen binding site and epitope are complementary to each other, so that the concavities of the one are filled by the convexities of the other. The complementarity is not only physical but also chemical in nature. The contacts of binding site and epitope include van der Waals forces, hydrogen bridge bonds between polar groups and ion pair bonds between differently charged side chains /4, 5/.

The constant regions of the heavy chains include domains (CH regions) with certain structural and antigenic differences that allow their classification into Ig subclasses.

In the IgG molecules, for example, the domains have the following functions /4, 5/:

• The first (C_H1) and the second domain (C_H2) form the hinge region. Because of this region, the Ig molecule is capable of transforming from the T form, in which it is in solution, into the V form, which it assumes during antigen binding.
• The C_H2 domain binds the complement protein C1q, which activates the classic pathway of the complement system
• The third domain C_H3 mediates binding of Fc fragment to Fc receptor of immune cells such as granulocytes, monocytes/macrophages and antigen dependent cytotoxic cells (ADCC)
• The combination of C_H2 and C_H3 acts as an additional domain for binding, for example, to granulocytes and natural killer (NK) cells.
• Papain cleaves the Ig molecule into three fragments: Two identical Fab fragments consisting of the complete L-chain, the variable region and a portion of the constant region of the H-chain
• A fragment consisting of the constant regions of the two heavy chains, linked via disulfide bridges. Since this fragment crystallizes, it is also referred to as Fc fragment (fragment crystallizable).

Refer to Fig. 18.9-1 – T form of Ig molecule in free solution and V form during antigen binding:

18.9.1.1 L-chain

Kappa- and lambda-L-chain types are differentiated. Each Ig molecule has either two kappa- or two lambda-L-chains since a B cell can only synthesize one type of L-chain. L-chains have a molecular weight of about 22 kDa and consist of an amino-terminal, variable region and a carboxy-terminal, constant region. The L-chain is connected to the heavy chain via a disulfide bond between a cysteine molecule in the constant regions of both chains. B cells produce approximately twice as many kappa chains as lambda chains. The term monoclonal free light chain or, if detectable in urine, Bence Jones protein refers to L-chains of one type that are not bound to heavy chains and are synthesized and secreted by malignant B-cell proliferation. Because of the cysteine molecules, they are prone to dimerization.

Polyclonally synthesized free light chains are of the kappa and the lambda types and are detected in about equal amounts in a sample; traces are present in the urine (e.g., associated with infectious diseases).

18.9.1.2 H-chain

The structure of the H-chain determines the Ig class of an Ig molecule. Differences between individual Ig classes concern their amino acid sequence, molecular weight, carbohydrates, antigenicity, allotypic heterogeneity and electrophoretic mobility. Five major Ig classes are distinguished, with each of them displaying two identical H-chains: IgG has γ-heavy chains, IgA has α-heavy chains, IgM has μ-heavy chains, IgD has δ-heavy chains and IgE has ε-heavy chains.

18.9.1.3 J-chain

The J-chain is a peptide connecting two Ig molecules of the same class (connection piece). Ig molecules of M and A classes exhibit polymorphism. IgM occurs mostly as a pentamer and IgA as a dimer. IgM monomers as well as IgA monomers are each only connected via a single J-chain. J-chains are glycoproteins with a molecular weight of 15 kDa and bind via disulfide bonds near the carboxy-terminal end of the heavy chains (Fig. 18.9-2 – Structure of immunoglobulins).

18.9.1.4 Secretory component

As a dimer, secretory IgA, which is synthesized by plasma cells of the respiratory, genitourinary and gastrointestinal tracts and released into secretions, contains a secretory component that protects the Ig molecule from peptidase-mediated degradation (Fig. 18.9-2 – Structure of immunoglobulins). The secretory component is a glycoprotein with a molecular weight of 60 kDa. Dimeric IgA is not bound to the secretory component until it penetrates the epithelial layer, where the secretory component is synthesized by epithelial cells. This secretory component is detectable in secretory IgA deficiency.

18.9.1.5 Antibody heterogeneity

The heterogeneity of antibodies can be subdivided into isotypic, allotypic and idiotypic variations.

Isotypic variation

Isotypes have the same constant domains. Isotypic variations consist of differences in H-classes, L-types and domains, which are present in all healthy members of a given species. The synthesis of isotypes is encoded by genes, which are present in all human beings.

Allotypic variation

Allotypic variation is defined as an allele-induced variation of the Ig within a species. Allotypes usually occur as variants of the constant region of the H-chains. For instance, Gm factor is responsible for the regulation of the constant H-chain region of IgG. More than 25 different Gm (genetic marker) allotypes are known. Allotypic variants are often the result of an amino acid substitution within the H-chains.

Idiotypic variation

Idiotypes have the same variable domains. The idiotypic variation relates to the diversity at the antigen binding site. The Ig synthesized by a plasma cell clone is normally completely identical (i.e., it consists of a uniform idiotype). An alteration (e.g., an amino acid exchange in the variable region of the antigen binding site) may lead to idiotypic variation. Such antibodies differ even if they belong to the same isotype and allotype. Many monoclonal components (para proteins) are probably idiotypic variations.

18.9.2 Ig classes

Ig classes IgG, IgA, IgM, IgD and IgE are present in descending order of concentration in the serum of healthy individuals /1, 2/. Within the IgG class, the subclasses IgG₁, IgG₂, IgG₃ and IgG₄ are differentiated, and the class IgA includes the subclasses IgA₁ and IgA₂, while the IgM class contains the subclasses IgM₁ and IgM₂. The physicochemical and biological properties of the individual Ig classes are listed in Tab. 18.9-1 – Physicochemical and biological properties of the Ig classes.

18.9.2.1 IgG antibodies

IgG consists of two identical H-chains with a molecular weight of about 50 kDa and two identical L-chains with a molecular weight of about 22 kDa. In primary infections (primary antibody response), IgG are usually secondary antibodies and, in repeat infection with the same organism, primary antibodies (secondary antibody response). Approximately one half of total body IgG is present in plasma, while the other half is distributed in the body fluids. When performing serum protein electrophoresis, IgG is located in the γ-globulin fraction. Under standardized conditions, the subclasses IgG₂ and IgG₄ migrate toward the anode, whereas IgG₁ and IgG₃ migrate toward the cathode. Fetal IgG originates from maternal blood and during the first 20 weeks of gestational age has a concentration corresponding to approximately 10% of that found in the mother.

Fetal infections during this time period do not cause an increase in IgG. Between the 22nd and the 28th week of gestational age, placental permeability markedly increases, thus accounting for the fact that at this point of time the fetal IgG concentration is equal to that in maternal blood. Concentrations of IgG₁, IgG₃ and IgG₄ are the same, whereas fetal IgG₂ concentration is lower than the maternal one. After delivery, maternal IgG decreases in the circulation of the newborn with a half-life of approximately 30 days, thus resulting in a residual concentration of only 3.5–4.0 g/L by the 3rd to 4th month. As infant’s own IgG synthesis gets under way, serum IgG concentration slowly increases, reaching a concentration of 7–8 g/L by the end of the first year and adult levels prior to the age of 16 years (Fig. 18.9-3 – Course of immunoglobulin serum levels during fetal period and in childhood) /2/.

The synthesis of IgG subclasses during the course of an immune response depends on the nature of antigens, their site of entry and the duration of antigen exposure. The antibody response is directed against:

• Protein antigens, such as bacteria and viruses, mediated by IgG₁ and IgG₃ and induced by CD4⁺T-cells
• Polysaccharide antigens (e.g., encapsulated bacteria such as Pneumococcus, Streptococcus group A but also H. influenzae) mostly mediated by IgG₂ and partly IgG₁ and not induced by CD4⁺T-cells
• Polyvalent antigens such as snake venoms, parasites, insects and food components in the case of chronic antigen stimulation and mediated by IgG₄. The IgG₄ antibodies, like IgE, bind to surface receptors of mast cells.
• Native DNA, mediated by IgG₁ and IgG₃ autoantibodies.

The IgG subclass response is regulated and modulated by interleukins; the number of existing B-cell sub populations in a tissue region also plays an important role. For instance, in the blood of healthy individuals, B cells with cytoplasmic IgG₁ and IgG₂ dominate, whereas in the tonsils B cells with IgG₁ and IgG₃ are predominant.

Because of its binding to the Fc receptor of the immune cell, the Fc fragment of the IgG molecule is of significant importance in the immune response. The Fc fragment mediates:

• The uptake of IgG coated bacteria by macrophages. The Fc fragment of the IgG molecule is bound by the Fc receptor of the macrophage; the bacterium is engulfed and incorporated into the macrophage in a zipper-like manner
• Clearance of IgG-containing immune complexes according to the aforementioned mechanism
• The antibody-dependent cellular cytotoxicity (ADCC) via effector cells such as monocytes/macrophages, granulocytes and lymphocytes. These cells have Fc receptors to which the Fc fragment of the IgG-loaded target cell binds. These cell is subsequently destroyed.

The IgG catabolism is proportional to the plasma concentration (i.e., enhanced in the case of a high IgG level and low in the case of a low concentration). Accordingly, the half-life is 70 days in case of a low IgG synthetic rate, but may be shortened to 11 days if the IgG synthetic rate is high.

18.9.2.2 IgM antibodies

IgM circulates in the serum as a pentamer, has a molecular weight of 971 kDa and consists of five monomers that are covalently linked via disulfide bonds and connected via five connection pieces (J-chains). Small amounts of monomers and hexamers are also present in the circulation. Each μ-chain contains one V region and four C regions.

Serum IgM comes in two flavors, pre-immune without exposure to exogenous antigen also known as natural IgM that is spontaneously produced, and the second type is exogenous antigen induced or immune IgM antibodies /3/.

The majority of IgM in serum is comprised of natural IgM antibodies. The two prominent features of natural IgM are poly reactivity and auto reactivity. The natural IgM antibodies are reactive with many conserved epitopes that are shared by microbes and self antigens. The production of natural IgM appears to be triggered by interaction with self antigens. In addition to providing early defense against microbes, natural IgM plays an important role in immune homeostasis, and provides protection from consequences of autoimmunity and inflammation.

Immune IgM is the first antibody secreted during an initial immune response to an exogenous antigen. Mature naive B cells in response to antigens undergo clonal expansion and differentiation into Ig secreting cells.

75–80% of IgM are located intravascularly. In serum protein electrophoresis, IgM migrates between the γ-globulin and the β-globulin fraction. Because of slight differences in the μ-chain, two IgM subclasses are distinguishable. The pentameric form of the IgM molecule has ten antigen binding sites, but only five of these are usable for antigen binding due to steric hindrance. The pentameric IgM can be split into monomers by SH group cleaving reagents.

Besides pentameric and monomeric IgM, a secretory IgM with a secretory component is present in body secretions much like IgA. The essential functions of IgM in immune response are the agglutination of pathogens and the activation of the classic complement pathway.

Maternal IgM does not cross the placental barrier. The healthy fetus as well as the newborn have an IgM concentration that is about 10% of the adult level and is produced by the fetus itself.

After birth, IgM synthesis rises markedly: 50% of the adult IgM concentration are reached after the first 4 months of life and the adult level is reached at the age of 8–15 years (Fig. 18.9-3 – Course of immunoglobulin serum levels during fetal period and in childhood). The fetus by itself can synthesize IgM in larger quantities from the 20th gestational week. Intrauterine infection can, therefore, cause a steep increase in the IgM concentration, and IgM levels > 0.2 g/L in cord blood are a criterion pointing to such an infection.

The IgM class includes the natural antibodies such as AB0 blood group isohemagglutinins, cold agglutinins (anti-i, anti-I), heterophilic antibodies, saline erythrocytic antibodies as well as antibodies to IgG (e.g., rheumatoid factors).

The catabolism of IgM is independent of the serum concentration.

18.9.2.3 IgA antibodies

IgA antibodies occur as serum IgA and as secretory IgA.

Serum IgA

Approximately 90% of IgA are present in a monomeric form with a molecular weight of 160 kDa and 10% in a polymeric form. Serum IgA tends to form complexes, especially with albumin, but also with enzymes, thus forming macroenzymes. Half of the serum IgA is located intravascularly and occurs in two subclasses. The ratio of the two IgA isotypes IgA₁/IgA₂ is 9/1. In serum protein electrophoresis, IgA migrates in the cathodic part of the β-globulin and the anodic part of the γ-globulin fraction.

Since IgA does not cross the placental barrier, it is not present in fetal blood. After birth, the synthesis of IgA begins slowly; by the end of the first year, the infant has about 25%, by 3.5 years of age approximately 50% and by 16 years of age 100% of the adult serum level (Fig. 18.9-3 – Course of immunoglobulin serum levels during fetal period and in childhood). The function of serum IgA is not known in further detail. It activates complement via the alternative pathway and has specific antibody functions. The catabolism of IgA is independent of the serum concentration.

Secretory IgA

This IgA molecule is composed of a unit of two IgA molecules, which are connected via a J-chain and have a secretory component (Fig. 18.9-2 – Structure of immunoglobulins). Secretory IgA is produced by plasma cells that are located in the lamina propria of mucous membranes. It is synthesized independently from serum IgA. Therefore, deficiency in serum IgA does not necessarily imply deficiency in secretory IgA. Secretory IgA is the predominant Ig of body secretions such as saliva, tears, colostrum, nasal secretions, tracheobronchial mucus, gastrointestinal secretions and breast milk. Newborns and infants are supplied with IgA via breast milk and, hence, are passively immunized against gastrointestinal infections. Essential functions of secretory IgA are: the binding of microorganisms on mucous membranes, activation of the alternative complement pathway and activation of inflammatory reactions. Inside epithelial cells, IgA is thought to neutralize intracellular microorganisms. In the lamina propria of mucous membranes, IgA binds antigens in the form of immune complexes and secretes them on the mucomembranous surfaces. This prevents the circulation from being overloaded with immune complexes. Individuals with secretory IgA deficiency are found to suffer more commonly from mucosal infection, atopy and autoimmune disease.

18.9.2.4 IgD antibodies

IgD has a high carbohydrate content with numerous oligosaccharide chains. Due to the fact that the two δ-chains are only held together via disulfide bonds and due to the abundance of lysine and glutamic acid in the hinge region, the molecule is prone to proteolysis.

Together with IgM antibodies, IgD antibodies are located on the cell membrane of B cells as antigen receptors. In cord blood, IgD is not detectable or only detectable in minimal concentrations; adult levels are reached at 2–5 years of age.

The distribution of IgD in the body is identical to that of IgM. The exact function of IgD is unknown, but this Ig class is thought to include antinuclear antibodies (ANA) as well as antibodies to insulin and penicillin. The higher the serum level of IgD, the slower its catabolism (i.e., the plasma concentration of IgD is inversely related to IgD catabolism).

18.9.2.5 IgE antibodies

The ε-heavy chain, like the μ-chain of the IgM, possesses five domains, which explains the molecular weight of 72.5 kDa. IgE binds via the domains Cε3 and Cε4 to the high-affinity receptor FcεRI located on mast cells and basophil granulocytes. Moreover, it binds to the low-affinity receptors FcεRII or CD23 present on monocytes, lymphocytes and eosinophil granulocytes.

IgE antibodies are also referred to as reagins. Their distribution in the body is identical to that of IgA and their catabolism, at a half-life of 2.5 days, is high. The serum IgE level does not represent the effective IgE load of the organism since the predominant IgE synthesis sites are located in the respiratory tract, gastrointestinal tract and lymph nodes. Because of its affinity to mast cells, some IgE is bound to the IgE receptors of these cells.

IgE antibodies mediate type I hypersensitivity reaction of the immediate type. Harmless, polyvalent antigens, such as pollen or house dust mites, stimulate mucosal B cells at the site of entry to synthesize specific IgE. This process is mediated by CD4⁺T cells. Specific IgE binds via Fc receptors to mast cells that are now sensitized. During the next contact of the polyvalent antigen with the sensitized mast cell, bound IgE antibodies are cross-linked, the cell is de granulated and mediators are released, which cause, for example, symptoms of hay fever, asthma and atopic eczema.

18.9.3 Quantitative determination of immunoglobulins

IgG, IgA, IgM, IgD

Immunoassay and immunonephelometric or immunoturbidimetric measurement /6/.

IgE

Immunoassays such as enzyme, fluorescence or luminescence immunoassays.

IgG subclasses

Immunoassay and immunonephelometric or immunoturbidimetric measurement mostly using monoclonal antibodies (Section 18.11 – Cryoglobulins and cryofibrinogen).

IgM in cord blood

Latex agglutination test for rapid determination of IgM; latex particles are coated with anti-IgM.

Secretory IgA

Radial immunodiffusion, rocket immunoelectrophoresis, radioimmunoassay and enzyme immunoassay /7/.

If secretory IgA levels are measured in saliva by employing radial immunodiffusion and specific plates (LC-partigen plates), which contain antiserum to serum IgA, the measured levels are multiplied by 3.25 in order to compensate for the molecular weight. Reducing pretreatment of saliva using dithiotreitol is recommended /8/.

18.9.3.1 Specimen

IgG, IgA, IgM, IgD, IgE

Serum, body fluids: 1 mL

IgG subclasses: same as for IgG.

Secretory IgA: saliva, tear fluid, intestinal juices and/or feces.

If immunoglobulin determination has to be performed in a neonate, as much cord blood should be collected as possible.

18.9.3.2 Reference interval

Refer to Ref. /1/ and Tab. 18.9-2 – IgG, IgA, IgM reference intervals in serum.

• IgD in serum: 0.03–0.14 g/L /9/
• IgE in serum: Below 0.24 mg/L (100 KU/L)

1 U IgE = 2.4 ng

Secretory IgA in saliva /7/: 0.08–0.20 g/L.

18.9.3.3 Clinical significance

Changes in serum Ig concentrations are usually classified into /2/:

• Hypogammaglobulinemias that may be associated with numerous diseases. The decrease in Ig can be due to reduced synthesis, increased loss or hyper catabolism of Ig or a combination of causes.
• Polyclonal gammopathies that may be due to an increase in antibodies of one or several Ig classes. The spectrum of diseases causing polyclonal gammopathy is broad and includes, for example, infections, chronic liver diseases, autoimmune disorders.
• Monoclonal gammopathies that are characterized by a narrow band (M-gradient) in the γ-globulin fraction in serum protein electrophoresis. M-gradients are caused by excessive proliferation of a B-cell clone, which synthesizes Ig of one class and one type (Chapter 22 – Monoclonal plasma cell proliferative diseases).

18.9.3.3.1 Hypogammaglobulinemia

If hypogammaglobulinemia is present, one should

• Determine the extent of the antibody deficiency by measurement of the Ig of each class and subclass
• Perform more detailed examinations concerning differentiation of the antibody deficiency, see Chapter 21 – Immune system.

Extent of antibody deficiency

Ig deficiencies can be of varying extent:

• One or all Ig classes or Ig subclasses are absent or strongly decreased
• The Ig of one Ig class or Ig subclass are moderately reduced as compared to an age-matched group of healthy individuals
• The Ig of the Ig classes and of some of the Ig subclasses are normal but, within a certain subclass, an antibody response cannot be established. For instance, a selective antibody synthesis defect can be present in the IgG₂ subclass involving only antibodies to pneumococcal polysaccharide.

The behavior of Ig in primary immunodeficiency diseases is presented in Chapter 21 – Immune system.

The behavior of Ig in secondary immunodeficiency diseases is shown in Tab. 18.9-3 – Laboratory findings in secondary immunoglobulin deficiency.

A decrease in serum Ig level due to hyper catabolism occurs in hyper metabolic states. For example, such a state is present in patients with hyperthyroidism; it can be associated with a reduction in Ig of all Ig classes. In patients with myotonic dystrophy, serum Ig are also reduced because of a shortened half-life. Antibodies to Ig are also rarely a cause for the rapid elimination of Ig from the circulation.

18.9.3.3.2 Polyclonal and oligoclonal gammopathies

With an influx of antigens into the organism, clonal selection operates to activate those B cells with antigen receptors for which the antigen has a high affinity. Usually, after primary contact of the immune system with such an antigen, several B cells are activated to a varying extent. They proliferate and mature into antibody-producing plasma cell clones /2/.

During an infection with complex agents (e.g., microorganisms); many antigens are produced because of antigen processing and presentation to the B cells. The selection of several T-cell stimulated B cells leads to their clonal proliferation and specific antibody production. This results in polyclonal immune response.

Polyclonal immune response

Polyclonal immune response leads to an increase in serum Ig, involving either one or several Ig classes and both Ig types. A broad-based increase in the γ-globulin fraction is detectable in serum protein electrophoresis.

Oligoclonal immune response

The oligoclonal immune response results from limited activation of B cells, also referred to as limited heterogeneity. Possible causes include: nature of the antigen, lack in reactivity of the immune system or contact of the antigen with tissues that are poor in immunocompetent cells (e.g., the central nervous system).

Diagnosis of polyclonal and oligoclonal gammopathies

The diagnosis of polyclonal or oligoclonal Ig increases is based on screening by serum protein electrophoresis; a more differentiated diagnosis is possible by quantitative determination of Ig classes and Ig subclasses or, in cerebrospinal fluid, by isoelectric focusing and subsequent immunofixation.

In diseases causing hypergammaglobulinemia, quantitative Ig determination in conjunction with the clinical picture as well as serological and clinical chemistry findings can provide results that contribute to a diagnosis, differential diagnosis, disease monitoring and formulating a prognosis.

This may be the case for:

• Acute and chronic infections
• Liver diseases
• Diseases of the central nervous system
• Intrauterine and perinatal infections.
• Ig determination in hypergammaglobulinemia

The indications are limited, in which the differentiation of hypergammaglobulinemia by means of quantitative Ig determination is clinically useful for the diagnosis of a disease. Usually, especially in infectious diseases, an increase in one or several Ig classes (with a similar Ig pattern) occurs /2/. No specific Ig pattern exists that occurs exclusively in a certain disease and by itself is of diagnostic value. However, the Ig pattern may be an important supplemental finding and may, in combination with the overall clinical picture, contribute to differential diagnosis, assessment of the course of the disease and formulating a prognosis.

18.9.3.3.3 Differential diagnostic value of Ig determination in hypergammaglobulinemia

An isolated polyclonal Ig increase in one Ig class or a more pronounced increase in one Ig class within the Ig pattern are valuable in differential diagnosis.

Isolated IgM increase

In combination with the clinical picture and especially if present for several days, an isolated IgM increase is a sign for an initial infection of the organism by a pathogen (primary reaction).

In newborns, an IgM increase in cord blood is considered to be a nonspecific sign of an infection acquired in utero.

Isolated IgG increase

In an acute infectious disease with normal or only mildly elevated IgM, an isolated increase in IgG is a sign of secondary response of the immune system to an already known pathogen. Chronic infections elicit primarily to an isolated IgG increase, while chronic active infections cause an increase in IgG, occasionally accompanied by elevated IgM and/or in IgA.

Isolated IgA increase

In liver diseases, a relatively higher IgA increase or an isolated IgA elevation suggests toxic damage (e.g., due to alcohol, hormonal contraceptives, antidepressants).

Ig pattern

The Ig pattern is not very conclusive if two or more Ig classes are increased. While increases in all three Ig classes are often found in liver cirrhosis, conclusions concerning the etiology of chronic liver disease can only be reached to a limited extent based on the Ig pattern (Tab. 18.9-4 – Pattern of IgG, IgA, IgM in liver diseases).

18.9.3.3.4 Monitoring of hypergammaglobulinemia

The assessment of the course of an inflammatory event that employs quantitative Ig determination should be based on the Ig pattern. Persistently elevated Ig concentrations argue for a sustained assault on the organism by the antigen, and the Ig will decline in the course of overcoming the infection process and the normalization for eliminating the extracellular antigen. The magnitude of the IgG concentration is a measure of the activity of the inflammatory process, especially in virally induced chronic liver disease, chronic bacterial infection, connective tissue disease and other autoimmune diseases. In toxic liver damage, the intensity of inflammation correlates with IgA concentration /10/.

During the course of infectious diseases affecting certain organs, the Ig concentration, and especially that of IgM, is of prognostic value. Persistence of elevated IgM levels at a time when a decline is expected suggests the transition to a chronic process, while continuously rising IgG levels suggest the transition to a chronic active process. In some infections such as borreliosis, patients can have persistent IgM antibodies for years without any clinical symptoms.

18.9.3.3.5 Screening for prenatally acquired infections

Because of the transplacental transfer of IgG, the newborn has the same IgG antibody pattern as the mother. An infection of the newborn cannot be recognized by methods that only detect IgG antibodies because maternal IgG antibodies are not distinguished from those produced by the fetus as result of an infection /11/.

From the 20th week of gestational age, the fetus is capable of producing IgM antibodies and from the 30th week it can also synthesize IgA. From this time point, intrauterine infections, due to various viruses (rubella, cytomegaly, herpes simplex, varicella, mumps, measles, influenza, hepatitis B, parvovirus B19), treponema pallidum or toxoplasma gondii are detectable by elevated IgM and/or IgA concentrations in the newborn blood (cord blood). The incidence of infections in newborns is reported to be 2–4% /11/.

Because of the simple and rapid measurement, the IgM concentration in cord blood serum is considered to be a good screening test. A concentration > 0.20 g/L is regarded to be an indicator of an infection.

A significant percentage of cases with intrauterine infection remains unrecognized because of the lack of clinical manifestations in the newborn or exhibits only a slight IgM increase.

In the presence of elevated IgM, clinical symptoms are only found in one third of newborns. The reason for the low diagnostic specificity is either placental leakage or contamination of cord blood with maternal blood. An infection is unlikely if the IgA concentration is also elevated and if, during a second test after 5 days, the levels of IgA and IgM in neonatal blood have markedly decreased (half-life: 5 days).

A persistence or increase in IgM indicates the presence of an infection acquired in utero or during the perinatal period. In such a case, infection-specific IgM or IgA antibodies should be determined by enzyme-linked immunosorbent assay (ELISA).

18.9.3.3.6 IgE increase

The increase in IgE is a characteristic sign of atopic diseases (see Chapter 23 – Atopy and allergy). Diseases associated with an IgE increase that is not induced by atopic diseases are presented in Tab. 18.9-5 – IgE increases in non-atopic diseases.

18.9.3.3.7 Oligoclonal Ig increase

The Ig increase is not infrequently limited to increases in certain antibody populations. These antibodies may belong to one or several Ig classes or to only one Ig subclass although they are still of polyclonal origin (i.e., they are present as kappa- and lambda-light chain types). In serum protein electrophoresis on cellulose acetate but more commonly on agarose, one or several distinct bands are visible against the diffuse background of the γ-globulin band (sawtooth pattern).

Diseases and conditions leading to oligoclonal increases in Ig in serum are listed in Tab. 18.9-6 – Causes of oligoclonal IgG increases.

Oligoclonal Ig in cerebrospinal fluid is described in Chapter 46 – Laboratory diagnosis of neurological diseases.

18.9.3.3.8 Monoclonal gammopathy

Refer to Section 22.3 – Monoclonal gammopathy.

18.9.3.4 Comments and problems

Standardization and quality assurance

Refer to Section 18.1.13 – Quality assurance.

Method of determination

Radial immunodiffusion: this method is relatively resistant to interferences. An antigen excess phenomenon must be kept in mind, recognizable by the presence of fuzzy precipitation along the margin. Aggregated Ig simulate falsely low Ig concentrations, while Ig fragments simulate falsely high ones.

Immunonephelometry: interferences in this methodology include light-scattering contaminants such as micro clots, cells from inadequately centrifuged samples, particles derived from proteins of the cerebrospinal fluid and microbial contaminations. In principle, problems should be anticipated in samples after deep-freezing or in hyperlipidemic samples. Nephelometric assays properly detect antigen excess and are approximately 10 times more sensitive than turbidimetric assays that are not latex particle-enhanced.

Immunoturbidimetry: turbidimetric Ig determinations, usually measured with clinical chemistry analyzers, are prone to interference by samples with high absorbance, as seen in hyperbilirubinemic, hemolytic or hyperlipidemic sera. Antigen excess is easily overlooked and inappropriately low Ig levels are determined.

References

1. Bienvenu J, Whicher J, Chir B, Aguzzi F. Immunoglobulins. In: Ritchie RF, Navolotskaia O. Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996: 11.01-1–16.

2. Ritzmann SE, Daniels JC, eds. Serum protein abnormalities. Diagnostic and clinical aspects. New York: AR Liss, 1982.

3. Gupta S, Gupta A. Selective IgM deficiency: an underestimated primary immunodeficiency. Frontiers in Immunology 2017: 8: article 1056.

4. Sheehan C, ed. Clinical immunology. Principles and clinical diagnosis. Philadelphia: Lippincott, 1990.

5. Kalden JR, Burmester GR. Das Immunsystem des Menschen. Göttingen: Edition SK + F, 1984.

6. Thomas L. Quantitative immunchemische Plasmaproteinbestimmung mittels Nephelometrie und Turbidimetrie. Lab Med 1990; 14: 313–20.

7. Johnson jr RB, Liu J. The application of enzyme immunoassay to the study of salivary IgA. J Immunoassay 1982; 3: 73–89.

8. Alaluusua S, Grönblad EA, Tölö H. Quantitation of IgA in human whole saliva: a comparison of three immunoassays. Acta Odontol Scand 1981; 39: 155–61.

9. Warsy AS, Bahakim AM. Immunoglobulin D – What do we know about it? Saudi Med J 1986; 7: 106–15.

10. Fateh-Moghadam A. Die Bedeutung der Immunglobuline G, A, M und E in der Diagnostik von Lebererkrankungen. Bayr Internist 1982; IV: 1–5.

11. Rosanelli K. Present diagnostic value of IgM-determination in newborns. In: Bethke K, Riegel K, Belohradsky BH, eds. Diagnostics in perinatal infections. Marburg: Med Verlagsges 1984: 174.

12. IgE, allergies and helminth parasites: a new perspective on an old conundrum. Immunology and Cell Biology 1996; 74: 337–45.

13. Renner ED, Belohradsky BH, Grimbacher B. Hyper-IgE-Syndrom. Monatsschr Kinderheilkd 2002; 150: 1186–79.

14. Einsele H, Saal JG, Dopfner R, et al. Hochmalignes Non-Hodgkin-Lymphom bei Hyper-IgE-Syndrom. Dtsch Med Wschr 1990; 115: 1141–4.

15. Heremans JF, Masson PL. Specific analysis of immunoglobulins. Techniques and clinical value. Clin Chem 1973; 19: 294–8.

18.10 Immunoglobulin G subclasses

Lothar Thomas

Immunoglobulin G (IgG) accounts for about 10–20% of plasma proteins. IgG can be further divided in 4 subclasses, named, in order of decreasing abundance IgG₁, IgG₂, IgG₃, and IgG₄. Although they are more than 90% identical on the amino acid level, each subclass has a unique profile with respect to antigen binding, immune complex formation, complement activation triggering of effector cells, half-life, and placental transport /1, 2/. Differences in the properties are presented in Tab. 18.10-1 – Properties of the IgG subclasses :

18.10.1 Indication

• Suspicion of a defective immune response in patients with frequent infections
• Monitoring of immunotherapy with inhalative antigens
• Suspicion and monitoring of IgG₄-related disease

18.10.2 Specimen

Serum, plasma (heparin, EDTA), body fluids (cerebrospinal fluid, bronchoalveolar lavage): 1 mL

18.10.3 Method of determination

The most common method measuring IgG subclasses is immunonephelometry There are two major vendors. Some laboratories use the IgG subclasses LC-MS/MS method. The methods are calibrated to the international reference material ERM-DA470K.

18.10.4 Reference interval

Refer to Ref. /3, 4/ and Tab. 18.10-2 – IgG subclass reference intervals.

18.10.5 Clinical significance

Quantitation of the amount of each IgG subclass in a given serum sample allows identification of selective IgG subclass deficiencies and IgG subclass increase i.e., IgG₄ related disease.

18.10.5.1 IgG subclass deficiency

IgG subclass deficiency is present if the concentration of one or several IgG subclasses decreases below the age-related reference interval values. In childhood, IgG subclass deficiencies are three times more common in boys than in girls. This changes during puberty so that in adults the female/male ratio is 2 : 4. In children, IgG₂ deficiency occurs most commonly, while in adults IgG₁ and IgG₃ deficiencies are the most common /5/.

Most patients with IgG subclass deficiency suffer from frequent respiratory tract infections /6/. Therefore, IgG subclass analysis is part of the routine diagnostic examination in patients who are susceptible to respiratory tract infections. The most important diseases associated with IgG subclass deficiency are described in Tab. 18.10-3– Diseases commonly associated with IgG subclass deficiency. IgG subclass deficiencies may occur in an isolated form or associated with other immune defects (IgA, IgM, IgG, complement deficiencies, and T-cell defects, ataxia teleangiectatica).

More commonly, a certain Gm phenotype is associated with low IgG subclass concentrations. Individuals who are homozygous for G3m(21) have very low IgG₃ concentrations /7/. G2m(23)-negative individuals not only have low IgG₂ levels but, after vaccination with polysaccharides (pneumococci), also show poorer vaccination response as compared to heterozygote carriers of this defect /8/. Occasionally, familial clusters are encountered.

In many cases, decreased IgG subclass concentrations may be clinically manifest as infectious diseases and may also occur secondarily after therapy with steroids, sulfasalazine and carbamazepine /9, 10/.

As in IgA deficiency, many patients with IgG subclass deficiency are healthy. Especially in children, a temporary decrease (delayed maturation) of IgG₂ is often present /11/. For diseases associated with IgG subclass deficiency, see Tab. 18.10-4 – IgG subclass deficiency associated diseases.

In many cases, the cause for IgG subclass deficiency is a regulatory defect in the immune response. In some patients with IgG₂ deficiency, an impaired synthesis of interferon has been described /12/. Deletions on chromosome 14 have been found but in very rare cases in the area of the gene cluster encoding the constant region of the H chain /13/.

Further testing to characterize the immune deficiency

IgG subclass deficiency is primarily considered to be an indicator of impaired immune response /6, 7/. Further, more detailed examinations are required in order to characterize the underlying immune defect and to estimate its clinical relevance /14/.

For instance in patients with IgG subclass deficiency:

• The synthesis of specific antibodies to proteins (tetanus, diphtheria) is usually not affected
• The production of polysaccharide-specific antibodies (e.g., pneumococcal antigen) is reduced in some patients. Detection of naturally acquired polysaccharide antibodies is not very conclusive for the identification of patients with an actual specific immune defect. Vaccination against pneumococci is recommended.

Assessment of functional activity of the immune system following pneumococcal vaccination

An adequate pneumococcal vaccination response is present if the pneumococcal antibody titer in the global test (ELISA, 23-valent vaccine as antigen) rises to > 1,000 U/mL 4–6 weeks after the vaccination or if a significant vaccination response (> 1 μg/mL, calibrated according to the WHO standard 89 SF) is detectable involving five examined pneumococcal serotypes /15/. An adequate vaccination response is detectable in the majority of the patients with IgG₂ subclass deficiency /16, 17/. If a vaccination response < 500 U/mL is measured in the global test, no significant vaccination response to any of the individual serotypes is usually detectable. If a titer < 500 U/mL is confirmed by repeat vaccination, relevant IgG subclass deficiency is diagnosed including a defect of polysaccharide-specific immunity. Depending on the clinical presentation, such a disorder necessitates prolonged therapy (antibiotic prophylaxis or immunoglobulin replacement therapy).

18.10.5.2 Increases in IgG subclasses

Abnormally increased IgG subclasses are common in chronic antigen stimulation, for instance, patients with infections. For further information, see Tab. 18.10-5 – Increases in IgG subclasses.

18.10.5.3 IgG₄-related disease

IgG₄-related disease (IgG₄-RD) was described originally in the pancreas as sclerosing pancreatitis, now referred to as type 1 IgG₄-related autoimmune pancreatitis (AIP). Shortly thereafter, however, the identification of a variety of extra-pancreatic organ involvement linked by unique histopathological features led to the recognition that AIP was part of a systemic condition /18, 19/.

The concept of IgG₄-RD encompasses a wide range of organs sharing two characteristics /20/:

• A set of unique histopathological features. The hallmarks are lymphoplasmacytic infiltrate, storiform fibrosis, obliterative phlebitis, and mild to moderate tissue eosinophilia.
• Elevations in serum IgG₄ concentrations.

IgG₄-RD can involve almost any organ e.g., the pancreas biliary tree, salivary glands, periorbital tissues, kidneys, lungs, lymph nodes, meninges, aorta, breast, prostate thyroid, pericardium, and skin /20/.

There is no evidence that the IgG₄-autoantibodies described so far in IgG₄-RD contribute directly to pathogenesis. The role of IgG₄ itself in the disease process remains unclear. IgG₄-RD tends to form tumefactive lesions. As a result, patients are often suspected of having malignancy.

IgG₄-RD presents in a subacute fashion in most patients, without rapid onset of constitutional symptoms such as fever. The clinical presentation is usually indolent, with signs and symptoms becoming evident over months or even years. High spiking fevers and other manifestations of systemic inflammation that mimic infections are classically absent. IgG₄-RD typically comes to medical attention because of single-organ involvement, but more widespread disease is often observed following a detailed workup. Involvement by IgG₄-RD of different organs can occur either simultaneously or metachronously, with the emergence of one newly affected organ following another. IgG₄-RD has a predilection for middle-aged to elderly men /18, 20/.

Diagnosis of IgG₄-RD

Diagnosis of IgG₄-RD requires both histopathological confirmation and clinicopathological correlation. Serological findings are largely non-specific. Acute phase reactants such as erythrocyte sedimentation rate and C-reactive protein are usually elevated to a moderate degree. Peripheral blood eosinophilia and increased serum IgE occur in almost 30% of patients. Some patients have positive low-titre anti-nuclear antibodies. Although serial measurement of serum IgG₄ is often useful in the assessment of disease activity, they should never be used as the sole determinant of treatment decisions /18/.

High serum IgG₄ concentrations occur in 60–70% of patients, typically in those with multi-organ involvement. Unfortunately, elevation of IgG₄ can be associated with conditions other than IgG₄-RD e.g., systemic vasculitides, connective tissue disease, infections and malignancies /21/. Some IgG₄-RD patients have normal serum IgG₄ concentrations despite histopathologic and immunohistochemical findings in tissue /20/.

According to Japanese study groups a definitive diagnosis of IgG₄-RD is obtained if the patient fulfills the following criteria /22, 23/:

• Elevated serum IgG₄ > 135 mg/dL
• Histopathology: IgG₄⁺/IgG⁺ cell ratio > 0.4
• Swelling or damage of affected organ
• Histopathology: more than 10 IgG₄-positive cells per high power field
• Even in the absence of elevated serum IgG₄, as long as there is organ involvement plus more than 10 IgG₄-positive cells per high power field, and the IgG₄⁺/IgG⁺ cell ratio > 0.4, a diagnosis of IgG₄-RD may still be made.

18.10.6 Comments and problems

The sum of the individual IgG subclasses should not deviate by more than 10% from the measured total IgG (plausibility check). If this is not the case, in an abnormal distribution of the IgG subclasses, a control of both measured parameters (IgG subclasses and total IgG) should be performed.

Method of determination

LC-MS/MS should be the preferred method for measurement of IgG subclasses. In a study /24/, using immunonephelometric IgG subclass reagents analytic errors were noted in patients with increases in IgG₄. The sum of the individual IgG subclasses was substantially greater than the measured total IgG concentrations, and the IgG₄ concentration was always less than the IgG₂ concentration. Using a tryptic digest LC-MS/MS method as reference to quantify the IgG subclasses biases of the immunonephelometric measurements compared with the correspondent LC-MS/MS measurements was observed. The biases potentially reflect two analytical phenomena caused by immunonephelometry:

• Cross reactivity of sample IgG₄ with IgG₂ reagents
• Measurement of IgG₁ and IgG₂ that represent an aggregate of the target Ig and nonspecifically IgG₄ (IgG₄ bound to either the target Ig or the reagent Ig).

In each case, these proposed phenomena would explain the observation of IgG₄-dependent positive biases with immunonephelometric IgG₁ and IgG₂ concentration measurements as compared with the corresponding LC-MS/MS measurements /24/.

Using immunonephelometric methods contaminations inducing light-scattering interfere with this method (e.g., micro clots, cells from inadequately centrifuged samples, particles derived from proteins of the cerebrospinal fluid and microbial contaminations). In principle, problems should be anticipated in samples after deep-freezing or in hyperlipidemic samples.

Reference intervals of IgG subclasses

The reference intervals of the IgG subclasses for children are age-dependent. By 6 months of age, IgG₁ and IgG₃ concentrations are about 50% of the adult level which is reached by 3 years of age. IgG₂ and IgG₄ are produced in a delayed fashion; during the 1st year of life, their concentrations are 25% and during the 3rd year of life 50% of the adult level.

Subclass assays yield different results. Whereas the reference intervals for IgG₁ and IgG₂ from Siemens and The Binding Site are similar /25/, there is a marked difference in those for IgG₃ and IgG₄. This is due to the fact that the standards established by The Binding Site are based on the material ERM-DA470k, while those established by Siemens are based on WHO 67/97, later replaced by Sanquin M1590. Hence, the reference intervals of a given manufacturer should be used consistently in a given case.

18.10.7 Pathophysiology

The function of the 4 IgG subclasses is to eliminate invading pathogens and their products. The Ig structure is adapted to these functions. Ig have the following functional units:

• A Fab portion with a variable region for antigen detection
• An Fc portion mediating the effects of the molecule. The Fc portion binds complement and reacts with Fcγ -receptors on the surface of the defense cells such as polymorphonuclear granulocytes and monocytes/macrophages. This results in the inactivation and/or elimination of the Ig-bound antigen. The fact that 4 IgG subclasses do not have a uniform Fc portion causes their functional differences /1, 2/.

The four IgG subclasses have the following functional differences (Tab. 18.10-1 – Physicochemical and biological properties of the IgG subclasses).

IgG₁

Antibody response to membrane proteins and soluble proteins (T cell-dependent antigens such as, for example, viral and bacterial antigens) primarily induce IgG₁ and with lower levels IgG₃ and IgG₄. IgG antibodies against proteins (tetanus toxin) and viral antigens are predominantly IgG₁ and IgG₃. IgG₁ deficiency is associated with recurrent infections. IgG₁ reaches an adult serum level at the age of 1–4 years; the other Ig subclasses reach approximately 50% of the adult level at this age. Adult levels are finally reached in the adolescent period.

IgG₂

T cell independent antigens like the polysaccharide capsule of H. influenzae and S. pneumonia lead mostly to an IgG₂-restricted antibody response. Antigens are pneumococcal antigen, teichoic acids, dextran HIB-PrP. Low concentrations of IgG₂ often occur in association with a deficiency in IgG₄ and/or IgA1 and IgA2.

IgG₃

Being a potent pro-inflammatory antibody IgG₃ is particularly effective in the induction of effector functions. Decreased IgG₃ levels are frequently associated with other subclass deficiencies.

IgG₄

Allergens are often good inducers of IgG₄ and IgG₁ in addition to IgE. In a non-infectious setting IgG₄ antibodies are often formed following repeated or long term exposure to antigens. Allergen-specific antigens (bees’ venom) under conditions of hyposensibilization stimulate antibody production primarily in the IgG₄ subclass. Helminth or filarial parasite infections may result in the formation of IgG₄ antibodies.

IgG₄-RD is a chronic inflammatory condition that affects almost any organ in the same patient. Molecular mimicry has been proposed to play a role. The target antigens for these IgG₄ antibodies seem evolutionarily conserved, because purified patient IgG₄, but not IgG₄ from healthy donors are deposited at the base of acini or pancreatic duct cells /23/.

References

1. Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: from structure to effector functions. Frontiers in immunology 2014; 5: article 520.

2. Maguire GA, Kumararatne DS, Joyce HJ. Are there any clinical indications for measuring IgG subclasses? Ann Clin Biochem 2002; 39: 374–7.

3. Schauer U, Stemberg F, Rieger CHL, Borte M, Schubert S, Riedel F, et al. IgG subclass concentrations in certified reference material 470 and reference values for children and adults determined with the Binding Site reagents. Clin Chem 2003; 49: 1924–9.

4. Lepage N, Huang SHS, Nieuwenhuys E, Filler G. Pediatric reference intervals for immunoglobulin G and its subclasses with Siemens immunonephelometric assays. Clin Biochem 2010; 43: 694–6.

5. Herrod HG. Clinical significance of IgG subclasses. Curr Opin Pediatr 1993; 5: 696–9.

6. Oxelius VA, Carlsson AM, Hammarstrom L, Bjorkander J, Hanson LA. Linkage of IgA deficiency to Gm allotypes; the influence of Gm allotypes on IgA-IgG subclass deficiency. Clin Exp Immunol 1995; 99: 211–5.

7. Preud’homme JL, Hanson LA. IgG subclass deficiency. In: Rosen FS, Seligman M, eds. Immunodeficiencies. Chur; Harwood 1993: 61–76.

8. Sarvas H, Rautonen N, Käyhty H, Kallio M, Mäkelä O. Effect of Gm allotypes on IgG₂ antibody responses and IgG₂ concentrations in children and adults. Int Immunol 1990; 4: 317–22.

9. Ito J, Nakagawa T, Morita Y, Okudaira H. Immunologic analysis of steroid-dependent asthma. Annals of Allergy 1989; 62: 15–20.

10. Leickly FE, Buckley RH. Development of IgA and IgG₂ subclass deficiency after sulfasalazine therapy. J Pediatr 1986; 108: 481–2.

11. Zielen S, Ahrens P, Kotitschke R, et al. IgG-Subklassenspiegel bei gesunden Kindern. Monatsschr Kinderheilkd 1990; 138: 377–80.

12. Inoue R, Kondo N, Kobayashi Y, Fukutomi O, Orii T. IgG₂ deficiency associated with defects in production of interferon-gamma; comparison with common variable immunodeficiency. Scand J Immunol 1995; 41: 130–4.

13. Plebani A, Ugazio AG, Meini A, et al. Extensive deletion of immunoglobulin heavy chain constant region genes in the absence of recurrent infections: when is IgG subclass deficiency clinically relevant? Clin Immunol Immunopathol 1993; 68: 46–50.

14. Warshaw BL, Check IJ. IgG-subclasses in children with nephrotic syndrome. Am J Clin Pathol 1989; 92: 68–72.

15. Rijkers GT, Sanders LAM, Zegers BJM. Anticapsular polysaccharide antibody deficiency states. Immunodeficiency 1993; 5: 1–21.

16. Gross S, Blaiss MS, Herrod HG. Role of immunoglobulin subclasses and specific antibody determinations in the evaluation of recurrent infection in children. J Pediatr 1992; 121: 516–22.

17. Macdermott RP, Nash GS, Auer IO, Shlien R, Lewis BS, Madassery J, Nahm MH. Alterations in serum immunoglobulin G subclasses in patients with ulcerative colitis and Crohn’s disease. Gastroenterol 1989; 96: 764–8.

18. Della-Torre E, Lanzilotta M, Doglioni C. Immunology of IgG₄-related disease. Clinical and Experimental Immunology 2015; 181: 191–206.

19. Stone JH, Zen Y, Deshpande V. IgG₄-related disease. N Engl J Med 2012; 366: 539–50.

20. Mahajan VS, Mattoo H, Deshpande V, Pillai SS, Stone JH. IgG₄-related disease. Annu Rev Pathol Mech Dis 2014; 9: 315–47.

21. Carruthers MN, Khosroshahi A, Augustin T, Despande V, Stone JH. The diagnostic utility of serum IgG₄ concentrations in IgG₄-related disease. Ann Rheum Dis 2014; 74: 14–8.

22. Umehara H, Okazaki K, Masaki Y, Kawano M, Yamamoto M, Saeki T, et al. Comprehensive diagnostic criteria for IgG₄ related disease (IgG₄-RD), 2011. Mod Rheumatol 2012; 22: 21–30.

23. Kawa S, Sköld M, Ramsden DB, Parker A, Harding SJ. Serum IgG₄ concentration in IgG₄-related disease. Clin Lab 2017; 63. 1323–37.

24. Van den Gugten G, Demarco ML, Chen LYC, Chin A, Carruthers M, Holmes DT, Mattman A. Resolution of spurious immunonephelometric IgG subclass measurement discrepancies by LC-MS/MS. Clin Chem 2018; 64: 735–42.

25. Wilson C, Ebling R, Henig C, Adler T, Nicolaevski R, Barak M, et al. Significant, quantifiable differences exist between IgG subclass standards WHO 67/97 and ERMDA470k and can result in different interpretation results. Clin Biochem 2013; 46: 1751–5.

26. Bartmann P. IgG-Subklassenmangel. Allergologie 1990; 13: 398–403.

27. French MA, Denis KA, Dawkins R, Peter JB. Severity of infections in IgA deficiency: correlation with decreased serum antibodies to pneumococcal polysaccharides and decreased serum IgG₂ and/or IgG₄. Clin Exp Immunol 1995; 100: 47–53.

28. Buckley RH. Immunoglobulin G subclass deficiency: fact or fancy? Current Allergy and Asthma Reports 2002; 2: 356–60

29. Oehling AK, Sanz ML, Resano A. Importance of IgG₄ determination in in vitro immunotherapy follow-up of inhalant allergens. Invest Allergol Clin Immunol 1998; 8: 333–9.

30. Hamano H, Shigeyuki K, Horiuchi A, Unno H, Naoyuki F, Akamatsu T, et al. High serum IgG concentrations in patients with sclerosing pancreatitis. N Engl J Med 2001; 344: 732–8.

31. Detlefsen S, Drewes AM. Autoimmune pancreatitis. Scand J Gastroenterol 2009; 44: 1391–1407.

32. Weindorf SC, Frederiksen JK. IgG4-related disease. Arch Pathol Lab Med 2017; 141: 1476–83.

18.11 Cryoglobulins, cryofibrinogen

Lothar Thomas

Cold reacting and cold precipitating proteins are differentiated into cold agglutinins, cryoglobulins and fibronectin-containing immuno complexes /1/. Cold agglutinins are proteins capable of agglutination and hemolysis of erythrocytes below body tempeature.

18.11.1 Cold agglutinins

Cold agglutinins /2/ are capable of agglutination and hemolysis of erythrocytes via complement activation (cryohemolysin) at temperatures below 37 °C and redissolve on rewarming. Cold agglutinins belong to the IgM class. The IgM antibody-antigen complex, a trigger of the classic complement pathway, generates the C3 convertase C4bC2a. The convertase catalysis C3 proteolysis into C3a and C3b, which in turn triggers opsonization and extravascular hemolysis in the liver. Cold agglutinin disease is caused by cold agglutinins, which are IgM antibodies that bind to I antigen on red cells when temperatures are below or at 37 °C. A titer < 1 : 128 is significant and a definite temperature below or at 37 °C (temperature amplitude). A lower titer or a temperature amplitude higher than 25 °C are not clinically. Patients with cold agglutinin disease have chronic anemia, fatigue, acute hemolytic crisis, agglutination-mediated acrocyanosis and a higher risk of thromboembolism. Cold agglutinin disease is distinguished from cold agglutinin syndrome:

• Cold agglutinine syndrome is transient and secondary to infections and overt malignant autoimmune conditions.
• Cold agglutinine disease is a low-grade B cell lymphoproliferative disorder that can be detected in blood or bone marrow. There is no clinical or radiologic evidence of malignancy.

The following therapies exist for cold agglutinin disease /2/:

• Rutiximab depletes B cells and induces partial responses in about 50%
• Sutimlimab, a humanized monoclonal antibody prevents cold agglutinin-mediated complement deposition on erythrocytes.

Refer also to

• Section 27.5.3.2 – Cold agglutinins
• Figure 24-1 – Activation and regulation of the complement system.

18.11.2 Cold agglutinin syndrome

The patient presented with dark urine, jaundice, and anemia after an upper respiratory tract infection 1 week earlier. Laboratory studies showed anemia and findings consistent with hemolysis. In the peripheral blood smear red cells, reticulocytes, and neutrophils coated with agglutinated erythrocytes, forming rosettes, were found. Phagocytosis of erythrocytes in macrophages and neutrophils were also found. The cold agglutinin titer was 1 : 1024, the serology for antibodies of Mycoplasma pneumoniae, Epstein-Barr virus and other common respiratory viruses was negative. The diagnosis of autoimmune hemolytic anemia from cold agglutinin syndrome associated with an upper respiratory infection was made /21/.

18.11.3 Cryoglobulins

Cryoglobulins are immunoglobulins or immunoglobulin complexes that precipitate at temperatures below 37 °C and redissolve on rewarming (Tab. 18.12-1 – Types of cryoglobulins).

Cryoglobulins are classified into three types /1, 3/:

• Type I (monoclonal). This type represents individual monoclonal immunoglobulins, generally associated with lymphoproliferative diseases.
• Type II (mixed type). Type II consists of mixed immunoglobulins with a monoclonal component and is strongly associated with hepatitis C infection.
• Type III (polyclonal). This type is constituted by a mixture of polyclonal immunoglobulins and is associated mainly with a wide range of infectious, autoimmune, and liver disease.

18.11.4 Indication

Suspected cryoglobulinemia

Skin symptoms: Raynaud’s phenomenon, purpura, livido reticularis, ecchymosis, ulceration, ischemic necrosis.

Clinical complaints: symptom complex consisting of weakness, arthralgia, neurological disorder, glomerulonephritis, arthritis, sicca syndrome, chronic hepatitis C /4/.

18.11.5 Method of determination

Workflow

The workflow for the evaluation and interpretion of cryoglobulins includes different stages /5/.

• Sampling: blood samples for the determination of cryoglobulins are collected in three 4–5 mL tubes without gel anticoagulant to obtain a total of 4–6 mL of serum after centrifugation
• Detection: a minimum of 2 hours for clotting and centrifugation at 37 °C, serum than is decanted into conical-bottomed tubes and placed at 4 °C for 7 days.
• Differentiation of precipitated cryoglobulins. The goal of cryoglobulin differentiation is their classification into types I–III. The cryoglobulin type allows conclusions regarding etiopathogenesis and, thus, facilitates the interpretation of clinical symptoms.

Refer to Table 18.12-2 - Measurement and differentiation of cryoglobulins.

Procedure:

1. For the detection of cryoglobulins, serum is incubated into a tube at temperatures of 4 °C until 15 days. Precipitate is visible for most type I cryoprecipitates within 24 hours, type II and type III cryoglobulins can take several days. Precipitates appear as a white and translucent pellet, as fine or thick flakes, as cryogels or as crystals.

2. For the characterization the cryoprecipitate is isolated by cold centrifugation, the supernatant is discarded and for quantification of the cryoglobulin the cryocrit is determined. For further investigations the cryoprecipitate is washed with cold isotonic NaCl or PBS-buffer and suspended in 0.5 mL NaCl. The sample is placed at 37 °C for 2 hours to dissolve the precipitate. For removal of precipitated proteins, like albumin, three washes in cold NaCl are recommended and the precipitate placed at 37 °C for 2 hours to dissolve the cryoglobulin.

3. For further characterization laboratory testing is carried out using /6/:

• Immunonephelometric investigation of IgA, IgG and IgM to determine the protein components of the cryoglobulin. A serum concentration of about 33 mg/L (diagnostic sensitivity of 73% with a diagnostic specificity of 70%) has been reported for the diagnosis of clinical manifestations /6/.
• Immunofixation electrophresis; determination of the monoclonality and the cryoglobulin isotypes (IgG, IgA, IgM, kappa or lambda) and hence clonality of the precipitate concentration become visible after centrifugation
• Rheumatic factor activity in the cryoprecipitate
• Determination of complement proteins or of CH50 in the serum of the patient. Hypocomplementemia is often associated with type I or type II cryoglobulinemia.

Spectrophotometric detection of cryoglobulins

Two methods are published:

• An assay was described /7/ based on the spectrophotometric detection of a difference in optical density between two aliquots of serum sample incubated at 4 °C and 37 °C respectively.
• In a second study /2/ the increase of turbidity during the cryoglobulin aggregation was determined. Measurements were performed using a dual-beam spectrophotometer with a cell whose temperature was maintained at 10 °C.

18.11.6 Specimen

• Whole blood without an anticoagulant: 10 mL

A blood sample is collected in a 8–10 mL prewarmed tube.

18.11.7 Reference interval

18.11.8 Clinical significance

The incidence of cryoglobulinemia is much higher than that of cryofibrinogenemia. For instance, in the Sheffield Protein Reference Unit, 887 samples were analyzed for cryoglobulinemia in 2004–2008 /9/. As a result of the analysis, 188 cryoglobulinemias and 5 cryofibrinogenemias were detected. The prevalence of types is listed in Tab. 18.12-3 – Cryoglobulinemias at the Sheffield Protein Reference Unit.

Negative evidence of cryoglobulin does not exclude cryoglobulinemia because, in many cases, improper handling of the samples yields false-negative results. The concentrations of C4 and CH50 are surrogate markers.

Hypocomplementemia is often associated with:

• Cryoglobulin-induced immune complex activity
• Type I or mixed cryoglobulinemia

Normal complement concentrations do not allow any conclusions.

About 16–70% of patients with chronic hepatitis C have cryoglobulinemia and positive rheumatoid factor /10/.

18.11.8.1 Cryoglobulinemia

Cryoglobulin-induced diseases are associated with immunoglobulin precipitation during cold exposure. Physiologically, small amounts of cryoglobulins are produced and eliminated by the hepatocytes via a specific receptor. Cryoglobulin deposits in the glomeruli are removed by mono­cytes/macro­phages /11/. Precipitation of cryoglobulins in the vessels causes the development of vasculitis, especially in the skin, kidneys and peripheral nervous system. Clinical manifestation of cryoglobulinemia depends on the organs affected. Histologically, a leucoclastic vasculitis secondary to vascular deposition of immune complexes is seen /3/. This leads to ischemia, necrosis and purpura. About 50–70% of symptomatic patients with cryoglobulinemia have liver involvement, arthralgia and asthenia, and about 25% have renal involvement. The incidence of nervous system involvement is 36%.

There is a lack of correlation between serum cryoglobulin concentration, and severity of disease. Many patients have cryoglobulinemia without symptoms even when serum cryoglobulin levels are high /12/. Cryoglobulins in most of the patients exist in low concentration (100–300 mg/L).

Cryoglobulins undergo reversible condensation upon changing temperature and concentration. Various morphologies of IgG cryoglobulin condensates (e.g., including crystals, amorphous aggregates and gels) exist in different patients /13/.

Mixed cryoglobulinemia, an autoimmune disease characterized by the formation of cold-precipitable cryoglobulin complexes composed of immunoglobulins, is recognized as the most common extrahepatic disease induced by hepatitis C infection.

Based on the immunoglobulin composition mixed cryoglobulinemia has two types of immunoglobulins: type II cryoglobulins consisting of polyclonal IgG and monoclonal IgM with rheumatoid factor (RF) activity and type III cryoglobulins characterized by polyclonal IgG with polyclonal IgM /15/. Mixed type II and polyclonal type III are generally associated with diseases. Approximately 95% of cryoglobulins are immune complexes that contain rheumatoid factor. Usually the RF is monoclonal or polyclonal IgM, although other immunoglobulins may be found. Type II and type III cryoglobulins account for about 50–60% and 25–30%, respectively. Mixed type II is generally associated with diseases where there is a chronic infection such as HCV, chronic hepatitis B or human immunodeficiency virus infection, and in autoimmune diseases such as Sjögren syndrome. More than 90% of patients with mixed type II cryoglobulinemia are infected with hepatitis C virus /14/.

In HCV-related mixed cryoglobulinemia, the majority of patients have asymptomatic cryoglobulinemia, and only 10–15% of these patients will develop cryoglobulinemic symptoms characterized by small vessel vasculitis, glomerulonephritis, and neuropathy due to immune complex deposition and activation of the complement cascade in small blood vessels. The prevalence of cryoglobulin is 2–4 times higher in HCV than in HBV /14/.

A differentiation is made between monoclonal and mixed forms of cryoglobulinemia. The mixed forms (type II and type III) account for up to 90% of cryoglobulinemias, the monoclonal form (type I) accounts for approximately 10%. About 95% of the mixed forms are based on chronic hepatitis C, the remaining forms are of different nature.

18.11.8.1.1 Monoclonal cryoglobulinemia (type I)

Monoclonal cryoglubilinemia is usually associated with plasma cell dyscrasia such as multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), Waldenstroems disease and accounts for 10% of cryoglobulins.

Type I cryoglobulins precipitate at higher temperatures (≤ 32 °C) than mixed cryoglobulins. Concentrations can range between 60 mg/L and 60 g/L.

Clinical manifestations are hyper viscosity syndrome with symptoms of peripheral vascular obstruction, purpura or dermal manifestation (Raynaud’s phenomenon). Type I is rarely associated with vasculitis. Joint manifestations are seen if the cryoglobulin (usually IgG₃) precipitates in crystallized form.

Laboratory findings

The cryocrit is usually high and protein concentrations are > 1 g/L in many cases. Examination for evidence of monoclonal IgG or IgM. The rheumatoid factor is negative, complement decreases are not continuously detectable.

18.11.8.1.2 Mixed cryoglobulinemias (types II and III)

Mixed cryoglobulinemia, an autoimmune disease characterized by the formation of cold-precipitable cryoglobulin complexes composed of immunoglobulins, is recognized as the most common extrahepatic disease induced by hepatitis C infection.

Based on the Ig composition mixed cryoglobulinemia has two types of immunoglobulins /15/:

• Type II cryoglobulins consisting of polyclonal IgG and monoclonal IgM with rheumatoid factor (RF) activity
• Type III cryoglobulins characterized by polyclonal IgG with polyclonal IgM.

Mixed type II and polyclonal type III are associated with diseases. Approximately 95% of cryoglobulins contain rheumatoid factor (RF). Type II and type III cryoglobulins account for about 50–60% and 25–30%, respectively. Mixed type II is generally associated with diseases where there is a chronic infection such as hepatitis C, chronic hepatitis B or human immunodeficiency virus (HIV) infection, and in autoimmune diseases such as Sjögren syndrome. A shared feature of these disorders is chronic inflammation, high antigen load and antigen-driven dysregulation of the B-cell system /16/. More than 90% of patients with mixed type II cryoglobulinemia are infected with hepatitis C virus /14/. According to a study /15/ higher frequencies of TH1 cells and activated memory B cells were associated with type II asymptomatic mixed cryoglobulinemia in HCV infection.

In HCV-related mixed cryoglobulinemia, the majority of patients have asymptomatic cryoglobulinemia, and only 10–15% of these patients will develop cryoglobulinemic symptoms characterized by small vessel vasculitis, glomerulonephritis, and neuropathy due to immune complex deposition and activation of the complement cascade in small blood vessels.

If the RF is monoclonal the mixed cryoglobulinemia is referred to as type II. If the RF is polyclonal, the cryoglobulinemia is referred to as type III. Infections can cause either type II or type III cryoglobulinemia. Associated diseases with type III cryoglobulinemia are post streptococcal glomerulonephritis, chronic infections and essential cryoglobulinemia /17/.

Diseases and symptoms associated with mixed cryoglobulins are listed in Tab. 18.12-4 – Diseases and conditions associated with mixed cryoglobulinemia.

Laboratory findings

As a rule, mixed cryoglobulins precipitate at temperatures ≤ 23 °C. Positive evidence of rheumatoid factor in type II, complement decrease is continuously detectable.

18.11.8.1.3 Heat-insoluble cryoglobulinemia

In the presence of this cryoglobulinemia, the precipitated protein dissolves at temperatures higher than 37 °C, for example at 56 °C. These forms of cryoglobulinemia are rare but have been described in association with multiple myeloma, Sjögren’s syndrome and cryoglobulin-occlusive membranoproliferative glomerulonephritis /11/.

18.11.9 Comments and problems

Temperature control

Temperature control is particularly important in the preanalytical phase of type I cryoglobulins because these cryoglobulins precipitate at higher temperatures and can also be present at concentrations > 5 g/L. High concentrations of monoclonal cryoglobulins tend to precipitate earlier at higher temperatures. For type III cryoglobulins, temperature control is not crucial because they slowly precipitate over a period of days /9/.

Cryoglobulin detection using the precipitate method is due to false negative results because the standard pre analytical procedures to collect blood (tube preheating, transport in container, sedimentation, and/or centrifugation at 37 °C) are not exist in many laboratories /4/.

Practical details

Highly concentrated cryoglobulins can precipitate at any sample kept at temperatures higher than 4 °C.

A negative cryoglobulin result in the presence of a suggestive clinical context (vasculitis, Raynaud phenomenon, peripheral neuropathy, arthralgia, glomerulonephritis) should be repeated because 10% of samples with a negative initial negative test are found to be positive with subsequent tests.The patient should come directly to the laboratory for cryoglobulin determination to avoid preanalytical transport problems /5/.

Cryoglobulins: Washing of the cryoprecipitate

The cryoprecipitate must be washed at cold temperature prior to protein determination to remove contamination by other serum proteins.

Practical recommendations for detection and characterization of cryoglobulins are listed in Ref. /20/.

References

1. Merlini G, Aguzzi F, Whicher JT, Chir B, Navolotskaia O. Cryoglobulins. In: Ritchie RF, Navolotskaia O, eds. Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996: 11.05-1–05.

2. Röth A, Barcellini W, D’Sa S, MiyakawaY, Broome CM, Michel M, et al. Sutimlimab in cold agglutinin disease. N Engl J Med 2021; 384 (14): 1323–34.

3. Voma CB, Levinson SS. Analysis, detection and quantitation of mixed cryoglobulins in HCV infection. Brief review and case examples. Clin Chem Lab Med 2016; 54: 1853–9.

4. Brouet JC, Clauvel JP, Danon F, Klein M, Seligman F. Biologic and clinical significance of cryoglobulins. Am J Med 1974; 57: 775–88.

5. Kalopp-Sarda MN, Miossec P. Practical details for the detection and interpretation of cryoglobulins. Clin Chem 2022; 68(2): 282–90.

6. Amdo TD, Welker JA. An approach to the diagnosis and treatment of cryofibrinogenemia. Am J Med 2004; 116: 332–7.

7. Kalovidouris AE, Johnson RL. Rapid cryoglobulin screening and aid to the clinician. Ann Rheumat Dis 1978; 37: 444–8.

8. Blain H, Cacoub P, Musset L, et al. Cryofibrinogenemia: a study of 49 patients. Cin Exp Immunol 2000; 120: 253–60.

9. Sargur R, White P, Egner W. Cryoglobulin evaluation: best practice? Ann Clin Biochem 2010; 47: 8–16.

10. Cicardi M, Cesana B, Del Nino E, et al. Prevalence and risk factors for the presence of serum cryoglobulins in patients with chronic hepatitis C. J Viral Hepat 2000; 7: 138–43.

11. Meng QH, Chibbar R, Pearson D, Kappel J, Krahn J. Heat insoluble cryoglobulin in a patient with essential type II cryoglobulinemia and cryoglobulin-occlusive membranoproliferative glomerulonephritis: case report and literature review. Clin Chim Acta 2009, Aug 406; 170–3.

12. Cesur S, Akin K, Kurt H. The significance of cryoglobulinemia in patients with hepatitis B and C virus infection. Hepatogastroenterology 2003, 50: 1487–9.

13. Wang Y, Lomakin A, Hideshima T, Laubach JP, Ogun O,Richarson PG, et al. Pathological crystallization of human immunoglobulins. PNAS 2012; 109: 13359–61.

14. Minopetrou M, Hadziyannis E, Deutsch M, Tampaki M, Georgiadou A, Dimopoulou E, et al. Hepatitis C virus (HCV) related cryoglobulinemia: cryoglobulin type and anti-HCV profile. Clin Vaccine Immunol 2013, 20: 698–703.

15. Kong F, Zhang W, Feng B, Zhang H, Rao H, Wang J, et al. Abnormal CD4+ T helper (Th) 1 cells and activated memory B cells are associated withtype III asymptomatic mixed cryoglobulinemia in HCV infection. Virology J 2015; 12: 100. doi: 10.1186/s12985-015-0324-2.

16. Charles ED, Dustin LB. Hepatitis C virus-induced cryoglobulinemia. Kidney International 2009; 76: 818–24.

17. Agarwal A, Clements J, Sedmak DD, Imler D, Nahman NS Jr, Orsinelli DA, et al. Subacute bacterial endocarditis masquerading as type III essential mixed cryoglobulinemia. J Am Soc Nephrol 1997; 8: 1971–6.

18. Amdo TD, Welker JA. An approach to the diagnosis and treatment of cryofibrinogenemia. Am J Med 2004; 116: 332–7.

19. Smith RT. A heparin-precipitable fraction of human plasma. II. Occurence and significance of the fraction in normal individuals. J Clin Invest 1957; 36: 605–16.

20. Kalopp-Sarda, Miossec P. Practical details for the detection and interpretation of cryoglobulins. Clin Chem 2022; 68 (2): 282–90.

21. Fukushima H, Matsumoto M. Cold agglutinin syndrome. N Engl J Med 2023; 389 (7): 642.

18.12Cryofibrinogen

Cryofibrinogenemia is diagnosed when EDTA plasma that is clear at 37 °C develops a precipitate when it is cooled to 4 °C. Cryofibrinogenemia is subdivided into an essential (primary) and a secondary form  /1/. The primary form is rare. The prevalence of secondary cryofibrinogenemia without clinical symptoms is said to be 13% in hospitalized patients. Primary cryofibrinogenemia often develops spontaneously in healthy individuals, while secondary cryofibrinogenemia is associated with an underlying chronic inflammatory disorder in the context of a malignant tumor, diabetes mellitus, inflammation, collagen vascular disease or active infection/1/. The female/male ratio is 1.4 : 1.

Suspected cryofibrinogenemia

In the presence of the following symptoms and/or conditions/diseases: cutaneous ischemia such as purpura, livido reticularis, ecchymosis, ulceration, ischemic necrosis and gangrene (rare).

Clinical significance

Cryofibrinogen can be detected in healthy asymptomatic individuals as many as 2% to 9%. The main clinical symptoms in patients with cryofibrinogenemia are cold intolerance, cutaneous ischemia such as purpura, livido reticularis and acral skin ulcerations. These patients live in colder climate zones and report a temporal association between cold exposure and the onset of symptoms /2/. According to one of the few cases described to date, the laboratory-diagnostic findings in essential cryofibrinogenemia included a cryocrit of 5% and a protein concentration of the cryoprecipitate of 850 mg/L. The cryofibrinogen precipitate must be distinguished from the heparin-precipitable fraction, which also forms in normal individuals during cold exposure if heparinized plasma is used instead of EDTA plasma /3/.

References

1. Amdo TD, Welker JA. An approach to the diagnosis and treatment of cryofibrinogenemia. Am J Med 2004; 116: 332–7.

2. Natali P, Debbia D, Cucinelli MR, Trenti T, Amati G, Spinella A, et al. Analysis of cryoproteins with a focus on cryofibrinogen: a study on 103 patients. Clin Chem Lab Med 2022; 60 (11): 1796–1803.

3. Smith RT. A heparin-precipitable fraction of human plasma. II. Occurence and significance of the fraction in normal individuals. J Clin Invest 1957; 36: 605–16.

18.13 β₂-microglobulin (β₂-M)

Lothar Thomas

β₂-M forms the non-variable light chain of the class I major histocompatibility complex (MHC) and is found on the surfaces of nearly all nucleated cells (Fig. 18.14-1 – HLA antigens on the cell membrane of nucleated cells). When MHC is degraded, the MHC associated β₂-M is released into circulation. This results in a constant production rate of free β₂-M of 2.4 mg/kg/day. β₂-M is a non-glycosylated polypeptide with a molecular mass of 11.8 kDa. Due to its small size, β₂-M is present in the glomerular filtrate of the kidney and other body fluids. Elevated β₂-M serum levels are measured in chronic renal failure, lymphoproliferative disorders, conditions with high cell turnover, inflammations and infections.

18.13.1 Indication

• Monitoring and assessment of therapy in lymphoid neoplasia, especially non Hodgkin lymphoma, Hodgkin’s lymphoma and multiple myeloma
• Assessment of the glomerular filtration rate, especially in children
• Diagnosis and monitoring in tubulointerstitial kidney damage
• Monitoring of β₂-M in dialysis patients
• Assessment of renal function after kidney transplantation and early detection of Cytomegalovirus infection
• Detection of a rejection episode after allogenic bone marrow transplantation
• Monitoring of disease progression in patients with HIV infection
• Diagnosis of fetal infections.

18.13.2 Method of determination

Immunoassay and immunonephelometric or immunoturbidimetric assays /1/.

18.13.3 Specimen

Serum, plasma: 1 mL

Random urine sample to which 0.5 mL of 2n NaOH is added in order to obtain a pH > 6. 10 mL of this urine sample need to be delivered to the clinical laboratory. It is used, for example, as part of occupational-medical examinations.

Urine collection, 6–8 h collection period; collection to be performed only during the daytime. This allows monitoring of the urine pH and, if necessary, alkalinization of the urine by adding 2n NaOH. The urine collection over the specified period of time is indicated for detection of acute, toxic injury to tubules.

18.13.4 Reference interval

Refer to references /2, 3, 4, 5, 6/ and Tab. 18.14-1 – Reference intervals for β₂-microglobulin.

18.13.5 Clinical significance

The concentration of β₂-M in serum and its excretion in urine provide valuable information only:

• In the presence of specific clinical problems
• If other diseases that may also have an impact on synthesis or excretion of β₂-M, have been ruled out first. For example, if the glomerular filtration rate is to be estimated, no lymphoid neoplasia should be present or the presence of a tubulointerstitial renal disease should be ruled out when the therapy of a lymphoid neoplasia is to be assessed.

Refer to Tab. 18.14-2 – β₂-microglobulin in various diseases

In the presence of constant serum levels, an acute rise in concentration or fractional urinary excretion of β₂-M indicates tubular damage. In serum concentrations > 6 mg/L and normal GFR, the tubular reabsorption capacity is exceeded and the concentration in urine cannot be used as an indicator of tubular damage /7/. In such cases, it is better to determine the excretion of α₁-microglobulin. Refer to Section 12.9.6.8.3 – α₁-microglobulinuria. This is recommendable in general because α₁-microglobulin in urine is more stable than β₂-M.

18.13.6 Comments and problems

Stability

In the presence of a pH < 6.0, β₂-M is denatured within a period of 2 hours /6/, even in the bladder. As a result of such degradation, it can no longer be immuno-chemically determined. Consequently, examination of the urine should not involve the first morning urine (usually with a pH < 6.0) but instead should employ a random daytime urine sample or a urine sample collected during the daytime. The pH must be checked after voiding and, if necessary, alkalinization should be performed by adding a few drops of 2n NaOH.

Reference interval

Children have slightly higher serum concentrations than adolescents and adults as well as individuals over the age of 60.

18.13.7 Pathophysiology

β₂-M is a peptide of 100 amino acids. The peptide chain is linked via a disulfide bond between amino acids 25 and 81 (cysteine in each case) /25/.

β₂-M is the light chain protein of the MHC class I antigens and thus present on the membrane of all nucleated cells. It is located on the outside of the cell membrane and is not covalently bound to the heavy chain of the MHC class I antigen. Thus, it is free to exchange with β₂-M in the body fluids, where more than 98% are present in the form of a free monomer.

The expression of MHC class I antigens and, thus, of β₂-M on the cell membranes of immune defense cells (especially of lymphocytes) is stimulated by cytokines of the hemolymphatic system. The β₂-M production is increased in all diseases associated with activation of the immune system such as bacterial infections, certain viral infections and autoimmune diseases (e.g., rheumatoid arthritis). The main synthesis site for β₂-M is lymphocytes. Healthy individuals with a body weight of 70 kg synthesize 9 mg of β₂-M/hour. The half-life is 40 min. The kidney is the main elimination site and glomerular filtration is approximately 210 μg/min. and 99.9% of β₂-M are reabsorbed in the proximal tubules.

Changes in serum β₂-M level or β₂-M excretion are caused by increased production, reduction in GFR or tubular reabsorption.

Since the lymphatic system is the main synthesis site of β₂-M, all conditions with an increased proliferation rate of lymphocytes are associated with elevated serum concentrations; this applies, in particular, to multiple myeloma, Hodgkin’s lymphoma, chronic lymphocytic leukemia and other malignant non Hodgkin lymphomas. In these diseases, the determination of β₂-M is a good indicator for monitoring the disease and assessing the treatment outcome. Monoclonal Ig level and serum β₂-M concentration show a parallel course in about 70% of the patients. The β₂-M concentration is not elevated in some patients despite marked monoclonal gammopathy. This is explained by changes in renal function in myeloma patients /26/. Other diseases associated with pronounced activation of the cellular immune response and elevated serum β₂-M are certain autoimmune diseases, infectious mononucleosis, and transplant rejection episodes /8/.

A reduction in GFR prolongs the half-life of β₂-M, and serum level increases exponentially i.e., is inversely related to GFR (this relationship applies to the entire filtration range). An increase in serum concentration may occur with a GFR decline to below 80 [mL × min^–1 × (1.73 m²)^–1]. Reports concerning a correlation between abnormal serum creatinine and serum β₂-M concentrations differ. The usefulness of β₂-M for assessment of GFR is limited by the fact that other diseases may also cause an elevation /12/.

β₂-M is reabsorbed in the brush border of the proximal tubule, followed by its catabolism into amino acids. Excretion of β₂-M is increased in tubular damage (e.g., due to bacterially induced interstitial nephritis, cadmium nephropathy and amino glycoside-induced tubular necrosis). Patients with amino glycoside-related nephrotoxicity have a urinary excretion > 10 mg in an 8-h urine collection.

In cadmium-exposed individuals, a relationship exists between exposure duration, the cadmium concentration in blood and β₂-M excretion. In individuals with prolonged occupational exposure to cadmium, increased β₂-M excretion should be expected after an exposure period of about 10 years /3/.

In Cytomegalovirus infection, an increase in CD8⁺ T lymphocytes occurs, which is responsible for an increase in serum β₂-M concentration.

Increased serum β₂-M serum levels in malignant lymphomas are presumably due to increased cell turnover. Another theory /26/ is based on the fact that in malignant lymphomas the cell membrane of lymphocytes carries less β₂-M than in healthy individuals. This is thought to be caused by a defect in the α-chain of MHC class I antigens, which subsequently binds less β₂-M. Because an intact MHC class I antigen structure is a prerequisite for recognition of altered cells by cytotoxic lymphocytes, these altered cells are not recognized and, hence, they are able to proliferate.

References

1. Terrier N, Bonardet A, Descomps B, Cristol JP, Dupuy AM. Dermination of β₂-microglobulin in biological samples using an immunoenzymometric assay (chemiluminescence detection) or an immunoturbidimetric assay: comparison with radioimmunoassay. Clin Lab 2004; 50: 675–83.

2. Evrin PE, Wibell L. Serum levels and urinary excretion in apparently healthy subjects. Scand J Clin Lab Invest 1972; 26: 69–74.

3. Lammers M, Gentzer W, Reifferscheidt G, Schmidt B. Dermination of β₂-microglobulin with a particle enhanced immunonephelometric assay. Clin Chem 2002; 48: A-119 (abstract).

4. Schaller KH, Gonzales J, Thürauf J, Schiele R. Früherkennung von Nierenschäden durch Blei, Quecksilber und Cadmium bei beruflich exponierten Personen. Zbl Bakt Hyg, I Abt Orig 1980; B 171: 320–35.

5. Jonasson LE, Evrin PE, Wibell L. Content of β₂-microglobulin and albumin in human amniotic fluid. Acta Obstet Gynec Scand 1974; 53: 49–58.

6. Schardijn J, v Eps LWS, Swaak AJG, et al. Urinary β₂-microglobulin in upper and lower urinary tract infections. Lancet 1979 I; 805–7.

7. Grützner FJ. Diagnostik mit β₂-Mikroglobulin. Inn Med 1982; 9: 45–56.

8. Späti B, Child JA, Kerruish SM, Cooper EH. Behaviour of β₂-microglobulin and acute phase reactant proteins in chronic lymphocytic leukaemia. Acta Haemat 1980; 64: 79–86.

9. Litam P, Swan F, Cabanillas F, et al. Prognostic value of serum β₂-microglobulin in low-grade lymphoma. Ann Int Med 1990; 114: 855–60.

10. Child JA, Späti B, Illingworth S, et al. Serum beta 2 microglobulin and C-reactive protein in the monitoring of lymphomas. Cancer 1980; 45: 318–26.

11. Bien E, Balcerska A. Serum soluble interleukin-2 receptor, β₂-microglobulin, lactate dehydrogenase and erythrocyte sedimentation rate in children with Hodgkin’s lymphoma. Clinical Immunology 2009; 70: 490–500.

12. Diem H, Fateh-Moghadam A, Lamerz R. Prognostic factors in multiple myeloma: role of β₂-microglobulin and thymidine kinase. Clin Investig 1993; 71: 918–23.

13. Kult J, Lämmlein Ch, Röckel A, Heidland A. β₂-Mikroglobulin im Serum, ein Parameter des Glomerulumfiltrates. Dtsch Med Wschr 1974; 99: 1686–8.

14. Portman RJ, Kissane JM, Robson AM. Use of β₂ microglobulin to diagnose tubulo-interstitial renal lesions in children. Kidney International 1986; 30: 91–8.

15. Drüeke TB, Massy ZA. Beta2-microglobulin. Semin Dial 2009; 22: 378–80.

16. Steinhoff J, Feddersen A, Wood WG, et al. β₂-Mikroglobulinurie bei Cytomegalievirus-Infektionen nach Nierentransplantation. Dtsch Med Wschr 1991; 116: 1008–12.

17. Uthmann U, Geisen HP. Beta-2-Mikroglobulin. Dtsch Med Wschr 1981; 106: 782–6.

18. Hümpfner A, Heidegger H. β₂-Mikroglobulinurie und Lysozymurie: Bedeutung als diagnostischer und therapeutischer Kontrollparameter bei Dilatation der oberen Harnwege in der Schwangerschaft. Nieren- und Hochdruckkrankheiten 1989; 18: 545–51.

19. Moss AR, Bacchetti P, Osmond D, et al. Seropositivity for HIV and the development of AIDS or AIDS-related condition: three year follow up of the San Francisco General Hospital cohort. Brit Med J 1988; 296: 745–50.

20. Norfolk DR, Forbes MA, Cooper EH, Child JA. Changes in plasma β₂ microglobulin concentrations after allogeneic bone marrow transplantation. J Clin Pathol 1987; 40: 657–62.

21. Nishimaki S, Shima Y, Sato M, An H, Hashimoto M, Nishiyama Y, et al. Urinary β₂-microglobulin in premature infants with chorioamnionitis and chronic lung disease. J Pediatr 2003; 143: 120–2.

22. Dreux S, Rousseau T, Gerber S, Col JY, Dommergues M, Muller F. Fetal β₂-microglobulin as a marker for fetal infectious diseases. Prenat Diagn 2006; 26: 471–4.

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18.14 Serum sickness

Lothar Thomas

The condition occurs after the therapeutic administration of nonhuman protein. Serum sickness is a type-III-hyper-sensitivity reaction due to the formation of antigen-antibody complexes.

Clinical findings: fever, nonpruritic morbilliform rash across the torso, arms, and on the legs.

Laboratory findings: neutropenia, C-reactive protein (> 80 mg/L) elevated, low C3 and C4 concentrations, negative blood culture.

Reference

1. Shanshal M, Ebadian M.Serum sickness. N Engl J Med 2023; 389 (8): 749.

18.15 IgG4-related diseases

Löthar Thomas

IgG4-related diseases are recognized as a chronic and multisystemic disorder. It is a matter of systemic fibro-inflammatory disease characterized by lymphoplasmacytic infiltration of IgG4 positive cells in tissues and often elevation of IgG4 concentration in serum.

18.15.1 Indication

• Swelling and lymphoplasmacytic infiltrates in the body, e.g. pancreas, thyroid gland, salivary glands, and in liver
• Chronic and multisystemic disorder
• Involvement of serous membranes in organs.

18.15.2 Clinical findings

IgG4 related disease is a chronic and multisystemic autoinflammatory disorder causing swellings ore massive lesions that can affect either a single organ or multiple organs of head and neck, chest and abdomen, the genitourinary body area, and the central nervous system /1/.

• Head and neck: thyroid gland, salivary glands, and lacrimal glands
• chest and abdomen: pancreas, lung, and the liver /2, 3/.
• genitourinary: kidney (nephritis, membranous glomerulonephritis)
• central nervous system: hypophysitis.

Other manifestations are type 1 diabetes, autoimmune pancreatitis, Ridel’s thyroiditis, nephritis, mediastinal and retropritoneal fibrosis.

The involved tissues often show painless swelling, enlargement and dysfunction.

18.15.3 Diagnosis

Elevated concentrations of IgG4 can be measured in most cases. However, diagnosis is made due to the presence of histological results in mass lesions and painless swellings.

References

1. Zhang X, Xing J, Guan L, Lin X, Li X. IgG4-assoziierte Erkrankungen mit gastrointestinaler Beteiligung; Fallberichte und Literaturübersicht. Kompass Autoimmun 2022; 4 (3): 149–55.

2. Minaga K, Watanabe T, Chung H, Kudo M. Autoimmune hepatitis and IgG-related disease. World J Gastroenterol 2019; 25 (19): 2308–14.

3. Zuo A, Liu X, Chung H, Guo Z, Jiang Y, Lu D. IgG4-related diseases involving pleura: a case report and literature review. Frontiers in Medicine 2023. doi: 10.3389/fmed.2023.1247884.