Clinical Significance of Plasma Proteins
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Clinical significance of plasma proteins
Prealbumin
α2-macroglobulin
Complement C3 fraction
γ-globulins
PLASMA PROTEINS
Plasma proteins comprise a heterogeneous group of proteins, each with its own specific or multiple functions. They are subject to concentration changes in a variety of physiological and pathological conditions.
The biosynthesis of these proteins is strictly and specifically regulated by genetically controlled mechanisms, and their normal plasmatic concentration may vary from a few micrograms up to several grams per litre.
The quantification of plasmatic protein concentration is thus a valid tool for the management of a variety of pathophysiological conditions.
· Separation and quantification methods are prevalently based on the molecular, chemical, physical and functional aspects of the analyte. The same molecule may often have different names according to the method employed. This is the case of macroglobulin, also named immunoglobulin M, γ-globulin or glycoprotein.
· Therefore, it is of great importance to study the structural, metabolic and functional characteristics of plasma proteins, as well as their genetic variations.
· Likewise, the clinical value relative to changes of single elements or to multiple variations is important as far as diagnosis and monitoring of pathological conditions is concerned.
CHARACTERIZATION OF PLASMA PROTEINS
A) Chemical properties : simple proteins, glycoproteins, lipoproteins
B) Physical-chemical properties: solubility, sedimentation, electrophoretic migration
C) Functional characteristics: enzymes, carrier proteins, coagulation factors, complement cascade factors, growth factors and differentiation factors, immunity, factors involved in metabolic processes.
FRACTIONAL PRECIPITATION BY MEANS OF ORGANIC SOLVENTS
By means of water-soluble solvents
Alcohols
Acetone
Dioxane
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The addition of solvents mixed to water cause
DEHYDRATION OF PROTEIN MOLECULES:
PROTEIN AGGREGATION
In order to prevent protein denaturation, fractionation must be performed at low temperatures (0 ° C) as well as in environments capable of quickly absorbing heat generated by exothermic reaction caused by mixing organic solvents with water (ice bath).
SEDIMENTATION
Molecular masses of proteins can be determined by means of sedimentation by ultracentrifugation
Svedberg: the sedimentation coefficient is given by the rate of sedimentation in a force field(speed / centrifugal force)
The sedimentation coefficient of a molecule depends on:
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MOLECULAR MASS
SHAPE (ellipsoid, sphere, cylindrical)DENSITY (compact sphere , empty ball)
SIZE
DENSITY AND VISCOSITY OF SURROUNDING MEDIUM
Friction Coefficient
It can be calculated by Stokes's equation (for spherical molecules)
The law no longer meets requirements when the shape departs from compact spherical shapes and at temperatures> 20 ° C
SUMMARY: the sedimentation speed of a particle is proportional to its mass. Denser molecules sediment faster than molecules with lower densities (in solutions at constant densities). Furthermore, friction coefficient greatly increases when considering either ellipsoid shapes or cylindrical/ stick shaped molecules.
ELECTROPHORETIC MIGRATION
Electrophoresis refers to the force that moves charged, soluble ions and molecules (with different sizes) in an electric field.
Soluble proteins are easily ionizable to give R-COO- or R-NH3+ ions.
Analytes
Zone Electrophoresis
The electrophoretic profile displays regions (or zones) where specific proteins will migrate
SOLID PHASE
AGAROSE,
(Cellulose acetate)
LIQUID PHASE
BUFFER-FILLED CAPILLARY
Factors affecting electrophoretic mobility
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
1. The analysis of electrophoretic patterns is among the few laboratory tests that does not rely on the comparison of numerical reference ranges established within a "healthy" cohort.
2. Visual inspection and interpretation of an electropherogram is affected by method-related, instrumental and operator-dependent variability.
3. For these reasons, close collaboration as well as active communication is highly encouraged between the operating staff and professionals performing visual inspection.
PLASMA PROTEINS
Fresh samples are recommended for analysis.
Serum should be collected according to standard clinical laboratory testing procedures.
Serum must be refrigerated (2-8 °C) and stored up to one week.
When longer storage periods are required, samples should be kept at -20°C (up to one month)
SAMPLES THAT SHOULD BE AVOIDED
· Haemolyzed serum samples should not be used
· Haemolysis leads to the appearance of extra peaks in alpha 2 or beta fractions.
· Avoid plasma samples
· Avoid old and poorly stored samples, as well as lipemic specimen
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
· The information provided by electrophoresis is limited to SOME of the over 100 serum proteins, as they are quantitatively more represented within the bands .
· This information may be of great clinical importance in some contexts (such as in the case of monoclonal components, alpha 1-antitrypsin deficiency, nephrotic syndrome etc.).
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
1. Plasma proteins are in a state of dynamic equilibrium that is modulated by the combined effect of synthesis speed and catabolism rate of the single protein entities.
2. Any event that is capable of affecting any of the two speeds may cause a shift in serum protein levels.
3. Plasma proteins are prevalently synthesized by the liver, with the exception of immunoglobulins (synthesized by plasma B cells), of complement factors (predominantly synthesized by macrophages) and of lipoproteins (synthesized by intestinal cells).
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
Total blood proteins can be quantified on both plasma and serum samples.
Nevertheless, due to automatized instrument issues, protein quantification is almost exclusively performed on serum samples.
Normal reference values for serum proteins range from 60 e 84 g/L. Plasma protein values, on the other hand, are 3-5% higher due to the presence of fibrinogen.
Serum values do not stay constant throughout life. Total serum proteins at birth amount to 55,2 g/L, whereas the albumin/globulin ratio is elevated (2,10).
Paediatric serum protein concentrations are lower compared to adult serum protein levels, with women showing lower values compared to men.
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
Factors that may contribute to variations of protidemia (other than age and gender) are:
1. physiological and conspicuous daily variations,
2. seasonal variations (with November peaks and drops in June),
3. increases after physical exercise,
4. decreases in bed-ridden subjects.
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
· Physiological plasma protein concentrations depend on protein synthesis and catabolism, as well as on decreases in protein concentrations from various compartments and from external losses.
· These factors should be accounted for when suspecting the development of a pathological condition. The concomitant variations of conditions in such contexts (such as increased synthesis and increased catabolism) may determine normal serum protein concentrations.
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
· Plasma proteins (especially albumin, fibrinogen and globulins) are synthesized mainly in the liver, with the exception of gamma globulins (produced by plasma cells).
· The intestinal cells can also contribute to lipoprotein production, along with macrophages, which synthesize complement factor proteins.
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
· Protein metabolism is rapid and intense. The total protein pool undergoes daily renewal in physiological conditions. 9% of all proteins are synthesized by the liver, as well as 10-25% of the circulating pool of proteins.
· The liver plays a key role also in catabolic processes.
· In physiological conditions, protein excretion (external losses) occurs through the gastrointestinal apparatus (all proteins), through renal excretion (selective), exocrine glands, respiratory system and genital organs.
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
· Proteins may be classified according to their functional properties:
1) Immunoglobulins with antigen-binding properties
2) Carrier proteins
3) Complement factors
4) Protease inhibitors
5) Various functions: acute-phase proteins, coagulation factors, lipoproteins, glycoproteins, etc.
RESOLUTION CRITERIA FOR EF
F. Aguzzi, D. Fenili, N. Montalbetti, C. Petrini, F. Salvatore, M. Tarantino
Official Recommendations of the SIBioC Committee. Serum Protein Electrophoresis 1° Biochimica Clinica 9; 1985
SOLID PHASE EF STAINING
Cellulose Acetate
+
Ponceau S-stain
= 0.14 g/L
Agarose
+
Acid Blue
= 0.04 g/l
The use of agarose gel or cellulose acetate DIAFANIZATION
Electrophoretic gels are quantified by DENSITOMETRIC SCANNING: staining intensities are graphed according to band intensity and size. Intensities are depicted as peaks where height and width of the peak are dependent on intensity and width of the coloured band (qualitative-semiquantitative quantification)
Protein fraction percentage values (for a 5-fraction electrophoresis) in normal serum are the following:
albumin 55-65%
α1 2-5%
α2 7-11%
β 9-13%
γ 14-20%
Detects only qualitative variations of serum proteins.
Peak intensity does not correspond to protein concentration since different stains perform differently with regards to protein affinity.
Proteins that are more represented within a given band are not necessarily the only ones to migrate in that position (with the sole and unique exception of albumin).
Visual (qualitative) inspection of the gel is thus preferable.
Zone Electrophoresis (ZEF)
ZEF is a separative technique that enables the detection of proteins that migrate in an electric field on the basis of their different pIs. However, it does not give any specific indication concerning the type of protein that compose one zone.
It comprises three analytical steps:
· Separation (usually performed at high pH in alkaline buffers) .
· Revelation with protein staining solution (sensitivity varies according to the type of stain used).
Rosso Ponceau-Amido black 1000mg/L
Coomassie brilliant blue 75-300mg/L
Crystal Violet 30mg/L
Colloidal Gold 1-6mg/L
Gold+silver enhancement 0,8-1mg/L
· Reading and interpretation
CLINICAL SIGNIFICANCE OF PLASMA PROTEINS
· Electrophoretic analysis is a valuable tool in medical diagnostics:
· It offers a global perspective over the protein panorama that plays a role in the most relevant serum protein processes.
· Enables the detection of variations in single protein concentrations or within groups of proteins. It also enables to detect heterogeneity within fractions (as in the case of genetic variants) as well as the detection of paraproteins.
Indications for testing
“normal electrophoretic run”
Data is reported upon result elaboration
Report indicating qualitative and quantitative (increase or decrease) description of altered protein fraction
Normal
SERUM PROTEINS
Normal concentration: 65-80 g/l
Liver
Origin:
Lymphoid Tissue
Hyperprotidemia: increased levels of specific proteins (dysglobulinemia), haemoconcentration
Hypoprotidemia: decreased protein levels (absorption disorders, intake disorders, protein loss, liver failure) haemodilution
Agarose gel and capillary serum protein electrophoresis
PREALBUMIN BAND
· PREALBUMIN is the largest band with the most anodal electrophoretic mobility.
· Detectable on non-diaphanized electrophoretic gels. Visual inspection is sufficient to confirm a decrease in prealbumin concentration.
· Prealbumin is a carrier protein. Along with albumin and transferrin, it is considered a very sensitive indicator of the organism's nutritional status. ;
· its very short half-life (1,9 days) enables the rapid evaluation of variations in the protein synthesis rate in liver.
Prealbumin
ALBUMIN BAND
ˑ ALBUMIN is the major protein component of serum, accounting for 60% of the total mass of plasma proteins (equivalent to 40-50 g/L).
ˑ Albumin plays two important roles: it acts as carrier protein (for fatty acids, bilirubin, hormones, drugs) and is the main determinant of colloid osmotic pressure (80% of the total colloid osmotic pressure is related to albumin);
ˑ sharp decreases in serum albumin concentration indicated an imbalance between hydrostatic and oncotic pressures. Plasma will move across vascular membranes and into tissues causing oedema.
ˑ Albumin inheritable genetic variants - known as alloalbumins or bisalbumins - may appear as bifid albumin peaks by serum protein electrophoresis, due to a higher or lower electrophoretic mobility of the variant compared to the normal albumin fraction. These variants have no clinical significance and their encounter is often occasional.
ALBUMIN BAND
ˑ Splitting: genetically acquired or transient bisalbuminemia.
ˑ Decrease: due to synthesis deficiency (liver diseases); due to increased losses (nephrotic syndrome, protein-losing enteropathies, burns); modest decrease during acute inflammation and pregnancy.
ALPHA 1 BAND
ˑ Reflects serum levels of ALPHA1-ANTITRYPSIN (AAT), physiologically the most important protease inhibitor (produced by neutrophil granulocytes).
It contains inflammatory processes by means of massive neutrophil intervention, by limiting the destructive action of proteolytic enzymes released by their granules or with the in situ death of neutrophils themselves.
ˑ Genetic regulation of synthesis and excretion of this molecule is very complex, controlled by a large number of alleles. This results in a great variety of phenotypes and protein variants within the general population most of which have no effects on the functional properties of the protein..
ALPHA 1 BAND
ˑ The presence of AAT protein variants give rise to two bands in the alpha 1 fraction of the electrophoretic lane: this finding must be reported.
ˑ The expression of some alleles results in a deficient protein synthesis (Null allele) or in release deficiency (Z allele), with very important clinical outcomes.
ALPHA 1 BAND
· Severe AAT deficiency implies that the activity of proteases, secreted by granulocytes and macrophages (during microinflammatory processes) are not inhibited. This causes destructive alterations of connective and elastic tissues of alveoles, thus initiating the processes which lead towards the clinical condition known as emphysema.
· In some cases, the protein is synthesized by hepatocytes, but it is not secreted. This causes significant intracellular accumulation consequently inducing serious liver injury with progression to cirrhosis, which may occur in early childhood settings.
ALPHA1 BAND
ˑ Increases in alpha 1 antitrypsin concentrations may typically be observed in inflammatory conditions in tissues of epithelial origin. For this reason, it used to be included within the group of "acute phase proteins".
ˑ Notably, in some (infrequent) cases, the presence of two alpha 1 bands is due to elevated levels of alpha1-fetoprotein. This protein is used as a tumor marker to detect tumors of disembriogenic origin, primary liver cancer or evolving hepatitis after the age of two. These conditions should be considered upon the encounter of a double alpha 1 band.
ALPHA 1 BAND
· Decrease: genetically induced deficiency of alpha1-antitrypsin in a homo- or heterozygous condition.
· Reported results should be followed by quantitative immunoassay
· Increase: acute inflammation
· Split Alpha 1 band: alpha 1-antitrypsin variant (non-pathological). Presence of alpha-fetoprotein?
ALPHA 2 BAND
· This band comprises two components: an anodal fraction and a cathodal fraction. The anodal fraction corresponds to alpha-2-Macroglobulin.
· Alpha2-Macroglobulin is among the largest plasma proteins found in the blood. It is a powerful protease inhibitor involved in defense mechanisms against endogenous proteases as well as in regulating homeostasis and balance during coagulation and fibrinolytic processes.
A2-Macroglobulin
ALPHA 2 BAND
ˑ This protein exerts its function by binding to proteases (previously cleared by inhibitors) and removing them from the blood circulation.
ˑ It carries hormones such as insulin and somatotropin: in relation to this function, concentrations (of this protein) are much higher in children than in adults.
ˑ Despite its highly functional activity, the clinical significance of this protein is poorly understood.
ALPHA 2 BAND
ˑ Decrease: protein-losing enteropathies, eclampsia, rheumatoid arthritis.
ˑ Physiological increase: childhood, pregnancy.
ˑ Pathological increase: nephrotic syndrome, acute phase
ALPHA 2 BAND
· The cathodal fraction of the alpha 2 band corresponds to the presence of HAPTOGLOBIN. This term indicates a family of plasma glycoproteins. Their structure is genetically determined by two autosomic codominant allels, namely Hp 1 and Hp 2, resulting in the expression of three phenotypes: Hp 1 - 1, Hp 1-2 e Hp 2-2.
· These proteins have different molecular weights which imply different electrophoretic speeds of migration: for this reason, the location of the haptoglobin fraction of the alpha2 band may vary slightly.
Haptoglobin
ALPHA 2 BAND
Th main role of haptoglobin is to bind free plasma haemoglobin.
Under physiological conditions, a small fraction of circulating red blood cells undergoes intravascular destruction (leading to haemolysis).
Free haemoglobin binds haptoglobin with a 1:1 molar ratio.
The haptpglobin-haemoglobin complex cannot pass through the glomerulus: it is therefore rapidly picked-up and removed by the histiocyte-macrophage system.
ALPHA 2 BAND
· Decreases:
- excessive consumption: haemolytic conditions
(haemolytic anemias, ineffective erythropoiesis, transfusion accidents, haemolysis associated with prosthetic heart valves, megaloblastic anemias, infectious mononucleosis, bruising, intra-tissue bleeding, prolonged exercise).
- synthesis disorders: reduction of functional liver parenchyma (hepatitis, cirrhosis, cancer).
The reduction should be reported and followed by a quantitative detection.
· Increases:
- inflammatory processes
- necrotic processes
- neoplastic processes
Other minor protein components of the Alpha-2 band are:
Ceruloplasmin: glycoprotein. Major copper-binding protein in the blood.
It is synthesized by the liver, but synthesis regulation occurs independently from the amount of circulating copper.
Each molecule binds to 6 copper atoms.
Copper is normally stored in the liver: it is transported to peripheral tissues, where it is used for the synthesis of a variety of enzymes.
Decreased ceruloplasmin levels are observed in Wilson's disease, characterized by elevated toxic deposits of copper in liver, brain, heart.
VITAMIN D BINDING PROTEIN: its specific function is unknown. It is the major vitamin D (and vitamin D metabolites) carrier protein. Synthesized by the liver.
BETA 1 BAND
· The beta 1 zone is mainly composed of transferrin. Disease statuses characterized by a genetically determined absence of transferrin (that is, lack of beta 1 band) have been reported in the literature.
· Structurally different transferrin variants all migrate in the beta 1 zone. Nevertheless, each individual only has one type of transferrin, rarely two types.
· This heterozygous condition is uncommon. Nevertheless, it determines splitting of the beta 1 band into two thin components, which may lead to misinterpretation of monoclonal components.
BETA 1 BAND
· Transferrin variants with low contents of sialic residues may cause band splitting. This may occur in patients with alcohol dependence or in patients affected by severe liver disease.
· This protein plays a main functional role in iron metabolism.
· Transferrin decreases have little diagnostic significance and are related to decreased hepatic synthesis.
· Along with prealbumin and albumin, transferrin is considered an indicator of the nutritional status of a subject.
BETA 1 BAND
ˑ Decreases: due to impaired synthesis:
ˑ Liver disease, chronic infections and neoplasms, acute anemia of chronic disease.
ˑ Renal loss: nephrosis, glomerulonephritis.
ˑ Physiological increases: pregnancy.
ˑ Pathological increases: iron-deficient anemia.
BETA 2 BAND
· The beta 2 zone is mainly composed of the C3 complement fraction.
· It may be particularly visible when FRESH SERUM is used. Aging of serum causes C3 conversion into its cleavage products (also true for serum stored at + 4 °C) that migrate to the beta 1 zone.
· C3 is the most important protein component of the complement system. It plays a central pivoting role in the activation of both the classical and the alternative pathways.
BETA 2 BAND
· C3 is the only component of the complement cascade present in sufficient concentration to allow its detection by serum protein electrophoresis. Visual inspection of beta2 band enables to draw meaningful information concerning the functional activity of this protein system, of crucial importance for the body's defense mechanisms.
BETA 2 BAND
ˑ A decreased intensity of the beta2 band, as well as the absence of the band, indicates decreased C3 concentration.
ˑ This data should always be confirmed by nephelometric quantification of the protein and results must be reported.
BETA 2 BAND
· Decreases:
- Genetically acquired variants: due to overconsumption:
glomerulonephritis, LES, bacteremia, arthritis
due to decreased synthesis: liver diseases.
-Genetically acquired variants (very rare)
"old" serum
· Increases:
- Old inflammatory processes
- Infectious processes
- Tumor processes
- Myocardial infarction
- Biliary obstruction
C REACTIVE PROTEIN
· C Reactive Protein is found in the serum of patients affected by inflammatory disorders. As ESR, it is extremely non-specific. Upon elevated concentrations, it can appear on the electropherogram as a narrow band in the cathodic area, simulating a monoclonal component.
GAMMA ZONE
· This fraction is mainly composed by immunoglobulins (antibodies). This is a very heterogeneous group of proteins showing electrophoretic mobility ranging from the beta region to the gamma region and, in some cases, even more anodally into the alpha zone.
· Immunoglobulins are synthesized by plasma cells. They play a key role in antigen recognition and elimination (or neutralization) within the host (humoral immunity).
· The antigens may be known molecules (as in infectious diseases), suspicious or questionable (as in autoimmune diseases), or difficult to define (as in chronic liver disease or sarcoidosis)
Gamma globulins
GAMMA ZONE
ˑ A high electrophoretic resolution, particularly of the gamma zone, is to be preferred since visual inspection is required in order to appreciate even small qualitative alterations of diagnostic relevance that may otherwise escape densitometric scanning.
ˑ Concentrations of the five immunoglobulin classes (G, A, M, D, E) vary widely across biological fluids, according to their functions and structural characteristics. Serum concentrations depend on the rate of synthesis/breakdown, although this relationship varies for each class and subclass according to intrinsic factors (such as age, gender, intensity of antigenic stimulation) as well as external factors (such as the use of drugs).
GAMMA ZONE
· Immunoglobulin analysis can detect a variety of alterations, that may be grouped as follows:
1) quantitative decreases.
2) quantitative increases.
3) qualitative alterations.
GAMMA ZONE
· Congenital or acquired immunodeficiency syndromes are characterized by increased susceptibility to infections, as well as to increased incidence of autoimmune diseases and lymphoreticular neoplasms (more rarely).
· Hypogammaglobulinemia is normally observed during childhood. However, this condition may persist until adolescence with reversible features (transient hypogammaglobulinemia of infancy).
GAMMA ZONE
· Primary deficiency disorders characterized by chromosomal location of the defect have a variable incidence (such as sex-linked agammaglobulinemia: 1150000, selective IgA deficiency: 11600).
· Hypogammaglobulinemia involving the three major immunoglobulin classes (or the IgG class) is easily identified by electrophoretic analysis.
· Selective IgA deficiency is associated with respiratory, gastrointestinal and urogenital tract infections (where IgA antibodies are most abundant). It is not easily detectable since other immunoglobulin classes may mask the effect, as they are present in normal or even increased amounts (compensatory mechanisms).
GAMMA ZONE
· Secondary hypogammaglobulinemias (not linked to birth defects of the immunocompetent system) are very frequent:
· due to decreased synthesis or increased catabolism (malnutrition, lymphoproliferative disorders, immunosuppressive drugs, radiation therapies, monoclonal gammopathies);
· due to increased excretion (nephrotic syndrome, protein-losing enteropathies, burns).
· Hypogammaglobulinemia detection in adults or in the elderly should always be checked for the possible existence of immunoproliferative disorders (lymphoma, micromolecular myeloma).
GAMMA ZONE
· Excess immunoglobulin production with the involvement of a large number of immunocompetent cell lines is the body's physiological humoral response to external pathogens.
· Hypergammaglobulinemia in acute infections is only visible on the electropherogram after a considerable amount of time - about 20 days - following antigenic contact.
· Chronic diseases associated with increases polyclonal immunoglobulin increases (chronic liver diseases, collagen diseases) are often due to abnormal antigen-antibody reactions with autoantigens against which the host immune system has lost tolerance.
GAMMA ZONE
· Increases in IgG concentration are most common, with the exception of liver diseases (which show predominant IgA increases) as well as parasitic infectious diseases (characterized by increased IgMs).
· Biliary cirrhosis is characterized by increased "background" levels of the total gamma-globulin area. This is due to the involvement of different classes and subclasses of immunoglobulins, with different electrophoretic mobilities.
GAMMA ZONE
· Particular profiles of the gamma zone can indicate the existence of specific immunoglobulin classes. Laboratory staff should be aware of the following profiles:
IgG increases cause spreading of the band over a wider area of the gamma zone (if only some subclasses are involved the band may be more anodal or cathodal)
IgA increases localize in the Beta-Gamma region, giving rise to the so-called "beta-gamma bridge",
IgM increases may be seen as a thick band on the application point.
GAMMA ZONE
· Though very unspecific, increased polyclonal Igs can be useful for evaluating the clinical course of some morbid affections (such as lupus, rheumatoid arthritis, acute viral hepatitis).
· Visual inspection of the electrophoretic pattern is a useful tool in the differential diagnosis of chronic liver disease. Indeed, increased IgA levels are indicative of alcoholic cirrhosis, whereas increased IgMs and higher IgG concentrations are typically seen in primary biliary cirrhosis and chronic active liver disease, respectively.
GAMMA ZONE
· Serum protein electrophoresis is the only available analytical method capable of detecting qualitative alterations due to the presence of monoclonal gammopathies (MGUS).
POLYCLONAL
MONOCLONAL
GAMMA ZONE
· Decreases: due to congenital or acquired hypogammaglobulinemia
· Polyclonal increases: due to physiological antibody response to pathogens. It has pathological features in pathological contexts such as in chronic diseases with immune disorders (as in chronic liver diseases, collagen affections and autoimmune disorders).
· Monoclonal increases: monoclonal gammopathies..
Lab Routine Techniques
Available laboratory routine techniques enable the identification of some of the possible protein (PT) variants or abnormalities.
· PT analysis requires three fundamental elements:
· Quality
· Concentration
· Function
Quality
· Comprises two discriminating characteristics:
1. Antigenic or individual composition
2. Electrophoretic mobility or distribution
A separative technique such as EF (electrophoresis) shows how protein fractions of a given sample are distributed relatively to normal sample distribution, but it is not able to detect protein composition.
Immunometric assays, such as nephelometry, detect protein presence and concentration. Nevertheless, it is not capable of describing the distribution, as in the case of separative techniques.
Concentration
Separative techniques provide estimates of total protein concentrations within each individual fraction.
Immunological techniques provide single protein concentrations namely the antigenic individuality.
Estimates of the concentration of individual PTs within single fractions, both techniques need to be used.
Methods
LIQUID PHASE QUANTIFICATION: requires the use of nephelometry or turbidimetry.
Nephelometry: antigens react with highly specific antibodies, with elevated protein binding affinity.
Antigen-specific complexes are formed, even in the presence of other proteins.
The light beam nephelometer is deflected (scatter). The deviation of the beam is proportional to the size and amount of complexes, in other words, to the protein concentration.
TURBIDIMETRY: technique based on the use of normal absorption spectrophotometer. It relies on the as assumption of a linear relationship between absorbance and protein concentration.
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