Electrophoresis Part 3 PDF

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Summary

Disease Screening with SPE- MLTs use SPE to screen for various diseases, the main ones being A1A deficiency, Cirrhosis, Nephrotic Syndrome and Acute Phase Reactions. - Electrophoretic patterns are very predictable and they act as a strong diagnostic tool.Alpha 1 Antitrypsin Deficiency- Most of the A...

Description

MLSC-3111, Clinical Biochemistry II Disease Screening with SPE -

MLTs use SPE to screen for various diseases, the main ones being A1A deficiency, Cirrhosis, Nephrotic Syndrome and Acute Phase Reactions. Electrophoretic patterns are very predictable and they act as a strong diagnostic tool.

Alpha 1 Antitrypsin Deficiency -

Most of the Alpha 1 fraction is made up of 90% A1A. This deficiency is associated with emphysema. A1A helps to maintain the elasticity of the alveoli through inhibiting proteases that break down elastin.

Nephrotic Syndrome -

Kidney damage is seen as a loss of proteins in the urine. In this syndrome, a loss of lower molecular weight proteins is seen since the kidney is losing its ability to filter fine/small proteins in the blood. Results in a decrease in albumin and IgG in the blood as they are filtered into the urine. A retention of higher molecular weight molecules is seen, causing high levels of alpha-2macroglobulin and beta-globulin in the blood. Typically, a pattern of low albumin and increased alpha-2 and beta fractions is seen in these patients.

Cirrhosis -

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Most identifiable SPE pattern of all. Beta-gamma globulin bridging is seen where the presence of fast-moving gamma globulins prevents the resolution between the beta and gamma fractions. The fast-moving gamma-globulins are there because cirrhosis is associated with increased IgA which likes to migrate to the dip between the beta and gamma fractions. This creates a visual “bridging” effect on the SPE stain. Typically, a pattern of decreased albumin, alpha-1, alpha-2 with increased beta and gamma fractions is seen in these patients. Beta-Gamma bridging must be reported.

MLSC-3111, Clinical Biochemistry II Acute Phase Reaction (APR) -

Also known as inflammation as a non-specific response to tissue damage/sensitization in the body. Serves to protect the body. It is accompanied by a series of local and systemic clinical, biochemical and cellular changes that cause an APR. The body favours the production of protective proteins against infection/injury over the non-protective proteins. Negative APR Proteins, these are markedly decreased and include albumin, transferrin and prealbumin. Positive APR Proteins, these are markedly increased and include fibrinogen, antitrypsin, haptoglobin, C-Reactive protein, etc. Typically, a pattern of decreased albumin (Neg APR Proteins) and increased alpha-1, alpha-2 and beta fractions. Gamma fraction does not change.

Terminology -

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Hypogammaglobulinemia, decreased serum concentration of one or more classes of normal immunoglobulins. Refers only to the gamma band. Can be either primary or secondary. o Primary Hypogammaglobulinemia, combined immunodeficiency. o Secondary Hypogammaglobulinemia, caused by another underlying disorder (multiple myeloma, leukemia, amyloidosis, or nephrotic syndrome). Little to no staining is seen in hypogammaglobulinemia. Cancer patients will show hypogammaglobulinemia since they are immunosuppressed from chemotherapy and have a reduced production of antibodies. Hypergammaglobulinemia, increased gamma band concentration. Can arise from either a polyclonal or monoclonal source. o Polyclonal Increase, elevation in all immunoglobulins. Can be a normal response to inflammation and is a non-malignant increase. A broad and diffuse increase in the gamma zone is seen. o Monoclonal Gammopathy, a disease process in which a single beta lymphocyte clone is being created in excess due to a neoplastic transformation. Only one lymphoid cell is affected, and one abnormal protein is created. The clone is referred to a paraprotein, M-protein or an M-spike. o The proteins and can be intact Ig or free Ig light/heavy chains. These proteins can be found in serum or urine and can be benign to highly malignant. o The gamma globulin band is increased in this disorder, but this is not considered as a true hypergammaglobulinemia. Only one individual immunoglobulin is elevated. o In a normal condition, a variety of Ig are circulating in the body while abnormal conditions have a single abnormal Ig being cloned. Causes a large discrete band and peak in the gamma fraction.

MLSC-3111, Clinical Biochemistry II o Disorders with paraproteins are also referred to as dyscrasias. o SPE serves to screen for monoclonal bands using quantitation through densitometry, providing clinically relevant information. Production of a single paraprotein supresses the production of all other Igs. An increase in Ig concentration means an increase in neoplasm growth. o SPE alone cannot identify the type of paraprotein as it can be IgG/M/A/D/E or a kappa/lambda light chain. Other Methods of Immunoglobulin Measurement Quantitation Immunoglobulins -

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Nephelometric Assays, assays for quantitation of IgGs and other proteins. This is a light scattering technique. It indicates the total concentration of IgG/M/A/D/E but do not distinguish between presence of monoclonal or polyclonal increase. Bands must be identified if present to aid in diagnosis and monitoring.

Immunofixation Electrophoresis (IFE) -

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Used to identify the type of paraprotein seen. This is a qualitative method since it only identifies the abnormal protein by adding known antisera to the sample. Routine IFE uses antisera for IgG/A/M, kappa and lambda. IgD and IgE is not included because D and E monoclonal proteins are very rare and making antiseras to them is very expensive. As a result, there is no need or budget to run IgD and IgE antisera on IFE. There are multiple steps to running an IFE o Step one, the paraprotein seen on the SPE is already quantitated and is put on the IFE. o Step 2, the sample is run on the IFE for ID of the paraprotein. We do not type the paraprotein every time we see one if it is the same patient having a test repeated because it is expensive. New automated staining systems may produce a “blush” across several lanes that look like bands. Biclonal Gammopathy, involves 2 paraproteins. Oligoclonal Gammopathy, involves more than 2 paraproteins. Always list the paraproteins in order of greatest to smallest concentration. Fibrinogen always migrates between the beta and gamma bands. If a plasma sample is put on instead of serum, it will cause a band that is inconsistent with the antisera bands.

Heavy and Light Chains -

Sometimes a heavy chain gene deletion results in the production of excessive free light chains.

MLSC-3111, Clinical Biochemistry II -

Normal plasma cell synthesis produces more light chains than needed to combine with heavy chains. Most free light chains (FLC) are reabsorbed with the rest being excreted into the urine A single immunoglobulin gene mutation can produce excessive gene mutation which can produce excessive FLC. Bence Jones Proteins, another name for FLCs. Igs have either kappa or lambda LC but never both. There is always a 2:1 kappa to lambda ratio. In Bence Jones proteinuria there are free LC found in urine. These LCs can damage the kidneys and usually lead to end-stage renal failure. BJPs are highly soluble at 37 degrees but make the urine cloudy at 40 degrees. At 60 degrees, the proteins precipitate out of solution and upon boiling they disappear. However, when urine is cooled down the BJPs returned into solution. A sample containing only FLCs will not be positive for proteins on the urine dipstick because the dipstick only detects albumin.

Urine Immunofixation Electrophoresis -

The preferred sample is a 24-hour urine but the first morning urine sample is acceptable. The urine must be concentrated because the electrophoresis is not sensitive enough to detect the BJPs in undiluted urine. A urine IFE is run when the serum SPE band is greater than 30 g/L, serum IFE monoclonal protein is detected or in the presence of hypogammaglobulinemia. If a urine sample isn’t available you can request a urine collection.

Nephelometric FLC Analysis -

Uses antibodies that recognize specific epitopes on kappa and lambda LCs. Eliminated the detection of whole Igs, provides direct quantitative value for light chains only and has a faster turnaround time.

Paraproteinemias -

Paraproteinemias are suspected with calcium elevation, kidney insufficiency, anemia and bone lesions.

Multiple Myeloma (MM) -

Considered a cancer due to proliferation of abnormal plasma cells that metastasize in the bone marrow. It is also considered an anemia due to suppression of normal RBC production. It is a neoplastic proliferation of a single clone of plasma cells. Plasma cells usually proliferate throughout the bone marrow and occasionally form plasmacytomas. Can also produce bone lesions.

MLSC-3111, Clinical Biochemistry II -

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Patients present with deep bone pain and bone fragility. Typically an elderly disorder and rouleaux of RBCs is seen. Diagnosis is made if two of these three conditions are met. o Production of Paraproteins, detected in the serum (> 30 g/L) or urine. o Plasma Cells, more than 10% in the bone marrow. o Bone Destruction, from either osteolytic lesions or extreme osteoporosis. Myeloma cells and antibodies cause serious medical problems. o Hypercalcemia, from bone destruction. o Thrombocytopenia, from myeloma cells suppressing normal cell production with its monoclonal production. o Hypogammaglobulinemia, from myeloma cells suppressing normal Ig production with its monoclonal production. o Normochromic Normocytic Anemia, cancer cells prevent RBC production. o Kidneys, excess antibody protein will damage the kidneys during filtration. Most patients die of kidney failure from paraproteins in the nephrons. Multiple Myeloma is associated with IgG and IgA paraproteins. Also, if the patient has the rarer IgD multiple myeloma or FLC multiple myeloma they will show UBJ in the urine 100% of the time. Non-secretory Multiple Myeloma, no paraproteins are excreted in serum/urine but bone marrow plasma clonal cells are greater than 10% with related organ/tissue impairment. Smoldering Multiple Myeloma, an asymptomatic condition where there are M-Proteins > 30 g/L and bone marrow plasma cells are > 10%. May progress into a symptomatic MM. MGUS, monoclonal gammopathy of undetermined significance. Paraprotein is present but patient has no symptoms. Up to 85% of monoclonal gammopathies are MGUS. Paraproteins in this condition are considered benign. Paraproteins are < 30 g/L, plasma cells are > 10% and there are no FLCs or organ injuries. MGUS are associated with increased risk of MM. Patients should be followed for a minimum of 5 years. The disease progresses from MGUS, to Smoldering to MM. Waldenstrom’s Macroglobulinemia, cancer of the immune system usually associated with IgM paraprotein. Increased blood viscosity is seen. Less malignant than MM but still requires monitoring. Lymphoid infiltration is slower and can be treated. The IgM concentration in this condition is usually > 30 g/L. Amyloidosis, pathological process in which proteins are deposited in tissues as aggregates that form beta sheet structures. The ID of light chains on SPE/IFE is not diagnostic and requires tissue analysis. Enlarged tongue is a sign of this (macroglossia). Cryoglobulinemia, circulating Ig complexes that precipitate at low temperatures and dissolve when warm. o Type I, monoclonal IgG/M/A. o Type II, monoclonal IgM with a polyclonal IgG mixture. o Type III, polyclonal Ig.

MLSC-3111, Clinical Biochemistry II -

Samples are collected in tubes at 37 degrees to avoid precipitation. They are heated in a glove with hot water in the fingers. Blood is left to clot, centrifuged and then serum is decanted for precipitate investigation. If precipitates are found, the cryocrit is determined (% precipitate of total aliquot volume). Cryoglobulins will redissolve while cryofibrinogen will not. Cryoglobulins show up in all IFE lanes.

Spinal Fluid Electrophoresis -

SF contains less than 1% of the total protein found in serum. 10% of it is prealbumin. Increased TP in SF can be due to either blood-Sf permeability or increased IgG synthesis in SF. The proteins are produced locally in the spinal fluid cavities and not the blood. This electrophoresis is usually done on people suspected of multiple sclerosis. Multiple Sclerosis (MS), a chronic relapsing and remitting disease characterized by diffuse demyelination of the nervous system. 90-95% of MS patients show a characteristic pattern of oligoclonal banding on SF electrophoresis. Nothing is seen in the serum electrophoresis because the proteins are produced locally in the CSF. Oligoclonal banding also appears in CNS syphilis and meningitis, so the band is not definitive of MS....