Presentation on theme: "High Resolution Protein Electrophoresis"— Presentation transcript:
1 High Resolution Protein Electrophoresis A Clinical Overview with Case StudiesByLawrence M. Killingsworth, Ph.D.
2 Human ProteinsOnly ~200 of the vast array of human proteins have been characterized.Clinical knowledge is limited to 25 – 30 relatively high concentration components of plasma, CSF, urine and other fluids.Of these, 15 or so can be visualized by high resolution agarose electrophoresis.
3 Normal Control Pre-albumin Albumin 1-Acid Glycoprotein 1-Antitrypsin -Lipoprotein1-AntichymotrypsinHaptoglobinC-3IgAIgGAlbumin1-Antitrypsin2-MacroglobulinTransferrin-LipoproteinFibrinogenIgM
4 Monoclonal Gammopathies Uncontrolled proliferation of a single clone of plasma cells at the expense of other clones.Protein analysis is valuable in diagnosing and monitoring lymphoproliferative diseases.
5 Evaluation of Monoclonal Gammopathies Serum and Urine High Resolution Protein Electrophoresis (24-hour urine preferred)Quantitative Serum ImmunoglobulinsSerum and Urine IFE
6 SPIFE ImmunoFixIFE is the method of choice to identify suspicious serum or urine electro-phoretic bands.Periodic evaluation by serum and urine electrophoresis and by quantitative Ig assay can help monitor therapy.
7 Monoclonal Gammopathies Variable mobility and band appearance.
8 Case 1: IgG Kappa Monoclonal ControlPatientPatient: 78 year-old maleHistory & Physical: Severe pain right leg and right lumbar region.SPE: Monoclonal band in gamma region.IFE: IgG Kappa.Hospital Course: Bulging disk surgically decompressed; referred to hematologist/ oncologist for follow-up & treatment.
9 Case 2: IgG Kappa Monoclonal Patient: 78 year-old maleHistory & Physical: Recent chemotherapy for lymphadenopathy.SPE: Hypoalbuminemia and monoclonal band in 2 region.IEP: IgG Kappa.Hospital Course: Treated with transfusions and plasmapheresis. Symptomatic improvement. Discharged for outpatient re-evaluation.ControlPatient
10 Case 3: IgA Lambda Monoclonal Patient: 74 year-old femaleHistory & Physical: Myeloma. Pain in lower thoracic and upper lumbar spine, right shoulder and left anterior ribs.SPE: Large monoclonal band in 2 region.IEP: IgA Lambda.Hospital Course: Radiation therapy. Transferred to hospital closer to home for continued radiation and chemotherapy.ControlPatient
11 Case 4: IgM Kappa Monoclonal Patient: 68 year-old maleHistory & Physical: Anemia, elevated IgM, edema in ankles, petechiae.SPE: Marked M-component in 2 region; also in urine.IEP: IgM Kappa in serum; free Kappa light chains in urine.Hospital Course: Bone marrow biopsy non-diagnostic. Discharged for out-patient treatment and followup for possible macroglobulinemia, chronic lymphatic leukemia or lymphoma.ControlPatient
12 Case 5: Lambda Light Chain Patient: 78 year-old maleHistory & Physical: Anemia, azotemia; admitted for dialysis.SPE: 1AT and Hp, pre-albumin, albumin & transferrin (consistent with acute inflammation); 2 small M-proteins in region. Urine: albumin,1AT, transferrin; large M-protein in region and smaller cathodal band.IEP: Monoclonal lambda light chain in urine and serum. No heavy chain.Hospital Course: Bone marrow biopsy confirmed multiple myeloma. Hemodialysis, plasmapheresis and chemotherapy improved symptoms.ControlPatient
13 Case 6: Lambda Light Chain Patient: 82 year-old maleHistory & Physical: Fractured left hip, suspected frontal lobe infarction.SPE: albumin, acute inflammation, Hp (consistent with in-vivo hemolysis or RBC turnover), mild hypogamma-globulinemia. Urine: Several small monoclonal bands in region.IEP: Monoclonal free lambda light chain in urine; no monoclonal proteins in serum.Hospital Course: Acute left cerebral infarction confirmed. B12 anemia treated. Died due to post-operative pneumonia following hip surgery.ControlPatientSerumUrine
14 Multiple Myeloma Clinical Presentation Bone pain, especially in spine, pelvis or ribsRenal failure of unknown etiologyRecurrent bacterial infectionsPhysical exam usually unremarkable – no lymphadenopathy or hepatosplenomegaly
15 Macroglobulinemia Clinical Presentation Fatigue Generalized weakness Skin and mucosal bleedingVisual disturbancesHeadacheOther neurological signs and symptomsCardiopulmonary abnormalities due to increased plasma volume and viscosityRecurrent bacterial infectionsPhysical exam may reveal purpura, lymphadenopathy and hepatosplenomegaly
16 1-Antitrypsin Deficiency Genetic Deficiencies1-Antitrypsin DeficiencyLinked to hepatitis and cirrhosis in neonates; chronic obstructive pulmonary disease and hepatic cirrhosis in adultsElectrophoresis useful in initial evaluationQuantitative immunochemical assays and phenotyping required
17 Immunoglobulin Deficiencies Genetic DeficienciesImmunoglobulin DeficienciesIsolated IgA deficiencyIsolated IgM deficiencyX-linked immunodeficiency with IgMWiskott-Aldrich SyndromeTransient hypogammaglobulinemia of infancyAtaxia TelangiectasiaSevere combined immunodeficiency (SKID)Common variable immunodeficiencyPan hypogammaglobulinemiaIgG and IgA deficiencyIsolated IgG deficiency
19 Chronic Hepatocellular Disease & Cirrhosis Liver DiseasesChronic Hepatocellular Disease & CirrhosisMost common pattern includes diffuse increase in IgG with proportionally greater increases in IgA and sometimes IgM1-antitrypsin is the most sensitive indicator for hepatocellular diseasePre-albumin is the most sensitive monitor in cirrhosis; 2-macroglobulin and ceruloplasmin also very elevatedAll other proteins usually normal or decreased
20 Case 7: Chronic Hepatocellular Disease Patient: 39 year-old maleHistory & Physical: Long-term alcohol abuse; ascites, leg swelling, shortness of breath, right side pain, enlarged liver and spleen.SPE: Hypoalbuminemia with normal migration. pre-albumin, -lipoprotein and transferrin, consistent with chronic disease. Diffuse IgA, IgG .Hospital Course: Ascites , right lung abcess treated. Liver tests normal at 4 weeks. Discharged in good condition.Pre-albuminAlbuminHaptoglobinTransferrinImmunoglobulinsC-32-Macroglobulin1-Antitrypsin-LipoproteinNormalPatient
21 Liver Diseases Hepatitis Often associated with acute phase inflammatory response in the early stagesDiffuse elevations in one or more of the immunoglobulins with chronic disease
22 Case 8: CirrhosisPre-albuminAlbuminHaptoglobinTransferrinImmunoglobulinsC-3-Lipoprotein1-Antitrypsin2-Macroglobulin-LipoproteinNormalPatientPatient: 54 year-old female History & Physical: Chronic alcoholism; deeply jaundiced, rapid pulse, hepatomegaly, splenomegaly. SPE: Hypoalbuminemia ( anodic mobility due to bilirubin binding). pre-albumin, -lipoprotein and transferrin, consistent with chronic disease. Diffuse IgA, IgG . Hospital Course: Rehydrated and stabilized. Discharged in good condition.
23 Protein Losing Disorders Selective Protein LossNephrosis can result in elevations in serum concentrations of large proteins with decreases in smaller components.Serum pattern shows:Increased 2-macroglobulin, -lipoprotein and polymeric forms of haptoglobin.Decreased pre-albumin, albumin, 1-acid glycoprotein, 1-antitrypsin, transferrin.IgM usually elevated, IgG usually decreased.
24 Case 9: Acute Renal Failure Patient: 57 year-old male History & Physical: Rapid onset abdominal pain and enlargement, edema, decreased urine output. SPE: albumin, 2-macroglobulin and -lipoprotein, consistent with selective protein loss due to glomerular-type proteinuria. Hospital Course: Received albumin and hemodialysis. Discharged in improved condition with limited outpatient dialysis. Final diagnosis: Acute renal failure due to tubular necrosis, possibly of a toxic nature.AlbuminHaptoglobinTransferrinImmunoglobulinsC-3-Lipoprotein1-Antitrypsin2-Macroglobulin-LipoproteinNormalPatient
25 Protein Losing Disorders Nonselective Protein LossWhole blood lossCongestive heart failureLiver failureHemodilutionMalnutritionProtein-losing enteropathies – greater decrease in immunoglobulins than other plasma proteins
26 Pregnancy & Hyperestrogenism* Moderate decreases in prealbumin, albumin, 1-acid glycoprotein and IgGLarge relative increases in 1-antitrypsin, ceruloplasmin, transferrin and fibrinogenModerate increase in -lipoproteinSlight increase in 2-macroglobulin and hemopexinHaptoglobin and C-3 essentially normal*Hyperestrogenism (i.e. contraceptive pills, estrogen medications) can mask pathological changes
27 Case 10: Infant w/ Hepatic Involvement Patient: 5 month-old female History & Physical: Previously healthy; 104º fever/24 hrs, jaundice, hepatomegaly. SPE: Hypoalbuminemia ( anodic mobility due to bilirubin binding), 1-AT, Hp, -lipoprotein, transferrin; consistent with acute inflammation. Age-appropriate hypogammaglobulinemia. Hospital Course: Blood culture positive for gram negative rods. Treated with antibiotics and discharged.Pre-albuminAlbuminHaptoglobinTransferrinImmunoglobulins-Lipoprotein1-Antitrypsin2-MacroglobulinC-3NormalPatient
28 Case 11: Acute Renal Failure Patient: 52 year-old male History & Physical: Weakness, progressive shortness of breath, tachycardia, anemia, azotemia, mild hepatomegaly, edema. SPE: Hypoalbuminemia, 1-AT, Hp, -lipoprotein, transferrin; consistent with acute inflammation. Low normal gammaglobulins. CRP? Hospital Course: Blood culture positive for Staph aureus. Vigorous antibiotic therapy. Died 5 days post-admission.ImmunoglobulinsC-reactive proteinPre-albumin2-MacroglobulinAlbuminHaptoglobinTransferrinC-3-Lipoprotein1-AntitrypsinNormalPatient
29 Case 12: Chronic Renal Failure Patient: 68 year-old male History & Physical: Chronic bladder outlet obstruction; abdominal pain, hematuria, urinary or bladder infection. SPE: Hypoalbuminemia with anodal mobility; 1-AT, Hp, transferrin; consistent with acute inflammation. Random urine pattern consistent with mixed glomerular-tubular proteinuria. Hospital Course: Continued antibiotics, hemodialysis.Pre-albuminHaptoglobinTransferrinC-3-Lipoprotein1AntitrypsinAlbumin2-Macroglobulin-LipoproteinImmunoglobulinsNormalPatient
30 Case 13: Acute & Subacute Inflammation Pre-albuminHaptoglobinTransferrinC-3-Lipoprotein1-AntitrypsinAlbumin2-MacroglobulinImmunoglobulins-LipoproteinNormalPatientPatient: 74 year-old maleHistory & Physical: Pneumonia, 2 weeks duration.SPE: Hypoalbuminemia with anodal mobility; 1-AT, Hp, transferrin, a-lipoprotein, and C-3; consistent with acute and subacute inflammation.Hospital Course: Antibiotics, discharged in good condition.
31 Case 14: Diffuse Hypergamma-globulinemia with Lymphoma Patient: 63 year-old female History & Physical: Pruritis, sweats and fatigue, multiple dermal nodules. SPE: Non-specific findings – diffuse increase in immuno-globulins, suggesting chronic inflammation. Hospital Course: Biopsy reports consistent with lymphoma; patient discharged for outpatient treatment and followup.2-MacroglobulinImmunoglobulinsPre-albuminHaptoglobinTransferrinC-3-Lipoprotein1-AntitrypsinAlbumin-LipoproteinNormalPatient
32 Urinary Proteins of Plasma Origin Normal Urinary Protein < 150 mg/dayPrimarily Filtered Plasma Proteins – albumin, low MW species, immunoglobulin componentsRemainder – derived from urinary tractElectrophoretic pattern of normal urine – trace albumin, sometimes transferrinUrineSerumAlbuminSometimes Transferrin
33 Urinary Proteins of Plasma Origin ProteinuriaGlomerular – results from increased passage of proteins through the glomerulus; characterized by loss of plasma proteins the size of albumin or largerTubular – results from decreased capacity of tubules to reabsorb proteins; characterized by inceased excretion of very small proteins such as 2-microglobulinSystemic – exercise, postural, pregnancy, overflow
34 Urinary Proteins of Plasma Origin High Resolution protein electrophoresis developed with a sensitive protein stainExcellent analytical techniqueEasily distinguishes & characterizes the various types of proteinuriaProvides useful insight on specific functions within the nephron“Biochemical biopsy”Differential diagnosis & monitoring of patients with renal dysfunction
35 Urinary Proteins of Plasma Origin Glomerular ProteinuriaRenal glomeruli are ultrafilters for macromoleculesDamage to renal glomeruli leads to increased urinary excretion of proteins (30,000 to 100, daltons) which are normally retainedSome selectivity remains – very large proteins (>500,000 daltons) still retained by glomerulusIn early disease, very LMW proteins (<15, daltons) are still reabsorbed by tubules and absent from urine
36 Urinary Proteins of Plasma Origin Glomerular ProteinuriaUrine Protein Pattern strong band of albumin strong, broad 1 zone due to 1-acid glycoprotein and 1-antitrypsin strong band of transferrin (1)Serum Protein Pattern marked decrease inalbumin marked decrease in 1-acid glycoprotein and 1-antitrypsin increases in large proteins: 2-macroglobulin, -lipoprotein
37 Urinary Proteins of Plasma Origin Severe Proteinuria / Nephrotic SyndromeTotal Protein > 3.5 g/dayHypoalbuminemia and hyperlipidemiaMassive edema
39 Case 1: Nephrotic Syndrome with Glomerular Proteinuria Patient: 45 year-old white maleHistory & Physical: Diabetes mellitus / nephrotic syndrome.SPE: pre-albumin, albumin and transferrin with 2-macro-globulin and -lipoprotein; consistent with selective renal protein loss.Urine Elp: albumin, 1-antitripsin and transferrin with trace pre-albumin and 2-components; consistent with sieving glomerular-type protein loss.Serum UrineTransferrinAlbumin1 acid glycoprotein1 AT1AT2-MacroHP-LippoC-3IgAIgMIgGNormal
40 Urinary Proteins of Plasma Origin Tubular ProteinuriaNormal tubules reabsorb and catabolize 95 to 99% of proteins from glomerular filtrateTubular disease reduces capacity to reabsorb and catabolize, resulting in increased urinary excretionCauses of tubular proteinemia: Chronic metal exposure (cadmium, gold, lead, mercury) Acute and chronic pyelonephritis Renal transplant rejection Toxicity due to aminoglycoside therapy Balkan nephropathy Uremic medullary cystic disease
41 Urinary Proteins of Plasma Origin Tubular ProteinuriaSerum Protein Pattern little or no change since LMW proteins are present in very low levelsUrine Protein Pattern faint albumin band double band in 2 area due to 2-microglobulin strong band in mid- region due to 2-microglobulin diffuse background in region due to free light chains
42 Urinary Proteins of Plasma Origin Mixed Glomerular/Tubular ProteinuriaChronic renal disease or renal failureCombined pattern with both “glomerular-type” and “tubular-type” proteins in the urine
43 Case 2: Heavy Metal Toxicity with Tubular Proteinuria Serum UrineNormalAlbumin2Micro- globulin2Micro globulin1-AT2-MacroHPTransferrin-LipoC-3IgAIgMIgGPatient: 52 year-old black maleHistory & Physical: Metal worker.SPE: Essentially normal.Urine Elp: Trace albumin, 2-microglobulin and 2-microglobulin; consistent with tubular-type proteinuria.
44 Urinary Proteins of Plasma Origin Other Conditions with Increased Urinary Protein ExcretionExercise Proteinuria – strenuous muscular exercise increases excretion of HMW and LMW proteinsPostural or Orthostatic Proteinuria – present/upright, absent/recumbent – benign or underlying cause?Pregnancy – usually transitory, may be associated with toxemia, delivery, UTI, or asymptomaticOverflow Proteinuria – increased plasma concen- tration of LMW proteins, e.g. BJP, myoglobin, hemoglobin, acute phase reactants
45 Case 3: Septicemia with Overflow Proteinuria Patient: 65 year-old white femaleHistory & Physical: High fever, chills, sweats, joint and muscle aches.SPE: pre-albumin, albumin, -lipoprotein and transferrin, 1-antitrypsin, haptoglobin, C3 & C-reactive protein; consistent with acute inflammation.Urine Elp: Trace albumin and transferrin, 1-acid glycoprotein, faint acute phase reactants; consistent with overflow proteinuriaAcute phasereactantsAlbumin1-acidglycoprotein1-AT1AT2-MacroHPTransferrinC-3IgAIgMIgG-LipoSerum UrineNormal
46 Cerebrospinal Fluid Proteins CSF Proteins versus Plasma Proteins CSF Total Protein: Much less than in plasma – 450 mg/L, ages 10 – 40 years, lumbar Slightly higher, ages Slightly higher for verticular & cisternal specimensCSF Production: Primarily ultrafiltration and active transport of proteins, ions, water and other components through the choroid plexus. Small amount produced within CNS
47 Cerebrospinal Fluid Proteins CSF Protein Composition Albumin – major protein present, 55 – 75% of the total1 – primarily 1-antitrypsin, -lipoprotein absent2 – essentially absent1 – transferrin detectable2 – carbohydrate-deficient “CSF-specific” transferrin – almost exclusively IgG, faint “-trace”Normal CSFAbnormalPatient CSFPre-albuminAlbumin1-AntitrypsinHaptoglobinTransferrinCSF TFOligoclonal Bands
48 Cerebrospinal Fluid Proteins Permeability of Blood-CSF Barrier Increased permeability caused by bacterial or viral menigitis neoplastic infiltration of meninges polyneuropathies disk herniations cerebral infarctionsIntegrity of blood-CSF barrier Total CSF protein 2-macroglobulin Protein ratios
49 Cerebrospinal Fluid Proteins Abnormal CNS Protein Production Demyelinating Diseases Increased IgG synthesis in Multiple Sclerosis and Subacute Sclerosing PanencephalitisIgG as Percentage of Total Protein Considers increased permeability vs. synthesis IgG >10% suspicious; >13% abnormal production likely.CSF/Serum Ratios Considers increased plasma concentration % of MS patients show values above reference range.Oligoclonal Banding Indicative of MS; new more sensitive procedure is IEF.
50 Cerebrospinal Fluid Proteins Oligoclonal BandingMultiple, restricted bands in the gamma fractionDetectable by high resolution electrophoresis and IEF methods90% of MS patients exhibit oligoclonal bandingNew CSF IEF methods have become the gold standard for diagnosing MS in Europe.
51 Cerebrospinal Fluid Proteins Isoelectric FocusingIEF followed by immunoblottingOligoclonal banding in unconcentrated CSF but not serum is diagnostic of MSGold Standard in EuropeS = Serum C = CSF
52 Cerebrospinal Fluid Proteins Laboratory Protocol to Rule Out MS Draw both CSF and serum samples.Note CSF color and appearance. Determine total protein, glucose, white cell count, differential, red cell count.Perform high resolution protein electrophoresis on concentrated CSF.Perform high resolution protein electrophoresis on serum for presence or absence of banding.In borderline cases, determine CSF and serum albumin and IgG ratios.
53 Cerebrospinal Fluid Proteins Laboratory Findings in Multiple Sclerosis NSCPatient SerumPatient CSFNormal CSFCSF Appearance: ClearLeukocytes: Usually normalTotal Protein: Usually normalGlucose: NormalElectrophoresis: Oligoclonal banding in 90% of casesIgG Ratio: Elevated in 90% of cases
54 Cerebrospinal Fluid Proteins Clinical Manifestations of MS 40% of patients present with optic neuritis60 to 70% present with evidence of spinal cord or brainstem lesion
55 Case 1: Multiple Sclerosis Patient: 33 year-old femaleHistory & Physical: left-side numbness - 9 months; fuzzy vision - 2 weeks.SPE: Normal.CSF: Clear, colorless. Cell count, differential, glucose and total protein all normal.CSF Elp: Increased gamma globulins, strong oligoclonal banding. Consistent with demyelinating disease.Normal CSFPatient CSFPatient SerumNSC
56 Case 2: Multiple Sclerosis Patient: 31 year-old maleHistory & Physical: Unsteady walk, leg numbness, inability to concentrate, irritability, slurred speech, disturbed vision, urinary urgency.SPE: Normal.CSF: Clear, colorless. Normal glucose. 29 WBC/mm3 (9% lymphocytes, 1% moncytes). Total Protein 55 mg/dL.CSF Elp: Gamma globulin increase, strong oligoclonal banding; consistent with MS.Normal CSFPatient CSFPatient SerumNSC
57 Case 3: Multiple Sclerosis Patient: 36 year-old femaleHistory & Physical: numbness in legs – 1 month; urinary urgency and blurred vision – 4 months; tires easily.SPE: Normal.CSF: Clear, colorless. Normal total protein, WBC count, and glucose.CSF Elp: Profound gamma globulin increase, very strong oligoclonal banding. (IgG & ratios markedly elevated.) Consistent with MS.Normal CSFPatient CSFPatient SerumNSC
58 Case 4: Suspected Viral Meningitis Patient: 25 year-old white femaleHistory & Physical: Progressive headache, lethargy, altered mental status; “flu” – 4 weeks prior.SPE: Borderline hypoalbuminemia.CSF: Clear, colorless. Normal glucose. 158 WBC/mm3 (1oo% moncytes). Total Protein 83 mg/dL.CSF Elp: Increased gamma globulins, prominent oligoclonal banding. Considering total protein; consistent with viral encephalitis. Discharged for outpatient followup.Normal CSFPatient CSFPatient SerumNSC
59 Case 4: Suspected Viral Meningitis (cont’d) Patient: Readmitted 8 days later with redeveloped headache.Normal CSFPatient CSFPatient SerumNSCCSF: Clear, colorless. Normal glucose and protein. 103 WBC/mm3 (90% lymphocytes, 10% neutrophils).CSF Elp: Gamma globulins and oligoclonal banding decreased from previously. Transient increase consistent with CNS infection / inflammation.
60 SPIFE 3000 AnalyzerAutomated sample application, separation and staining of visible analytesAutomated reagent application for enzymatic assaysOn-board cooling for excellent separation and resolutionOn-board availability of two stains