High Resolution Protein Electrophoresis

1
High Resolution Protein Electrophoresis
A Clinical Overview with Case Studies

• By
• Lawrence M. Killingsworth, Ph.D.

2
Human Proteins

• Only 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
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 Immunoglobulins
• Serum and Urine IFE

6
SPIFE ImmunoFix

• IFE 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.

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Monoclonal Gammopathies
Variable mobility and band appearance.
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Case 1 IgG Kappa Monoclonal
Patient 78 year-old male History 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.
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Case 2 IgG Kappa Monoclonal
Patient 78 year-old male History 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.
10
Case 3 IgA Lambda Monoclonal
Patient 74 year-old female History 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.
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Case 4 IgM Kappa Monoclonal
Patient 68 year-old male History 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.
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Case 5 Lambda Light Chain
Patient 78 year-old male History 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.
13
Case 6 Lambda Light Chain
Patient 82 year-old male History 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.
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Multiple Myeloma
Clinical Presentation

• Bone pain, especially in spine, pelvis or ribs
• Renal failure of unknown etiology
• Recurrent bacterial infections
• Physical exam usually unremarkable no
  lymphadenopathy or hepatosplenomegaly

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Macroglobulinemia
Clinical Presentation

• Fatigue
• Generalized weakness
• Skin and mucosal bleeding
• Visual disturbances
• Headache
• Other neurological signs and symptoms
• Cardiopulmonary abnormalities due to increased
  plasma volume and viscosity
• Recurrent bacterial infections
• Physical exam may reveal purpura, lymphadenopathy
  and hepatosplenomegaly

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Genetic Deficiencies
?1-Antitrypsin Deficiency

• Linked to hepatitis and cirrhosis in neonates
  chronic obstructive pulmonary disease and hepatic
  cirrhosis in adults
• Electrophoresis useful in initial evaluation
• Quantitative immunochemical assays and
  phenotyping required

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Genetic Deficiencies
Immunoglobulin Deficiencies

• Isolated IgA deficiency
• Isolated IgM deficiency
• X-linked immunodeficiency with ? IgM
• Wiskott-Aldrich Syndrome
• Transient hypogammaglobulinemia of infancy
• Ataxia Telangiectasia
• Severe combined immunodeficiency (SKID)
• Common variable immunodeficiency
• Pan hypogammaglobulinemia
• IgG and IgA deficiency
• Isolated IgG deficiency

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Inflammatory Response
CHRONIC
ACUTE
SUBACUTE
Pre-albumin ? ?? ? ?
Albumin ? ? ? ?
ALPHA-1 ?-lipoprotein ? ? ?
?1-acid glycoprotein ? ? ? ?
?1-antitrypsin ? ? ? ?
ALPHA-2 Cerulopasmin ? ? or N
Haptoglobin ? ? ? ?
BETA-1 Transferrin ? ? ? ? ?
BETA-2 C-3 ? ? or N
Fibrinogen ? ? ? ?
IgA ?
GAMMA IgM ?
IgG ?
CRP ? ? ? ? ? ? ?
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Liver Diseases
Chronic Hepatocellular Disease Cirrhosis

• Most common pattern includes diffuse increase in
  IgG with proportionally greater increases in IgA
  and sometimes IgM
• ?1-antitrypsin is the most sensitive indicator
  for hepatocellular disease
• Pre-albumin is the most sensitive monitor in
  cirrhosis ?2-macroglobulin and ceruloplasmin
  also very elevated
• All other proteins usually normal or decreased

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Case 7 Chronic Hepatocellular Disease
Patient 39 year-old male History 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.
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Liver Diseases
Hepatitis

• Often associated with acute phase inflammatory
  response in the early stages
• Diffuse elevations in one or more of the
  immunoglobulins with chronic disease

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Patient 54 year-old femaleHistory 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.
Case 8 Cirrhosis
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Protein Losing Disorders
Selective Protein Loss

• Nephrosis 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.

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Patient 57 year-old maleHistory 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.
Case 9 Acute Renal Failure
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Protein Losing Disorders
Nonselective Protein Loss

• Whole blood loss
• Congestive heart failure
• Liver failure
• Hemodilution
• Malnutrition
• Protein-losing enteropathies greater decrease
  in immunoglobulins than other plasma proteins

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Pregnancy Hyperestrogenism

• Moderate decreases in prealbumin, albumin,
  ?1-acid glycoprotein and IgG
• Large relative increases in ?1-antitrypsin,
  ceruloplasmin, transferrin and fibrinogen
• Moderate increase in ?-lipoprotein
• Slight increase in ?2-macroglobulin and hemopexin
• Haptoglobin and C-3 essentially normal

Hyperestrogenism (i.e. contraceptive pills,
estrogen medications) can mask pathological
changes
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Patient 5 month-old femaleHistory 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.
Case 10 Infant w/ Hepatic Involvement
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Patient 52 year-old maleHistory 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.
Case 11 Acute Renal Failure
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Patient 68 year-old maleHistory 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.
Case 12 Chronic Renal Failure
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Case 13 Acute Subacute Inflammation
Patient 74 year-old male History 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.
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Patient 63 year-old femaleHistory 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.
Case 14 Diffuse Hypergamma-globulinemia with
Lymphoma
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Urinary Proteins of Plasma Origin

• Normal Urinary Protein lt 150 mg/day
• Primarily Filtered Plasma Proteins
  albumin, low MW species, immunoglobulin
  components
• Remainder derived from urinary tract
• Electrophoretic pattern of normal urine
  trace albumin, sometimes transferrin

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Urinary Proteins of Plasma Origin

• Proteinuria
• Glomerular results from increased passage of
  proteins through the glomerulus
  characterized by loss of plasma proteins the
  size of albumin or larger
• Tubular results from decreased capacity of
  tubules to reabsorb proteins characterized by
  inceased excretion of very small proteins
  such as ??2-microglobulin
• Systemic exercise, postural, pregnancy,
  overflow

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Urinary Proteins of Plasma Origin

• High Resolution protein electrophoresis developed
  with a sensitive protein stain
• Excellent analytical technique
• Easily distinguishes characterizes the
  various types of proteinuria
• Provides useful insight on specific functions
  within the nephron
• Biochemical biopsy
• Differential diagnosis monitoring of patients
  with renal dysfunction

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Urinary Proteins of Plasma Origin

• Glomerular Proteinuria
• Renal glomeruli are ultrafilters for
  macromolecules
• Damage to renal glomeruli leads to increased
  urinary excretion of proteins (30,000 to
  100,000 daltons) which are normally
  retained
• Some selectivity remains very large proteins
  (gt500,000 daltons) still retained by
  glomerulus
• In early disease, very LMW proteins (lt15,000
  daltons) are still reabsorbed by tubules and
  absent from urine

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Urinary Proteins of Plasma Origin

• Glomerular Proteinuria
• Urine 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
  in?albumin - marked decrease in ?1-acid
  glycoprotein and ?1-antitrypsin -
  increases in large proteins ?2-macroglobulin,
  ?-lipoprotein

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Urinary Proteins of Plasma Origin

• Severe Proteinuria / Nephrotic Syndrome
• Total Protein gt 3.5 g/day
• Hypoalbuminemia and hyperlipidemia
• Massive edema

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Urinary Proteins of Plasma Origin

• Disorders Associated with Nephrotic Syndrome
• Glomerular diseases
• Proliferative glomerulonephritis
• Other diseases - Infections - Drugs
  - Neoplasia - Multisystem diseases -
  Miscellaneous - Hereditary disorders

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Case 1 Nephrotic Syndrome with Glomerular
Proteinuria
Patient 45 year-old white male History
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.
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Urinary Proteins of Plasma Origin

• Tubular Proteinuria
• Normal tubules reabsorb and catabolize 95 to
  99 of proteins from glomerular filtrate
• Tubular disease reduces capacity to reabsorb
  and catabolize, resulting in increased
  urinary excretion
• Causes 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

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Urinary Proteins of Plasma Origin

• Tubular Proteinuria
• Serum Protein Pattern - little or no
  change since LMW proteins are present
  in very low levels
• Urine 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

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Urinary Proteins of Plasma Origin

• Mixed Glomerular/Tubular Proteinuria
• Chronic renal disease or renal failure
• Combined pattern with both glomerular-type
  and tubular-type proteins in the urine

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Case 2 Heavy Metal Toxicity with Tubular
Proteinuria
Patient 52 year-old black male History
Physical Metal worker. SPE Essentially
normal. Urine Elp Trace albumin, ??
?2-microglobulin and ?2-microglobulin
consistent with tubular-type proteinuria.
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Urinary Proteins of Plasma Origin

• Other Conditions with Increased Urinary Protein
  Excretion
• Exercise Proteinuria strenuous muscular
  exercise increases excretion of HMW and LMW
  proteins
• Postural or Orthostatic Proteinuria
  present/upright, absent/recumbent benign or
  underlying cause?
• Pregnancy usually transitory, may be
  associated with toxemia, delivery, UTI, or
  asymptomatic
• Overflow Proteinuria increased plasma concen-
  tration of LMW proteins, e.g. BJP, myoglobin,
  hemoglobin, acute phase reactants

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Case 3 Septicemia with Overflow Proteinuria
Patient 65 year-old white female History
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 proteinuria
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Cerebrospinal Fluid Proteins

• CSF Proteins versus Plasma Proteins
• CSF Total Protein Much less than in
  plasma 150 450 mg/L, ages 10 40 years,
  lumbar Slightly higher, ages 40
  Slightly higher for verticular cisternal
  specimens
• CSF Production Primarily ultrafiltration
  and active transport of proteins, ions,
  water and other components through the
  choroid plexus. Small amount produced within
  CNS

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Cerebrospinal Fluid Proteins

• CSF Protein Composition
• Albumin major protein present, 55 75
  of the total
• ?1 primarily ?1-antitrypsin,
  ?-lipoprotein absent
• ?2 essentially absent
• ?1 transferrin detectable
• ?2 carbohydrate-deficient CSF-specific
  transferrin
• ? almost exclusively IgG, faint
  ?-trace

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Cerebrospinal Fluid Proteins

• Permeability of Blood-CSF Barrier
• Increased permeability caused by -
  bacterial or viral menigitis - neoplastic
  infiltration of meninges - polyneuropathies
  - disk herniations - cerebral infarctions
• Integrity of blood-CSF barrier - Total CSF
  protein - ?2-macroglobulin - Protein
  ratios

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Cerebrospinal Fluid Proteins

• Abnormal CNS Protein Production
• Demyelinating Diseases Increased IgG
  synthesis in Multiple Sclerosis and
  Subacute Sclerosing Panencephalitis
• IgG as Percentage of Total Protein
  Considers increased permeability vs. synthesis.
  IgG gt10 suspicious gt13 abnormal production
  likely.
• CSF/Serum Ratios Considers increased
  plasma concentration. 86 of MS patients
  show values above reference range.
• Oligoclonal Banding Indicative of MS new
  more sensitive procedure is IEF.

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Cerebrospinal Fluid Proteins
Oligoclonal Banding

• Multiple, restricted bands in the gamma
  fraction
• Detectable by high resolution
  electrophoresis and IEF methods
• 90 of MS patients exhibit oligoclonal
  banding

New CSF IEF methods have become the gold standard
for diagnosing MS in Europe.
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Cerebrospinal Fluid Proteins
Isoelectric Focusing

• IEF followed by immunoblotting
• Oligoclonal banding in unconcentrated CSF but not
  serum is diagnostic of MS
• Gold Standard in Europe

S Serum C CSF
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Cerebrospinal Fluid Proteins
Laboratory Protocol to Rule Out MS

1. Draw both CSF and serum samples.
2. Note CSF color and appearance. Determine
   total protein, glucose, white cell count,
   differential, red cell count.
3. Perform high resolution protein electrophoresis
   on concentrated CSF.
4. Perform high resolution protein
   electrophoresis on serum for presence or
   absence of banding.
5. In borderline cases, determine CSF and serum
   albumin and IgG ratios.

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Cerebrospinal Fluid Proteins
Laboratory Findings in Multiple Sclerosis
CSF Appearance Clear Leukocytes Usually
normal Total Protein Usually normal Glucose
Normal Electrophoresis Oligoclonal banding in
90 of cases IgG Ratio Elevated in 90 of cases
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Cerebrospinal Fluid Proteins
Clinical Manifestations of MS

• 40 of patients present with optic
  neuritis
• 60 to 70 present with evidence of
  spinal cord or brainstem lesion

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Case 1 Multiple Sclerosis
Patient 33 year-old female History 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.
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Case 2 Multiple Sclerosis
Patient 31 year-old male History 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.
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Case 3 Multiple Sclerosis
Patient 36 year-old female History 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.
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Case 4 Suspected Viral Meningitis
Patient 25 year-old white female History
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.
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Case 4 Suspected Viral Meningitis (contd)
Patient Readmitted 8 days later with redeveloped
headache.
CSF 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.
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SPIFE 3000 Analyzer

• Automated sample application, separation and
  staining of visible analytes
• Automated reagent application for enzymatic
  assays
• On-board cooling for excellent separation and
  resolution
• On-board availability of two stains

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SPIFE Assays

• Cholesterol Profile
• Serum Proteins
• Split-Beta SPE
• Urine Proteins
• Immunofixation
• Urine Immunofixation
• Hi Resolution Proteins
• Alkaline Hemoglobins
• Acid Hemoglobins
• CK LD Isoenzymes
• Alk Phos Isoenzymes
• CSF IgG IEF
• Lipoproteins

Pre-market approval pending 4/02
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SPIFE Hi Res Proteins

• Excellent separation
• Active cooling preserves pattern integrity
• Acid violet stain for added sensitivity

Urine
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SPIFE IFE 15, 9, 6 3

• Up to 210 profiles in 8 hours

• Highest sensitivity antisera

• Acid violet for best sensitivity
• Built in QC wells

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