Objectives

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Diagnostic Hematology Disorders of Hemoglobin
and Gammopathies
Morey A. Blinder, M.D. Associate Professor of
Medicine and Pathology Immunology
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Hemoglobin structure
Hgb A tetramer
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Globin chain synthesis
Development period
Globin chain component
a cluster - chromosome 16
of adult Hgb
Hgb name
a1
a2
z
z2e2 Gower 1 z2g2 Portland Embryonic a2e2 Gower
II a2g2 F Fetal lt1 a2d2 A2 1.5-3.5 Ad
ult a2b2 A gt95
e
Gg
d
b
Ag
b cluster - chromosome 11
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Thalassemia

• Heterogenous group of disorders due to an
  imbalance of a and b globin chain synthesis
• a thalssemia a-globin chain production
  decreased
• b thalassemia b globin chain production
  decreased
• The globin chains that are produced are normal
• Quantitative deficiency
• bo thalassemia No b-globin chain is made
• b thalassemia decreased b-globin chain is made
• With 4 a genes and 2 b genes there is wide
  phenotypic variation

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Alpha Thalassemia

• Inadequate production of alpha chains
• Hemoglobin analysis normal can be detected by a
  globin gene analysis
• Absence of 1-2 alpha chains
• Common
• Asymptomatic
• Does not require therapy
• Absence of 3 alpha chains
• Microcytic anemia (Hgb 7-10)
• Splenomegaly
• Absence of 4 alpha chains
• Hydrops fetalis (non-viable)

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Laboratory Findings in Alpha Thalassemia
? chains Hgb (g/dl) MCV (fl) RDW ??/?? Norm
al Normal Normal ??/-? 12-14 75-85 Normal
?-/?- or - -/?? 11-13 70-75 - -/- ?
7-10 50-60 - -/- - - -
-
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Beta Thalassemia
Inadequate production of b chains
Clinical Syndrome Genotype Hemoglobin (g/dl)

• Minor (Trait) ?/ ? or ?/ ? 10-13
• Intermedia ?/? 7-10
• Major ?/? or ?/? lt 7

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Beta Thalassemia - Hgb analysis
Hemoglobin analysis Increased levels of Hgb A2
and Hgb F
Clinical Syndrome Genotype A A2 F

• Minor (Trait) ?/ ? or ?/ ? 90-94 3.5-8 1-10
• Intermedia ?/? 5-60 2-8 20-80
• Major ?/? 2-10 1-6 gt85
• ?/? 0 1-6 gt94

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Approach to Beta Thalassemia

• Screening/counseling
• RBC transfusion therapy
• Agents to increase hemoglobin F (Hydroxyurea)
• Bone marrow transplantation

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Clinical Presentationsof Abnormal Hemoglobins

• Sickling disorder
• Thalassemia or microcytic anemia
• Cyanosis
• Erythrocytosis
• Hemolytic anemia
• Asymptomatic (screening or family study)

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Relative Frequency of Hemoglobin Variants
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Sickle Cell Disease

• Inherited as autosomal recessive
• Point mutation in beta globin (?6 Glu Val)
• Gene occurs in 8 of African-Americans

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Screening for Sickle Cell Trait and Disease

• RBC lysate with concentrated phosphate buffer and
  sodium hydrosulfite
• Incubate 10-20 min

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Hemoglobin electrophoresisMethodology

• Separates hemoglobins on solid support media
• Cellulose acetate (Alkaline gel)
• Citrate agar (Acid gel)
• Inexpensive and quickly prepared
• Sharp resolution of major hemoglobin bands
• Electrophoretic variability based on charge

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Hemoglobin electrophoresis
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Hemoglobin electrophoresis Variants of sickle
cell anemia
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Hemoglobin electrophoresis Identification of
abnormal hemoglobins
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High Pressure Liquid Chromatography (HPLC)

• Separates hemoglobins by a cation exchange column
• Resolution of various hemoglobins including Hgb F
  is excellent
• Procedure can be automated leading to reliable
  interpretation
• Hemoglobin fractions can be quantified

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HPLC Normal Adult Hemoglobin
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HPLC Sickle cell trait
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HPLC Sickle cell anemia (Hgb SS)
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HPLC Hgb SC disease
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Monoclonal gammopathies

• Laboratory evaluation of gammopathies
• Diseases associated with gammopathies
• Common clinical syndromes

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Properties of human immunoglobulins
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Clinical indications for the evaluation of
immunoglobulins

• Normochromic normocytic anemia
• Nephrotic syndrome in a non-diabetic patient
• Osteolytic lesions
• Lymphadenopathy
• Non-ischemic heart failure
• Elevated total serum protein
• Hypercalcemia

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Free light chains

• Have been detected in urine for gt50 years
• Polyclonal antibody against free LC
• Purified so no cross-reactivity and does not bind
  to intact immunoglobulin
• Bound to latex beads - detected by a variety of
  techniques (turbidity)

Korngold and Lapiri Cancer (1956) 9262-272
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Representative sensitivity levels
Kappa Lambda SPEP 500-2000 mg/L 500-2000
mg/L (.05-.2 g/dl) IFE 150-500 mg/L 150-500
mg/L (.015-.05 g/dl) Free light chains 1.5
mg/L 3.0 mg/L (.0015.003 g/dl)
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Comparison of FLC measurements in serum and urine
in healthy individuals
l FLC (mg/L)
k FLC (mg/L)
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Serum free light chain concentration in various
diseases
l FLC (mg/L)
k FLC (mg/L)
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Potential uses of serum free light chains

• Sensitive marker for diagnosing monoclonal
  lymphoproliferative diseases
• k/l ratio may be a prognostic marker for MGUS
• Useful marker in non-secretory myeloma or
  patients with only
• Bence-Jones proteinuria
• Marker to follow disease

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Lymphoproliferative Disorders Commonly Associated
with a Monoclonal Gammopathy

• Monoclonal gammopathy of undetermined
  significance (MGUS)
• Multiple myeloma
• Waldenstroms macroglobulinemia
• Amyloidosis

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Monoclonal Gammopathy of Undetermined
Significance (MGUS)

• Commonly found on serum protein electrophoresis
• Occurs in 2 of persons gt 50 years of age
• Characteristics
• Low serum monoclonal protein concentration (lt3
  g/dl)
• Less than 5 plasma cells in bone marrow
• Little or no monoclonal protein in urine
• Absence of lytic bone lesions
• No anemia, hypercalcemia, or renal insufficiency

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Benign Monoclonal Gammopathy Course of MGUS in
241 Patients
Am J Med 1978 64814-26
N Engl J Med 2002346564-9 (Updated)
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Patterns of Monoclonal Protein Increase
Multiple myeloma Pattern No. patients
() Stable with sudden increase 19 (25) Stable
with gradual increase 9 (12) Gradual
increase 9 (12) Sudden increase 11
(15) Stable 10 (13) Indeterminate 17
(23)
N Engl J Med 2002346 564-9
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Predictors of progression to myeloma or related
conditions

• Concentration of monoclonal protein
• Monoclonal protein 20-year
• concentration risk of progression
• 0.5 g/dl 14
• 0.6-1.5 g/dl 25
• 1.6-2.0 g/dl 41
• 2.1-3.0 g/dl 49
• Immunoglobulin type
• IgA or IgM monclonal gammopathy 2-3x risk of
  progression

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Stratifying Risk in MGUS Patients

• Risk
• Low High
• FLC ??? ratio Normal Abnormal
• Ig concentration lt1.5 g/dl gt1.5 g/dl
• Ig type IgG Other

Rajkumar, S.V. et al., Blood 2005 106 812
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Identifying risk in MGUS patients
Rajkumar, S.V. et al., Blood 2005 106 812
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Recommended testing in patients with suspected
MGUS

• Recommended in all patients
• History and Physical exam
• CBC
• BMP (serum calcium and creatinine)
• Protein studies
• SPEP, UPEP, Immunofixation
• Detection of serum FLC
• (?) Quantitative immunoglobulins
• Recommended in patients with monoclonal protein
  gt1.5g/dl
• Bone marrow exam
• Skeletal survey
• Not recommended
• B-2 microglobulin

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SummaryMonoclonal gammopathies of uncertain
significance

• Monoclonal proteins rarely disappear
  spontaneously (lt5)
• MGUS is a risk factor for multiple myeloma and
  related disorders
• Risk of progression to multiple myeloma or
  related disorders is increased with higher
  initial monoclonal protein levels
• Risk of progression is 1 per year

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Multiple Myeloma Incidence and Etiology

• 13,000 cases/year in USA
• Median age - 65 yrs.
• Incidence in African-Americans is two-fold other
  ethnic groups
• Familiar clusters are rare
• Environmental/occupational exposures have been
  implicated

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Multiple Myeloma Clinical Manifestations

• Bone pain/skeletal involvement
• Fatigue/anemia
• Renal insufficiency
• Hypercalcemia
• Neurologic symptoms
• Infections

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Laboratory evaluation

• CBC with peripheral smear
• Chemistry panel (Include calcium and
  creatinine)
• SPEP/UPEP (immunofixation electrophoresis)
• Urinalysis/24 hr urine for protein
• Bone marrow exam
• Skeletal survey
• LDH and b2-microglobulin
• Serum viscosity

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Peripheral smear Plasma cell
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Bone marrow aspirate Plasma cell infiltrate
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Diagnostic Criteria for Multiple Myeloma

• Major criteria
• I. Bone marrow plasmacytosis gt 30
• II. Histologic diagnosis of plasmacytoma
• III. Serum paraprotein IgG gt 3.5 g/dl or IgA
  gt 2.0 g/dl

• Minor criteria
• a. Bone marrow plasmacytosis 10-30
• b. Serum paraprotein less than major
  criteria
• c. Osteolytic lesion
• d. Hypogammaglobulinemia

• One major criteria and one minor criteria
• Minor criteria a b and one other

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Smoldering Myeloma
Asymptomatic proliferation of plasma cells

• Criteria
• Presence of
• Plasmacytosis gt10
• Serum monoclonal protein gt3 g/dl

Absence of end organ damage Hemoglobin gt 10
g/dl Normal renal function Normal serum
calcium No osteolytic lesions Little or no urine
monoclonal protein
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Clinical course and prognosis in smoldering
myeloma
Probability of progression to active myeloma or
primary amyloidosis

• Risk of progression
• 5 yrs 10/yr
• 10 yrs 3/yr
• 20 yrs 1/yr

Kyle, RA, N Engl j Med 20073562580
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Characteristics of Multiple Myeloma and its
Precursors
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Staging Classification for Multiple Myeloma

• Stage Criteria
• I All of the following
• Hemoglobin gt 10 g/dl
• Normal serum calcium
• No generalized osteolytic lesions
• Low paraprotein level
• IgG lt 5 g/dl
• IgA lt 3 g/dl
• Urine light chains lt 4g/24 hours
• II Intermediate between stage I and III
• III One or more of the following
• Hemoglobin lt 8.5 g/dl
• Serum calcium gt 12 g/dl
• Diffuse osteolytic lesions
• High paraprotein level
• IgG gt 7 g/dl
• IgA gt 5 g/dl
• Urine light chains gt 12 g/dl

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Waldenstroms MacroglobulinemiaIncidence and
clinical features

• 1,500 cases/year in USA
• Median age -, 63 yrs
• Presenting symptoms
• Weakness and fatigue 44
• Hemorrhagic manifestations 44
• Weight loss 23
• Neurologic symptoms 11
• Visual disturbances 8
• Raynauds phenomenon 3

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Waldenstroms MacroglobulinemiaClinical Features

• Tumor infiltration
• Bone marrow 90
• Splenomegaly 38
• Lymphadenopathy 30
• Circulating IgM
• Hyperviscosity syndrome 15-20
• Cryoglobulinemia 5-15
• Cold agglutinin disease 5-10
• Bleeding disorders 10
• Tissue IgM
• Neuropathy 10-20

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Amyloidosis Classification and Biochemical
Composition

• Primary amyloidosis
• Immunoglobulin light chain (AL)
• Secondary amyloidosis
• Amyloid A protein (AA)
• Synthesized by liver as an acute phase reactant
• Hereditary amyloidosis
• Transthyretin-derived amyloid (ATTR)

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Primary Amyloidosis Clinical Features

• Nephropathy
• Renal function loss 80
• Proteinuria 75
• Cardiomyopathy
• Heart failure 40-50
• Neuropathy
• Polyneuropathy 36
• Orthostatic hypotension 26
• Carpal tunnel syndrome 8
• Enteropathy
• Hepatomegaly 57
• Macroglossia 32
• Diarrhea Malabsorption 8

involved
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Primary Amyloidosis Histopathology
Tongue (Macroglossia)
HE
Congo Red
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Primary amyloidosisKey points

• 1. Suspect amyloidosis when a patient has
  unexplained
• Nephrotic range proteinuria with or without renal
  insufficiency
• Cardiomyopathy manifested by fatigue or CHF
• Peripheral neuropathy
• Hepatomegaly
• 2. Pursue diagnosis if
• A monoclonal protein is detected in serum or
  urine
• 3. Confirm diagnosis with Congo red stain of
• Bone marrow
• Subcutaneous fat
• Other affected tissue
• 4. Perform echocardiogram to assess prognosis
• 5. Begin systemic treatment

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Common clinical syndromesassociated with
monoclonal gammopathies

• Bleeding disorders
• Hyperviscosity
• Cryoglobulinemia
• Peripheral neuropathy

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Hemostatic defects associated withMonoclonal
proteins

• Effect on hemostasis Assay
• Inhibition of platelet aggregation PFA Bleeding
  time
• Inhibition of fibrin polymerization Thrombin
  time
• Acquired von Willebrand disease VWF activity and
  antigen
• Acquired factor X deficiency Factor X activity

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Acquired factor X deficiency

• Low factor X levels (lt50)
• Severe bleeding with activity lt10
• Associated with amyloidosis
• Factor X binds to amyloid deposits in tissues
• Treatment
• Underlying amyloidosis
• Splenectomy
• Large volumes of FFP/plasma exchange

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Hyperviscosity syndrome

• Associated with Waldenstroms macroglobulinemia
  (15-20 of patients)
• Measure serum viscosity (normal lt1.8)
• Clinical syndrome of hyperviscosity occurs gt4.0
• Symptoms
• Headaches
• Other neurologic symptoms (dizziness, mental
  status changes
• Blurry vision
• Easy bleeding

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Cryoglobulinemia

• Type I (monoclonal) cryoglobulin
• Associated with any lymphoproliferative disorder
• Waldenstroms macroglobulinemia 10-20
• Symptoms
• Raynaud phenomenon
• Purpura
• Renal insufficiency
• Arthralgia
• Blood handling is difficult
• Collect blood in 37 C tube
• Transport and centrifuge at 37 C
• Chill serum to 4 C for 48 hrs
• Assay for cryoglobulin

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Peripheral smear Cryoglobulinemia
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Identification of monoclonal proteinby
SPEP/immunofixation at 37C
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Neuropathies associated withmonoclonal protein
disorders

• Associated with any lymphoproliferative disease
• Target antigens are occasionally identified (MAG
  myelin associated glycoprotein)
• Symmetric, distal, sensory or sensorimotor
• May simulate CIDP (Chronic inflammatory
  demyelinating polyneuropathy)
• Associated with any class of monoclonal protein

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Summary

• Lymphoproliferative disorders associated with
  monoclonal proteins are common
• Diagnosis may be difficult
• Treatment requires identification of underlying
  disease and any associated clinical syndromes