Download Diagnostic Hematology

Diagnostic Hematology:
Disorders of Hemoglobin and
Gammopathies
Muhammad Shoaib Khan
GM Centre - 1
Hemoglobin structure
Hgb A tetramer
a globin
b globin
b globin
a globin
Globin chain synthesis
a cluster - chromosome 16
z
a2
a1
z2e2
Gower 1
z2g2
Portland
a2e2
Gower II
a2g2
F
a2d2
A2
a2b2
e
Gg
Ag
d
b cluster - chromosome 11
b
Embryonic
Fetal
<1%
1.5-3.5%
Adult
A
>95%
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
Incidence of Thalassemia
• ~100,000 patients with homozygous b-thalassemia
world-wide
• Found in Mediterranean countries, South Asia and
Far East
• Prevalence in the United Sates is increasing due to
population migration
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)
Laboratory Findings in Alpha Thalassemia
a chains
Hgb (g/dl)
MCV (fl)
RDW
aa/aa
Normal
Normal
Normal
aa/-a
12-14
75-85
Normal
a-/a- or - -/aa
11-13
70-75
- -/- a
7-10
50-60
- -/- -
-
-
-
Beta Thalassemia
Inadequate production of b chains
Clinical
Syndrome
Genotype
Minor (Trait)
b/ b+ or b/ b°
10-13
Intermedia
b+/b+
7-10
Major
b+/b° or b°/b°
<7
Hemoglobin (g/dl)
Beta Thalassemia - Hgb analysis
Hemoglobin analysis: Increased levels of Hgb A2 and Hgb F
Clinical
Syndrome
Genotype
Minor (Trait)
b/ b+ or b/ b°
90-94 3.5-8 1-10
Intermedia
Major
b+/b+
b+/b°
b°/b°
5-60 2-8
2-10 1-6
0
1-6
A
A2
F
20-80%
>85
>94
Approach to Beta Thalassemia
• Screening/counseling
• RBC transfusion therapy
• Agents to increase hemoglobin F (Hydroxyurea)
• Bone marrow transplantation
Clinical Presentations of Abnormal Hemoglobins
•
•
•
•
•
•
Sickling disorder
Thalassemia or microcytic anemia
Cyanosis
Erythrocytosis
Hemolytic anemia
Asymptomatic (screening or family study)
Sickle Cell Disease
• Inherited as autosomal recessive
• Point mutation in beta globin (b6 Glu Val)
• Gene occurs in 8% of African-Americans
Relative Frequency of Hemoglobin Variants
Screening for Sickle Cell Trait and Disease
• RBC lysate with
concentrated phosphate
buffer and sodium
hydrosulfite
• Incubate 10-20 min
Hemoglobin Electrophoresis: Methodology
• 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
Hemoglobin electrophoresis
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Hemoglobin electrophoresis:
Variants of sickle cell anemia
Hemoglobin electrophoresis:
Identification of abnormal hemoglobins
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
HPLC: Normal Adult Hemoglobin
A0
A1C
HPLC: Sickle cell trait
HPLC:
Sickle cell anemia
(Hgb SS)
Hb F
A2
HPLC:
Hgb SC disease
Monoclonal Gammopathies
• Laboratory evaluation of gammopathies
• Diseases associated with gammopathies
• Common clinical syndromes
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
Free light chains
• Have been detected in urine for >50 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) 9:262-272
Representative sensitivity levels
Kappa
Lambda
SPEP
500-2000 mg/L
500-2000mg/L
IFE
150-500 mg/L
150-500 mg/L
Free light chains
1.5 mg/L
3.0 mg/L
Comparison of FLC measurements in serum and urine
in healthy individuals
_ FLC
(mg/L)
(mg/L)
l FLC
100
10
Normal serum
Normal urine
1
0
0.1
1
10
 FLC(mg/L)
(mg/L)
 FLC
100
Serum free light chains
100000
10000
(mg/L)
l FLC(mg/L)
l FLC
SPE Sensitivity
Normal sera
1000
κ LCMM
λ LCMM
100
IIMM
High pIgG
AL Amyloidosis
10
Renal impairment
NSMM
1
IFE Sensitivity
0.1
0.1
1
10

100
1000
10000
100000
FLC (mg/L)
Composite Figure of serum free light chain concentrations in various diseases
Potential uses of serum free light chains
• Sensitive marker for diagnosing monoclonal lymphoproliferative
diseases
• /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
Lymphoproliferative Disorders Commonly Associated
with a Monoclonal Gammopathy
• Monoclonal gammopathy of undetermined
significance (MGUS)
• Multiple myeloma
• Waldenstroms macroglobulinemia
• Amyloidosis
Monoclonal Gammopathies of Undetermined
Significance (MGUS)
• Commonly found on serum protein electrophoresis
• Occurs in ~2% of persons > 50 years of age
• Characteristics
– Low serum monoclonal protein concentration (<3 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
“Benign Monoclonal Gammopathy” Course of MGUS in
241 Patients
Group
Description
Median follow-up
22 years
No.
%
1
No substantial increase of serum
or urine monoclonal protein (benign)
46
19
2
Monoclonal protein ≥3.0g.dl but
no myeloma or related disease
23
10
3
Died of unrelated causes
113
47
4
Development of myeloma,
amyloidosis or related disease
59
24
241
100
Total
Am J Med 1978; 64:814-26
N Engl J Med 2002;346:564-9 (Updated)
Patterns of Monoclonal Protein
Increase
Pattern
Stable with sudden increase
Stable with gradual increase
Gradual increase
Sudden increase
Stable
Indeterminate
N Engl J Med 2002:346; 564-9
Multiple myeloma
No. patients (%)
19 (25%)
9 (12%)
9 (12%)
11 (15%)
10 (13%)
17 (23%)
Summary:(MGUS)
• Monoclonal proteins rarely disappear spontaneously (<5%)
• 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
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
Multiple Myeloma: Clinical Manifestations
•
•
•
•
•
•
Bone pain/skeletal involvement
Fatigue/anemia
Renal insufficiency
Hypercalcemia
Neurologic symptoms
Infections
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
Peripheral smear: Plasma cell
Bone marrow aspirate: Plasma cell infiltrate
Diagnostic Criteria for Multiple Myeloma

Major criteria
I. Bone marrow plasmacytosis > 30%
II. Histologic diagnosis of plasmacytoma
III. Serum paraprotein IgG > 3.5 g/dl or IgA > 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
Waldenstroms Macroglobulinemia
Incidence and clinical features
•
•
•
1,500 cases/year in USA
Median age -, 63 yrs
Presenting symptoms
–
–
–
–
–
–
Weakness and fatigue
Hemorrhagic manifestations 44%
Weight loss
Neurologic symptoms
Visual disturbances
8%
Raynauds phenomenon
44%
23%
11%
3%
Waldenstroms Macroglobulinemia:
Clinical Features
• Tumor infiltration
– Bone marrow
– Splenomegaly
– Lymphadenopathy
90%
38%
30%
• Circulating IgM
–
–
–
–
Hyperviscosity syndrome
Cryoglobulinemia
Cold agglutinin disease
Bleeding disorders
15-20%
5-15%
5-10%
10%
• Tissue IgM
– Neuropathy
10-20%
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)
Primary Amyloidosis: Clinical Features
• Nephropathy
% involved
– Renal function loss
– Proteinuria
80
75
– Heart failure
40-50
• Cardiomyopathy
• Neuropathy
– Polyneuropathy
– Orthostatic hypotension
– Carpal tunnel syndrome
• Enteropathy
– Hepatomegaly
– Macroglossia
– Diarrhea ± Malabsorption
36
26
8
57
32
8
Primary Amyloidosis: Histopathology
Tongue
(Macroglossia)
H&E
Congo Red
Primary amyloidosis
Key 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
Common clinical syndromes
associated with monoclonal gammopathies
• Bleeding disorders
• Hyperviscosity
• Cryoglobulinemia
• Peripheral neuropathy
Hemostatic defects associated with
Monoclonal 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
Acquired factor X deficiency
•
•
•
•
•
Low factor X levels (<50%)
Severe bleeding with activity <10%
Associated with amyloidosis
Factor X binds to amyloid deposits in tissues
Treatment
– Underlying amyloidosis
– Splenectomy
– Large volumes of FFP/plasma exchange
Hyperviscosity syndrome
•
•
•
•
Associated with Waldenstroms macroglobulinemia (15-20% of patients)
Measure serum viscosity (normal <1.8)
Clinical syndrome of hyperviscosity occurs >4.0
Symptoms
–
–
–
–
Headaches
Other neurologic symptoms (dizziness, mental status changes
Blurry vision
Easy bleeding
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
Peripheral smear: Cryoglobulinemia
Neuropathies associated with
monoclonal 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
Summary
• Lymphoproliferative disorders associated
with monoclonal proteins are common
• Diagnosis may be difficult
• Treatment requires identification of
underlying disease and any associated
clinical syndromes