Diagnostic Hematology: Disorders of Hemoglobin and Gammopathies Muhammad Shoaib Khan GM Centre - 1

Hemoglobin structure a globin  globin  globin Hgb A tetramer

Globin chain synthesis  cluster - chromosome 16  cluster - chromosome 11  Gower 1  PortlandEmbryonic  Gower II  FFetal <1%  A % Adult  A>95% Globin chain component Hgb name Development period % of adult Hgb   GG AA

Thalassemia Heterogenous group of disorders due to an imbalance of  and  globin chain synthesis –  thalssemia:  -globin chain production decreased –  thalassemia:  globin chain production decreased The globin chains that are produced are normal Quantitative deficiency: –  o thalassemia: No  -globin chain is made –  + thalassemia: decreased  -globin chain is made With 4  genes and 2  genes there is wide phenotypic variation

Incidence of Thalassemia ~100,000 patients with homozygous  -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  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)

 chainsHgb (g/dl)MCV (fl)RDW  /  NormalNormalNormal  /-  Normal  -/  - or - -/  /-  / Laboratory Findings in Alpha Thalassemia

Beta Thalassemia Minor (Trait)  /  + or  /  ° Intermedia  + /  Major  + /  ° or  °/  ° < 7 Clinical SyndromeGenotypeHemoglobin (g/dl) Inadequate production of  chains

Beta Thalassemia - Hgb analysis Minor (Trait)  /  + or  /  ° Intermedia  + /  % Major  + /  ° >85  °/  ° 01-6>94 Clinical SyndromeGenotypeAA2F Hemoglobin analysis: Increased levels of Hgb A2 and Hgb F

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 (  6 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 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

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 A1C A0A0A0A0

HPLC: Sickle cell trait

HPLC: Sickle cell anemia (Hgb SS) A 2 Hb F

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:

Representative sensitivity levels KappaLambda SPEP mg/L mg/L IFE mg/L mg/L Free light chains1.5 mg/L3.0 mg/L

Comparison of FLC measurements in serum and urine in healthy individuals  FLC (mg/L) FLC (mg/L)

Serum free light chains 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  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 N Engl J Med 2002;346:564-9 (Updated) Am J Med 1978; 64:814-26

Patterns of Monoclonal Protein Increase Multiple myeloma Pattern No. patients (%) Stable with sudden increase19 (25%) Stable with gradual increase 9 (12%) Gradual increase 9 (12%) Sudden increase11 (15%) Stable10 (13%) Indeterminate17 (23%) N Engl J Med 2002:346; 564-9

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  2-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 fatigue44% –Hemorrhagic manifestations44% –Weight loss23% –Neurologic symptoms11% –Visual disturbances 8% –Raynauds phenomenon 3%

Waldenstroms Macroglobulinemia: Clinical Features Tumor infiltration – Bone marrow90% – Splenomegaly38% – Lymphadenopathy30% Circulating IgM – Hyperviscosity syndrome15-20% – Cryoglobulinemia5-15% – Cold agglutinin disease5-10% – Bleeding disorders10% Tissue IgM – Neuropathy10-20%

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 – Renal function loss80 – Proteinuria75 Cardiomyopathy – Heart failure40-50 Neuropathy – Polyneuropathy36 – Orthostatic hypotension26 – Carpal tunnel syndrome8 Enteropathy – Hepatomegaly57 – Macroglossia32 – Diarrhea ± Malabsorption8 % involved

Primary Amyloidosis: Histopathology H&ECongo Red Tongue (Macroglossia)

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 hemostasisAssay Inhibition of platelet aggregationPFA; Bleeding time Inhibition of fibrin polymerizationThrombin time Acquired von Willebrand diseaseVWF activity and antigen Acquired factor X deficiencyFactor 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