Diagnostic Hematology: Disorders of Hemoglobin and Gammopathies

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

2. a globin b globin b globin a globin Hemoglobin structure Hgb A tetramer

3. Development period a cluster - chromosome 16 Globin chain component % of adult Hgb Hgb name z a2 a1 Globin chain synthesis z2e2 Gower 1 z2g2 Portland Embryonic a2e2 Gower II a2g2 F Fetal <1% a2d2 A2 1.5-3.5% Adult a2b2 A >95% e Gg Ag d b b cluster - chromosome 11

4. 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

5. 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

6. 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)

7. Laboratory Findings in Alpha Thalassemia  chains Hgb (g/dl) MCV (fl) RDW / Normal Normal Normal /- 12-14 75-85 Normal -/- or - -/ 11-13 70-75 - -/-  7-10 50-60 - -/- - - - -

8. Minor (Trait) / + or / ° 10-13 Intermedia +/+ 7-10 Major +/° or °/° < 7 Beta Thalassemia Inadequate production of b chains Clinical Syndrome Genotype Hemoglobin (g/dl)

9. Minor (Trait) / + or / ° 90-94 3.5-8 1-10 Intermedia +/+ 5-60 2-8 20-80% Major +/° 2-10 1-6 >85 °/° 0 1-6 >94 Beta Thalassemia - Hgb analysis Hemoglobin analysis: Increased levels of Hgb A2 and Hgb F Clinical Syndrome Genotype A A2 F

10. Approach to Beta Thalassemia • Screening/counseling • RBC transfusion therapy • Agents to increase hemoglobin F (Hydroxyurea) • Bone marrow transplantation

11. Clinical Presentations of Abnormal Hemoglobins • Sickling disorder • Thalassemia or microcytic anemia • Cyanosis • Erythrocytosis • Hemolytic anemia • Asymptomatic (screening or family study)

12. Sickle Cell Disease • Inherited as autosomal recessive • Point mutation in beta globin (6 Glu Val) • Gene occurs in 8% of African-Americans

13. Relative Frequency of Hemoglobin Variants

14. Screening for Sickle Cell Traitand Disease • RBC lysate with concentrated phosphate buffer and sodium hydrosulfite • Incubate 10-20 min

15. 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

16. Hemoglobin electrophoresis

17. Hemoglobin electrophoresis: Variants of sickle cell anemia

18. Hemoglobin electrophoresis: Identification of abnormal hemoglobins

19. 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

20. A0 A1C HPLC: Normal Adult Hemoglobin

21. HPLC: Sickle cell trait

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

23. HPLC: Hgb SC disease

24. Monoclonal Gammopathies • Laboratory evaluation of gammopathies • Diseases associated with gammopathies • Common clinical syndromes

25. 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

31. 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

32. 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

33. l FLC (mg/L) k FLC (mg/L) Comparison of FLC measurements in serum and urine in healthy individuals

34. l FLC (mg/L) Serum free light chains Composite Figure of serum free light chain concentrations in various diseases

35. 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

36. Lymphoproliferative Disorders Commonly Associated with a Monoclonal Gammopathy • Monoclonal gammopathy of undetermined significance (MGUS) • Multiple myeloma • Waldenstroms macroglobulinemia • Amyloidosis

37. 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

38. “Benign MonoclonalGammopathy” Course of MGUS in 241 Patients Am J Med 1978; 64:814-26 N Engl J Med 2002;346:564-9 (Updated)

39. 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 2002:346; 564-9

40. 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

41. 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

42. Multiple Myeloma: Clinical Manifestations • Bone pain/skeletal involvement • Fatigue/anemia • Renal insufficiency • Hypercalcemia • Neurologic symptoms • Infections

43. 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

44. Peripheral smear: Plasma cell

45. Bone marrow aspirate: Plasma cell infiltrate

47. 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

48. 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%

49. Waldenstroms Macroglobulinemia:Clinical 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%