Diagnostic Hematology:

Disorders of Hemoglobin and Gammopathies

Muhammad Shoaib Khan GM Centre - 1

a globin

Hemoglobin structure

Hgb A tetramer b globin b globin a globin

Globin chain synthesis

a cluster - chromosome 16 z a2 a1 z2e2 Gower 1 z2g2 a2e2 a2g2 a2d2 a2b2 Portland Gower II F A2 A Embryonic Fetal Adult e

G

g

A

g d b cluster - chromosome 11 b <1% 1.5-3.5% >95%

Thalassemia

• Heterogenous group of disorders due to an imbalance of a globin chain synthesis – – a b thalssemia: a -globin chain production decreased thalassemia: b globin chain production decreased and b • The globin chains that are produced are normal • Quantitative deficiency: – – b o 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 analysis globin gene 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

aa

/

aa aa

/-

a a

-/

a

- or - -/

aa

- -/-

a

Hgb (g/dl) Normal 12-14 11-13 7-10 MCV (fl) Normal 75-85 70-75 50-60 RDW Normal Normal - -/- -

Beta Thalassemia

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

Beta Thalassemia - Hgb analysis

Hemoglobin analysis: Increased levels of Hgb A2 and Hgb F Clinical Syndrome Genotype A A2 F 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 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 ( b 6 Glu Gene occurs in 8% of African-Americans Val)

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 A1C A 0

HPLC: Sickle cell trait

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

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

SPEP IFE Free light chains Kappa 500-2000 mg/L 150-500 mg/L 1.5 mg/L Lambda 500-2000mg/L 150-500 mg/L 3.0 mg/L

Comparison of FLC measurements in serum and urine in healthy individuals

100 10 1 Normal serum Normal urine 0 0.1



1 10 100

Serum free light chains

100000 10000 SPE Sensitivity 1000 100 10 Normal sera κ LCMM λ LCMM IIMM High pIgG AL Amyloidosis Renal impairment NSMM 1 IFE Sensitivity 0.1

0.1

1 10 100 1000



FLC (mg/L) 10000 100000

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 1 2 Description No substantial increase of serum or urine monoclonal protein (benign) Monoclonal protein ≥3.0g.dl but no myeloma or related disease 3 4 Died of unrelated causes Development of myeloma, amyloidosis or related disease Total

Am J Med 1978; 64:814-26 N Engl J Med 2002;346:564-9 (Updated)

Median follow-up 22 years No. % 46 23 113 59 241 19 10 47 24 100

Patterns of Monoclonal Protein Increase

Pattern Multiple myeloma No. patients (%) Stable with sudden increase Stable with gradual increase Gradual increase Sudden increase Stable Indeterminate

N Engl J Med

2002:346; 564-9

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 b 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 fatigue Hemorrhagic manifestations 44% Weight loss Neurologic symptoms Visual disturbances Raynauds phenomenon 8% 44% 23% 11% 3%

• • •

Waldenstroms Macroglobulinemia:

Clinical Features Tumor infiltration – Bone marrow – Splenomegaly – Lymphadenopathy Circulating IgM – Hyperviscosity syndrome – Cryoglobulinemia – Cold agglutinin disease – Bleeding disorders Tissue IgM – Neuropathy 90% 38% 30% 15-20% 5-15% 5-10% 10% 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 – Renal function loss – Proteinuria Cardiomyopathy – Heart failure Neuropathy – Polyneuropathy – – Orthostatic hypotension Carpal tunnel syndrome Enteropathy – Hepatomegaly – – Macroglossia Diarrhea ± Malabsorption 36 26 8 8 % involved 80 75 40-50 57 32

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 Inhibition of platelet aggregation Inhibition of fibrin polymerization Acquired von Willebrand disease Acquired factor X deficiency Assay PFA; Bleeding time Thrombin time VWF activity and antigen 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