Abnormalities of Blood Cells ..2Pathophysiology and Laboratory Diagnosis of Anemias and other Blood Cell Disorders

Anand Shreeram Lagoo, MD, PhDProfessor of PathologyDirector, Clinical Flow Cytometry Laboratory

Phone: 668-0921, Pager 970-2903

Abnormalities of blood cells ..Lecture 2Lecture Outlinen Anemias of reduced red cell production

¨ Reduced hemoglobin synthesisn Iron deficiency (Case 2), Pathophysiology of iron metabolism. Additional tests.)n Anemia of chronic inflammation (Case 3). Hepcidin and control of iron metabolism.n Reduced globin synthesis – Thalassemias

¨ Disturbed maturation / production of RBCn Megaloblastic anemia (Case 4), Folate and Vit B12 metabolism and actions.n Aplastic anemia (Case 5)

n White blood cells¨ Myeloproliferative neoplasms (MPN)

n CML (Case 6). Philadelphia chromosome.n Other MPNs. Polycythemia vera (Case 7). Jak2 and other mutations in MPN.

¨ Acute myeloid leukemia¨ Myelodysplastic syndromes (MDS): Morphology and genetics

n Platelets¨ Thrombocytopenia.

n ITP (Case 8)n Other causes

Etiologic Classification of Anemias ...2n Impaired RBC production

¨Defective hemoglobin synthesisn ↓ Heme synthesis – iron deficiencyn ↓ Globin synthesis – Thalassemia

¨Disturbed proliferation / Maturation of RBCn Anemia of chronic diseasen Defective DNA synthesis – Folate / B12 deficiencyn Anemia of chronic renal disease (↓erythropoietin)

¨Disturbed stem cells (aplastic anemia)¨Marrow replacement by neoplasms – AML, MDS¨Marrow infiltration: Metastatic tumors, granulomas

etc

Anemia

MicrocyticHypochromic

Case 2n 59 yo caucasian mann Presents with fatigue and headache for 4

monthsn He has noted some upper abdominal

distressn Physical examination is normaln Lab data:

¨Hct: 27 %¨Hb: 8.9 gm/dL¨MCV: 67 fL¨MCH: 22.6 pg¨Platelets: 600,000¨WBC 4,900/cu mm

Thrombocytosis

Inappropriately low

Case 2: Microcytic, hypochromic anemia(continued)n 59 yo caucasian man with Microcytic anemia and

thrombocytosis¨ Hct: 27 %¨ Hb: 8.9 gm/dL¨ MCV: 67 fL¨ MCH: 22.6 pG¨ Platelets: 600,000¨ WBC 4,900/cu mm

n Reticulocyte: 30,000/mm3

n Peripheral Blood Film-¨ WBC differential:

n Neutrophils 65% n lymphocytes 33% n monocytes 2%

¨ Abnormal RBC morphology

Case 2- Peripheral Blood FilmMicrocytic hypochromic anemia

Anisocytosis Poikilocytosis

Hypochromia

RDW=19.6

Microcytic hypochromic anemia:Etiological differential diagnosisn Iron deficiency anemia

¨Most frequent cause of anemian Thalassemiasn Anemia of chronic inflammationn Sideroblastic anemian Lead poisoning

Understanding iron metabolism:

n The body has no mechanism to excrete excess iron

n Absorption of dietary iron is strictly controlled to maintain total iron in the body

n Free iron is toxic, therefore it is bound to proteins –¨Specific binding to transferrin and apoferritin¨Non-specific binding to albumin

Understanding iron metabolism:n Transferrin is the primary transport molecule for

iron. ¨ Blood transferrin level is referred to as “Total Iron

Binding CapacityӬ Proportion of transferrin molecules bound to iron = %

saturation of iron binding capacity¨ This iron is most readily available for Hb synthesis

n Some iron binds to another protein called apoferritin to form a water soluble molecule called ferritin¨ Ferritin is present in blood and ferritin iron can be easily

delivered for Hb synthesis. ¨ Ferritin is increased in inflammation (acute phase

reactant)n Excess iron is stored in

bone marrow as water insoluble Hemosiderin

Low blood Ferritin = Low/ Absent storage iron*

*Note: Ferritin levels increase due to inflammation, even when iron stores are low. Therefore, normal or high Ferritin does NOT guarantee normal storage iron.

Trasferrin levels increasewhen iron stores decline

Total Iron Binding Capacity (TIBC) increases but it is less

saturatedHeme iron – 20% absorbed Non-

Heme Iron- Only 1-2% absorbed

Absorbed in Duodenum

Control of Iron absorption in Duodenum

Divalent Metal Transporter

When storage iron is adequate –Most absorbed iron lost through

shedding of duodenal cells

When ion stores are low/ absent –

Most absorbed iron transferred to plasma

Case 2 continued

n Additional laboratory tests:n Serum Iron: 10 (low) n Iron binding capacity: 450 (high) n Transferrin saturation: 2% (low) n Serum ferritin: 10 ng/mL (low) n Diagnosis: Iron deficiency anemia

Must investigate causes of chronic blood loss in iron deficiency anemia in older adults. Dietary iron deficiency more common in children and reproductive age females.

n Stool samples positive for occult blood

Laboratory Results in Microcytic Hypochromic Anemias

Serum Fe TIBC % Saturation Serum ferritin

Marrow Fe

Iron Deficiency

↓↑ ↓ ↓ ↓

Thalassemia N N N N ↑Anemia of Chr Dz

↓ N - ↓ ↓ N - ↑ N - ↑

Sideroblastic anemia

↑ ↓ ↑ ↑ ↑

Case 3n 23 yo woman

¨ Fatigue, arthralgias, skin rash for several months¨ PE: Malar rash

n Lab data:¨ Hct 29 %¨ Hb 9.2 gm/dl¨ MCV 82 fl¨ Platelets: 150,000¨ WBC: 4,900

n Blood film: ¨ Normochromic, normocytic RBCs¨ WBC diff: Normal

n Retic: 60,000/cu mm (inappropriately low)

Case 3- Peripheral Blood Film

Normochromic, normocytic RBCs

Normocytic - Normochromic Anemia and Low Retic Count: differential diagnosis n Secondary to systemic illness

¨Anemia of chronic inflammation¨Renal insufficiency¨Endocrine disorders

n Primary BM (stem cell) disorders¨Aplastic anemia¨Pure Red Cell aplasia¨ Infiltrative disorders

Case 3: Additional Testsn ESR: 80 mm/hrn BUN: 42n Creatinine 2.0n Anti Nuclear Antibody 1:1256n Complement C3/C4 Lown Anti-ds DNA Positive

n Diagnosis¨ Systemic Lupus Erythematosus (SLE)¨ Renal insufficiency¨ Anemia of Chronic Disease (= anemia of inflammation)

n Possibly worsened by low erythropoietin

Control of Iron absorption in Duodenum

Divalent Metal Transporter

Chronic inflammation → IL1 →

Increased hepcidin production from Liver

Hepcidin blocks exit of iron from duodenal lining cells and from storage pool in

macrophages

Other Molecules Involved In Iron Absorptionn Hereditary mutations in these molecules →

excess iron absorption → Hemochromatosis¨ These molecules are required for appropriate synthesis /

action of Hepcidin

n Hemochromatosis (HFE) gene¨Mutations cause adult hemochromatosis

n Hemojuvelin¨Mutations cause a severe hemochromatosis in

childrenn Transferrin receptor 2

Case 4n 54 yo man

¨ Presents with nausea, poor appetite, mild diarrhea¨ PE: Normal

n CBC:¨ Hct: 35 %¨ Hb: 12 gm/dl (Anemia) ¨ MCV: 115 fl (Macrocytosis) ¨ Retic: 65,000/ cu mm (not elevated, relatively low) ¨ Platelets: 200,000¨ WBC: 4,000

n Blood film: Macrocytosis, WBC differential is normal

n Normal upper and lower GI studies

Macrocytic Anemias with low Retics: Megaloblastic or Normoblastic? n Megaloblastic (specific morphological change in red cell

precursors in bone marrow)¨ Vit B12 deficiency¨ Folate deficiency¨ Myelodysplastic syndromes¨ Drug-induced

n Normoblastic¨ Hypothyroidism¨ Liver disease¨ Alcohol

Megaloblastic

Case 4- Peripheral Blood Film

Hypersegmented neutrophil

Case 4 continued

n Several months later -¨Paresthesias of hands and feet¨Difficulty using the clutch and gas pedals while

drivingn PE:

¨Mild scleral icterus¨Absent position and vibratory sensation¨Diminished two-point discrimination

Case 4 continued

n Diagnostic laboratory evaluation-¨Serum B12 level- 30 (normal > 180) ¨Anti-intrinsic factor antibodies positive

n Diagnosis- B12 deficiencyPernicious anemia

Back to the Basics…

Cobalamin Pteroyl glutamic acid

B12 Folate

Folate and Vit B12Folate Vitamin B12

Dietary source Nearly all foods Abundant in all animal derived foods

Effect of cooking Destroyed ResistantProcessing before absorption

Polyglutamate to monoglutamate/Blocked by acidic foods

Complex (see next slide)

Absorbed in Upper third of intestine Distal ileumBody stores Last 3 months Last >5 yearsMain causes of deficiency

DietaryIncreased demand

Problem of absorption

Clinical findings Megaloblastic anemia Megaloblastic anemia, neuropathyLaboratory Dx Serum and RBC folate, B12

levelsSerum and RBC folate, B12 levels

Dietary B12 (cobolamine, Cbl) released by peptic digestion →

bind to salivary protein haptocorrin

Intrinsic Factor (IF) -Secreted by gastric parietal cells-Required for absorption of B12

Autoantibodies in pernicious anemia:1. Block binding of B12 to IF2. Parietal cell3. Prevent B12-IF complex binding to cubulin Trancobolamine

(TC) transports Cbl to tissues

Actions of B12 and Folate:n Folate is directly required for Purine (DNA)

synthesis, B12 is indirectly involved through folate metabolism¨ Only tetra-hydro folate (THF) can participate in purine

synthesis¨ Dietary folate is converted to THF and then to methyl-THF¨ Methyl-THF can be converted back to THF if B12 is present

n Only B12 can transfer the methyl group from Methyl-THF to homocysteine

¨ In the absence of B12, most folate is “trapped” as methyl-THF , levels of THF decline, and DNA synthesis suffers

Anemia due to B12 or Folate Deficiencyn Treatment with folate will correct anemia due to folate

deficiency or B-12 deficiencyn Mitochondrial action of B12: (Folate independent)

¨ Adenosyl-Cbl acts as coenzyme for conversion of methylmalonyl-CoA to succinyl-CoA

¨ ? Associated with myelin formation and etiology of neuropathy observed in B12 deficiency

n Neuropathy of B12 deficiency may be aggravated by folate administration

n B12 administration will not correct anemia due to folate deficiency

Case 5n 22 yo mechanic

Admitted with fever, sore throat and numerous bruisesn PE - Purulent tonsillitis, petechiae and ecchymosesn CBC:

¨ Hb: 6.1 gm/dl¨ MCV: 106 fl¨ Retic: 5,000/ cu mm¨ Platelets: 5,000 / cu mm¨ WBC: 1,900¨ WBC diff: Neutrophils 10% ¨ Lymphs: 88% (relative lymphocytosis)¨ Monos: 2%

n Blood Smear: No immature cells. Severe neutropenia andthrombocytopenia confirmed. RBCs normal

Pancytopenia

Differential Diagnosis of Pancytopenian Reduced Production:

¨ Hematologic malignancy – Acute leukemiaMyelodysplasiaMyelofibrosis

¨ Aplastic anemia¨ Bone marrow suppression

n Drugs, radiation, infections, toxins¨ Metastatic tumor in marrow ¨ B12/folate/copper deficiency

n Increased destruction:¨ Paroxysmal nocturnal hemoglobinuria¨ Hemophagocytic syndrome¨ Hypersplenism

Case 5- Bone marrowNormal BMBiopsy Aspirate

Diagnosis: Aplastic Anemia

Myeloid Differentiation

Bone marrow Peripheral blood

Blast Promyelocyte Myelocyte Metamyelocyte Band PMN

Case 6n 42 yo dentist

¨ Turned down as a blood donor because of Hb of 11.5n PE Splenomegaly 4cm below left costal marginn Further testing revealed:

¨ WBC: 47,000/ cu mm¨ WBC diff:Neutrophils 40%¨ Bands: 20%¨ Metamyelocytes:16%¨ Myelocytes: 8%¨ Promyelocytes:6%¨ Blasts: 2%¨ Eos: 2%¨ Basos: 4%¨ Monos: 2%¨ Platelets: 680,000/ cu mm

Immature myeloid cells

Case 6- Peripheral Blood Film

Leukocytosis with left shift

Myelocyte

Metamyelocyte

Blast

Causes of Leukocytosisn Reactive

¨Neutrophilia: Infection, tissue necrosis, burns¨Eosinophilia: Allergies, parasites, drug reactions,

certain lymphomas, collagen-vascular disorders¨Basophilia: Rare (suspect neoplastic condition)¨Monocytosis: chronic infections, SLE, IBD¨Lymphocytosis: Chronic infections, viral infections

n Neoplastic¨Acute leukemias – myeloid vs lymphoid¨Chronic leukemias – myeloid vs lymphoid¨Myeloproliferative neoplasms

Case 6 continued

n Diagnostic evaluationCytogenetics- Philadelphia chromosome +(due to translocation between chromosomes 9 and 22, producing an abnormal product by splicing ABL and BCR genes)

n Diagnosis: Chronic myelogenous leukemia (CML)¨ A type of chronic myeloproliferative neoplasm

Chronic Myeloproliferative Neoplasms (MPN)n Chronic myelogenous leukemia (CML) –

↑Neutrophils, basophilsn Polycythemia vera (PV) - ↑RBCn Essential thrombocythemia (ET) - ↑Pltn Idiopathic myelofibrosis (MF) - ↑Fibrosis

Chronic Myeloproliferative Neoplasms: Clinical Featuresn Enlarged spleen (except in Essential Thrombocythemia)n Present with abnormal WBC, RBC, or platelet countn Thrombosis and bleeding ® ? Platelet dysfunctionn Must be distinguished from a reactive state, i.e.,

¨ ­ RBC ® due to: Hypoxic stimulation Excess Erythropoietin¨ ­ Plts ® due to: infection, inflammation¨ ­ WBC

n Natural history evolve over years. ie. not acuten Usually NOT associated with fever, night sweats etc

Case 7n 60 yo woman

Presents with pruritus, headache and early satiety n PE Splenomegaly 5cm below left costal margin

n CBC¨ Hb: 20 gm/dL¨ MCV: 88 fl¨ Platelets: 580,000 / cu mm¨ WBC: 18,500¨ WBC diff: Normal

n Smear: No immature cells. Neutrophilia. Thrombocytosis

Polycythemia

Case 7 continued

n Differential Diagnosis of Polycythemia¨Secondary

n Smokingn Excessive erythropoietin

¨Primary = Polycythemia veran Diagnostic test:

¨Mutation analysis of JAK2 gene - POSITIVE¨DIAGNOSIS - Polycythemia Vera

JAK-2 mutation results in activation of JAK-STAT pathway in absence of ligand – “cytokine independent constitutive activation”

n NOTES:¨ Other mutations seen in MPNs: Calreticulin Receptor (CALR) and

MPL (thrombopoietin Receptor¨ The presence of mutation does not discriminate between the various

MPNs

Acute Leukemiasn Acute lymphoblastic leukemias

¨B-cell and T-cell typen Acute myeloid leukemias

¨With recurrent cytogenetic abnormalitiesn t(15;17) (cases acute promyelocytic leukemia), t(8;21),

t(9;11) and inv(16)n With molecular mutations: Flt3, NPM1, CEBPAn AML with multilineage dysplasian Therapy related AMLn AML not otherwise specified (AML)

Anand S. Lagoo, MD, PhD, 2016

Bone marrow

Normal promyelocyte

Anand S. Lagoo, MD, PhD, 2016

Anand S. Lagoo, MD, PhD, 2016

Distribution of cytogenetically and molecularly defined subsets of AML

Blood. 2016 Jan 7;127(1):29-41

Myelodysplastic Syndromesn Clonal proliferation of myeloid progenitor

¨ Retain capacity to mature into erythroid, myeloid, and megakaryocytic lineages

¨ But maturation is ineffective and disordered (dysplastic)¨ Disease of older adults, rare in children¨ Variable cytopenias- anemia, leukopenia, and/or

thrombocytopenia¨ Marrow is hypercellular (due to ineffective hematopoiesis)

and cells are dysplastic¨ Variable number of myeloid blasts (but <20%)¨ Cytogenetic abnormalities¨ Median survival 9 to 29 months¨ Cause of death: Progression to AML, hemorrhage, infection

Erythroid dysplasia

Anand S. Lagoo, MD, PhD 2016

Megakaryocytic dysplasia

Anand S. Lagoo, MD, PhD 2016

Case 8n 29 yo woman, previously healthy

Presents with heavy menstrual bleeding, numerous bruises

PE: Petechiae and ecchymoses. No splenomegalyn Lab data:

¨ Hb: 13.4 gm/dL¨ MCV: 85 fl¨ Platelets: 5,000 / cu mm¨ WBC: 10,500¨ WBC diff: Normal ¨ Smear: No immature cells. Thrombocytopenia. No

schistocytesn DIAGNOSIS: Immune thrombocytopenic purpura (ITP)

Differential Diagnosis of Thrombocytopenian Impaired production

n Accelerated destruction

n Disorder of distribution (hypersplenism)

n Multifactorial

Differential Diagnosis of Thrombocytopenian Impaired production

¨Drugs¨ Infections¨Aplastic anemia¨Hematologic malignancy¨Myelophthisis¨Myelodysplasia¨B12/folate deficiency

Differential Diagnosis of Thrombocytopenian Impaired productionn Accelerated destruction

¨ ITP¨ Drugs, including Heparin¨ Collagen vascular diseases¨ Infections including HIV¨ Disseminated intravascular coagulation (DIC)¨ TTP/HUS¨ Alcohol¨ Inherited platelet disorders¨ Post-transfusion purpura¨ Non-Hodgkin lymphomas

n Disorder of distribution (hypersplenism) n Multifactorial

Summaryn CBC and peripheral blood smear are the mainstay of diagnosing

disorders of blood cellsn Anemia is very common worldwide and has many causesn Anemias are classified based on red cell morphology followed

by an etiological classification using special testsn Leukocytosis is often reactive but various leukemias must be

consideredn Immune destruction of platelets is a common cause of

thrombocytopenia but decreased production due to bone marrow abnormalities must also be considered.