Abnormalities of Blood Cells
Transcript of Abnormalities of Blood Cells
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.