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Foundations Unit

Foundations Unit. Diagnostic Tools in Hematology. Ruth Padmore, MD, FRCPC, PhD Staff Hematopathologist, The Ottawa Hospital Associate Professor, University of Ottawa. Objectives.

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Foundations Unit

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  1. Foundations Unit Diagnostic Tools in Hematology Ruth Padmore, MD, FRCPC, PhD Staff Hematopathologist, The Ottawa Hospital Associate Professor, University of Ottawa

  2. Objectives • Identify the major components of the complete blood count (CBC), including the red and white cell parameters and platelet count. • Discuss the utility of a peripheral blood film. • Recognize the indications for a bone marrow examination. • Describe what is involved in a bone marrow examination. Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  3. CBC = complete blood count, one of the most diagnostically useful and cost effective tests in medicine Sample collection: one mauve-top tube, containing anticoagulant EDTA, which prevents blood from clotting during analysis The sample is analyzed using an automated hematology analyzer Objective 1. The components of the CBC Mauve-top tube for CBC Automated hematology analyzer Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  4. Objective 1. The components of the CBC Results of CBC from automated hematology analyzer displayed in electronic patient chart (OACIS). Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  5. Objective 1. The components of the CBC • The three cellular components blood, reported in the CBC are: • Red blood cells (RBC): • 6 – 8 um in diameter, red colour, no nucleus • Contain hemoglobin for oxygen transport • White blood cells (WBC): • 12 -15 um in diameter, have nucleus • Important for immunity • Platelets (PLAT): • Small, 2um in diameter, no nucleus • Important for blood clotting Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  6. Objective 1. The components of the CBC Photomicrograph of Peripheral Blood showing the Cellular Components Platelets → ← Platelets WBC (neutrophil) → Red cells → Red cells → WBC (monocyte) ↑ Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  7. Objective 1. The components of the CBC • The Red blood cells and White blood cells have parameters which are measured by the automated hematology analyzer: Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  8. Objective 1. The components of the CBC 1. How many cells? WBC, RBC, PLAT Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  9. Objective 1. The components of the CBC • Parameter: How many cells in the blood? • The automated hematology analyzer counts the number of cellular components in the blood. • Red blood cell count (RBC) • Normal RBC range = 3 – 5 x 1012/L • White blood cell count (WBC) • Normal WBC range = 3 – 10.5 x 109/L • Platelet count (PLAT) • Normal PLAT range = 125 – 400 x 109/L Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  10. Objective 1. The components of the CBC 2. Red cell parameters measured by the automated hematology analyzer Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  11. Objective 1. The components of the CBC • Red blood cells parameters • The automated hematology analyzer measures two red cell parameters: 1. Hemoglobin (HGB) • Hemoglobin content in the blood • Normal adult female HGB = 115 – 155 g/L • Normal adult male HBG = 130 – 170 g/L 2. Mean corpuscular value (MCV) • Average size (volume) of the red blood cells • Normal MCV = 80 – 100 fL (femtoliter, 10-15L) Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  12. Objective 1. The components of the CBC 3. Red cell parameters calculated by the automated hematology analyzer Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  13. Objective 1. The components of the CBC Red blood cells parameters The automated hematology analyzer calculates four red cell parameters: 1. Hematocrit (HCT) the proportion of the blood occupied by the red blood cells HCT = RBC x MCV Normal HCT = 0.35 – 0.50 L/L Hematocrit may also be measured manually, by centrifuging whole blood in a capillary tube (spun hematocrit, packed cell volume), see image to the right. Plasma = Liquid portion of unclotted blood – 55 mm – Red cells Hct = 0.45 L/L – 45 mm – Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  14. Objective 1. The components of the CBC • Red blood cells parameters • The automated hematology analyzer calculates four red cell parameters: 2. Mean corpuscular hemoglobin (MCH) • the content of hemoglobin in an average red blood cell • MCH = HGB/RBC • normal MCH = 27 – 34 pg (picogram, 10-12 grams) 3. Mean corpuscular hemoglobin concentration (MCHC) • concentration of hemoglobin in a volume of packed red blood cells • MCHC = HBG/HCT • normal MCHC= 315 – 365 g/L Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  15. Objective 1. The components of the CBC Red blood cells parameters The automated hematology analyzer calculates four red cell parameters: 4. Red cell distribution width (RDW) a measure of the variation in red cell size RDW = red cell volume within one standard deviation (SD) of the mean cell volume/mean cell volume normal RDW = 11.5 – 15.5% RDW % red cells Mean cell volume Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  16. Objective 1. The components of the CBC 4. Platelet parameter measured by the automated hematology analyzer Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  17. Objective 1. The components of the CBC • Platelet parameter • Mean platelet volume (MPV) • Average size of the platelets • Normal MPV = 6 – 12 fL Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  18. Objective 1. The components of the CBC 5. Types of WBC identified in the 5 – part differential count Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  19. Objective 1. The components of the CBC • White blood cell (WBC) differential count: • There are 5 different types of WBC in the peripheral blood of a healthy person: • Neutrophils • Lymphocytes • Monocytes • Eosinophils • Basophils Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  20. Objective 1. The components of the CBC eosinophil  segmented neutrophil  Cells identified in the five-part differential count lymphocyte  basophil →  monocyte Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  21. Objective 1. The components of the CBC White blood cell (WBC) differential count: The WBC differential count can be performed in two ways 1. Automated differential count: performed by the automated hematology analyzer instrument Automated hematology analyzer counts the number of cells in each category Automated hematology analyzer is able to recognize different types of WBC using electronic signals, optical methods and special stains Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  22. Objective 1. The components of the CBC White blood cell (WBC) differential count: The WBC differential count can be performed in two ways 2. Manual differential count: performed by the hematology technologist, counting 100 consecutive white blood cells on the peripheral blood film The peripheral blood film is prepared by placing a drop of blood on a glass slide, and spreading it, to make a film of blood on the slide, followed by staining with Wright’s/Giemsa stain Glass slide with peripheral blood film Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  23. Objective 1. The components of the CBC White blood cell (WBC) differential count: Manual differential count: After counting 100 consecutive white blood cells, the percentage of cells in each category is calculated The absolute count of each cell type is calculated by multiplying the percentage of each type of cell by the total WBC Example of calculation of the manual differential count Step 1. 100 cells counted 64 neutrophils, 30 lymphocytes 6 monocytes, 0 eosinophils, 0 basophils Step 2. Multiply percentage of cells in each category by WBC count WBC = 8.2 x 109/L Absolute neutrophil count = 64% x 8.2x109/L = 5.2x109/L Absolute lymphocyte count = 30% x 8.2x109/L = 2.5x109/L Absolute monocyte count = 6% x 8.2x109/L = 0.5x109/L Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  24. Objective 1. The components of the CBC • White blood cell (WBC) differential count: • Normal ranges of the 5 different types of WBC in the peripheral blood of a healthy person: • Neutrophils 2 – 7.5 x 109/L • Lymphocytes 1 – 4 x 109/L • Monocytes 0.1 – 1.0 x 109/L • Eosinophils 0.0 – 0.5 x 109/L • Basophils 0.0 – 0.1 x 109/L Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  25. Objective 1. The components of the CBC CBC parameters in different gender/age/ethnic groups Normal adult males have higher hemoglobin (HGB) and red cell amounts (HCT, RBC) than normal adult females Adult Female Adult Male Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  26. Objective 1. The components of the CBC • Normal reference intervals for newborns • Hemoglobin and MCV of newborns are higher than in adults • Newborn HGB = 135 – 195 g/L, newborn MCV = 98 – 118 fL • Normal reference intervals for children • Lymphocyte count range of children is higher than in adults • Normal lymphocyte count in a 4 year old child = 2.0 to 8.0 x 109/L • Normal reference intervals in Africans/Asians: • Normal neutrophil count range is lower: 1.5 – 7.5 x109/L CBC parameters in different gender/age/ethnic groups Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  27. Objective 1. The components of the CBC Reticulocytes are immature non-nucleated red cells in the peripheral blood Reticulocytes still contain RNA, and are identified by their bluish colour (polychromasia) on the blood film, and larger size They can also be identified by the automated hematology analyzer Appropriate reticulocyte response to anemia? Yes: consider hemolysis or hemorrhage No: check MCV and explore differential diagnosis based on MCV result Reticulocyte Count Reticulocyte → Example of increased reticulocytes due to hemolysis from autoimmune hemolytic anemia Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  28. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film • The CBC and Peripheral Blood Film are basic and useful tests and should be part of initial investigation of most patients Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  29. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film Numerical abnormalities detected by the CBC Cell Type Too few Too many Red cells anemia polycythemia WBC leukopenia leukocytosis Neutrophils neutropenia neutrophilia Lymphocytes lymphopenia lymphocytosis Platelets thrombocytopenia thrombocytosis Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  30. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film Red cell parameter abnormalities detected by the CBC (examples) Red cell parameter Too low Too high MCV microcytosis macrocytosis • Low HGB, low MCV • Microcytic anemia • Causes: iron deficiency, thalassemia, anemia of chronic disease • Low HGB, high MCV • macrocytic anemia • Causes: Vitamin B12/folate deficiency, liver disease, myelodysplastic syndrome, hypothyroidism, drugs, ethanol Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  31. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film Normal red cells are biconcave discs, with round nuclear contour, and an area of central pallor on the peripheral blood film Assessment of red blood cell morphology on the peripheral blood film Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  32. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film Schistocytes: red cell fragments Microangiopathic hemolytic anemia: Schistocytes with thrombocytopenia Formed by slicing of the red cells by fibrin strands in the microvasculature Seen in thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC) Macroangiopathic hemolytic anemia: Schistocytes with normal platelet count Formed by mechanical damage to red cells by leaky prosthetic heart valve Assessment of red blood cell morphology on the peripheral blood film  Schistocyte Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  33. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film Spherocytes: are small, hyper-dense red cells, with loss of central pallor Formed by repair of red cell membrane, with partial loss of the red cell membrane Seen in: Autoimmune hemolytic anemia Hereditary spherocytosis Assessment of red blood cell morphology on the peripheral blood film Spherocyte → Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  34. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film Sickle cells: are elongated, pointed, sickle-shaped red cells Formed by polymerization of abnormal hemoglobin S Seen in sickle cell disease, caused by a homozygous presence of amino acid change in position 6 of the beta globin chain of hemoglobin, Glutamic acid (Glu) (normal) to Valine (Val) (sickle cell) Assessment of red blood cell morphology on the peripheral blood film Sickle cell → Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  35. Objective 2. Diagnostic Utility of the CBC/Peripheral Blood Film Useful in the diagnosis of leukemia and lymphomas, characterized by circulating neoplastic cells Photomicrograph of a blast cell with Auer rod (arrow) in the peripheral blood in acute myeloid leukemia Assessment of white blood cell morphology on the peripheral blood film Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  36. Objective 3. Indications for a bone marrow examination • Abnormal peripheral blood counts/morphology, if cause cannot be determined by other means • Re-evaluation of patients who do not respond to therapy as expected (e.g. presumed idiopathic thrombocytopenic purpura) • Assessment of neoplastic hematological disorders • Diagnosis of neoplastic hematological disorders (leukemia, lymphoma, plasma cell myeloma) • Staging on neoplastic disorders (especially lymphoma) • Monitoring of therapy in patients with neoplastic hematological disorders • Bone marrow assessment prior to autologous bone marrow transplant • Work-up for infectious diseases and/or fever of unknown origin (FUO) (tuberculosis, Q-fever) • Diagnosis of storage diseases (e.g. Gaucher’s disease) Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  37. Objective 4. What is involved in a bone marrow examination The procedure: Marrow is obtained from the posterior iliac crest in adult patients MARROW ASPIRATE: Marrow is aspirated with rapid suction The marrow aspirate is directly smeared on a glass slide, and examined for adequacy (are marrow particles present?) The aspirate smear is stained with Wright’s/Giemsa stain, and examined by microscopy Marrow particle → Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  38. Objective 4. What is involved in a bone marrow examination MARROW BIOPSY The marrow is biopsied using a biopsy needle The biopsy is removed from the hollow biopsy needle using a stylet The biopsy obtained should be at least 1cm in length The marrow biopsy is fixed in formalin, paraffin-embedded, surface decalcified, cut to obtain 4um thick sections, and stained with H&E Processing of the marrow biopsy takes a minimum of 24 hours Bone marrow and fat cells   Bone trabeculae Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  39. Objective 4. What is involved in a bone marrow examination • ANCILLARY TESTS IN BONE MARROW EXAMINATION • Ancillary tests are useful in the diagnosis, prognosis and follow-up of hematological neoplastic disorders, such as leukemia and lymphoma • Extra marrow aspirates are obtained, and transferred to appropriate vacutainer tubes • Discuss with staff and technologists ahead of time, what ancillary testing is required • Types of ancillary tests: • 1. Flow cytometry immunophenotyping • 2. Cytogenetic karyotyping and FISH analysis • 3. Molecular studies • 4. Microbiology culture Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

  40. Objectives • Identify the major components of the complete blood count (CBC), including the red and white cell parameters and platelet count. • Discuss the utility of a peripheral blood film. • Recognize the indications for a bone marrow examination. • Describe what is involved in a bone marrow examination. Foundations Unit – Diagnostic Tools in Hematology – Dr. Padmore – October 15, 2008

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