7/15/2018 Complete blood count (CBC) • Usual components • WBC • RBC Subtleties of the CBC • Hemoglobin • Hematocrit Meghan East • Indices Salisbury University • Platelet count • Optional: Microscopic evaluation • Alternate name: PBC for Peripheral Blood Count 4 Outline Complete blood count (cont.) • Review: what is included in a CBC • Usually performed on automated instrument • Common changes seen on the CBC and relevance • 5 part diff most common to patient care • 3 part diff on smaller instruments • “New-ish” tests in the CBC & how to use clinically • Can also be performed manually • Hemocytometer to count cells • Effects of improper collection and transportation • Manual hemoglobin on CBC results • Micro hematocrit • Case studies • Calculations for indices 5 Components of Blood Complete blood count (cont.) • Specimen from correct patient • Volume • Patient identified prior to obtaining sample • 4—6 liters • Properly labeled sample and requisition • Approximately 8% of total body weight • Usual specimen is EDTA whole blood. • Plasma 55%--liquid portion • EDTA in liquid form; chelates Ca++ and prevents clotting • Water • Ideal peripheral smears made within 3 hours • Solutes: proteins, electrolytes, hormones • Within 1 hour highly preferred • Cellular elements 45%--cellular portion • > 5 hours after collection results in artifacts • Red blood cells: carry oxygen and carbon dioxide • Smear from direct finger puncture (un-anticoagulated) must be • Example diseases- anemia, polycythemia made immediately • White blood cells: immunity • Stability of specimen for counts • Example diseases- leukemia, infection • Perform on room temp blood within 6 hours • Platelets: primary hemostasis (initiate clot) • Perform on refrigerated blood within 24 hours • Example diseases- thrombosis, bleeding disorders 6 hubpages.com 1 7/15/2018 Peripheral blood distribution of Complete blood count (cont.) WBC types • Unacceptable CBC specimens • Neutrophils 40-85% • Too old • Neutrophils 40-80% Reversed In young • Macroscopic or microscopic clotting • Bands 0-5% children* • Clotting activates & consumes coagulation factors • Binds blood cells within fibrin mesh • Lymphocytes 25-35% • erroneous for all assays • Monocytes 2-10% * So-called “Reverse Diff” • clogs apparatus of automated analyzers • Non-fasting specimen • Eosinophils 0-5% • Improper collection from line with TPN • • Hemolysis Basophils 0-1% 7 Absolute concentrations of CBC– component detail (cont.) leukocytes • RBC • Much better value than % in evaluation of whether a • Cells per cubic millimeter (microliter), expressed in millions (106) OR particular cell type is elevated • Cells per liter, expressed in 1012 cells • Reference ranges • Examples • Neutrophils 1.8 - 7.0 x 109/L • 5.00 X 106 cells per μL OR • Bands 0 - 0.7 x 109/L • 5.00 X 1012 cells per L • Lymphocytes 1.0 – 4.8 x 109/L • Reference ranges • Monocytes 0.1 - 0.8 x 109/L 6 • Adult male = 4.50 – 5.90 X 10 cells per μL • Eosinophils 0 - 0.4 x 109/L • Adult female = 3.80 – 5.20 X 106 cells per μL • Basophils 0 – 0.2 x 109/L 8 CBC– component detail (cont.) Leukocyte function (cont.) • WBC • Granulocytes = non-specific arm of the immune 3 system (along with macrophages) phagocytosis • Cells per microliter, expressed in thousands (10 ), OR & digestion of pathogens 9 • Cells per liter, expressed in 10 units • Neutrophils – bacteria • Examples • Eosinophils – weak phagocytes; parasite toxins, allergy • 5.1 X 103 cells per μL abatement 9 • Basophils – phagocytosis irrelevant; inflammatory response • 5.1 X 10 cells per L from IgE 3 • Reference range = 4.0 – 11.0 X 10 cells per μL • Lymphocytes = specific immune system arm • Monocytes & macrophages = non-specific arm of the immune system phagocytosis & antigen presentation to T helper cells 9 2 7/15/2018 Neutrophil distribution Basophil function • Origin in marrow, 7-8 days total: • 2-3 days in “Mitotic pool” = myeloblast myelocyte • Can also phagocytize • 5-7 days in “Storage pool” = metamyelocyte • Major function is mediators of inflammatory segmented stage responses • Released into PBS, stay for 7-10 hours • Have receptors for IgE • Marginal pool – adhere to vessel walls OR • When bound to IgE, histamine is released from • Circulating pool – free in plasma granules • Constant dynamic change between pools • Histamine release immediate hypersensitivity • Circulating pool can be increased during stress via mobilization of marginal pool by epinephrine • Also recruit eosinophils and neutrophils • Diapedesis into tissues for functions • Also function in the coagulation system via heparin granules Monocyte distribution and Neutrophil function function (cont.) • Participate in innate immune response • Participate in both immune systems • Phagocytosis by neutrophils • Innate • Ingest and digest invaders • Other functions of neutrophils • Act as “vacuum cleaners” to clean up debris and old cells • Secrete substances which activate parts of the • Adaptive coagulation system • Antigen-presentation of digested material to T helper cells • Interact with the kinin system, an ancillary pathway • Cytokines stimulate various lymphoctyes in the AIR (adaptive associated with coagulation immune response) • Secrete the pyrogen, IL-1, which induces fever Eosinophil functions Lymphocytes • Similar to neutrophils • Mature lymphocytes • Size VERY variable! (from 7-10 μm to as large as a monocyte) • Capable of phagocytosis • Three categories • Also secrete granular contents outside the cell • Small mature lymphocyte better defense against parasites, especially • Larger normal lymphocyte helminths • Variant lymphocyte • Are also increased in response to allergy, asthma, • Current preferred term and generalized inflammation • Historic/other terms used • Reactive lymphocyte • Atypical lymphocyte Courtesy and copyright © of the Clinical Chemistry and Hematology Laboratory, Wadsworth Center, NY State Department of Health (http://www.wadsworth.org/chemheme, with permission 3 7/15/2018 T-cell functions (cellular Complete blood count – immunity) component detail (cont.) • Delayed type hypersensitivity • Platelet count • Cytotoxic reactions • Cells per microliter, expressed in thousands (103) OR 9 • Killing virus-infected cells • Cells per liter, expressed as 10 cells • Rejection of transplanted organs • Examples • 150 X 103 cells per μL OR • Regulator T-cells • 150 X 109 cells per L • CD4+ helper is central player • Reference range = 150 – 450 X 103 cells per μL • Interact with other T-cells, B-cells and macrophages • CD8+ suppressor shuts off system • Normal ratio of CD4+ to CD8+ = 2:1 22 B-cell functions CBC– component detail (cont.) (humoral immunity) • Hemoglobin = concentration • g/dL OR • Unstimulated, carry immunoglobulin on cell • g/L surface • Example: 13.5 g/dL = 135 g/L • Antibody release • Reference ranges • StimulusTransform into plasma cells Antibody • Adult male = 13.5 – 17.5 g/dL secretion for current stimulus • Adult female = 12.0 – 16.0 g/dL • Stimulus Memory B-cells that can secrete antibody if stimulus encountered later 23 Complete blood count – Natural Killer (NK cell) functions component detail (cont.) • Part of the innate/non-specific arm of the immune • Hematocrit = percentage or proportion of system plasma that is RBC’s • Kill without any previous sensitization or immune • 45% = 0.45 L/L response • Reference ranges • Tumor cells Adult male = 42-52% • Virus-infected cells Adult female = 36-46% • Antibody coated cells hubpages.com 4 7/15/2018 CBC– component detail (cont.) CBC– component detail (cont.) • Indices = mathematical calculations with • MCH = mean corpuscular hemoglobin erythrocyte values • Calculation: • Size and hemoglobin content of RBC’s • Each is an average (except RDW) Hgb MCH = X 10 e.g. 14.2 X 10 = 29.1 pg • MCV = mean corpuscular volume RBC* 4.88 • MCH = mean corpuscular hemoglobin • MCHC = mean corpuscular hemoglobin concentration *Expressed as 106 /uL • RDW = red cell distribution width • Indices used to help classify anemias • Reference Interval = 26.0-34.0 pg (1 picogram = 10-12 gram) 25 28 Complete blood count – CBC– component component detail (cont.) detail (cont.) • MCV = mean corpuscular volume • Calculation: • MCH = mean corpuscular hemoglobin (cont.) Hct e.g. 42.6 • Cell “color” between 26.0-36.0 pg = normochromic MCV = X 10 X 10 = 87.3 fL RBC* 4.88 • Cell “color” < 26.0 pg = hypochromic • Cell “color” > 36.0 pg = hyperchromic *Expressed as 106 /uL • Large cells (macrocytes) have more hemoglobin but are a normal color; NOT called hyperchromic • Spherocytes abnormal cells that are darkly colored for their size • Reference Interval = 80.0-100.0 fL so could be called hyperchromic (1 femtoliter = 10-15 Liter) • Some advocate never using the term 26 29 http://departments.weber.edu/chpweb/3e/Acute%20Lymphocytic%20Leukemia/cbcresults2%20.html http://www.dwickes.com/anemia http://www.corbisimages.com/stock-photo/rights-managed/42-23595743/severe-iron-deficiency-anemia-showing-hypochromic-pale CBC– component detail CBC– component detail (cont.) (cont.) • MCHC = mean corpuscular hemoglobin concentration • MCV (cont.) • Calculation: • Using MCV to classify RBC’s • Cell volume 80-100 fL = MCHC = Hgb e.g. 14.2 X 100 X 100 = 33.3 g/dL normocytic HCT 42.6 • Cell volume < 80 fL = microcytic • Reference Interval = 32.0-36.0 g/dL • Cell volume >100 fL = • This is the better parameter for assessing cell color macrocytic • MCH doesn’t take cell size into account • Larger cells = more hemoglobin • Hemoglobin concentration/cell better 27 30 5 7/15/2018 http://departments.weber.edu/chpweb/3e/Acute%20Lymphocytic%20Leukemia/cbcresults2%20.html