Hematology - 01 Intro to Hematology

HEMATOLOGY INTRODUCTION TO HEMATOLOGY OUTLINE • • • • • • • Hematology History of Hematology Blood Common Affixes used in Hematology Vocabulary Hematology Laboratory Specimen Collection INTRODUCTION TO HEMATOLOGY HEMATOLOGY • • • Haima - blood; Logos - study/science Discipline that studies the development and diseases of blood; studies blood cells Includes o Analyses of concentration, morphology, and function of cells in the blood o Precursors of the bone marrow; blood cell development ▪ Bone marrow - produces majority of blood cells; precursors for RBC, WBC, platelets o Hemostasis and thrombosis ▪ Hemostasis - ability to maintain blood fluid; balance between anticoagulants & procoagulants o Sp. gravity - 1.055 o Thick and viscous - 3.5-4.5x thicker than water o 75-85 mL per Kg body weight o 20 grams of solids/100mL of blood Functions of Blood o Respiratory ▪ Blood carries oxygen to cell, carries CO2 from cells to lungs ▪ Gas transport o Nutritional ▪ Carries analytes (biologic solutes) that are dissolved in blood ▪ Analytes - proteins, nutrients, minerals ▪ Transports nutrients o Excretory ▪ Carries metabolic waste products from cell to excretory organs (kidneys, liver) o Buffering action ▪ Vital to maintain normal blood pH o Body temperature o Transport of hormones o Defense Carbohydrate Methodologies ▪ Immune defense ▪ Due to presence of WBCs ▪ WBC - fight infection COMPONENTS OF BLOOD HISTORY OF HEMATOLOGY • • • • • • Athanasius Kircher (1657) - described ‘worms’ in the blood Anton van Leeuwenhoek (1674) - gave an account of RBCs; discovery began curiosity of scientists to study blood William Hewson (1770) - description of different cell types o His and Leeuwenhoek’s discoveries birthed hematology o Father of Hematology Giulio Bizzozero (1800’s) describe platelets as “petites plaques” James Homer Wright (1902) development of Wright stain o Provided a new technique for visual examination in microscopy Joseph and Wallace Coulter o First automated cell counter o “Coulter counters” BLOOD • • • Red liquid circulating in the heart, veins, arteries, and capillaries Main fluid for transporting nutrients, waste products, gases, hormones through the circulatory system Characteristics o Fluid (in vivo - inside the body) o Red (due to hemoglobin protein) o Slightly alkaline ▪ Arterial - 7.35-7.45 pH ▪ Venous - 7.38-7.48 Ph o o • Normal blood volume - 4-6L Other solutes: proteins, carbohydrates, lipids, blood gases o WBCs Plasma vs Serum o Plasma ▪ Liquid part of anticoagulated or unclotted blood ▪ Hazy ▪ All coagulation factors are present o Serum ▪ Liquid part of clotted blood ▪ Clearer ▪ Clotted factors consumed during coagulation: • FI (Fibrinogen) • FV (Proaccelerin) • FVIII:c (Anti-Hemophilic Factor A) • FXIII (Fibrin Stabilizing Factor) • FII (Prothrombin) - less than 20% present • • Red Blood Cells o Erythrocytes o Size: 6-8 um o Shape: biconcave disk o With central pallor area (almost pale center) ▪ 1/3 of central part RBC ▪ Due to biconcave disk shape o Thickness: 2-8 um o Average life span: 120 days o Primary function: hemoglobin synthesis ▪ Carries oxygen to the peripheral tissues ▪ Carries carbon dioxide to the lungs ▪ Provides respiratory function of blood o Diseases ▪ Anemia - decrease in Hemoglobin ▪ Polycythemia - decrease in blood cells o Laboratory assessment ▪ RBC count ▪ Hemoglobin, Hematocrit ▪ RBC indices White Blood Cells o Leukocytes o Primary function: provides immune reaction to pathogens by phagocytosis or antibody production o Diseases ▪ Leukocytosis - increase in WBCs ▪ Leukopenia - decrease in WBCs ▪ Leukemia - cancer of mature WBCs o Laboratory assessment ▪ WBC Count ▪ Differential WBC Count COMMON AFFIXES USED IN HEMATOLOGY VOCABULARY PREFIX a- /an- lack, without, absent, decreased unequal, dissimilar micro- small myel(o)- before wrinkled cell abnormal, difficult red Iron pertaining to the blood panphlebphagopoikiloPolyPrePykno- (1) from bone marrow; (2) spinal cord all, overall, all-inclusive vein Eat, ingest Varied, irregular Many (pro-) Before Dense Reticulo· Netlike hyperiso- e leuk(o)macromegameta- beneath, under, deficient, decreased above, beyond, extreme equal, alike, same white large, long large, giant (1) after, next; (2) change -algia -ase -cide pain along a nerve an enzyme The killer of -oma -opathy -osis -crit -cyte -ectomy To separate Cell Incision and removal -emia -itis Blood Inflammation -penia -phil(ic) -plasla (-plastic) -poiesis -lysis Destruction or dissolving anisoantecrenacytdyserythroferrhemo(hemato-) hvpo- SchisScierSideroSpleenThromboXanth- Split Hard Iron Spleen Clot, thrombus Yellow SUFFIX -poietin Swelling or tumor Disease (1) Abnormal increase; (2) disease Deficiency, decreased Attracted to, affinity for Cell production or repair Cell production, formation, & development Stimulates production HEMATOLOGY LABORATORY • o • Five types ▪ Granulocytes • Cytoplasm have granules • Neutrophils (Segmented, bands) (B) • Eosinophils (D) • Basophils (E) ▪ Agranulocytes • Cytoplasm with invisible granules • Lymphocytes (F) • Monocytes (G) Platelets o Thrombocytes o Size: 1-4 um o Life Span: 9-10 days o Primary function ▪ Maintains blood vessel integrity by instigating vessel wall repair ▪ Control hemostasis • Functions o Establish or rule out a diagnosis o Confirm a physician’s clinical impression of a possible hematological disorder o Detect an unsuspected disorder o Monitor the effects of therapy o Detect minimal residual disease following therapy Hematology Laboratory Safety o Applicable safety practices required by OSHA standards ▪ OSHA standards - ensures safe and helpful work practices and environment • Proper labeling • Handling of chemicals/specimens o Standard Precaution ▪ “Universal Precaution” ▪ All blood, body fluids, and unfixed tissues are to be handled as though they were potentially infectious o Blood-borne Pathogens ▪ Microorganism in the blood that can cause disease ▪ HIV, HBV, HCV, Malaria, Syphilis ▪ SPECIMEN COLLECTION • • Patient Identification o Considered as the most critical step in specimen collection o Identify patient by asking him to state and spell their full name (including middle name) o If patient if not able to speak: verify patient’s identity from relative, attending nurse, or attending physician Physiologic factors affecting Test Result o Posture - shift in posture may affect levels of lipids, enzymes, and proteins o Diurnal rhythm - during afternoon, increased eosinophils, and decreased levels of ACTH and cortisol; physiologically normal o Stress - increased WBC count, and coagulation factors (I, V, VIII:c, XIII) o Exercise - increase WBC, platelets, Creatine kinase, Lactate dehydrogenase o Diet - after fatty meal: increased Alkaline phosphatase, hemoglobin (cyanmethemoglobin method - uses spectrophotometry) ▪ Plasma become turbid; affects spectrophotometry o Smoking - increased WBC, and cortisol ▪ Chronic smoking: may also increase RBC, Hemoglobin, Hematocrit SOURCES OF BLOOD FOR HEMATOLOGY TESTING • • Skin/Capillary Puncture o Used only when small quantities of blood are required o Blood obtained contains mixture of blood from venules, areterioles, capillaries, and interstitial fluids o Heel warmer - increases blood flow by 7 folds o Recommended depth of skin puncture - < 2mm o Avoid milking/squeezing ▪ Milking - introduces increased interstitial fluid o Discard first drop of blood (due to high levels of interstitial fluid) o Indication - infants, geriatrics, obese patients, burn patients o Collection sites ▪ Infants (<1 age) - lateral or medial plantar area ▪ Children (>1 age), adult palmar surface of the distal portion of the 4th or 3rd finger o Order of draw for Skin punture ▪ Tube for Blood Gas analysis ▪ Slides ▪ EDTA microcollection tube ▪ Other anticoagulated tubes ▪ Serum microcollection tube Venipuncture o Major veins (antecubital area) ▪ Cephalic vein - located at the thumb side of the hand; away from the body; 2nd choice ▪ Basilic vein - located ate the little-finger side of the hand • Final vein of choice due to proximity to nerve endings and arteries Median Cubital vein - connects the basilic and cephalic vein • Vein of choice of venipuncture • Well anchored, larger among the 3 o • • Important considerations ▪ Angle: 15-30° ▪ Tourniquet application • Length of time: <60 seconds o Prolonged can result in longer coagulation times • Distance: 3-4 inches (7.5-10 cm) from the puncture site ▪ Needle length: 1-1.5 inches ▪ Needle gauge: 21g Common Causes of Hemolysis o Hemolysis - release of RBC components to the whole blood ** o Prolonged tourniquet application ▪ Cause hemoconcentration o Moisture or contamination of blood collection tube ▪ Water may rupture RBC o Using needles with too small bore o Excessive agitation during mixing of samples o Frothing of blood sample (bubbles) during collection Capillary vs Venous Blood o Capillary blood ▪ Lower RBC count, hematocrit, hemoglobin, and platelet count ▪ Higher WBC count (15-20% or up to 1000/ul) ▪ Higher glucose level (by 10-20 mg/dL) ▪ Should be noted when used ADDITIVES • • • • Subtances that are added in blood collection tubes for various purpose Antiglycolytic agents - Prevent glycolysis Clot activators - promote clotting o Glass or silica particles - activate clotting factors o Thrombin - enzyme that converts fibrinogen to fibrin Anticoagulants - prevents clotting o Disadvantages of EDTA blood ▪ Platelet Satellitism • Platelets adhere on WBC surfaces (neutrophils) • May cause pseudothrombocytopenia (false decreased count of platelet) • Correction: recollect blood sample using 3.2% Sodium citrate (L.Blue top) then recount platelets • Correction Formula: platelet count x 1.1 ▪ EDTA-induced Platelet Clumping • Platelet clumps as large as WBC • May cause pseudothrombocytopenia (falsely decreased platelet), pseudoleukocytosis (falsely elevated WBC) • Correction: recollect blood sample using 3.2% Sodium citrate (L.Blue top) then recount platelet and WBC • Correction Formula o Platelet count x 1.1 o WBC count x 1.1 Additive Color Code Notes Excessive EDTA (underfilled tube): • Falsely decreased HCT, ESR • Falsely increased MCHC, platelet count • Degenerative changes in WBC NOTES: • CBC & reticulocyte count -can be performed on EDTA blood stored up to 4 hrs (RT) • WBC Count, Hematocrit, Platelet count - can be performed on ETA blood stored up to 24 hrs (4C) • ESR - should be performed within 2 hrs (stored at RT) or 6 hrs (4C) • Blood Smear - should be made within 2 hrs of collection Inversion: 8 times Heparin Platelet Satellitism o o o Green Top Mechanism of action: inhibits thrombin EDTA-induced Platelet Clumping Ammonium heparin - may falsely elevate ammonia Sodium heparin - may falsely elevate sodium Lithium heparin - may falsely elevate lithium Additive Color Code Anticoagulant of choice for Osmotic Fragility Test Forms of Heparin: Ammonium heparin, Sodium heparin, Lithium heparin Notes Antiglycolytic agents Sodium fluoride Gray top Preserve glucose (3 days) Not used for blood smear preparation • May cause morphologic distortion of platelets and leukocytes • May cause bluish discoloration of background of blood film For blood glucose and blood alcohol determination Anticoagulant: Potassium oxalate Inversion: 8 times Lithium lodoacetate Gray top Preserve glucose (24 hours) Not used for coagulation studies (inhibits thrombin) For blood glucose and blood alcohol determination May cause errors in automated cell counting Anticoagulant: Potassium heparin Inversion: 8 times Clot Activators Glass tube Optimal anticoagulant concentration: 15-20 units/mL of blood Red Top (Glass) Non-additive tube Glass or Silica particles Red Top (Plastic) Used for STAT serum determination Thrombin Orange top Used for STAT serum determination Inversion: 8 times 3.2% Sodium Citrate Light Blue Top Critical Blood-to-Anticoagulant Ratio: 9:1 Mechanism of Action: binds to calcium in an unionized form No inversion Used for coagulation testing (PT, APTT, D-dimer, etc.) Inversion: 5 times Important Consideration: forceful mxig and excessive inversion can activate platelets and shorten clotting times Mode of Action: Thrombin converts fibrinogen to fibrin Inversion: 3-4 times Inversion: 5 times • Separator Tube Thixotropic Gel Gold Top An inert material that undergoes temporary change in viscosity during centrifugation process Separated packed RBC from serum Inversion: 5 times Anticoagulants EDTA (Ethylene- Lavender/ diaminetePurple top traacetic acid) Optimal anticoagulant concentration: 1.5-2.2 mg/mL of blood Order of draw o To prevent additive carry over o Blood culture - first Blood Collection Tube Blood Culture Tubes (SPS) Tubes for coagulation testing (3.2% sodium citrate) Serum tubes Used for most routine hematological studies (CBC, Reticulocyte count, mod, Westergren ESR) Heparin tube EDTA tube Sodium fluoride tiube Insufficient EDTA (overfilled tube): presence of clots 8 3-4 0 Mechanism of action: chelates calcium Forms of EDTA: K2EDTA, K3EDTA, LiEDTA, Na2EDTA Inversion 5 8 8 8 Color Code Yellow top Light Blue top Red (Glass) - no additive Red (plastic) - glass or silica particles Green top Lavender top Gray top • Other blood collection tubes Blood Collection Tube Yellow Top Tan Top Royal Blue Top White Top Black Top Pink Top (with special crossmatoh label approved by AABB) Additive Uses Sodium polyanethol sulfonate Blood Culture Action: • Prevent phagocytosis • Prevent complement activation • Neutralizes some antibiotics Acid Citrate Dextrose Blood bank testing, paternity testing, HLA testing, DNA testing K2EDTA Lead determination • Certified to contain less than 0.01 ug/ml of Lead K2EDTA Trace elements, Nutritional chemistry • Contains very low levels of trace elements K2EDTA wilh gel separator Molecular tests 3 8% Sodium citrate Westergren method of Blood-to-anticoagulant ESR determination ratio: 4:1 K2EDTA Blood bank test (crossmatching) SAMPLE PREPARATION USED IN HEMATOLOGY LAB • • • • • • • Whole blood - Used in CBC, OFT, ESR Plasma o Platelet-poor Plasma (<10,000 platelet/uL) - used for coagulation tests ▪ Centrifugate whole blood at 1500-200 g for 10 minutes o Platelet-rich Plasma (200-300 10x9/L platelets - used for platelet function tests ▪ Centrifuge whole blood at 50-100g for 10 minutes Serum - for prothrombin consumption test o Whole blood is allowed to clot and then centrifuged Blood smear - for PBS examination, WBC differential count, Reticulocyte count Buffy coat smear, - for WBC differential count (leukopenic patient), abnormal cell morphology studies, borneborne/blood-borne parasitic infection studies (Leishmania spp.) Defibrinated blood - used for special hematological tests o Blood is collected in a flask with glass beads or paper clips. The flask is continuously agitated until clot forms on the surface of the beads and then removed Fresh blood from Skin puncture - for bleeding time and micromethod of clotting time