Hematology Lab - 03 WBC and Platelet Count

HEMATOLOGY LABORATORY DILUTING FLUID COMPLETE BLOOD COUNT: WBC & PLATELET COUNT • OUTLINE • Introduction o Complete Blood Count o Materials o RBC and WBC Count • WBC Differential Count o WBC Differential Count o Peripheral Blood Smear o Nucleated RBC • Platelet Clount o Platelets o Indirect manual o Direct Method • • • INTRODUCTION COMPLETE BLOOD COUNT (CBC) • • • • • aka Full Blood Count, Full Blood Exam Most frequent blood test Provides a detailed information about the cells in the blood Screening test for most disease o Diseases affect blood cell levels Sample: EDTA Blood o Unclotted is preferred because clotted may have false negatives • • Includes: o Red Blood Cell Count o Hemoglobin o Hematocrit o Red Blood Cell Indices o White Blood Cell Count o White Blood Cell Differential Count o Platelet Count MATERIALS • • • • • Compound microscope Thoma pipettes o RBC pipette o WBC pipette Diluting fluid Counting chamber/Hemacytometer Coverslip This lessen the concentration of blood sample in order to disperse the blood cells and facilitate its counting under the microscope Criteria: o Has good preservative property o Does not initiate growth of molds/yeasts o High specific gravity o With buffering action o Cheap and easy to prepare RBC Diluting Fluids o Isotonic ▪ Preserve morphology of RBCs ▪ Any change in morphology cause misidentification o Examples: ▪ Dacie’s Fluid/Formol citrate - best RBC diluting fluid ▪ NSS – used in case of excessive rouleaux formation and autoagglutination • Prevents rouleaux and autoagglutination ▪ 3.8% Sodium citrate ▪ Hayem’s ▪ Gower’s ▪ Toissons’s ▪ Bethel’s WBC Diluting Fluid o Hypotonic ▪ Lyses RBCs & precursors except nucleated RBC ▪ Eliminate non-WBC cells o Examples: ▪ 2% (v/v) Glacial Acetic Acid ▪ 1-3% Acetic acid with Gentian Violet • Glacial acetic acid (2.00 mg) - lyses cell • Gentian Violet (1.00 mL) - stain • Distilled water (100 mL) - solvent ▪ 1% Hydrochloric acid • Hydrochloric acid (1.00 mL) - lyses cell • Distilled Water (100 mL) - solvent ▪ Turk’s Solution • Glacial acetic acid (2.00 mL) - lysing agent • Methyl violet (1.00 mL) - stains nucleus • Distilled Water (100 mL) - solvent • Can enhance WBC nuclear definition RBC AND WBC COUNT • Procedure: 1. Draw blood up to the 0.5 mark of the thoma pipette (RBC/WBC) 2. Wipe the tip of the thoma pipette 3. Draw diluting fluid up to the mark above the bulb of the pipette (RBC: 101; WBC: 11) 4. Shake the pipette for 5 minutes 5. Discard few drops 6. Charge the counting chamber 7. Stand for 3 minutes 8. Count cells on both counting area ▪ RBC count: 5 tertiary RBC squares (4 corners, 1 central) ▪ WBC count: 4 secondary WBC squares (4 corners) • • • • • o NOTE: o Count WBC or RBC on both counting area (primary square) of the hemacytometer and get the average. o Allowable difference between two chambers: ▪ RBC: 15-16 counts ▪ WBC: 10-12 counts General Formula o Cell count = # of cell counted x dilution factor Depth x Area o Depth: 0.1 mm o Conventional Unit: cells/mm3 o SI Unit (RBC): x 1012/L ▪ cells/mm3 1,000,000 o SI Unit (WBC): x 109/L ▪ cells/mm3 1,000 Area of Squares o 1° square: 9.0 mm2 o 2° square: 1.0 mm2 o RBC 3° square: ▪ 25 per central 2° square ▪ Area: 0.04 mm2 ▪ RBC 4° square: • 16 per RBC 3° square • Area: 0.0025 mm2 o WBC 3° square: ▪ 16 per 1 secondary square ▪ Area: 0.0625 mm2 Reference Value: o Red Blood Cell Count (x 1012/L): ▪ Male: 4.2 – 6.0 ▪ Female: 3.6 – 5.6 ▪ Newborn: 5.0 – 6.5 o White Blood Cell Count (x 109 /L): ▪ Adult: 4.5 – 11.0 ▪ Newborn: 5.0 – 21.0 o Reported as above, within, or below reference range WBC DIFFERENTIAL COUNT WBC DIFFERENTIAL COUNT • • • Percentage of each type of WBC in the blood: o Neutrophils ▪ 2 forms (band/stab/staff or segmented) o Lymphocytes o Monocytes o Eosinophils o Basophils Specific WBC type is counted using peripheral blood smear (100x objective/OIO) Specimen: EDTA Blood Smear (stained) Materials: o Compound microscope o Oil immersion (e.g., cedarwood) o Microscope slides ▪ Spreader slide ▪ Film slide o Differential counter ▪ Each button correspond to a specific WBC PERIPHERAL BLOOD SMEAR WEDGE TECHNIQUE • • • • • • aka Double slide, spreader slide, or push smear The most convenient and most commonly used PBS preparation method. Should be made within 2-3 hours of drawing specimen Requires two 3 x 1 inch glass slides (film slide, spreader/pusher slide) Fixative: Methanol Procedure: 1. A drop of blood is placed 0.25 inch from one end of the film slide (2-3 mm diameter) 2. The spreader slide is held at 30-45° angle on the film slide (not touching the blood) 3. Draw back the slide until it touches the drop of blood, allow the blood to spread across the width of the slide. 4. Push the slide quickly towards the other end to create a wedge film (even pressure) 5. Allow to air dry 6. Fix the blood film in the slide using methanol • Features of a well-made Peripheral Blood Film: 1. The film is 2/3 to 3⁄4 of the length of the slide 2. The film is finger shaped (very slightly rounded feathery edge) 3. The lateral edges are visible 4. The film is smooth (no irregularities, holes, streaks, etc) 5. Presence of “rainbow” appearance in the feathery edge of the film CRITERIA FOR WBC IDENTIFICATION 1. 2. 3. 4. • Procedure: 1. Prepare a stained blood smear from a well-mixed EDTA whole blood. 2. Perform LPF (10x) scan ▪ Verify stain quality ▪ Examine erythrocyte distribution pattern and shape - area where RBC are close to each other but only few are overlapping 3. Place a drop of cedarwood in the thin area of the smear. 4. Perform OIF (100x) exam ▪ Platelet estimate ▪ WBC estimate ▪ WBC differential count Cell size Nuclear-cytoplasmic ratio Cytoplasmic characteristic Nuclear characteristic WBC Cell Size Cytoplasm Nucleus Granulocytes Segmented 9-15 um Multilobed (2-5) Neutrophils/ connected by PMN (mature) thin filaments With fine lilacpink Band/Stab/ 9-15 um Indented nucleus granules Staff (>1/2 of nuclear (youngest diameter) granulocytic S, C, Z shaped precursor) Sausage-shaped Eosinophil 9-15 um With coarse reddishBilobed orange granules Basophil 10-16 um With coarse bluish-black granules that Multilobes (3-4) obscure the nucleus Agranulocytes Small 7-10 um Sky-blue (Robin Compact, large Lymphocyte egg blue) and round No granules Large/Big/ 11-25 um Sky-blue (Robin Immature looking Atypical egg blue) (round, oval, or Lymphocyte No granules slightly Indented by RBC indented) Monocyte 14-20 um Blue-gray with Kidney-shaped , ground glass horshoe/coffee appearance bean with brainwith/out vacuoles like convolutions • Lymphocytes are only counted as one regardless of size • Segmented Neutrophils • o In the OIF: 1. Classify 100 WBC 2. Report result as percentage of all WBC counted 3. Keep separate count of nRBC, if present 4. Note and report abnormal WBC morphology 5. Grade abnormal erythrocyte morphology 6. Identify miscellaneous abnormal cells Band/Stab/Staff • Eosinophils MANNER OF REPORTING • • • Basophil • • Small Lymphocyte • Relative Count o The number of specific WBC type per 100 WBCs o Formula: Relative count % = # of specific WBC x 100 100 WBC counted Absolute Count o Gives the number of specific WBC type per cubic millimeter of blood o More informative than Relative count o Formula: Absolute Count = Relative count % x WBC count Sample Problem: A peripheral blood smear was prepared using an EDTA-whole blood. A WBC Differential count was performed by classifying 100 WBC. The identified WBC are as follows: o 65 Segmented Neutrophils/100 WBC = 65% o 25 Lymphocytes/100 WBC = 25% o 5 Monocytes/100 WBC = 5% o 2 Eosinophils/100 WBC = 2% o 1 Basophil/100 WBC = 1% o 2 Bands/100 WBC = 2% Reference Value RELATIVE ABSOLUTE COUNT (%) COUNT(/mm3) NEUTROPHIL 47 – 77% 1,600 – 7,260 BAND/STAFF/STAB 2 – 6% 350 - 700 LYMPHOCYTE 20 – 40% 960 – 4,400 MONOCYTE 2 –10% 180 – 880 EOSINOPHIL 0 – 6% 45 – 440 BASOPHIL 0 – 1% 45 – 110 NUCLEATED RBC • • Large Lymphocyte • • • aka Orthochromic normoblast/Orthochromic erythroblast/Metarubricyte/pyknotic erythroblast/acidophilic normoblast Not lysed by WBC diluting fluid Identified in peripheral blood smear Reference value: o Adult: <5 nRBC/100WBC o Newborn: <10 nRBC/100 WBC CORRECTED WBC COUNT • • Monocyte • • Performed if there are ≥5 nRBC (adult), ≥10 nRBC (newborn) seen in 100 WBCs. Formula: Round final answer to nearest hundred Sample Problem: o WBC Count: 15,000/mm3 o 12 nRBCs seen (in 100 WBC) PLATELET COUNT PLATELETS • • • • • • • aka Thrombocytes Cytoplasmic fragments of mature megakaryocytes o Megakaryocyte - largest cell in the bone marrow o Not true cell because they are fragments Anucleated Size: 2-5 um (1-4 um) Life span: 9-10 days On wright-stained smear: o Ellipsoidal or Disc-shape (round to irregular) o Light blue to purple granular cytoplasm Primary function: o Maintains blood vessel integrity by instigating vessel wall repair o Controls hemostasis DIRECT METHOD DAMESHEK METHOD • • • • • • • • • PLATELET COUNT • • • Sample: Anticoagulated EDTA-Whole Blood Venous blood is preferred than capillary blood o Capillary blood may have increased platelet count Methods: o Direct o Indirect o Platelet Estimate REES-ECKER METHOD • • • Hemacytometric method Diluent: Rees-Ecker Procedure: 1. Rinse RBC pipet first with diluting fluid ▪ Aspirate and release diluting fluid repeatedly 2. Draw blood up to 0.5 mark of RBC pipet 3. Draw diluting fluid to 101 mark (0.5:101 or 1:202) 4. Shake the pipet for 3-5 minutes 5. Discard few drops (1:200) 6. Charge the counting chamber and let it stand for 3-5 minutes 7. Count platelets in the 4 WBC secondary squares (as in WBC count) 8. Formula: • Sample Problem: A 1:200 dilution is made from an EDTA whole blood. A total of 460 platelets are counting using the 4 WBC secondary squares. Compute for the platelet count. o Dilution: 1:200 o Platelets counted: 460 o Depth: 0.1 mm o Area: 4 mm2 (1.0 mm2 x 4) MATERIALS • • • • • RBC Thoma pipette Slides Diluting fluid Counting chamber Compound Microscope INDIRECT MANUAL • • • Platelets are counted in relation to red blood cells on a fixed-stained smear. Platelets are counted per 1000 RBCs. Not reliable. Same procedure as Fonio’s method Perform RBC count also Uses Rees-Ecker Fluid o Diluting fluid for platelet o Cannot lyse RBC & WBC Computation: Platelet count = RBC Count x platelets counted 1000 RBCs Unit: cells/mm3 SI Unit: cells/mm3 x 0.001 Normal value: o 250,000 – 500,000/mm3 o 250 – 500 x 109/L Composition of Rees-Ecker Fluid Brilliant 0.15g Dye Cresyl Blue Sodium 0.4g Prevents aggregation Citrate of platelets Distilled water 100 mL Formalin 3 drops in Preservative 1:10 dilution Sucrose 8.0g INDIRECT METHOD • Fonio’s Method: 1. Mix 1 drop of blood and 1 drop of 14% MgSO4 in a glass slide. ▪ MgSO4 prevent aggregation of platelet 2. Air dry then stain (Wright stain) 3. Count platelets per 1000 RBCs 230 x109/L GUY AND LEAKE METHOD AND BRECHER-CRONKITE METHOD • • • • Guy and Leake Method o Light Microscope Method o Diluent: Crystal Violet, Sodium citrate, Distilled water, formalin (40%) Brecher-Cronkite Method o Phase Contrast Microscope Method o Diluent: 1% ammonium oxalate o Most accurate method ▪ Platelet can be identified by debris/artifacts ▪ Ammonium oxalate hemolyzes RBCs ▪ Swells up platelets ▪ 11-15% error Guy and Leake Method, Brecher-Cronkite Method Procedure: 1. Collect blood in an EDTA evacuated tube 2. Rinse TWO RBC pipet with the diluent. 3. Prepare a 1:200 dilution by aspirating blood to the 0.5 mark of the RBC pipet and aspirate diluting fluid up to 101 mark. Make two preparations using the same procedure (0.5:101; 1:202) 4. Mix for 3-5 minutes 5. Discard few drops 6. Charge one side of the hemacytometer using one preparation and charge the other side using the 2nd preparation 7. Stand for 3 minutes 8. Count platelets on both counting area in the entire RBC square (25 RBC tertiary square) using HPO. Get average from both counting area. ▪ Allowable error - 10% 9. Formula: Sample Problem: A 1:200 dilution was prepared using EDTA Whole Blood. An average of 190 platelets were counted from RBC secondary square of both counting areas. What is the platelet count? o Dilution: 1:200 o # of platelets counted: 190 o Depth: 0.1 mm o Area: 1 mm2 SI = 380,000 x .001 = 380 x 109/L PLATELET COUNT ESTIMATE • • • • Platelets are counted in stained peripheral smear using 10 consecutive oil immersion field. Procedure: 1. Count platelets per 10 consecutive oil immersion field. 2. Compute for the average platelet counted 3. Formula: Estimated Platelet Count per uL = ave. platelets countedΤuL x 20,000 Reporting o The result is reported as quailitatively Platelet Estimate Reporting 0 – 49,000/uL Marked decrease 50,000 – 99,000/uL Moderate decrease 100,000 – 149,000/uL Slight decrease 150,000 – 199,000/uL Low Normal 200,000 – 400,000/uL Normal 401,000 – 599,000/uL Slight increase 600,000 – 800,000/uL Moderate increase >800,000/uL Marked increase Sample Problem: A peripheral blood smear was prepared using an EDTA anticoagulated whole blood. A platelet estimate was performed and the platelets counted per 10 consecutive oil immersion field are as follows: Field 1: 12 Field 6: 14 Field 2: 15 Field 7: 17 Field 3: 15 Field 8: 12 Field 4: 17 Field 9: 18 Field 5: 14 Field 10: 15 Compute and report the platelet count estimate.