Manual Platelet Count Methods PDF

HEMA312 PRELIM LABORATORY TOPIC #2 - Equipment used: Hemacytometer and Thoma pipet (RBC or WBC) MR. MIKHAIL KRISTOFFER GONONG - Uses phase contrast microscope...

HEMA312 PRELIM LABORATORY TOPIC #2 - Equipment used: Hemacytometer and Thoma pipet (RBC or WBC) MR. MIKHAIL KRISTOFFER GONONG - Uses phase contrast microscope - Diluting fluid: 1% ammonium oxalate (also used for manual WBC MANUAL PLATELET COUNT | DIRECT AND INDIRECT METHOD counts; lyses RBC’s and allows platelets to form pseudopods) - Count 25 RBC squares (area is 1mm2) DIRECT METHOD - If low counts: PRE-ANALYTICAL PHASE ✓ use WBC pipet (1:20 dilution) Platelets ✓ RBC pipet: 1:100 dilution - smallest formed elements in the blood from megakaryocyte cytoplasm; 2-4 um TOCANTIN’S METHOD - round or biconvex shape - Equipment used: Hemacytometer and Thoma pipet (RBC or WBC) - MPV: 8-10 fL - Uses light microscope - fragments of cytoplasm that have broken off platelet precursors. - Diluting fluid: Rees and Ecker - Platelets function in the coagulation of the blood. - Brilliant Cresyl blue (BCB): dye - Neutral formalin: prevents platelet clumping Platelets are difficult to count due to: - Sodium citrate: anticoagulant ✓ Small size – often hard to differentiate from bacteria and debris - Count 25 RBC squares (area is 1mm2) ✓ Adhesiveness – affinity for adhering to glass ✓ Aggregation – tendency to clump together PROCEDURE A. DILUTION OF BLOOD SAMPLE PRINCIPLE 1. The capillary bore of the RBC pipette is rinsed with Rees-Ecker ✓ Whole blood is diluted with Rees-Ecker diluting fluid which makes diluting fluid. All excess fluid should be removed from the pipette platelets readily visible under the bright-field microscope before blood is drawn into the pipette. This step will ensure ✓ The platelets are then counted in the central large square of the platelet will not adhere on the wall of the capillary bore of the counting chamber (hemacytometer) pipette. ✓ Collection of the blood in the desired amount in the EDTA evacuated 2. Draw the blood into the pipette up to 0.5 mark. If excess blood tube by venipuncture was draw, adjust the blood level to the exact 0.5 mark with NSS moistened cotton. Clean the stem of the pipette first before any MATERIALS AND EQUIPMENT adjustment will be made. ✓ EDTA evacuated tube 3. The blood should be diluted immediately with the Rees-Ecker ✓ Microscope with LPO and HPO solution. Make a 1:200 dilution by filling the pipette with the ✓ Rees & Ecker Diluting Fluid solution up to 101 mark. Two pipettes should be use ✓ Hemocytometer and coverslip simultaneously, and each should be used to charge one side of the ✓ RBC pipette with rubber tubing counting chamber. and mouthpiece 4. After the dilution, the pipettes should be shaken immediately for ✓ Tally Counter or Cell Counter at least 60 seconds. This step will prevent platelet clumping and will ensure accurate counting. B. CHARGING OF SOLUTION ONTO THE COUNTING CHAMBER 5. After mixing, discard the first 5 drops from each pipette. ✓ Note: Do not induce or blot the tip of the pipette by any absorbent material to facilitate the process. The drop should drop freely by gravity alone. Each side of the hemocytometer should be charged with a different pipette 6. After charging the chambers, the hemocytometer should be kept in covered container for about 10 to 15minutes with wet gauze or cotton pads beneath the hemocytometer to prevent evaporation, this step will allow the platelets to settle aid in accurate counting later. C. COUNTING OF PLATELETS 7. Carefully place the hemocytometer on the microscope stage so as not to disturb the platelets. Count platelets in 5 RBC squares in the center of the counting chamber. Both sides of the counting chamber should be counted and the results averaged. Using the high power magnification count all the platelets, they appear as small, roundish, unevenly shaped structures. NOTE: o If 5 RBC squares are counted area should be 0.20mm² o If 25 RBC squares (central large square) are counted area should be 1 mm² IMPORTANT NOTE: ✓ Platelets should be counted on each side of the hemacytometer, and the difference between the totals should be less than 10% ✓ The accuracy of the manual platelet count should be verified METHODS FOR DIRECT PLATELET COUNT by performing a platelet estimate on a Wright-stained 1. Brecher-Cronkite, Tocantins/Rees-Ecker peripheral blood film made from the same specimen. 2. Guy and Leake’s 3. Nygard’s 4. Walker and Sweeney’s 5. Van allen’s 6. Unopette (calibrated) BRENCHER-CRONKITE METHOD - Reference method for manual platelet counts  COMPUTATION INDIRECT METHOD - Parameters: PRE-ANALYTICAL PHASE ✓ theaverage number of platelets per square millimeter ✓ the dilution factor (which is 200, just like the RBC PRINCIPLE count) ✓ A well-made smear is stained with Wright’s or Wright’s-Giemsa to ✓ the volume of the diluted blood counted, which is the visualize cellular elements of blood including the platelet area times depth (1mm² x 0.1 mm=0.1µl) ✓ Is done in the portion of the smear where the red cells start to overlap ✓ 10 OIO consecutive fields are examined for platelet and reported as NORMAL VALUE: 150,000-400,000 platelets/uL or 150-400 x 103 platelets/uL cells per microliter of blood FORMULA MATERIALS AND EQUIPMENT ✓ EDTA evacuated tube ✓ Microscope ✓ Glass slides (including spreader slide) ✓ Wright’s stain ✓ Tally counter ✓ Cedarwood oil PROCEDURE 1. Make a well-made blood smear and stain with Wright’s or Wright’s-Giemsa stain. See the product insert for the specific INTERPRETATION OF RESULTS instructions. Manufacturer has sometimes have modifications. BELOW NORMAL: Thrombocytopenia 2. Focus the slide using low-power magnification. Examine the smear - Aplastic anemia for areas that is suitable for counting. Care must be taken to stay - Pernicious anemia in an appropriate area of the slide. The RBC’s should barely be - Acute leukemia touching one another in the area of the estimate. - Idiopathic thrombocytopenia purpura 3. Using the high magnification, check the area of smear for suitability for counting the platelets. ABOVE NORMAL: Thrombocytosis 4. Shift the objective to the 1000x magnification. Examine the strip - Polycythemia vera of the film moving from one field to next systematically (See - Hemolytic anemia Figure 1.1) - Chronic myeloproliferative disorders 5. Count and record the platelets seen in then consecutive oil - Post-splenectomy immersion fields. SOURCES OF ERROR ✓ Light adjustment is critical. If the condenser is not lowered it will fade out the platelets. ✓ Bacteria and debris can be misinterpreted as platelets, this type of artifact is generally much more retractile than platelets. ✓ Clumping of platelets sometime occurs, and if so the specimen must be recollected. EDTA is the anticoagulant of choice for preventing platelet clumping. ✓ General hemacytometer errors, i.e. overloading chamber, counting wrong borders, etc. ✓ The phenomenon of “platelet satellitosis” may occur when EDTA anticoagulant is used. This refers to the adherence of platelets around neutrophils, producing a ring or satellite effect. Using sodium citrate as the anticoagulant should correct this problem. Because of the dilution in the citrate evacuated tubes, it is necessary to multiply the obtained platelet count by 1.1 for accuracy COMPUTATION - A platelet count should always be checked on the smear to make sure it looks accurate. To estimate a platelet count from a blood smear you regard each platelet seen per oil power field as being equivalent to 20,000 platelets/mm³ or uL - By determining the average number of platelets seen per oil field multiplying this number by 20,000 you can estimate the platelet count. For example, if you see 10 platelets per oil field your estimated could be: 10 x 20,000 or 200,000/mm³. - Usually 7-20 platelets per OIF represent a normal platelet count. FORMULA