Differential Count: HEMA 312 Lab Notes

HEMA 312 (LAB) Study Notes

Differential Count (Experiment 1)

• Hematology students should be able to explain hemostasis, coagulation, and fibrinolysis principles.
• They should appreciate lab assays for diagnosing disorders, perform assays with precision, and manifest values like integrity.
• A learner must differentiate leukocyte types, perform a differential count, and correlate results with diseases.
• The examination includes a quantitative and qualitative study of platelets, a differential count, and morphological characteristics of blood cells.
• Staining blood smears is a critical part of the blood examination.
• Technicians need to recognize characteristics of normal blood cells and biological variations.
• Morphological and histochemical characteristics differentiate blood cells, including size, shape, cytoplasmic granulation, and nuclear features.
• Experience is the best teacher, acquired through frequent differential analysis in hematology sections.
• Routine blood smears should be kept for physician review, typically for a week.
• Eosinophilia is correlated with increased eosinophils, which indicates allergies, parasitic infections, or hematologic disorder.
• Lymphocytosis is excess normal lymphocytes.
• Leukocytes are formed elements for immunity.
• Monocytosis is increased monocytes.
• Neutrophilia is increased neutrophils.
• Correct labeling of collected blood samples is required.

Differential Count Procedure

• Stained blood smear use aids leukocyte ID and erythrocyte/thrombocyte appearance study.
• 100 leukocytes are counted, and the proportion of each type is reported as a decimal fraction.
• Inspect the smear under low power magnification, locate the thin end with no overlapping erythrocytes.
• High power magnification is used to check white cell distribution.
• Oil immersion is used to identify and count 100 consecutive leukocytes; record each cell type.
• Staining order: Fixative (5x), Eosin stain (5x), New Methylene Blue (10x), Buffer (until stain is removed).
• Segmented Neutrophil: 9-15 um diameter, lilac pink granules, 2-5 lobed nucleus.
• Eosinophil: 9-15um diameter, reddish orange granules, bilobed.
• Basophil: 10-16 um diameter, bluish black granules, 3-4 lobes.
• Lymphocytes: 8-16um, round&compact nucleus.
• Monocyte: 15-20mm diameter, horseshoe shaped nucleus.
• Normal WBC values: 4.0-11.0x 10^9/L. At birth: 10.0-30.0x 10^9/L.

Counting Leukocytes

• If WBC count is between 20,000 and 50,000 per cu mm, classify 300 leukocytes.
• If the WBC count is greater than 50,000 per cu mm, classify 500 leukocytes.
• The proportion of each leukocyte type is expressed as a percent of the total number.
• Begin counting near the end of the smear where red cells start to overlap.
• Examine the film strip systematically, recording leukocyte types using a cell counter.

Platelet Count: Direct Method (Experiment 2)

• Hematology students should explain hemostasis, coagulation, and fibrinolysis principles.
• They should appreciate lab assays for diagnosing hemostatic disorders, perform assays with precision, and manifest values like integrity.
• A learner must perform the platelet count and correlate the results with diseases.
• Platelets aid in vasoconstriction, hemostatic plug formation, thromboplastic activity, and clot retraction.
• Platelets function in the coagulation of blood.
• Platelets are difficult to count because they are small, adhesive and tend to aggregate.
• Whole blood is diluted with Rees-Ecker diluting fluid, platelets are counted in the hemocytometer.
• Coagulation: process by multiple plasma enzymes and cofactors interact in sequence, forming an insoluble fibrin clot.
• Platelets are non-nucleated cells formed in the red bone marrow that help with coagulation (also called thrombocytes).
• Vasoconstriction: controls blood pressure, primary hemostasis, and distribution of blood throughout the body.
• The EDTA evacuated tube containing the collected blood sample is needed.

Platelet Count Direct Method: Phase

• The capillary bore of the pipette should be when platelet count too low.
• All excess fluid should be removed from the pipette before blood is drawn into the pipette ( to avoid platelet adhesion).
• Adjust the blood level to the exact 0.5 mark with NSS-moistened cotton.
• Two pipettes should be use simultaneously, and each should be used to charge one side of the counting chamber.

Platelet Count Modification

• discard the from each pipette after mixing.
• Both sides of the counting chamber should be mounted and the results averaged

Platelet Count Formula

• The following formula is usually used:
• Platelet count = of cells counted ⅹ Dilution Factor (0.20)= Platelet ⅹ 10/L
• Depth of counting chamber (0.10)
• • Note that you should NOT include in dividing since it is the area per millimeter

Platelet Count: Modified Indirect Method (Experiment 3)

• A blood smear stained with Wright's or Wright's-Geimsa stain helps to visualize the cells.
• Platelets are difficult to count because they are small, adhesive, and tend to aggregate.
• Always read lowfields (battlement method). Platelet count= number of platelets counted in 10 oil immersion field ⅹ 20,000 /10 Reference range: 150,000 to 350,000

Abnormal Platelet Morphology

• Platelet satellitosis is an in vitro phenomenon seen in EDTA blood films.
• Microthrombocytes are tiny platelets that indicate thrombocytopenic with Wiskott - Aldrich syndrome.
• Giant bizarre platelet are with cytoplasmic vacuolnation which occur in patient with MDS.
• Giant adendritic platelet exceeding size of background RBCE found in patient with May-Hegglin anomaly.

Bleeding Time: Duke & Ivy Methods (Experiment 4) and Errors

• Antineoplastic, anti-inflammatory drugs, aspirin and aspirin are needed for the tests.
• The bleeding time measures the duration of bleeding after a skin incision. Varies by method (template, Ivy, Duke).
• Bleeding time relies on blood vessel elasticity and platelet function.
• Duke Method: Parallel to the fingerprints on the finger(s) is crucial.
• Duke Method Reference Range: 1 to 3 minutes and Report by 3'00"
• The test is usually not recommended for patients with a platelet count of less than 75,000/µl.

Ivy Method

• The test is performed by making a skin puncture and applying and inflating a cuff to 40mmhg for the duration of the test.
• Use 3 fingers apart .

Sources of error during test

• Aspirin may prolong bleeding time for up to 2 weeks.
• Too little pressure for the device and the wound will can cause shallow or nonexistent results.
• Low skin temperature leads to decreased blood flow.
• Borderline test result is 6 to 11 minutes.
• 19. Results : Baggo magka-abnormal bleeding time result

Clotting Time: Slide, Wright's, and Lee & White Method (Experiment 5)

• Wright's Method :uipo ( wipe ist blood)
• Slide Method: uipo 1st blood
• The test involves assessing blood lost fluidity/setting into semisolid clot process.
• • In vivo vs. in vitro clot formation locations are inside and outside the blood vessels, respectively.
• In vivo : process that occurs within the living organism vs. in vitro: an artificial environment outside the living organism.

Clotting Time cont,,,

• Follow universal safety and disinfectant procedures.
• Perform aseptically a venipuncture using a 20-gauge needle syringe and withdraw 4ml blood ( follow universal safety procedures on the disposal of needles)
• Reference range: 4-9 minutes
• Agitation and handling speed up coagaulation and also a ref range

Capillary Fragility Test (Experiment 6)

Phases

• Check and Examine the forearm, hand and fingers to make certain that no petechiae are present.
• Apply a blood pressure cuff on the upper arm above the elbow, and take blood pressure reading.
• Inflate the blood pressure cuff to a point midway between the systolic and diastolic blood pressures for five minutes.
• Reference grading : 1+ = a few petechiae on the anterior part of the forearm and 2+ = many petechiae on the anterior part of the forearm"

Whole Blood Clotting Time (Experiment 7)

• 5. Place the three tubes in a 37* C water bath.
• At exactly 5 minutes, tilt test tube #1 gently to a 45* angle. Repeat the procedure every 30 seconds.