Hematology 2 [Lab] Midterms PDF

HEMATOLOGY 2 [LAB]​ MIDTERMS | MLSHEM2L | MS. VALERIE JANE VILLARIN, RMT, MSMLS SY. 2024-2025 rr LESSON 3: PLATELET COUNT (DIRECT) High Platelet Counts (Thrombocytos...

HEMATOLOGY 2 [LAB]​ MIDTERMS | MLSHEM2L | MS. VALERIE JANE VILLARIN, RMT, MSMLS SY. 2024-2025 rr LESSON 3: PLATELET COUNT (DIRECT) High Platelet Counts (Thrombocytosis) PLATELETS -​ May indicate inflammation or trauma, where the body -​ Maintain blood vessel integrity by initiating repairs increases platelet production to aid in healing. -​ Adhere to damaged vessel surfaces and aggregate -​ Essential thrombocythemia: A rare malignant with other platelets to form plugs condition with extremely high platelet counts and -​ Secrete proteins and small molecules to trigger uncontrolled production thrombosis (clot formation) Low Platelet Counts (Thrombocytopenia) -​ Play a central role in hemostasis: sealing wounds, repairing vessel walls, and maintaining vascular -​ Commonly caused by drug treatments patency -​ Can lead to easy bruising, uncontrolled hemorrhage, and emergency department visits CHARACTERISTICS -​ Thrombocytopenia disrupts hemostasis, increasing ​ Size: 2–4 µm in diameter, round or oval, anucleate, the risk of uncontrolled bleeding even with minor and granular injuries. ​ Despite their small size, they are essential for life COUNTING AND ANALYSIS ​ Often referred to as "cell fragments" due to the absence of a nucleus -​ Platelets are counted visually using a hemocytometer ​ Platelets are not full cells but fragments of larger cells or by automated blood cell analyzers called megakaryocytes, found in the bone marrow. -​ Automated analyzers provide more accurate counts and generate a Mean Platelet Volume (MPV) ​ Elevated MPV: suggests larger platelets, possibly indicating bone marrow regeneration SIGNIFICANCE -​ Accurate platelet counts are crucial for diagnosing and managing conditions like thrombocytopenia and thrombocytosis -​ Platelet disorders contribute to a variety of hematologic and vascular diseases HEMOCYTOMETER -​ Specialized counting chamber initially created for blood cell counts -​ Enables accurate estimation of cell concentration in CONDITIONS ASSOCIATED WITH PLATELETS samples -​ Useful in situations where automated cell counters are unavailable or impractical Overactivation Applications: -​ Linked to deep vein thrombosis, pulmonary emboli, ​ Microbiology: Used to quantify microbial populations heart attacks, strokes, peripheral artery disease, and in cultures, such as bacteria, yeast, or fungi. recurrent miscarriages ​ Cell culture: Essential in maintaining cell lines, -​ Deep vein thrombosis (DVT): Clots form in deep ensuring the correct cell density for experiments or veins, often in the legs. growth conditions. -​ Pulmonary embolism: A clot breaks loose and ​ Medical diagnostics: Assists in evaluating blood or blocks blood flow in the lungs fluid samples for diagnostic purposes, including -​ Heart attacks and strokes: Clots block blood flow to assessing cell counts in cases of disease or the heart or brain infection -​ Peripheral artery disease: Reduced blood flow to the limbs due to clots. HEMOCYTOMETER STRUCTURE -​ Recurrent miscarriages: Overactivation can cause -​ Thick, flat glass slide with a rectangular indentation placental clotting, disrupting pregnancy forming a chamber -​ Chamber features a grid etched on the surface ANIPAN, J.L.A. | MED228 1  HEMATOLOGY 2 [LAB]​ MIDTERMS | MLSHEM2L | MS. VALERIE JANE VILLARIN, RMT, MSMLS SY. 2024-2025 rr -​ Glass coverslip held at a fixed distance (0.1 mm) from -​ Coverslip, designed to fit the hemocytometer the grid for precise volume measurements precisely -​ Consistent volume determination -​ Grid contains nine large squares, each measuring 1 mm x 1 mm PROCEDURES IN USING A HEMOCYTOMETER Central Square Preparation -​ Divided into 25 medium squares, further subdivided -​ Dilute the sample if necessary to ensure cell into 16 smaller squares (400 total) concentration falls within the measurable range -​ Used for counting red blood cells (RBCs) -​ Mix the sample thoroughly to ensure even distribution of cells. Four Corner Square Loading the Sample -​ Each measures 1 mm² -​ Place a specialized coverslip on the hemocytometer. -​ Used for counting larger cells like white blood cells -​ Using a pipette, introduce the sample into the (WBCs) chamber via the small grooves, ensuring even Counting Rules distribution without overfilling. -​ Include cells touching specific borders to prevent Cell counting double-counting. -​ Place the hemocytometer under a microscope. ​ Inclusion Criteria: To avoid -​ Focus on the grid and identify the cells within the double-counting, only cells touching specific chosen squares. borders of the square, typically the top and -​ Count cells according to the established rules left edges, are counted. -​ Calculation ​ Exclusion Criteria: Cells touching the bottom and right edges are excluded DIRECT PLATELET COUNT (REESE AND ECKER METHOD) -​ Manual technique used for counting platelets in a blood sample -​ Performed using a hemocytometer, a specialized counting chamber for blood cells -​ Provides direct platelet counts, making it useful in settings where automated systems are unavailable -​ Method involves diluting whole blood with a reagent solution that preserves platelets and stains them for easier visualization under a microscope -​ Stained platelets are counted manually in a defined volume within the hemacytometer -​ Count is then converted to the number of platelets per microliter (µL) of blood MATERIALS AND EQUIPMENTS USED -​ Pipettes for sample dilution -​ Microscope for cell visualization -​ Diluting fluids ANIPAN, J.L.A. | MED228 2  HEMATOLOGY 2 [LAB]​ MIDTERMS | MLSHEM2L | MS. VALERIE JANE VILLARIN, RMT, MSMLS SY. 2024-2025 rr ​ Ideal for laboratories without automated platelet counters ​ Enables observation of platelet morphology alongside counting LIMITATIONS ​ Subject to human error, leading to variability in results ​ Requires meticulous sample preparation and manual counting ​ May be difficult to detect in cases of severe thrombocytopenia ​ Platelet clumps or debris may interfere with accuracy DIRECT PLATELET COUNT REAGENTS AND MATERIALS (UNOPETTE SYSTEM) ​ Reese and Ecker Fluid -​ Manual method for performing direct platelet counts ​ Hemocytometer and Coverslip and other hematologic analyses ​ Filter paper -​ Designed to simplify the process of diluting and ​ Petri dish counting blood cells, providing accurate results when ​ Microscope automated methods are unavailable or not feasible ​ RBC Pipette COMPONENTS -​ The Unopette system is a self-contained unit that includes the following: ​ Capillary Pipette ​ Diluent Reservoir ​ Protective Sleeve ​ Mixing Device PROCEDURE USING REESE AND ECKER 1.​ Fill red cell pipette with blood up to mark 0.5 2.​ Wipe-off blood on the pipette tip without affecting the column of blood in the pipette stem 3.​ Such Reese and Ecker diluting fluid to mark 101 4.​ Shake pipette thoroughly for 3 minutes 5.​ Charge hemocytometer PROCEDURE USING UNOPETTE SYSTEM 6.​ Place a water-moistened filter paper inside the petri 1.​ Using the protective shield on the capillary pipette, dish puncture the diaphragm on the reservoir 7.​ Place hemocytometer a top filter paper and cover 2.​ Remove the shield from the pipette assembly by petri dish twisting 8.​ Let stand for 5 minutes 3.​ Holding the pipette horizontally, touch the tip of the 9.​ Focus the white blood cells counting area under low pipette to the blood. Pipette will fill by capillary action. power Filling the cease automatically when the blood 10.​ Shift to high power. Count the platelets in 4 white cell reached the capillary pipette to remove excess blood, squares which could interfere with the dilution 11.​ Calculate platelet count using the formula 4.​ Squeeze reservoir slightly to apply negative pressure Platelet count = # of Cells x 10 x 200/4 thus removing some air inside Reference Range: 150,000 – 400,000 5.​ Cover the opening of the overflow chamber of the ADVANTAGES pipette with index finger and seat the pipette securely ​ Does not require advanced equipment in the reservoir neck ANIPAN, J.L.A. | MED228 3  HEMATOLOGY 2 [LAB]​ MIDTERMS | MLSHEM2L | MS. VALERIE JANE VILLARIN, RMT, MSMLS SY. 2024-2025 rr 6.​ Release pressure on the reservoir. Then remove CLINICAL SIGNIFICANCE finger from the pipette opening allowing blood to draw -​ Diagnosing Platelet-Related Disorders into the reservoir 7.​ Squeeze the reservoir several times to allow Thrombocytopenia (low platelet count) adequate mixing of blood and diluent ​ Bone marrow failure, immune thrombocytopenic 8.​ Place index finger over the upper opening and gently purpura (ITP), infections, or certain medications invert several times to thoroughly mix the diluent ​ Easy bruising, prolonged bleeding, petechiae, and 9.​ Let the mixture stand for 10 minutes potential internal hemorrhaging 10.​ Convert the system to dropper assembly Thrombocytosis (high platelet count) 11.​ Discard several drops (3-4) then charge properly the ​ Inflammation, infections, iron deficiency, or hemocytometer myeloproliferative disorders such as essential 12.​ Place the hemocytometer in a moist petri dish for 10 thrombocythemia minutes ​ Increased risk of clot formation (thrombosis), which 13.​ Mount the hemocytometer on the microscope and can lead to heart attacks, strokes, or pulmonary locate the RBC square embolisms 14.​ Count the platelets on all 25 tertiary square or the entire RBC square PLATELET MORPHOLOGY ASSESSMENT 15.​ Calculate platelet count using the formula: -​ Abnormal platelet morphology can indicate: Platelet count = # of Cells x 1 x 10 x 100 Macrothrombocytes (giant platelets) Reference Range: 150,000 – 450,000 ​ A rare bleeding disorder characterized by abnormally ADVANTAGES giant platelets and thrombocytopenia. ​ Provides precise counts when performed correctly ​ Associated with myeloproliferative disorders or ​ Uses a standard dilution factor for reproducibility inherited conditions like Bernard-Soulier syndrome ​ Cheaper than automated platelet counters, making it accessible in resource-limited settings ​ Pre-filled diluent reservoir simplifies the process ​ Minimizes sample handling errors ​ Suitable for other hematologic counts, such as white blood cell (WBC) and red blood cell (RBC) counts LIMITATIONS ​ Manual counting takes longer compared to automated Hypogranular platelets systems. ​ Accuracy depends on the skill and experience of the ​ Platelets with a rim of cytoplasm that contains less operator. than 20 granules. ​ Improper loading of the hemocytometer or uneven ​ Suggestive of conditions such as myelodysplastic mixing can lead to inaccurate counts. syndromes ​ May not detect very low platelet counts accurately. CLINICAL APPLICATIONS -​ Useful in diagnosing thrombocytopenia or thrombocytosis -​ Can serve as a backup or verification method for automated platelet counts -​ Often employed in hematological studies to evaluate platelet function and morphology (less commonly used in modern clinical settings due to automation, remains a fundamental manual technique in hematology) ANIPAN, J.L.A. | MED228 4