Hematology Lab - 06 Peripheral Blood Smear

HEMATOLOGY LABORATORY • PERIPHERAL BLOOD SMEAR • OUTLINE • Peripheral Blood Smear o Wedge Technique o Fixing and Staining o Well-made Wedge/PBS Smear o Normal vs. Poorly Made PBS o Optimal Assessment Area o WBC Estimate o Platelet Estimate • • Add a drop of blood places .25 inch from one end of the slide. Drop should be 2-3 mm in diameter. o Large drop - thicker smear o Small drop - thinner smear Place the spreader slide at a 30-45° angle in a place near the drop but not touching the blood. Draw back the slide until it touches the blood. Allow the blood to spread across the width of the slide. Push the slide quickly at the other end. Use even pressure when pushing. o Length of smear should 2/3 - ¾ of length of the slide. PERIPHERAL BLOOD SMEAR • The examination of the peripheral blood smear is an integral part of the routine examination of blood films. Essential information can be obtained from this examination. o Information regarding RBC, WBC, platelets o Determines health status WEDGE TECHNIQUE • • • • aka Double slide, spreader slide, or push smear The most convenient and most commonly used PBS preparation method. Uses: o WBC (HPF/40x) & Platelet Count Estimate (OIF/100x) o WBC Differential Count (OIF/100x) o nRBC count (OIF/100x) Specimen: EDTA Whole Blood o Prepared within 2-3 hours after collection o Heparin - not used, stains the smear too blue FACTORS AFFECTING PBS THICKNESS/THINNESS Thick Smear Thin Smear Size of Blood Drop Large drop Small drop Angle of Spreader Increased angle Decreased angle Slide Pressure exerted Heavy pressure Light pressure Speed Too fast Too slow Hematocrit Increased Hct Decreases Hct • Corrective action o ↑ Hct, decrease spreader angle or use small drop o ↓ Hct, increase angle of spreader or use large drop FIXING AND STAINING MATERIALS • • • • • Glass slides o Film slide o Spreader slide (smooth edged) o May be frosted (for labelling) Methanol (fixative) o Preserve morphology Romanowsky stain (nonvital polychrome stain) o Non-vital Stains dead cels o Polychrome - more than one stain o Uses acidic and basic stain o Eosin - acidic o Methylene blue - basic o Uses phosphate buffer (6.4 - 6.8 pH) Microscope (visualization) Oil immersion o Cedarwood Oil PROCEDURE • Procedure 1. Immerse slide with fixative (methanol) and air dry ▪ Alcohol allow fast air drying 2. Immerse in Methylene blue then in Eosin ▪ No need for washing before 3. Wash excess stain with water ▪ Wash at backside of slide 4. Air dry WELL-MADE WEDGE/PBS SMEAR MACROSCOPIC CHARACTERISTICS • • • • • The film is 2/3 to 3⁄4 of the length of the slide o Widely distributed Film is finger shaped (very slightly rounded feathery edge) The lateral edges are visible The film is smooth (no irregularities, holes, streaks, etc) o Ensures well-distributed cells Presence of “rainbow” appearance in the feathery edge of the film MICROSCOPIC CHARACTERISTICS • • • • • Pink to purple color of smear o Heparin make smear bluish RBC – orange to salmon pink WBC nuclei – pink to purple o Neutrophils - pink with violet or lilac granules o Eosinophils – bright orange refractile granules o Basophil – purplish black granules o Lymphocyte – robin’s egg blue cytoplasm, compact nucleus o Monocyte - faint blue gray cytoplasm Platelets – purple blue to lilac with cytoplasm containing red blue granules The area between cells should be colorless, clean, and free of stain precipitate • 100x Objective Examination (OIO) o Platelet Estimate Count (use 10 OIO fields) o WBC Differential Count (100 cells) ▪ Battlement Method (Back-and-forth Serpentine) OPTIMAL ASSESSMENT AREA • • • Macroscopically: o Area between the thick area and the very thin area (feathered edge) Microscopically: o RBCs are uniformly and singly distributed, with few touching or overlapping, and have their normal biconcave appearance (with central pallor) Approx. 200-250 RBC per OIF ACCEPTABLE VS UNACCEPTABLE PBS AREAS TO AVOID • • • • Chipped/rough edge - caused by spreader slide Streaks - due to uneven pressure Bubbles - may have increased lipids • POORLY STAINED SMEAR Problems RBCs - gray WBCs - too dark Eosinophil granules - gray WBCs - too pale or red WBCs - barely visible Causes • Stain or buffer too alkaline • Inadequate rinsing • Prolonged staining • Heparinized blood specimen • Short drying period • Too alkaline washing water • Old smear • Stains or buffer too acidic • Underbuffering • Excessive rinsing Feathered edge (thin area) o RBC: ▪ Macrocytic ▪ Lacks central pallor (spherocytic) ▪ Flattened o Distorted WBCs Thick part o RBC appear microcytic o Artifactual rouleaux (stacked coin pattern) feathered edge thick part WBC ESTIMATE MICROSCOPIC EXAMINATION • • 10x Objective Examination (LPO) o Assessment of overall film quality, color, and distribution of cells o Selection of optimal assessment area 40x Objective Examination (HPO) o WBC estimation (use 10 fields) • • • Count WBC in 10 consecutive HPF Check result of automated analyzer Formula: WBCΤuL = Ave. WBC counted x 2,000 # of WBCs/HPF Estimated WBC count 2 – 5 WBCs/hpf 4,000 – 7,000 WBCs/uL 4 – 6 WBCs/hpf 7,000 – 10,000 WBCs/uL 6 – 10 WBCs/hpf 10,000 – 13,000 WBCs/uL 10 – 20 WBCs/hpf 13,000 – 18,000 WBCs/uL PLATELET ESTIMATE • • • Agranulocyte Monocyte Count platelets in 10 consecutive OIF Formula: PltsΤuL = ave. # of platelets x 20,000 Platelet Estimate Reporting 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 WBC DIFFERENTIAL COUNT • • • Percentage of each type of WBC in the blood: o Neutrophils o Lymphocytes o Monocytes o Eosinophils o Basophils Specific WBC type is counted using peripheral blood smear (100x objective) • Basophil 9-15 um 9-15 um 10-16 um Cytoplasm With fine lilacpink granules With coarse reddishorange granules With coarse bluish-black granules that obscure the nucleus Nucleus Multilobed (2-5) connected by thin filaments Indented nucleus (>1/2 of nuclear diameter) S, C, Z shaped Sausage-shaped Relative Count o The number of specific WBC type per 100 WBCs o Formula: • Absolute Count o Gives the number of specific WBC type per cubic millimeter of blood o More informative than Relative count o Formula: • Reference Ranges: NEUTROPHIL BAND/STAFF/STAB LYMPHOCYTE MONOCYTE EOSINOPHIL BASOPHIL Cell Size 7-10 um • • Bilobed • Multilobes (3-4) Large/Big/ Atypical Lymphocyte 11-25 um Cytoplasm Nucleus Sky-blue (Robin Compact, large egg blue) and round No granules Sky-blue (Robin Immature looking egg blue) (round, oval, or No granules slightly Indented by RBC indented) RELATIVE COUNT (%) 47 – 77% 2 – 6% 20 – 40% 2 –10% 0 – 6% 0 – 1% ABSOLUTE COUNT(/mm3) 1,600 – 7,260 350 - 700 960 – 4,400 180 – 880 45 – 440 45 – 110 METHODS • Agranulocyte Small Lymphocyte Small & large lymphocyte are only counted as one • WBCs Cell Size 9-15 um Cytoplasm Nucleus Blue-gray with Kidney-shaped , ground glass horshoe/coffee appearance bean with brainwith/out vacuoles like convolutions MANNER OF REPORTING Procedure 1. Prepare a well-made and stained PBS 2. Using OIO, identify 100 WBC based on type 3. Express result as percentage or as absolute count. Granulocyte Segmented Neutrophils/ PMN (mature) Band/Stab/ Staff (youngest granulocytic precursor) Eosinophil Cell Size 14-20 um 100-cell differential – Routinely performed method of differential count 50-cell differential – performed only if WBC count is <1.0 x 109/L o Use buffy coat smear o In cases of leukopenia o Result is multiplied by 2 200-cell differential – performed only if: o Leukocytosis o Eosinophil = >10% o Basophil = >2% o Monocytes = >11% o Lymphocytes > Neutrophils (among adults)