Principles of Blood Smear, Differential Count, and Stains (PDF)

PRINCIPLES OF PERIPHERAL BLOOD FILMS PERIPHERAL BLOOD FILMS SPECIMEN: disodium or tripotassium EDTA ○ Blood films should be made within 4 hours of drawing the specimen. ○ After 5 hours at room temperature causes unacceptable blood cell artifacts: ech...

PRINCIPLES OF PERIPHERAL BLOOD FILMS PERIPHERAL BLOOD FILMS SPECIMEN: disodium or tripotassium EDTA ○ Blood films should be made within 4 hours of drawing the specimen. ○ After 5 hours at room temperature causes unacceptable blood cell artifacts: echinocytic red blood cells spherocytes necrobiotic leukocytes vacuolated neutrophils EDTA SPECIMEN CONSIDERATION 1. In vitro platelet satellitosis 2. Pseudoleukocytosis Corrective Action: ○ Recollect blood using sodium citrate tube ○ Reanalyze using hematology analyzer ○ Use the corrected dilution of anticoagulant: Blood is nine-tenths of the total tube volume (2.7 mL of and 0.3 mL of sodium citrate. The “dilution factor” is the reciprocal of the dilution (10/9 or 1.1). The WBC and platelet counts are multiplied by 1.1 PERIPHERAL BLOOD FILMS SPECIMEN: finger and heel puncture Limitations: 1. Some platelet clumping must be expected if made directly from the puncture or heparinized microhematocrit tubes. 2. Only few films can be made from skin puncture. Corrective Action: Use EDTA microcollection tubes SMEAR PREPARATION Manual wedge technique. It is the most convenient and most commonly used method. Requires at least two 3-inch x 1-inch (75- mm x 25-mm) clean glass slides. One slide serves as the film slide, and the other is the pusher or spreader slide. 1 drop of blood (2 to 3 mm) from skin puncture or microhematocrit tube (nonheparinized for EDTA-anticoagulated blood or heparinized for capillary blood) is placed at one end of the slide. SMEAR PREPARATION The size of the drop of blood is important: Too large a drop creates a long or thick film Too small a drop makes a short or thin blood film. The pusher slide Held securely in the dominant hand at about a 30- to 45-degree angle Blood is allowed to spread across the width of the slide Picked the entire blood and quickly and smoothly pushed forward to the end of the slide Moving too slowly accentuates poor leukocyte distribution When the hematocrit is higher than normal (60%), such as in patients with PCV or in newborns, the angle should be lowered (25 degrees). For extremely low hematocrits, the angle may need to be raised. Features of a Well-Made Wedge PBS 1. The film is two-thirds to three-fourths the length of the slide. Name of patient. 2. The film is finger shaped, very slightly rounded at the feather edge, not bullet shaped; this provides the widest area for examination. 3. The lateral edges of the film are visible. 4. The film is smooth without irregularities, holes, or streaks. 5. When the slide is held up to the light, the thin portion (feather edge) of the film has a “rainbow” appearance. 6. The whole drop of blood is picked up and spread. UNACCEPTABLE PBS a. Chipped or rough edge on spreader slide. b. Hesitation in forward motion of spreader slide. c. Spreader slide pushed too quickly. d. Drop of blood too small. e. Drop of blood not allowed to spread across the width of the slide. f. Dirt or grease on the slide; may also be due to elevated lipids in the blood specimen. g. Uneven pressure on the spreader slide. h. Time delay; drop of blood began to dry. DRYING OF SMEAR All blood films and bone marrow smears should be dried as quickly as possible to avoid drying artifact. Small fan is used to facilitate drying. Blowing breath on a slide is counterproductive because the moisture in breath causes RBCs to become echinocytic (crenated) or to develop water artifact (also called drying artifact). STAINING OF PBS and BM SMEAR Romanowsky Stain Pure Wright stain Wright-Giemsa stain  These are considered polychrome stains because they contain both eosin and methylene blue.  Giemsa stains also contain methylene blue azure. PURPOSE:  Staining of blood films simply to make the cells more visible and to allow their morphology to be evaluated. STAINING OF PBS and BM SMEAR Methanol fixes the cells to the slide. Oxidized methylene blue and eosin form a thiazine-eosinate complex, which stains neutral components. Buffer: staining reactions are pH dependent ○ 0.05 M sodium phosphate (pH 6.4) or ○ aged distilled water (distilled water placed in a glass bottle for at least 24 hours; pH 6.4 to 6.8). STAINING OF PBS and BM SMEAR Methylene blue is basic and stains acidic cellular components, such as ribonucleic acid (RNA). Eosin is acidic and stains basic components, such as hemoglobin and eosinophilic granules. ○ Neutrophils are so named because they have cytoplasmic granules that have a neutral pH and pick up some staining characteristics from both stains. STAINING METHOD 1. Manual technique.  Slides are placed on the rack, film side facing upward.  The Wright stain is poured directly from the bottle through a filter onto the slide.  It is important to flood the slide completely.  Stain should remain on the slide at least 1 to 3 minutes to fix the cells to the glass.  Approximately equal amount of buffer is added to the slide and a metallic sheen (or green “scum”) should appear on the slide if mixing is correct.  When staining is complete, the slide is rinsed with a steady but gentle stream of neutral-pH water. STAINING METHOD 2. Automated slide stainers.  Staining takes about 5 to 10 minutes.  The processes of fixing/staining and buffering are similar in practice to those of the manual method.  The slides may be automatically dipped in stain and then in buffer and a series of rinses.  Film quality and color consistency are usually good with any of these instruments. STAINING METHOD 3. Quick Stains  Quick stains, as the name implies, are fast and easy.  The whole process takes about 1 minute.  Stain is purchased in a bottle as a modified Wright or Wright-Giemsa stain.  The required quantity can be filtered into a Coplin jar or a staining dish.  Aged, distilled water is used as the buffer.  Stained slides are given a final rinse under a gentle stream of tap water and allowed to air- dry. FEATURES OF WELL STAINED PBS Macroscopically  a well-stained blood film should be pink to purple. Microscopically  RBCs should appear orange to salmon pink  WBC nuclei should be purple to blue  Cytoplasm of neutrophils should be pink to tan with violet or lilac granules.  Eosinophils should have bright orange refractile granules. PBS EXAMINATION Macroscopic Examination A film that is bluer overall than normal may indicate that the patient has increased blood proteins and rouleaux may be seen on the film. A grainy appearance to the film may indicate RBC agglutination, as found in cold hemagglutinin diseases. Holes all over the film could mean that the patient has increased lipid levels. Markedly increased WBC counts and platelet counts can be detected from the blue specks out at the feather edge. PBS EXAMINATION Microscopic Examination at 10x objective ○ Overall film quality, color, and distribution of cells can be assessed. ○ The feather edge and lateral edges should be checked quickly for WBC distribution (“snowplow” effect) ○ Presence of fibrin strands should be rejected ○ The film can be scanned quickly for any large abnormal cells, such as blasts, reactive lymphocytes, or even unexpected parasites. ○ The area available for suitable examination can be assessed. PBS EXAMINATION Microscopic Examination at 40x objective o Use to select an area for differential count and to evaluate cellular morphology. o WBC estimate can be performed (10 fields) HPF 40x: average number of WBCs x 2000 OIO 50x: average number of WBCs x 3000 Example: - average of 4 WBC in 10 fields - 4 x 2000 = 8,000 (8 x 109/L PBS EXAMINATION Microscopic Examination at 40x objective  Because of the variation in the field diameter among different microscopes, an estimation factor should be determined for each microscope in use, and that number should be used to obtain the WBC and PLT estimates when using that microscope. PBS EXAMINATION Microscopic Examination at 100x objective o WBC differential count is performed. o Performing the differential normally includes counting and classifying 100 WBCs to obtain percentages of WBC types. o The RBC, WBC, and platelet morphology evaluation and the platelet estimate are executed. o Nucleated RBCs (NRBCs) if present are counted and reported as NRBCs per 100 WBCs. PBS EXAMINATION Microscopic Examination at 50x objective o WBC differential and morphology examinations. o The larger field of view allows more cells to be evaluated faster. o Examination at this power is especially efficient for validating or verifying instrument values when a total microscopic assessment of the film is not needed. OPTIMAL ASSESSMENT AREA Fig. A. Ideal area: the RBCs are uniformly and singly distributed, with few touching or overlapping, and have their normal biconcave appearance (central pallor).  there are generally about 200 to 250 RBCs per 100x oil immersion field. Fig. B. Too thin: there are holes in the film or the RBCs look flat, large, and distorted, is unacceptable Fig. C. Too-thick: distorts the RBCs by piling them on top of one another like rouleaux. PERFORMANCE OF DIFFERENTIAL COUNT  Use of a back-and-forth serpentine, or “battlement,” track pattern is preferred.  100 WBCs are counted and classified through the use of push-down button counters.  To increase accuracy, it is advisable to count at least 200 cells when the WBC count is higher than 40 x 109/L.  If the WBC count is 100 x 109/L or greater, it would be more precise and accurate to count 300 or 400 cells.  Results are reported as percentages: Example:  58% segmented neutrophils  30% lymphocytes  9% monocytes  3% eosinophils. PERFORMANCE OF DIFFERENTIAL COUNT If present, WBC abnormalities such as toxic granulation, Döhle bodies, reactive lymphocytes, and Auer rods are evaluated and reported. ICSH recommendation: a. Add a comment on the presence of reactive lymphocytes and count them as a separate population of cells in the differential count if they are present in large numbers. b. Toxic granulation and Döhle bodies are graded as “slight” to “marked,” or 1+ to 3+. c. Auer rods are reported as “present” when seen. PERIPHERAL BLOOD FILMS Red blood cell morphology.  cell size (microcytosis, macrocytosis)  variability in size (anisocytosis)  cell color (hypochromia)  cell shape (poikilocytosis)  cellular inclusions May use specific terminology for reporting the degree of abnormal morphology, such as “slight,” “moderate,” or “marked,” or use a scale from 1+ to 3+. Simpler report use the term present for morphologic abnormalities that are clinically significant. PERIPHERAL BLOOD FILM. PERIPHERAL BLOOD FILMS. SUMMARY White Blood Cell Parameters 1. Total WBC count (WBCs 3 109/L) 2. WBC differential count values expressed as percentages, called relative counts 3. WBC differential count values expressed as the actual number of each type of cell (e.g., neutrophils 3 109/L), called absolute counts 4. WBC morphology SUMMARY White Blood Cell Count  Each relative number (e.g., neutrophils at 0.67) is multiplied by the total WBC count (13.6 x 109/L) to get the absolute WBC count. Absolute count: 0.59 x 13.6 = 8.0 x 109/L  The acronym for absolute neutrophil count is ANC.  The ANC is a very useful parameter for assessing neutropenia and neutrophilia. PEDIATRIC HEMATOLOGY Leukocytosis is typical at birth for full-term and preterm infants, with a wide reference interval. There is an excess of segmented neutrophils and bands and an occasional metamyelocyte, with no evidence of disease. PEDIATRIC HEMATOLOGY Neutrophilic Leukocytes Term and premature infants have a greater absolute neutrophil count than that found in older children. Band forms are also higher for the first 3 to 4 days after birth. Newborn girls have absolute neutrophil counts averaging 2000 cells/mL higher than those of boys; neonates whose mothers have undergone labor have higher counts than neonates delivered by cesarean section with no preceding maternal labor. The neutrophil counts in premature infants are similar to or slightly lower than the neutrophil counts in full-term infants during the first 5 days of life. PEDIATRIC HEMATOLOGY Neutropenia. Neutropenia is defined as a reduction in the number of circulating neutrophils to less than 1.5 x 109/L. Neutropenia accompanied by bands and metamyelocytes is often associated with infection, particularly in preterm neonates. Neutropenia represents a decrease in neutrophil production or an increase in consumption. Neutrophilia. Neutrophilia refers to an increase in the absolute number of neutrophils to greater than 8.0 x 109/L. Morphologic changes associated with infection include Döhle bodies, vacuoles, and toxic granulation. PEDIATRIC HEMATOLOGY Eosinophils and Basophils The percentages of eosinophils and basophils remain consistent throughout infancy and childhood. Lymphocytes Lymphocytes constitute about 30% of the leukocytes at birth and increase to 60% at 4 to 6 months. They decrease to 50% by 4 years, to 40% by 6 years, and to 30% by 8 years. Benign immature B cells (hematogones), although predominantly found in the bone marrow, can sometimes be seen in the peripheral blood of newborns. PEDIATRIC HEMATOLOGY Monocytes The mean monocyte count of neonates is higher than adult values. At birth the average proportion of monocytes is 6%. During infancy and childhood, an average of 5% is maintained, except in the second and third weeks, when the proportion increases to around 9%. The count reaches adult levels at 3 to 5 months.