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Blood Smear Examination CRITERIA FOR PERIPHERAL BLOOD SMEAR REVIEW Initial analysis of the peripheral blood picture is made in most clinical laboratories with an automated instrument. Samples are selected for further analysis when quantitative or qualitative abnormalities beyond a defined standard are found. The following are examples of quantitative RBC abnormalities that may prompt a blood smear review. Each laboratory, however, should develop its own guidelines: • Hgb: < 8 or >18 g/dL (<10 or > 21g/dL in a newborn) • Hct: <20% or > 60% in adults (<40% or >65% in a newborn) • MCHC: <29 g/dL • MCV: <69 femtoliters (fl) or >110fl • Flags generated by the hematology analyzer that indicate possible red cell abnormalities or spurious results Any of these findings should be followed up with a peripheral blood smear review. MATERIALS AND SUPPLIES • • • Prepared slides Manual cell counter designed for differential counts Microscope, immersion oil and lens paper EXAMINATION OF A BLOOD SMEAR Examination of a blood smear is an integral part of a hemogram. Blood smear analysis allows quantification of the different types of leukocytes (called the differential count), estimation of the platelet count, and detection of morphologic abnormalities that may be indicators of pathophysiologic processes. In some instances, a diagnosis may be evident. Deriving full value from blood smear examination requires a well-prepared, well-stained blood smear and some basic skills in the methods of assessment. Though some automated hematology analyzers provide a differential count as part of their output, this does not fully take the place of a microscopic exam by an experienced observer. A systematic approach is important so that all the available information is derived from each smear examined. Follow this protocol: 1. Scan the smear at low magnification (10x objective) o to locate the optimal area for examination at higher magnification, and check the feathered edge o to evaluate the distribution of leukocytes on the smear 2. Perform WBC estimate (x10 or x40 objective) 3. Perform the differential count (100x objective) 4. Assess platelet numbers (100x objective) 5. Perform a morphologic assessment of blood cells (100x objective) One goal of low power scanning is to locate the best area of the smear for examination at higher magnification. Distribution of cells A second goal of scanning is to check that leukocytes are uniformly distributed throughout the smear and not excessively concentrated at the feather edge. In some ill-made smears, most of the leukocytes are dragged to the end of the smear. If this is found to be the case, prepare another smear and move the spreader slide more quickly, which should Low magnification (100x) view of the feathered edge. Such ensure that white cells are poor distribution of leukocytes adversely affects the accuracy adequately distributed. of the differential count. Differential Counting When you have finished your scanning and have found the area of the smear where cells are optimally spread, turn to the 100X oil immersion objective and begin your differential count and morphology assessment. The high dry (40X) objective should never be used for this purpose since the magnification is too low to see adequately all the features of the cells. Begin the differential count by moving back and forth across the smear in a pattern that avoids covering the same territory. Battlefield Method Wandering Method Identify each leukocyte that is encountered until 100 cells have been counted and sorted by type. As you see each cell, record it in the proper category on the counter (using either a mechanical differential counter or the program on your computer in the dry lab). The percentage of each cell type that results is termed the relative differential count. The absolute differential is derived by multiplying the percentage of each cell type by the WBC to get the number of each type of leukocyte /µl. If nucleated red cells (NRBC) are encountered, they should be recorded separately from the leukocytes and reported as the number of nRBC/100 WBC. This number is then used to correct the apparent WBC (which is actually just a count of nucleated cells) so that it represents the number of leukocytes/µl. To help you learn to identify leukocytes and assess blood cell morphology, this module contains sections that display photomicrographs of the different kinds of leukocytes and some of the species variations. WBC Estimate The white cell count can be estimated from the peripheral smear by counting the number of WBC in 5-10 low power fields (x10; at this magnification one (1) WBC seen is = 200WBC/μL or 0.2 x 109/L), and then apply the calculation below : WBC estimation= Total number of leukocytes counted x 0.2 x 109/L 5 Example: Total number of leukocytes counted= 150 (5 field 10X) 150/5 X 0.2 x 109/L= 6.0 x 109/L This number should be within ±25% of the actual automated white cell count. If it is not within this range, the white cell count and the estimation should be repeated.  If completed on the high dry magnification (x40; one (1) WBC seen is = 2000WBC/μL or 2 x 109/L), and then apply the calculation below :  WBC estimation= Total number of leukocytes counted x 2 x 109/L 5 Platelet Estimate: After you have finished the differential count, continue your blood smear analysis with an estimate of the platelet count as low, normal, or high. To estimate platelet number, determine the average number of platelets in 5 to 10 oil immersion fields. At this magnification, each platelet is approximately equal to 15,000 – 20,000 platelets/µl or 20 x 109/L. The following guidelines are useful for this session: "Adequate" platelets / field Adult ~8-20/ field The platelet estimate should be done in the part of the smear used for differential counting and evaluation of morphology. This method of estimating platelets is less precise if platelet clumps are present, though an estimate based on the dispersed platelets in such a smear does provide a 'not less than' estimate. PLT estimation: = Total number of Plts counted x 20 x 109/L 5 Upper panel: normal platelet count. Lower panel: severe thrombocytopenia. On CBC's, the following reporting protocol is used: • • • • • Incr.: count clearly above reference range Adeq.: count clearly within reference range Low? : count near low end of reference range. If in fact below range, only mildly so. Low : count clearly below reference range. Very Low: count in range where risk of spontaneous hemorrhage is significant (ie, <25,000/microliter). Blood cell morphology To complete the blood smear analysis, assess the different types of blood cells for significant morphologic abnormalities and describe your findings. This part of the hemogram is the most subjective and difficult to quantitate, although there are published guidelines for grading morphologic abnormalities The following may serve as a guideline (examine thin edge of the smear): 1+ = 2 - 4/atypical cells/Oil Immersion Field (OIF) 2+ = 5 - 7/OIF 3+ = 8 - 10/OIF 4+ = >10/OIF. Grade slight, occasional or few 1+ 2+ 3+ 4+ Observation rare cells seen one or two cells are seen in every field moderately increased, normal cells can still be found, 34/field markedly increased in number; >5/field all cells are abnormal; reserved for extreme cases such as hereditary RBC abnormalities The terms few, moderate, many, and marked may be substituted for the 1+ - 4+ grading system. Additionally, if there is a slight increase in the number of polychromatophilic red cells in the smear, this should be reported as slight polychromasia. Normal blood cells and some common leukocyte abnormalities are shown later in this module, but a detailed discourse on red cell shape changes is beyond the scope of this laboratory session. Proficiency in recognizing and interpreting morphologic abnormalities comes only with practice and familiarity with normal features of each species. An important preliminary to gaining such expertise is knowledge of the common artifacts of preparation that affect blood smears: Stain Precipitate This microscopic field is sprinkled with dark purple granules of precipitated stain. Stain precipitate has many forms and can appear as granules smaller or larger than these. It can be distributed as patches or dispersed across large areas. It is important to identify this material correctly, so that it is not mistaken for bacteria or for red cell parasites. Stain precipitate and refractile artifact on smears of cat blood have led many people into a mistaken diagnosis of erythroparasites. When precipitate on smears becomes a problem, one should discard the stain, thoroughly clean and dry the staining jars, and refill them with fresh stain. Proper care of staining solutions is important for obtaining the best results. Smudged Cells This photo shows an intact neutrophil and a smudged cell. A few partly or completely disrupted leukocytes are found in any blood smear but may be extremely numerous in a badly made smear. Aging of blood in the tube results in degeneration and increased fragility of leukocytes resulting in excessive numbers of smudged cells. The nucleus in smudged cells is more magenta than the deep purple of intact nuclei. Minimal disruption can leave some cytoplasm around the nucleus but the cell border is indistinct. More complete smudging can strip the cytoplasm away from the nucleus, leaving only a blob of nuclear material. Most smudged cells are not included in the differential count unless enough remains for unmistakable identification, e.g. a nucleus surrounded by eosinophil granules can be safely counted as an eosinophil. Water Artifact The red cells shown here are crenated and many have multiple refractile "bubbles" across their surfaces. The refractility is often called water artifact because it results from exposure of the smear to water, either as high humidity in the air or as contamination of the fixative with water. If a smear is affected by water artifact, the fixative solution in the staining jar should be discarded and replaced with fresh fixative. Notice that some of the spicules on the red cell are pointing toward the viewer; this orientation of the spicules can mislead a novice into thinking there are inclusions in the red cell. Disadvantages of the Peripheral Blood Smear Peripheral blood smear examination provides information that cannot be obtained from automated cell counting. However, peripheral smear evaluation has some limitations and special considerations. These include: • • Experience is required to make technically adequate smears. There is a non-uniform distribution of white blood cells over the smear, with larger leukocytes concentrated near the edges and lymphocytes scattered throughout. • There is a non-uniform distribution of red blood cells over the smear, with small crowded red blood cells at the thick edge and large flat red blood cells without central pallor at the feathered edge.
Segs Band Lymph Monos Eos Baso Figure 1: White Blood Cell Morphology
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Biology Lab Report: Peripheral Blood Smear Examination

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Title: Peripheral Blood Smear Examination
The two major objectives for this experiment were;

To determine the estimated number of White Blood Cells (WBC) on a peripheral blood

To examine the clinical significance of the results obtained.

Peripheral smear testing has been a common procedure done in the field of hemogramology. In
this field, blood samples are spread on a smearing glass and a periscope is used to count the
number of white blood cells, platelets, and red blood cells. An estimate of all these is done and
compared to the normal number in a healthy body. If the numbers match, exceed, or drop by a
certain allowable percentage, then the blood retrieved from the person under study is clean,
otherwise the person is not healthy and they are most definitely suffering from a certain disease.
During the peripheral blood smear analysis procedure, before performing an estima...

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