mlt 210 - blood film and CBC

specimen collection

use EDTA tubes

ideally made within 4 hrs of collection

finger and heel sticks are also appropriate, but slides must be made at the bedside

platelet satellitosis

EDTA phenomenon that causes platelets to bind to neutrophils

decreases platelet count and increases WBC count

corrected by collecting the specimen in sodium citrate tubes and multiplying the result by the DF

slides stained with romanowsky stain, which contains eosin and oxidized methylene blue, methanol is added to fix cells, wright or wright-giemsa is added, buffer is added (0.05 sodium phosphate), slide rinsed with neutral pH water and allowed to dry

steps in slide staining

eosin

stain pH: acidic

cellular pH: basic

ex. cell component: hemoglobin and eosinophilic granules

color: red

thiazine-eosinate complex

stain pH: neutral

cellular pH: neutral

ex. cell component: neutrophilic granules

color: violet

methylene blue

stain pH: basic

cellular pH: acidic

ex. cell component: RNA and basophilic granules

color: blue

microecopic eval. - 40x or 50x

“high and dry”

proper area to do differential is selected

wbc estimate is peformed

microscopic eval. - 100x

performance of a wbc differential is done on this setting

rbc, wbc, and platelet morphology is evaluated

platelet estimate is performed

rbc and wbc inclusions are noted

nrbcs are counted per 100 wbcs

select a portion of the slide where rbcs begin to touch and overlap (not excessively), using 40x count the number of wbcs in 10 fields and take an average, multiply the average number of wbcs per field by 2000, compare result to instrument printout

performance of a wbc estimate

+/- 2.0 × 10³ cells per uL or 10% of the instrument value - whichever is greater

acceptable number for a wbc estimate

select portion of slide where rbcs are not touching but holes are observed, using 100x count number of platelets in 10 fields and take an average, multiply average number by 20000, compare result to instrument printout

performance of a platelet estimate

for instrument counts greater than 100.0 × 10³: within +/- 50.0 × 10³

for instrument counts less than 100.0 × 10³: within +/- 20.0 × 10³

acceptable number for a platelet estimate

performing a wbc differential

should always be done systematically to avoid double counting or entering unacceptable areas

a battlement (serpentine) track pattern should be performed from edge to edge

200 cell differential

wbc differential performed on a count greater than 40.0 × 10³

300 cell differential

wbc differential performed on a count greater than 100.0 × 10³

differential performed on a buffy coat smear

wbc differential performed on a count less than 2.0 × 10³

buffy coat smear

made by aliquoting a part of the sample, spinning it down, and removing the buffy coat layer from the top of the red cells to make a slide

ensure accuracy of count, evaluate total count, evaluate relative differential counts, evaluate absolute differential counts, note presence of immature cells, note abnormal morphology

steps to evaluate wbc parameters of a CBC

relative counts

refers to the percent of each cell line counted in the differential (ex: 56% of neutrophils)

absolute counts

may be a better indicator of which cell line is affected

obtained by multiplying the differential percentage by the total wbc count

evaluate hemoglobin and hematocrit, calculate and eval. MCV, calculate and eval. MCHC, evaluate RDW, note abnormal morphology, double check rbc count and MCH

steps to evaluate rbcs

evaluating hemoglobin and hematocrit

decreased hemoglobin: anemia, increased hemoglobin: polycythemia

rule of three: hemoglobin x 3 = hematocrit

calculating and eval. MCV

MCV less than 80 fL: microcytic

MCV 80-100 fL: normocytic

MCV greater than 100fL: macrocytic

calculating and eval. MCHC

MCHC less than 32 g/dL: hypochromic

MCHC 32-36 g/dL: normochromic

MCHC greater than 36 g/dL: occurs due to spherocytes, cold agglutinin, lipemia

eval. RDW

describes variability in volumes/sizes of rbcs

reported as CV or SD

increase above normal range is known as anicytosis

polychromasia

observed when reticulocyte numbers are increased

double check rbc count and MCH

rbc count not indicative of anemia, MCH should trend in the same direction as MCV

platelet count, mean platelet volume (MPV), platelet morphology

platelet parameters of a CBC

eval. platelet count

perform platelet estimate and compare to automated count

compare potential causes of discrepancies

consider platelet count in relation to all three cell lines by evaluating wbc count and hemoglobin

pancytopenia

a decrease in all three cell lines (leukemia and aplastic anemia)

pancytosis

increase in all three cell lines (polycythemia vera)

evaluating MPV

compare observed platelet site on the peripheral blood smear to the MPV

very large platelets (as big as wbc) are worth noting

eval. platelet morphology

size, granularity arrangement

size may be noted as large (2x normal size) or giant (bigger than 6 micrometers)

satelliting