Buffy Coat Film Preparation Procedure
Learning Objectives
- Prepare buffy coats for evaluation.
Principle:
The buffy coat is the fraction of an anticoagulated blood sample that contains most of the white blood cells and platelets following centrifugation. Centrifuging an aliquot of whole blood will separate the sample into three layers: packed RBC, plasma, and the buffy coat. By removing the plasma and skimming off the buffy coat layer, peripheral blood slides can be prepared with a greater concentration of WBC’s. A buffy coat is used to get a 100 WBC count differential on patient with an overall WBC count <1.0.
Materials
- 6 x 50mm Borosilicate Glass Tubes
- 9-inch glass Pasteur pipette and bulb
- 12 x 75 plastic aliquot tubes
- Glass Slides
- Applicator Sticks
- Centrifuge
Sample Type
Whole Blood: A whole blood sample collected in a tube containing EDTA (Lavender tube)
Procedure
- Gently mic the patient sample for two minutes
- Label a 6 x 50 glass tube with the patient identifying information.
- Using a 9” Pasteur Pipette, transfer about 0.75mL of the sample to the labelled tube.
- Place the tube in a 12 x 75 adapter tube. Centrifuge the sample for 10 minutes at 3000 RPM (1400RCF)
- Using a 9” Pasteur Pipette, remove as much of the plasma as possible from the sample without disturbing the buffy coat layer.
- Place a small drop of plasma, the size of a dime, onto a clean slide. Discard the remaining plasma.
- Using the same pipette, draw the buffy coat layer into the pipette by skimming the surface of the RBC layer.
- Disperse the sample from the pipette onto the slide containing plasma. Using an applicator stick, emulsify the buffy coat with the RBC’s.
- Prepare two blood films by pulling a drop of the buffy coat emulsion onto a new slide and spreading the drop as usual. Use the same technique and used when making a peripheral blood smear.
- Label the slides with the patient’s full name, MRN, Accession Number and “BC” encircled.
- Allow the films to air dry. Slides can be stained on the Midas III Plus in the same technique as peripheral blood smears.
Procedural Notes
A regular whole blood peripheral blood smear must accompany the buffy coat slides. The RBC morphology will be done on the whole blood smear and the WBC cell identification will be done on the buffy coat smear.