Prepare the fat and perivascular (F-PV) crush slide
Before the myeloid-erthroid (M-E)/concentrate/buffy coat smears can be processed, a F-PV crush slide should be prepared. Removal of the F-PV layers provides access to the plasma(P) and M-E layers. The F-PV slides are used for iron store evaluation when stained with the Prussian blue iron stain. To prepare the F-PV slides, pipette off both the fat and PV layers from the 1 mL Wintrobe tube. Dispense both of these layers onto one labeled slide. With another slide, prepare a "pull apart" smear. Place the F-PV labeled slide into a Coplin jar. In order to not disrupt the fat, alcohol fixation of this slide is not possible. To appropriately fix this slide, in the bottom of the Coplin jar, place a lint-free wipe that has been dipped in formalin. Cover the Coplin jar with the lid and allow the formalin fumes to fix the F-PV slide for 10 minutes. Remove the slide from the Coplin jar and place on your designated special stain bench.
Process the M-E/concentrate/buffy coat smears
After the F-PV crush slide is prepared, the M-E/concentrate/buffy coat smears can be processed. A clean pipette should be used to make the concentrate. After pipetting off the extra plasma into a glass culture tube, mix two parts plasma to one part of M-E in a separate glass culture tube. Use the pipette to gently mix the sample up before placing a small drop on the slide to smear. These slides can be used and/or stored for molecular genetics and immunophenotying studies, so preparing more than what will be stained with the Wright's stain is recommended. Customarily, approximately 10 slides should be prepared. Immediately place these slides under a fan for drying.
If there is not enough plasma for proper dilution, additional Wintrobe tubes can be prepared and spun to collect the adequate amount of plasma for a 2:1 ratio. Peripheral blood samples collected in an EDTA tube for complete blood count (CBC) testing can also be spun for collection of the patient's plasma. Likewise, if there is a trace of ME, multiple Wintrobe tubes can be prepared and spun to collect the adequate amount of M-E.
The M-E/concentrate/buffy coat slides allow for excellent evaluation as different cells can be found in this layer. All of these cells are evenly distributed, providing uniform staining as well as excellent cellular morphology. Clumps of normoblasts can skew differential counts. Mixing the sample well prior to smear preparation is the best way to eliminate clumping of the normoblasts.
Image 1: From left to right: RBC/M-E layers remaining in the Wintrobe tube, excess plasma (save if needed to dilute a thick or particle rich M-E/plasma mixture), mixture of two parts plasma and one part of M-E for M-E/concentrate/buffy coat smears.
Image 2: Immediately upon M-E/concentrate/buffy coat slide smear preparation, dry slides under a fan.