1. Freshly drawn blood samples received by the Clinical Pathology laboratory should be placed into heparinized tubes 10 cm. high and with a bore no wider than 5mm., centrifuged until the red blood cells, buffy coat and sera are segregated. Draw off the sera and replace it with 4% buffered glutaraldehyde carefully pipetted down the side of the tube on top of the buffy coat. Leave for at least two hours at 4º C.
2. With a diamond pencil score the circumference of the tube below the buffy coat,knock away the red blood cell portion of the tube and with the aid of an applicator stick plunge the buffy coat into a container of 0.1M sodium cacodylate buffer at 4º C. for at least 15 min. The specimen may be stored up to a week.The specimen may be cut into mm 3 blocks at this time.
The remainder of this procedure may be performed in a covered 5 ml. sealed glass container in a refrigerator and/or on a rotating stand or in a RMC EMP 5160 Ultraprocessor Tissue Processor .
3. Make three changes of 0.1M sodium cacodylate buffer for at least 15 min. each at 4º C.
4. Place in a covered container in 2% osmium tetroxide with 0.1M sodium cacodylate buffer for one hour at 4º C.
The remainder of this procedure may be done in an uncovered container at room temperature.
5. Place in 0.1M sodium cacodylate buffer for 5 min..
6. Place in in 50% ethanol/distilled water for 5 min.
7. Place in in 70% ethanol/distilled water for 10 min.
8. Place in in 90% ethanol/ distilled water for 10 min.
9. Place in three changes,20 min. each 100% reagent grade ethanol.
10. Place in 50% L.R.White/100% reagent grade ethanol for 30 min.
11. Place in 75% L.R. White/100% reagent grade ethanol for 30 min.
12. Place in two changes, 30 min. each in 100% L.R. White
13. Embed in L. R. White in covered gelatin capsules and allow to stand overnight at room temperature in a hood.
14. Place in a 60º C oven for 22 to 26 hours.
reviewed 8-24-06 CPD
