viral immunity & pathogenesis group
OUTLINE
We consider manual counts as less quick but more precize than authomatic (electronic) since in the latter case the counts are based on the size of particles, so everything that has the same size as platelet will be counted as an event. Anyway both give comparable results- thrombocytopenic probe in comparison with normal one remains thrombocytopenic no matter what type of counts performed. In our lab we use as a routine procedure manual counts and sometimes we use automatic cell counter in addition.
PROTOCOL
Obtain blood from plexus retroorbitalis by using sliconized microcapillaries (20ul)
Immediately dilute 1:100 in Unopette kits (Becton Dickinson)
Let the probe stay for 1.5 hrs RT for complete erythrocyte lysis
Count platelets under a phase contrast microscope at x400 magnification using Neubauer hemocytometer
SOLUTIONS
The Unopette Reservoir contains 1.98ml of diluent mixture: Ammonium oxalate 11.45gm, Sorensen`s phosphate buffer 1.0gm , Thimerosal 0.1gm, Purified water qs to 1L
ADDITIONAL INFO
For automatic counts you can use :
H-6000 or H*2 flow autoanalyzer (Bayer Co)
electronic cell counter model 2F Channelyser model 256 (Coulter Electronics)
REFERENCES
Bergmeier, W., Rackebrandt, K., Schroder, W., Zirngibl, H. and Nieswandt, B. (2000). Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies. Blood 95(3): 886-93.
Bizzaro, N. and Fiorin, F. (1999). Coexistence of erythrocyte agglutination and EDTA-dependent platelet clumping in a patient with thymoma and plasmocytoma. Arch Pathol Lab Med 123(2): 159-62.
Teeling, J.L., Jansen-Hendriks, T., Kuijpers, T.W., de Haas, M., van de Winkel, J.G., Hack, C.E. and Bleeker, W.K. (2001). Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia. Blood 98(4): 1095-9.
