A tetrazolium-based colorimetric assay for metabolic activity of stored blood platelets.
BACKGROUND
Much work has been carried out in an attempt to establish the optimum cryopreservation of platelets. To estimate the surviving numbers of stored platelets, the metabolic redox activity of resting platelets was measured and compared with the aggregation response.


METHODS
The activity was determined using a simple colorimetric assay based on reduction of a tetrazolium salt, WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-[4-nitrophenyl]-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt), to water-soluble formazan and its quantification with a spectrophotometer. The aggregation response of the platelets was measured at 37 degrees C with an aggregometer.


RESULTS
Colored WST-8 formazan was produced in proportion to the number of intact fresh platelets, but only a little or none was produced by dead platelets lysed by sodium dodecyl sulfate (SDS), by platelets in which glycolysis had been blocked by the inhibitor monoiodoacetic acid (MIA), and by frozen platelets. The assay in conjunction with platelet aggregometry could distinguish between platelets which had lost only the aggregation response and those which had lost both aggregation response and redox activity. Freezing platelets in the presence of cryoprotectant Banbanker resulted in approximately 70% fresh platelets with redox activity and aggregation response.


CONCLUSIONS
These results indicate that the WST-8 assay can be useful for identifying viable platelets independent of the aggregation response and may contribute to clarifying the mechanism of the loss of function of stored platelets.
Maekawa
Yuichi
Y
Department of Clinical Laboratory Science, Osaka Prefecture College of Health Science, 3-7-30 Habikino, 583-8555, Osaka, Japan. aac64810@pop02.odn.ne.jp
Yagi
Kyouko
K
Nonomura
Ayako
A
Kuraoku
Rie
R
Nishiura
Emi
E
Uchibori
Emi
E
Takeuchi
Kikuko
K
D016475:3T3 Cells; D000328:Adult; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001793:Blood Preservation; D002463:Cell Membrane Permeability; D002470:Cell Survival; D003124:Colorimetry / Q000379:methods*; D004396:Coloring Agents / Q000032:analysis*; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006019:Glycolysis; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D019807:Iodoacetic Acid / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction; D010974:Platelet Aggregation / Q000187:drug effects; D010976:Platelet Count; D010979:Platelet Function Tests / Q000379:methods*; D019810:Sodium Azide / Q000494:pharmacology; D013696:Temperature; D013778:Tetrazolium Salts / Q000032:analysis*; D014343:Trypan Blue / Q000032:analysis
C476329:2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt; D004396:Coloring Agents; D003451:Cryoprotective Agents; D004791:Enzyme Inhibitors; D007038:Hypotonic Solutions; D013778:Tetrazolium Salts; D019810:Sodium Azide; D014343:Trypan Blue; D019807:Iodoacetic Acid