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Factor Substitution Assays

From: Peracha,

Mr. Fritsma: We do not have facilities for factor assays at our laboratory. I need to prepare aged plasma for correction studies. Should aged plasma have a prothrombin time exceeding 90 seconds or should it be not more than 90 seconds?

Hello, Peracha, thank you for your question. We really don’t use substitution assays to detect single-factor deficiencies anymore. In fact, George King Biomedical, Inc, of Kansas City, Kansas was the last company to market the reagents, and according to their Laboratory director, Judy Gillison, they discontinued them in 1993.

Thus I would begin by urging that you find a way to perform factor assays. The factor-depleted plasmas you need are readily available from several distributors, including George King Biomedical and Precision BioLogic, Inc and your laboratory coagulometer can be readily adapted to perform the assay.

If you choose to perform substitution assays, you need two reagents, aged serum and BaSO4 or Al(OH)3 absorbed plasma. To prepare aged serum, collect blood, allow to clot, separate the serum and incubate at 37° C for 24 h to destroy factors V and VIII. Aliquot and freeze. Aged serum provides factors VII, IX, X, XI and XII. If prepared correctly, aged serum will generate no clot in either the prothrombin time (PT) or partial thromboplastin time (PTT) assay.

For absorbed plasma, collect fresh anticoagulated blood,separate, and add Amphogel (Al(OH)3) or dry BaSO4 to the plasma for a few hours to absorb the vitamin K-dependent factors. Mix occasionally, centrifuge, aliquot and freeze. Absorbed plasma provides fibrinogen and factors V, VIII, IX and XII.

You may then perform a PT and PTT on the test plasma with no reagent, aged serum, and absorbed plasma and interpret results using the attached table from Fritsma GA. Clot-based assays of coagulation. In Corriveau DM, Fritsma GA. Hemostasis and Thrombosis in the Clinical Laboratory, 1988, Lippincott.

Again, you may find that in the long run it is more effective to develop factor assays with commercially prepared factor-depleted plasmas, however whichever approach you decide on, I wish you great success.


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