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An Accurate Assay for Platelet Factor 3

An article by Roy E. Speck, inventor of the ellagic acid APTT test. The Spectra™ APCT test is a new test which improves on the APTT’s scope.

It is well established that there is a need to accurately measure platelet factor 3 (PF3). The uniform release of PF3 from the platelet is required for an accurate measurement of PF3 activity. This has been called the available PF3 activity. In the past, total PF3 release could be attained by using a freeze-thaw procedure¹, sonification and by using hypotonic solutions².

Other methods have been reported which were designed to measure PF3 these include: the thromboplastin generation time test^3-5, prothrombin consumption time^6,7, kaolin clotting time^8,9, adenine diphosphate (ADP) clotting time test¹⁰, activation of prothrombin¹¹, stypven clotting time test^11,12, and the use of chromogenic substrate¹³. These tests have serious limitations which included the need to adjust the platelet count, special procedures for releasing PF3, poor sensitivity, failure to fully activate plasma coagulation factors in the intrinsic pathway, prolonged incubation time (20-30 min.), variability of results, and failure to provide reproducible results.

A standard ellagic acid APTT is a rapid test that uniformly activates the plasma coagulation factors. The Spectra™ APCT reagent is designed to not only rapidly activated the plasma coagulation factors but to quantitatively release PF3 from platelet rich plasma (PRP) as well. The results are similar to those given by the ellagic acid APTT but are sensitive to PF3 as well.

A reagent that releases PF3 and activates the intrinsic factors has the following advantages: 1) the effectiveness of the patient’s own PF3 in supporting coagulation is estimated, 2) no special procedure is needed to release PF3, 3) no adjustment of platelet count is needed, 4) since the PF3 release and the activation of the plasma coagulation factors are rapid, incubation times of 1-5 min. can be used, 5) because the reagent is optically clear and truly soluble, the APCT can be performed on instruments with optical detectors, as well as other commonly used coagulation instruments, 6) the test is precise, with a well defined normal range and a high precision, 7) the results provide an overview of the entire intrinsic system without the addition of a platelet substitute.

Materials and Methods: The following materials were used: Spectra™ APCT reagent (patented), Spectra™ Platelet Depleted Plasma, B-D Vacutainers™, Aquasil™ and a coagulation analyzer model 600 (Boehringer-Mannheim). The procedure for siliconizing glassware is as follows: a 1% Aquasil™ solution in distilled water (vol/vol) was prepared. Items to be siliconized were immersed in this solution for 30 sec., making sure that the entire surface came into contact with the silconizing solution. The items were rinsed with distilled water and air-dried for 24 hours. Plasticware that is clean and appropriate to coagulation can also be used obviating the need to siliconize glassware.

Preparation of PRP: A clean venipuncture without hemostasis was performed using a siliconized Vacutainer containing 0.109 M buffered sodium citrate. Samples were mixed immediately and centrifuged for 5 min. at 500 RCF. The PRP was transferred to a siliconized test tube using a siliconized Pasteur pipet. Samples can be stored at room temperature for up to 2 hours. Plasma specimens with any trace of hemolysis were discarded.

Determination of activated plasma clotting time: APCT reagent (100ul) and PRP (100ul) were added to the reaction cuvette and mixed. After incubation for 3 min at 37 C, 0.02 M calcium chloride (100 ul) that had been pre-warmed for 3 minutes was added, and the clotting time was determined on the coagulation analyzer.

Determination of activation rate of APCT test: APCT tests were run on normal PRP with incubation times of 0, 1, 2, 3, 4 and 5 min.

Determination of APCT normal range: PRP samples were prepared from 46 adult donors who were taking no medication. The APCT tests were performed using these platelet rich samples. The 95% confidence limit was determined. The APCT were performed according to the procedure outlined in the “Materials & Methods” section.

Determination of normal with-in run precision: Forty replicate determinations were performed on an aliquoted pool of fresh, normal PRP. The mean standard deviation and 95% confidence limit are listed in “Results” section.

Determination of abnormal within-run precision: Forty APCT determination were performed on thrombocytopenic plasma samples. The mean, standard deviation, coefficient of variation and 95% confidence limit are listed in the “Results” section15.

A comparison was performed between the results of APCT with PRP, which had been frozen and thawed and unfrozen platelet rich samples. An aliquot of each sample was frozen and thawed three times. APCT tests were performed on both the untreated and frozen samples. The student t-test was applied and the significance determined.

Determination of PF3 reserve: The PF3 reserve is defined as that amount of PF3 that may be lost before abnormal clotting times occur. Dilutions of the patient’s platelet rich plasma were made using platelet depleted normal plasma. Use of platelet depleted plasma keeps the coagulation factors constant, making the PF3 concentration the only measured test variable. The PF3 activity was decreased until the critical level was attained. This is the percent dilution of the patient’s PRP giving an APCT result of 40 sec (the upper limit of our normal range). The difference between 100% and this value is the PF3 reserve.

The procedure was as follows: 1) Patient’s PRP was prepared. 2) A vial of platelet depleted normal plasma (CR-119) and a vial of Spectra™ APCT were reconstituted. 3) The following dilutions of the patient’s PRP using the reconstituted PDP (50, 25, 12.5, 6.25, 3.125 and 1.56%) were made. 4) the APCT test was prepared on the 100% sample and all of the dilutions. 5) The APCT times vs. percent PRP were graphed on linear graph paper. 6) The percent PRP at the 40 second point was determined. This is the critical point. The critical point percent was subtracted from 100% to obtain the PF3 reserve (100%-CP%= platelet reserve %).

Determination of normal range for PF3 reserve: Platelet rich samples were prepared from 20 normal adults who were not taking any medication. The PF3 reserve was determined for each sample. The mean, standard deviation, and 95% confidence limit were determined.

The determination of PF3 activity in platelet concentrates was as follows: 1) a uniform 1.0 mL sample from platelet concentrates was obtained. This sample was placed in a 10X75 mm siliconized test tube. 2) A 1:10 dilution was prepared using platelet depleted normal plasma. 3) The PF3 reserve procedure using the diluted sample as 100% activity was determined. The final result was multiplied by 10 to determine platelet reserve.

Results: The normal range for the APCT test was N=46; mean = 32.0 seconds; standard deviation (SD) = +/-4.4 seconds; 95% confidence limit = 23-40 seconds. The APCT test normal within run precision was N = 40; mean = 31.5 second; SD+/- 0.457 seconds; coefficient of variation (CV) = 3.1%; 95% confidence limit = 30.6-32.3 seconds. The APCT test abnormal within-run precision was N=40; mean =57.8 seconds; SD = +/- 1.8 seconds; CV - 3.1%; 95% confidence limit was 54.3-61.3 seconds. The normal range for PF3 reserve was N =20; mean 96.4%; SD = +/- 1.35%; 95% confidence limit = 93.8-99%.

Discussion: The APCT test is somewhat similar to the APTT test. The APTT test contains an activator of the intrinsic system with an added platelet substitute. The APTT uses platelet poor plasma as a sample. Therefore, the APTT cannot measure PF3. The APCT tests also has an activator for the intrinsic system but contains no phospholipid. The APCT test uses PRP as a sample and depends on the actual PF3 from the platelet as a source of phospholipid support for the coagulation system. This fundamental change allows the measurement of the entire intrinsic coagulation system including platelet function.

It has been demonstrated that the APCT test fully activates in less than 1 minute and is stable for longer than 5 minutes. There is no significant difference in clotting time between PF3 by careful freeze-thaw techniques and the PF3 released by the APCT reagent (t test [probability <0.001]). The PF3 reserve is a suitable parameter with which to express PF3 activity since it also relates to the patient’s clinical status. The PF3 reserve is a direct measure of how much platelet activity can be lost before the patient’s platelets can no longer support intrinsic pathway coagulation. It does not, however, quantitate the formation of the platelet plug or platelet adherence. The test is independent of platelet quantity, morphologies, or other factors. It measures only the platelet’s ability to activate phospholipid, which is necessary to support coagulation. The use of the bleeding time and platelet count of assessing PF3 activity have severe limitations. Platelet exhibit a wide range of activity with respect to their PF3 content. Some platelets contain virtually no PF3 activity, while others are rich in PF3. These and other considerations make the bleeding time highly variable and insensitive.

The advantages of the APCT test are: 1) The PF3 reserve test is specific for PF3 2) The PF3 reserve is indexed to the upper limit of normal of the APCT test. This is the point at which there is insufficient PF3 to support normal hemostasis. 3) Since the APCT test is precise, the PF3 reserve is also precise. 4) The PF3 reserve is easy to perform and can be automated.

Platelet transfusions are used to supply viable platelets to patients who have an insufficient number of platelets or platelets of inferior quality in regard to their being able to support hemostasis. At present, blood donors intended for apheresis and /or platelet concentrates are not screened for suitability regarding platelet quality. Platelet concentrates also are not tested for PF3. The patient receiving platelet concentrates is not tested to determine effectiveness of the platelet transfusion.

The use of this simple test to screen donors, test stored platelets, and monitor the progress of recipients should enhance critical health care. Donors whose platelets are inhibited or defective in PF3 need not be phlebotomized. Platelet concentrates that cannot support the intrinsic pathway clotting mechanism can be detected before infusion. Patients whose platelet reserve is high enough to maintain hemostasis need not be transfused. Patients who are being transfused with platelets can be monitored so that they can be maintained at an adequate level of PF3.

References:

1. Fantl P, Ward HA. The thromboplastin component of intact blood platelets is present in masked form. Exp J Biol Med Sci 1958: 36:499-504.
2. Ullutin ON, Karoca M A study of the pathogenesis of thrombopathia using the “platelet osmotic-resistance test.” Br J Haematol 1959; 5:302-6.
3. Biggs R, Douglas AS. The thromboplastin generation test. J Clin Pathol 1953; 6:23-9.
4. Bowie EJ, Thompson JH, Owen CA. Standardization of platelets in the thromboplastin generation test. Am Clin Pathol 1965; 44:673-7.
5. Weiss HF, Eichelberger JW. The detection of defects in patients with mild bleeding disorders. Use of quantitative assay for PF3. Am J Med 1962; 32:872-83.
6. Owen CA, Thompson JH. Soybean phosphatides in prothrombin consumption and thromboplastin generation tests. The use in recognizing “thromboplastic hemophilia.” Am J Clin Pathol 1960; 33; 197-208.
7. Polasek J, Duckert F. Quantitative determination of PF3. Thromb Diath Haemorrh 1971; 25:532-44.
8. Hardisty HI, Hutton RA. The kaolin clotting time of platelet rich PRP: a test of PF3 availability. BR J Haematol 1965; 11: 258-75
9. Spaet TH, Cintron J Studies on PF3 availability Br J Haematol 1965; 11:258-75.
10. Horowitz, HI, Cohen BD, Martinez P, et al. Defective ADP induced PF3 activation in uremia. Blood 1967; 30:331-41.
11. Joist JH, Dolzel G, Lloyd JV, Kinbrough-Rathbone RL, Mustard JF. PF3 availability and the platelet release reaction. J Lab Clin Med 1974; 84:474-82.
12. Hardisty, RM, Hutton RA Platelet aggregation and the availability of PF3. Br J Haematol 1966; 12: 764-76.
13. Sandbert H. Anderson LO. A highly sensitive assay of PF3 using a chromogenic substrate. Thrombosis Res 1979; 14:113-24.
14. Speck, RE Substance relating to testing of blood coagulation. US Pat # 3,486,981 (filed Mar 15, 1965).
15. Gryna FM. Basic statistical methods. In: Juran JM, Gyrna FM, Bingham RS, eds. Quality Control Handbook. New York: McGraw-Hill Book Co., 1974:22-70, 3rd ed.

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