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Oak Ridge Poster Sessions |
1 Thermogenesis Corp., Rancho Cordova, CA;
The CryoSealTM System (CSS; Thermogenesis Corp.) is a device that can prepare freeze–thaw cryoprecipitate (Cryo) generated from plasma in ~1 h. Traditional blood bank preparations of Cryo contain high concentrations of antihemophilic factor (plasma coagulation factor VIII) and fibrinogen, but require several days, numerous pieces of laboratory equipment, and considerable staff handling to prepare (1). We investigated whether CSS-prepared material is substantially equivalent in factor VIII and fibrinogen contents to the traditionally prepared Cryo material.
Whole blood drawn from volunteer donors (n = 16) at the Sacramento Medical Foundation Blood Center (SMFBC), with use of citrate–phosphate–dextrose–adenine anticoagulation, was centrifuged at 2500g for 5 min at 4 °C within 6 h of collection. The units of plasma were then used for preparation of Cryo in the CSS.
The CSS consists of a thermodynamic device featuring a temperature-controlled rocker plate and a clear, plastic, single-use plasma-processing container that rests on the rocker plate. Upon initiation of Cryo processing, the CSS automatically transfers the donor plasma from a transfer pack to the processing container by a peristaltic pump in a closed system of tubing. The CSS then lowers the temperature and sets the plate in motion to rapidly freeze the plasma to -27 °C. The rocker plate temperature is then raised to 2 °C to thaw the plasma while concurrently rocking the plate in a manner that causes the insoluble clotting and adhesive proteins of Cryo to agglomerate and migrate to the tip of the processing container for removal by the attached syringe. The residual Cryo-poor plasma is then automatically transferred back to the transfer pack to yield a Cryo volume of ~10.0 mL. This Cryo sample was submitted for factor VIII activity and fibrinogen analysis.
Immunodepleted factor VIII-deficient plasma (Dade International) was used for all factor assays. CryoCheck (Precision Biologicals) was the factor VIII calibrator used for donor samples, and US Standard Antihemophilic Factor Mega 1 was used as the calibrator for Cryo. All tests were performed on the MLA 1000C coagulation analyzer (Medical Laboratory Automation) with Actin FS (Dade) aPTT reagent. Each factor calibrator was automatically prepared by the MLA 1000C by sequentially diluting the calibrator seven times with buffered saline (barbital buffer; Dade) to dilutions from 1:5 to 1:320. The subsequent test samples were diluted 3 times to give dilutions from 1:5 to 1:20. Each calibrator dilution was tested in duplicate and the replicate results had to match within 5% to be accepted as a valid point. All factor results were reported in units/mL or total units (factor VIII units/mL multiplied by volume).
Fibrinogen was determined by the modified Clauss (2) method by using commercially prepared thrombin (Dade). College of American Pathologists Fibrinogen Reference plasma was used to calibrate the fibrinogen calibration curve. The fibrinogen calibrator was automatically prepared by the MLA 1000C by sequentially diluting the reference material 5 times with barbital buffer, creating dilutions from 1:3.5 to 1:40. The fibrinogen calibrator was analyzed only once before sample testing. Each fibrinogen calibrator and test plasma were analyzed in duplicate, with agreement within 5% required. Cryo samples were diluted 1:5 with buffered saline before analysis, and the final result was corrected for dilution. Fibrinogen results were reported in mg/dL (as specified by the American Association of Blood Banking) for donor plasma or total mg (fibrinogen mg/dL multiplied by volume).
Student's paired t-test was used for analysis, with P <0.05 determined to be statistically significant.
The mean total contents (total volume x fibrinogen/factor VIII
concentration) of factor VIII and fibrinogen in the CSS-generated Cryo
were 216 units and 231 mg, respectively, which represents recoveries
(total Cryo/total donor plasma) of 47% and 29%, respectively.
Quality-control assays over the previous 15 months of the traditionally
prepared Cryo material from SMFBC yielded an average factor VIII
concentration of 128 units per average volume (15.0 mL). Mean Cryo
fibrinogen contents were 100–350 mg (3). Thus
CSS-generated Cryo material was more than twice as concentrated in
factor VIII (16.9 units/mL) than conventional preparations (8.5
units/mL) (Table 1
).
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We conclude that CSS generated higher concentrations and yields of factor VIII and fibrinogen than did conventional preparations. This CSS process is highly automated and takes ~1 h to complete the freeze–thaw cycles, markedly less than traditional blood bank methods. The CSS method facilitates rapid preparation of Cryo for either immediate clinical use or long-term storage.
Footnotes
Univ. of California, Davis Med. Ctr., Sacramento, CA, and *address correspondence to this author at UC Davis Med. Ctr., 4625 2nd Ave., Rm. 3203, Sacramento, CA 95817: fax 916-734-3320,
References
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