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Technical Method Fresh Frozen Cryosupernatant in Assays

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Technical Method Fresh Frozen Cryosupernatant in Assays J Clin Pathol: first published as 10.1136/jcp.38.4.475 on 1 April 1985. Downloaded from J Clin Pathol 1985;38:475-477 Technical method Fresh frozen cryosupernatant in assays. The remainder was frozen for 2 h in metal containers in a blast freezer (Pritchard Laboratory place of fresh frozen plasma for Refrigeration) set at -60°C. The frozen platelet poor plasma was then thawed at 4°C for 12-16 h as broad spectrum coagulation for routine cryoprecipitate production. The cryo- factor replacement precipitate was sedimented by centrifugation at 3500 g for 10 min at 4°C and the excess cryosuper- natant was then siphoned into the second satellite JAF NAPIER, H BASS, R PENGILLEY bag. The cryoprecipitate, in about 15-20 ml plasma, Welsh Regional Transfusion Centre, Rhydlafar, was redissolved by placing it in a 37°C waterbath for St Fagans, Cardiff 10 min; aliquots of this and the cryosupernatant were then snap frozen in liquid nitrogen. The vol- umes cryoprecipitate, and cryosupernat- The use of fresh frozen plasma (FFP) has shown a of plasma, ant were calculated from their weights. dramatic increase in recent years. In the Welsh Reg- The factor content of platelet poor a population of 2-2 million, 294 units coagulation ion, covering plasma, cryoprecipitate, and cryosupernatant were were supplied in 1974, and this increased to over 5000 in 1983. examined as outlined below: clotting test (KCCT), one Much of the growth in fresh plasma use has been The kaolin cephalin time, factor V assay, and one for treatment or prevention of coagulation.disorders stage prothrombin VIII were based on the methods other than primary factor VIII deficiency or defibri- stage factor assays described by Austen and Rhymes.4 Cephalin and nation syndromes. In a recent local survey less than copyright. were a combined reag- 5% of all FFP issued was required for conditions in kaolin, however, replaced by which factor VIII values might be expected to be ent, activated cephaloplastin reagent (Dade), in the one factor VIII assay. Brain reduced. Most of the FFP was used during plasma KCCT test and stage was from the National UK exchange, massive transfusion, or cardiac surgery. thromboplastin obtained Previous examination of clotting, factor activity in Reference Laboratory, Manchester; factor V and cryosupernatant plasma has shown its potential factor VIII deficient substrate and 100% factor V were obtained from http://jcp.bmj.com/ value as a replacement fluid especially in large vol- and factor VIII plasma standard Immuno Ltd. The 3rd British Standard for Blood ume therapeutic plasma exchange and in severe fac- was tor V t-his evidence, FFP has Coagulation Factor VIII Concentrate (human) deficiency.'-3 Despite National Institute for continued to be the product of choice in a wide vari- supplied by the Biological ety of conditions. It seemed worthwhile, therefore, Standards and Control, London. Fibrinogen con- to re-examine the potential value of fresh frozen centrations were measured using a fibrinogen reag- in place of FFP in circumstances ent kit (Boehringer Mannheim) and fibrinogen cryosupernatant a latex kit (Mercia- on September 25, 2021 by guest. Protected such as fluid replacement for apheresis patients and degradation products by prophylaxis of coagulation abnormalities occurring Brocades Ltd). Fibronectin was assayed by during massive transfusion. immunoelectrophoresis using the Laurell rocket technique.5 In this technique, 187 ,ul of rabbit anti- Material and methods human fibronectin was added to 25 ml of 1% agar- ose ME Seakem (Miles Laboratories). The elec- Ten units of whole blood from healthy volunteer trophoresis buffer was 0-08 M Tris, 0-025 M Tricine buffer, pH 8-6. donors were collected into CPDA- 1 triple bags (Travenol PL146). Within 6 h of donation the blood- was centrifuged at 3500 g for 10 min at 4°C. The Results resultant platelet poor plasma was immediately expressed into a 300 ml satellite bag and each unit The percentages of factor V and factor VIII reco- of concentrated red cells was sealed off. Aliquots of vered in the cryosupernatant from the original platelet poor plasma were snap frozen in liquid nit- plasma were 75% and 18*6% respectively (Table). rogen for use as reference samples in coagulation The corresponding figures for fibrinogen and fibronectin were 64-6% and 45.3%. Accepted for publication 4 December 1984 Measurements of fibrinogen degradation products 475 J Clin Pathol: first published as 10.1136/jcp.38.4.475 on 1 April 1985. Downloaded from 476 Napier, Bass, Pengilley Coagulation factor activities of 10 units ofplatelet poor plasma before and after processing into cryoprecipitate and cryosupernatant plasma Platelet poor plasma Cryoprecipitate Cryosuperatant (Mean 228 ml) (Mean 17.4 ml) (Mean 210 ml) Kaolin cephalin clotting time(s)* 51-59 41-51 68-84 (54.0) (44.6) (74.0) One stage prothrombin time(s)* 14-15 5 135-16 15-19 6 (14.5) (15-1) (17-6) Fibrinogen degradation products (,ug/ml) <15 <15 <15 Fibrinogen % Yield 100-0 32-4 64-6 g/l 2-4 7-8 1-82 gpack 0-54 0-135 0-382 Range 0-43-0 65 0-1-0-27 0-30-47 Factor V % Yield 100-0 5 0 75-0 U/mi 0-91 0-66 0 74 U/pack 207-48 11-48 155-4 Range 108-269 525-24-3 944-191 Factor VIII % Yield 100-0 52-6 18-6 U/ml 1-08 7-47 0-21 U/pack 246-20 129-40 44-1 Range 1956-314.8 76-192 269-65 Fibronectin % Yield 100-0 49 35 45.3 U/ml 1-41 9-1 0-69 U/pack 321-48 158-3 144-9 Range 173-503-2 903-281-6 74-4-241-6 *Values given as range (mean). were below 15 ,ug/ml. The mean clotting time of the is used. Cryoprecipitate, fresh blood, or plasma are one stage prothrombin test was 17-6 s and that of best suited for management of the relatively copyright. the kaolin cephalin clotting test was 74 s. uncommon defibrination syndrome and these pro- ducts would also be preferred for broad spectrum Discussion haemostatic defects in neonates. Fresh frozen plasma should probably continue to be used for Fresh frozen plasma is a valuable source of material treatment of coagulation problems as a result of for factor VIII concentrate and its use where factor liver failure or the relatively uncommon congenital http://jcp.bmj.com/ VIII is not required is wasteful. Cryosupernatant coagulation deficiency states for which specific con- shares many of the features of fresh frozen plasma centrates are not available. Cryosupernatant seems but is relatively more economical. Despite this, the well suited to use in place of fresh frozen plasma use of fresh frozen plasma has grown dramatically, during plasma exchange and it has been used instead while cryosupernatant plasma has been neglected as of albumin for this purpose without adverse effects.' a possible alternative. There seems little reason why frozen cryosupernat- The coagulation factor composition of cryosuper- ant should not be substituted for fresh frozen plasma on September 25, 2021 by guest. Protected natant as measured in this study shows clearly that during major blood loss or cardiovascular surgery, even extensive transfusion of such fluid would not especially when it is used to prevent the develop- result in clinically significant coagulation abnor- ment of dilutional coagulation factor deficiencies. malities. Most factors are present in adequate con- This wasteful practice of using fresh frozen plasma centrations for haemostasis. Factors VII, IX, and X seems to have gained widespread acceptance even have already been shown not to be removed by the though it has been shown that clinically significant cryoprecipitation process.' It is probable that the deficiencies are found relatively infrequently.9 Pru- slightly prolonged KCCT times in this report reflect dent practice would entail more frequent use of the deficiency of factor VIII in cryosupernatant. platelet count and coagulation screening tests to Addition of FVIII in the form of NHS intermediate identify those patients with haemostatic defects who concentrate almost corrects the KCCT time to nor- may require more energetic replacement treatment. mal. The concentration of fibrinogen degradation Fibronectin deficiency is a possible though products is sufficiently low to be clinically unproved phenomenon of clinical importance,9 '° insignificant. Frozen cryosupernatant could there- and preliminary attempts at replacement treatment fore be substituted for fresh frozen plasma for many have been reported." Since cryosupernatant con- of the clinical purposes for which the latter material tains around 45% of the original fibronectin it seems J Clin Pathol: first published as 10.1136/jcp.38.4.475 on 1 April 1985. Downloaded from Cryosupernatant in replacement therapy 477 possible that some clinically useful replacement 2 Katz AJ, Morse EE. Factor V activity in fresh frozen and cryo- precipitate removed plasma. Vox Sang 1972;22:39-44. could be provided if several units of cryosupernatant Ahr J, Adjizian JC, Droulle Ch, et al. Cryoprecipitate depleted plasma were transfused. Clearly, cryoprecipitate plasma as a replacement fluid. Plasma Therapy 1981;2:243-6. would be required to rectify a major deficiency state. 4 Austen DEG, Rhymes IL. A laboratory manual ofblood coagu- From the data presented above cryoprecipitate lation. Oxford: Blackwell Scientific Publications, 1975. Laurell CB. Electroimmunoassay. Scand J Clin Lab Invest. supernatant appears to have a therapeutic potential 1972;29 (suppl 124):21-37. that is currently not fully exploited. This may be of 6 Monthony JF, Wallace EG, Allen DM. A non-barbital buffer for particular importance in areas where shortages of all immunoelectrophoresis in agarose gels. Clin Chem forms of plasma proteins for transfusion restrict the 1978;24: 1825-8.
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