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From www.bloodjournal.org by guest on August 9, 2017. For personal use only. Brief Report Factor VIII (AHF) Concentration pH 6.5 Citrate-Plasma By S A RESULT of ulants on the is the anticoagulant ACD platelets which vided as many are platelet S. MomusoN FmiNcIs recent survival investigations of transfused into for required platelet The the can suggested be most 6.5. Many laboratories the treatment on Factor ACD might VIII find it convenient hemophilia. ) concentration ( AHF \IETHODS The Factor housma VIII assay’ by as modified volumes Ten of whole After and blood the separation I. The PPP was partitioned the other contained ACD; samples the were fromil 7.0 2. was felt separately l)lmflering the the that of as platelet with at pH plasma for of additional effect of Langdell, three VIII ranged reduced pH normal by different the Wagner volunteers activity at procedures before from 6.3 to into centrifuging were tubes. One tube of Veronal buffer use and 1.5 in Brink. volumes 1800-1900 g. of for 30 used: contained (pH 7.3). 0.3 nil. Aliquots and after storage 6.5 while the buffered at -20 might affect additional of these C. The p11 plasma but the used British Medical and as (pH on Research 6.3-6.5). the the Council’s contained view of of ranged the assay then returned to ) was 7.0-7.2 standing assayed. VIII three was at The technic Haematology in determined of of approximately Experimental equal temperature was cryoprecipitation portions an treated room pH system this possibility as described Pool 2 ml. Research in Unit, England. in the it ( pH After Factor into Londoa, assay plasma and partitioned School, assertions reflecting based was to control plasma was PPP Medical opinions citrate prior The “acidified” The hospital construed additional immediately erythrocytes. method the Mary’s amid/or any effect on Factor VIII. In an attempt to circumvent of erythrocytes was utilized. The PPP was treated the piasmiia was again separated the incul)ation with ervthrocytes. after Another Time be of PPP, from obtained into two plastic the same volume paragraph, Shannon.’5”#{176} Fronz St. making investigated. metho(l collected was Factor fresh-washed and 3. the from capacity the same nianner for 15 minutes and of pro- MATERIALS one-stage (PPP) ACD added preceding volume before for with that to 7.2. It quite in assayed samples quantities Macpherson.’4 were plasma The plasma (PRP) ACD-plasma this the was AND the was Hardistv ACD#{176}.Platelet-poor nuinutes. used to store Therefore, anticoag- emerged for require reduction of pH of the platelet rich acid citrate; consequently, a by-product is fresh of has conveniently technics concentrates additional of classical effect fact transfusion. clinically concentrates.12 the platelets,1”2 of choice usually in this U. paper are S. Navy the author’s Departnuent own or time and are Naval not to Service at large’. First FliAxcis search submitted S. Council’s Jan. 18, 1966; NIORRISON, Experimental accepted LCDR, NI.C., for publication Haematology School, London. #{176}ACDNIH Formula A ( 0.8 per per cent hydrous dextrose). cent Visiting U.S.N.: Research citric acid, April 25, 1966. Investigator, Unit, 2.2 per St. cent British Mary’s sodium Medical Hospital citrate, Re- Medical and 2.45 479 BLOOD, VOL. 28, No. 3 (SEPTEMBER), 1966 From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 480 S. FRANGJ.S small plastic 0.2 nil., was containers. To the first was added 0.2 added to the second and third samples. -20 C. The first ice” mixture 2000 stored and and second were samples were allowed to then rapidly thaw ml. more ACD, while Verona! buffer, The third sample was then stored at frozen taken out of the freezer, samples was thawed resuspended at room in 2.2 ml. by immersion into an alcohol at were centrifuged at plasma the cold-precipitate to be done, all three samples was were overnight g for 20 minutes. After pouring off the supernatant at -20 C. for 1 to 10 days. When the assay was two MORRISON temperature, 4 and C. and the cold-precipitate in “dry the first buffer. RESULTS the 1. When the sample 54 per cent pH of plasma was with the the differed fractions by less which 52 per cent) were tion. There was were cold-precipitated, of additional than 0.1 unit. thawed 0.2 control Factor stored however, of each a pH sample ( average 96 per approximating pH been were ACD cent). of all three of the (average after paired samplasma cent immediately the sample samples 40 to 60 per C. separated, the additional concentration was having themi these preassay additional from with between pair differ of ( average without When -20 estimate cent and not VIII at the per samples final at 4 C. overnight sample 50 units. the the The difference, and did of of the of assay, erythrocytes ACD technic, one ACD to fresh cent in the no of the more per than of that concentration concentration cryoprecipitate time approximately additional by VIII at the was returned ACD Factor to be 90 to 100 3. With differed ACD VIII sample additional ples Factor the without found samples additional the assayed, was of the the ) of the ACD. 2. When the pH with separa- samples processed which in the presence 6.5. DiscussioN reduction A in plasma acidified plasma tion.7 However, pH and/or given. salt In the “acidified” pH It is well reaction known by buffer alters activity. assay. pH the the Factor However, Alternatively, fibrmnogen pH the assay of the conditions pendent of the question. lower of the VIII to assay. the salt concentration the reflect The rule out per not not the seemi assay of the when found to unin reduced pH the the a definitive be reduction influence of It diluted that physiologic under removal the of the as the to is greatly possibility at in reaction.17 in a reversible allow of assay was a reflectiomi mixture as well seen or cent salt concentrato readjust the method and known results dilution do 30 AHF was reversed is also data the on (Plasma so as to result the was determined assayed. improved to thrombin-fibrinogen was is not 25 effect VIII assay incubatiomi not molecule change. difference Factor actual does reduction and this being the nor prior system ) This may pH levels plasma assay. assay, cami influence the pH of to altered pH or attempt was made a comparable However, that the reaction,17 mental this of the during seen. that therefore, However, to here to physiologic mixture affected pH was concentration prior reported readjusted appeared, lower pH. VIII recently ascribed stated whether an concentration studies plasma was Factor has been it was not thrombin- given citrate in during experi- ion conclusion mdcon From www.bloodjournal.org by guest on August 9, 2017. For personal use only. FACrOR VIII The IN experiment hemophiliac. buffering in vivo these Factor iii 481 PLASMA the the Under reductiomi tional CiTRATE utilizing to simulate atteni1)t capacity events experimental VIII of erythrocytes attendant upon conditions concentration after was infusion there exposure was to, a simple of plasma no or into a significant storage addi- in, ACD. The and recent its development application in forward times Factor The tional ACD. with activity The the The plasma, precipitated VIII on also of product presence indicate protein the additional VIII step to he the means of the assay ACD had without ACD 30 In only as a factors in precipitated of Factor VIII large basis. of additional samples that method a is reported quantitative plasma Factor represent method was used the reduced pH as the cryoprecipitation end a in the as results of cryoprecipitation banking1#{176} may procurement. frozen plasma Factor same blood the cryoprecipitatiomi excess citrate and the system. technic the general VIII as study eliminating of to potemit as presemit fere 6.5 H does the addi- not inter- concentration. SUMMARY A by-product posed of methods, a useful source unaltered incubation same Factor of Factor that, assay the after system. Factor acidified plasma untreated plasma. found producto undo 6,5. secundari recentemente Viste activitate que iste to il esseva post incubation he mesme activitate citrato, su pH he as le addition essayage. preparate preparate Iste de de ab plasma ab plasma “acidified” acidificate to cent. However, plasma had the plasma is probably This prepared conclusion was supported by cryoprecipitation. as in as as de un esseva es per de plasma a In the from from utile fonte de con plachettas fresc de 6,5 es de Factor supportate Factor eliminate como de non minus VIII alte que de iste l)la51’’It cento. plasma post citratate In tal preparatos, in systerna le in Ic precipitatos in a Tamemi, habeva observationes factores Factor pH VIII, probabilemente VIII que per see- a un nonalterate cryoprecipitation. le concentration esseva in prepared concentratos comparation de un pH como fonte preparate effective factors prepared precipitates es citratate In plasma efficace nontractate. per he activitate amontava a 54 pro erythrocytos fresc, he “acidificate” VIII como le preparato de controlo. que be INTERLINGUA esser in illo. e le pH trovate 54 pro- might Compared are eliminated in the precipitates high methodos VIII citrato Esseva pH IN conclusion Factor 6.5 preparation poterea un minus ordinari. concentratos plasma trovate COIl eluite de factor Isto suggestiona infusion non plasma del investigate recently plasma investigated. the ACD-plasma. concentrates proponite esseva this to be erythrocytes SuI\fAnIo Un was found ACD and concentration VIII was by Because control. infusion, additional concentrates, 6.5. activity was fresh as the VIII as ordinary on Factor VIII preparatiomis platelet at pH activity washed activity suggests of VIII, the with VIII source of Factor by observations such preparation citrate-plasma citrate-plasma, after This the is fresh precipitatos con de From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 482 FRANCIS S. MORRISON ACKNOWLEDGMENTS The in author the is indebted preparation to Professor of this P. L. Mollison paper, and to Miss and Dr. Mary P. Barkhan Stevenson for helpful for advice capable technical assistance. REFERENCES 1. Kissmeyer-Nielsen, B.: Platelets 2. F., and Madsen, C. in blood stored in untreated and siliconised glass bottles and plastic bags. II. Survival studies. J. Clin. Path. 14:630, 1961. Aster, R. H., and Jandi, J. H.: Platelet sequestration in man. I. Methods. J. Clin. Invest. 3. Morrison, F. effect of 43:843, 1964. S.. Baldini, and M.: anticoagulants survival. Blood 25:612, The 1965 6. Cohen, P., Watrouse, P., and F. H.: Glycerol preservation concentrates blood. (Second Conf. Ridge, 7. Cohen, July, P., F. H.: on Blood Cooley, M. of ACD. 1965. H., 8. Eng. and ACD Abraharnsen, EDTA and A. F.: The Cr5’ survival of platelets. Scand. labelled J. brief review Sang. (In press.) Langdehl, 1. Chiu, J. A., Sullivan, S.: Transfusion M. of P., and platelet R. J. tests. Hardisty, R. D., Wagner, K. M.: factor Lab. NI., and Blood transfusion: practical of recovery platelets. 26:732, 1965. Morrison, F. S. : Platelet a aspects. R. Effect on Vox H., of one-stage Clin. Med. and anticlot- 41:637, J. Macpherson, C.: Haemorrh. 7:215, 1962. Pool, J. C., and Shannon, 372, 1965 (Abstract). A. E.: Production of high-potency concentrates of antihernophilic globulin in a closedbag system: assay in vitro and in vivo. New Eng. J. Med. 273:1443, 1965. 17. Biggs, man 9. Shively, on human 16. of 2:52, incompatibility transfused Diath. blood Haernat. prepared in acidified 27:449, 1966. Effect of anticoagulant Pool, J. C., and Shannon, A. E.: Simple production of high potency antihernophilic globulin (A.HG) concentrates in a closed bag system. Transfusion 5: and 1965. ABO E. J.: platelet 15. of effect on the recovery 5: A one stage Factor VIII (antihemophilic globulin) assay and its use on venous and capillary plasma. Thromb. Gardner, yields acidified Transfusion Flatow, F. A., Jr., and Freireich, The increased effectiveness of ting 1953. Oak derived from with EDTA and J. Med. 273:845, from plasma. (Abstract). Brinkhous, hernophihic III. Corn- radioactivity New 13. (Abstract) Platelets, preservation. platelet concentrates blood anticoagulated 12. 14. 1964). Platelet parison 1965 25:608, 1965 and (Ab- Gardner, rich concentrates plasma. Blood 1 1. Aster, R. H.: of platefrom ACD derived Blood 10. of stract) (Second Conf. on Blood Platelets, Oak Ridge, July, 1964). 4. Morrison, F. S., and Baldini, M.: The favorable effect of ACD on the viability of fresh and stored human platelets. Vox Sang. (In press.) 5. Morrison, F. S.: The effect of filters on the efficiency of platelet transfusion Transfusion. (In press) let 378, platelet on prepared concentrates platelet R., Blood adelphia, 31. and Macfarlane, Coagulation, F. A. Davis, R. 3rd 1962, C.: Hu- Ed. Phil- pp. 30- From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 1966 28: 479-482 Brief Report: Factor VIII (AHF) Concentration in pH 6.5 Citrate-Plasma FRANCIS S. 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