Letters to the Editor 257

Letters to the Editor

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Quality of Fresh-Frozen Plasma during StorageJ. Riggert a, S. Mörsdorf b, G. Pindur b, M. Köhler a, U.T. Seyfert b

aDepartment of Transfusion Medicine, University of Göttingen,b Department of Hemostaseology and Transfusion Medicine,University of the Saarland, Homburg/Saar, Germany

Fresh-frozen plasma (FFP) either prepared from whole blood orcollected by plasmapheresis plays an important role in the treatment ofcoagulation disorders. Normal storage conditions for FFP involvetemperatures varying between –30 C for 1 year and –40 C or below for2 years as an accepted standard, by which the quality of plasma pro-teins shows no deterioration [1–4]. In order to reduce transfusion-transmitted virus infections, in Germany FFPs are stored in quarantineand are released for transfusion only if the serological viral parameters(anti-HIV 1,2, HBsAg, anti-HCV) of the pertinent donor are negativeat a second examination 6 months after the donation [3]. This resultsin limited permitted storage times of FFPs. Therefore, we prospec-tively examined the effects of an extended storage period at –30 C forup to 2 years in 90 FFPs, which were frozen within 6 h after blooddonation and consecutive separation into components. For samplingpurposes, each unit was thawed after 15, 18, 21 and 24 months and re-frozen immediately. The content of total protein, protein C, protein S,antithrombin III, fibrinogen and the coagulation factors II, V, VII,VIII, IX, X, XI and XII were investigated. Coagulation factors weredetermined by one-stage assays and were calibrated using commercialplasma standards, which had been calibrated against the respectiveWHO preparations, when available. Results are expressed as percent-ages where 100% corresponds to 1 U/ml.

At the end of storage (24 months) we found only slight differencesin most of the parameters investigated in comparison with the valuesafter 18 or 21 months (table 1). In addition, the repeat thawing/freezingprocedures in order to obtain samples for testing were possibly respon-sible for reduced concentrations of coagulation factors II, VII andVIII:c at the end of storage, but with the exception of factor VII, thesedifferences were moderate. Moreover, contaminating cells in plasmamay have a negative influence on labile coagulation factors [5, 6]. In aprevious study, storage of FFP for 12 months at –30 C resulted in nodeleterious effect on factor VIII [7]. In our studies, however, factorVIII:c, which is accepted as the most sensitive coagulation factor tocontrol the quality of plasma, had adequate values at the end of theextended storage, which met the demands of the Council of Europe( 70% of the original factor VIII) for FFP [1]. Alternatives to quaran-tine storage combined with donor retesting are virus inactivation pro-cedures used to obtain plasma as safe as possible concerning the risk ofvirus transmission. Currently used virus inactivation procedures, i.e.solvent/detergent treatment or methylene blue photoinactivation, alsoresult in loss of plasma proteins [8]. Most of the coagulation parame-ters investigated in our study had similar or higher values when com-pared with solvent/detergent-treated plasma pools [9]. In spite of sev-eral thawing/freezing procedures and a storage temperature of only–30 C, sufficient amounts of plasma proteins were observed after24 months. From our data, storage time of FFP at –30 C can be extend-ed to this duration, thus facilitating the quarantining of FFP.

References

1 Council of Europe: Guide to the preparation, use and quality assurance ofblood components. New edition. Strasbourg Council of Europe Press, 1995.

2 Walker RH (ed): Technical Manual, ed 11 Arlington, American Associationof Blood Banks, 1993.

3 Vorstand und wissenschaftlicher Beirat der Bundesärztekammer (Hrsg):Leitlinie zur Therapie mit Blutkomponenten und Plasmaderivaten. Köln,Deutscher Ärzte Verlag, 1995.

4 Kakaiya RM, Morse EE, Panek S: Labile coagulation factors in thawedfresh frozen plasma prepared by two methods. Vox Sang 1984;46:44–46.

5 Pepper MD, Learoyd PA, Rajah SM: Plasma factor VIII, variables affectingstability under standard blood bank conditions and correlation with recoveryin concentrates. Transfusion 1978;18:756–760.

6 Mannucci PM, Lattuada A, Ruggeri ZM: Proteolysis of von Willebrand fac-tor in therapeutic plasma concentrates. Blood 1994;83:3018–3027.

7 Suontaka AM, Åkerblom O, Blombäck M, Eriksson L, Högman CF, PayratJM: Stability of blood coagulation factors and inhibitors in blood drawn intohalf-strength citrate anticoagulant. Vox Sang 1996;71:97–102.

8 Wieding JU, Hellstern P, Köhler M: Inactivation of viruses in fresh frozenplasma. Ann Haematol 1993;67:259–266.

9 Hellstern P, Sachse H, Schwinn H, Oberfrank K: Manufacture and in vitrocharacterization of a solvent/detergent-treated human plasma. Vox Sang1992;63:178–185.

Dr. J. RiggertDepartment of Transfusion MedicineUniversity of GöttingenRobert-Koch-Strasse 40D-37075 Göttingen (Germany)Tel. +49 551 39 8690, Fax +49 551 39 8691E-Mail jriggert med.uni-goettingen.de

Table1. Characterization of FFP after 18 and 24 months of storage at –30 C(n = 90)

Parameter 18 months 24 months

Total protein, g/dl 6.0 (5.0–6.5) 6.0 (5.5–7.0)Protein C, % 90 (70–110) 90 (70–110)Protein S, % 100 (65–140) 100 (80–140)Antithrombin III, % 90 (75–105) 95 (60–130)Antithrombin III, mg/dl 25.5 (21.4–31.2) 25.5 (20.2–31.3)Fibrinogen, mg/dl 241 (201–315) 236 (195–300)Factor II, % 100 (85–115) 93 (79–116)*Factor V, % 75 (60–95) 75 (65–108)Factor VII, % 100 (75–125) 80 (59–116)*Factor VIII:c, % 85 (65–100) 78 (60–96)*Factor IX, % 95 (75–115) 89 (70–110)Factor X, % 95 (80–120) 97 (78–120)Factor XI, % 95 (78–110) 92 (72–116)Factor XII, % 100 (76–128) 99 (65–120)

Median values and 10th and 90th percentiles are given in parentheses.*p 0.05 (Wilcoxon paired test) compared to the results obtained after

18 months.

258 Letters to the Editor

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Absence of Seroconversion to Hepatitis B and Camong Blood Donors with Elevated AlanineAminotransferaseE. Castro, R. Gonzalez, L. Barea, J. MuncunillCentro de Donación de Sangre de Cruz Roja Española, Madrid España

The alanine aminotransferase (ALT) activity measurement inblood donors was first proposed in 1959 to identify hepatitis carriers. Asignificant correlation was observed between ALT values in blood do-nors and the development of hepatitis in the recipients [1]. Neverthe-less, its value decreased after the introduction of HBsAg and HCVantibody tests in blood screening.

Recently it has been discontinued in the USA [2] and several re-search teams have reported the absence of hepatitis B and hepatitis Cvirus genome in seronegative donors with elevated ALT [3] as the onlymarker. Moreover, serum ALT levels, even among anti-HCV-positivedonors, fail to detect viremic carriers [4]. In this way, in a recent sero-conversion study performed by Busch et al. [5] among blood donorswith elevated ALT as the only marker, the seroconversion rate detectedwas 0% for HBsAg and 3 out of 1 million donations for HCV.

We review 28,152 consecutive donations – made by 20,331 blooddonors – in our institution from February 1994 to February 1995. Rou-tine blood donor screening includes HBsAg and anti-HCV tested bythe EIA 3.O version. ALT was measured by an enzyme-kinetic assay at37 C. The calculated upper normal value was 40 IU/l for males and 35IU/l for females, and 75 IU/l was taken as a high cutoff. We reenteredthose blood donors with normal values after 6 months. The subsequentdonations of all the blood donors identified with an elevated ALT werereviewed.

The prevalence of infectious markers during this period was 0.13%for HBsAg, 0.26% for anti-HCV and 0.8% with elevated ALT ( 75IU/l). We identifed 1,201 (5.9%) blood donors with ALT values 45IU/l as the only marker. Among these, 82% had an ALT between 45–75 IU/l and 18% 75 IU/l; 90% were male and 10% female; 39% firsttime and 61% repeat donors and 50% were between 25 and 40 yearsold.

We found 565 out of 1,201 (46.96%) blood donors with elevatedserum ALT who donated after the index donation. The average follow-up period was 11.94 months with 513 (91%) donating subsequently6 months after the index donation. None of these returning donors hadseroconverted neither to HBV (HBsAg) nor to HCV (anti-HCV)markers in the subsequent donations. Among returning donors, 135(24%) out of 564 had a persistent elevation of ALT 45 IU/l as anisolated marker.

Our results also advise against the maintenance of that surrogatemarker. Moreover, it generates great costs regarding reagents andequipment, as well as an important loss of otherwise valid units andcontributes to a needless anxiety among blood donors.

In summary, in geographical areas with a low prevalence of viralhepatitis in the blood donor population, the ALT measurement as apreseroconverting marker of hepatitis B or C has no value and wewould advise its elimination from the blood screening.

References

1 Aach RD, Szmuness W, Mosley JW, et al: Serum alanine aminotransferaseof donors in relation to the risk of non-A non-B hepatitis in recipients: Thetransfusion-transmitted viruses study. N Engl J Med 1981;304:989–994.

2 NIH Consensus Development Panel on Infectious Disease Testing for BloodTransfusion: Infectious disease testing for blood transfusions. JAMA 1995;274:1374–1379.

3 Sankary TM, Yang G, Romeo JM, Ulrich PP, Busch MP, Rawal BD, VyasGN: Rare detection of hepatitis B and hepatitis C virus genome by polymer-ase chain reaction in seronegative donors with elevated alanine aminotrans-ferase. Transfusion 1994;34:656–660.

4 Rossini A, Gazzola GB, Ravaggi A, Agostinelli E, Biasi L, Albertini A,Radaeli E, Cariani E: Long-term follow-up of and infectivity in blood do-nors with hepatitis C antibodies and persistently normal alanine aminotrans-ferase levels. Transfusion 1995;35/2:108–111.

5 Busch MP, Korelitz JJ, Kleinman SH, Lee SR, AuBuchon JP, Schreiber GB,Retrovirus Epidemiology Donor Study: Declining value of alanine amino-transferase in screening of blood donors to prevent postransfusion hepatitisB and C virus infection. Transfusion 1995;35:903–910.

Emma Castro, MDCentro de Donación de Sangre de Cruz Roja Española, MadridAvda. Reina Victoria 24, pta. bajaE–28003 Madrid (España)Tel. +34 1 534 63 85, +34 1 534 54 76, Fax +34 1 534 64 91E-Mail cds.madrid cruzroja.es

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Antibodies to Hepatitis C Virus in ChandigarhBlood DonorsSudarshan Kumar, S.K. AgnihotriDepartment of Blood Transfusion and Immunohaematology, PGIMER,Chandigarh, India

Donor sera were tested for HCV antibodies in order to know theseroprevalence amongst the Chandigarh voluntary and replacementblood donors. A total of 780 samples from random blood donors weretested with Ortho HCV 3.0 ELISA (Ortho Diagnostic System). Cut-offvalues were calculated according to the manufacturer’s instructions;the samples with optical densities equal to or greater than the cut-offvalues were interpreted as positive. The positive results were con-firmed on the same sample by repeat testing with Ortho HCV 3.0ELISA and then by Innotest HCV Ab 111separately. All first-time pos-itive samples showed positive results on repeat testing. The results ofthe study are presented in table 1. All these donations were also testedfor HIV, hepatitis B surface antigen and syphilis. One anti-HCV-posi-tive donor also showed HBsAg positivity. The ø2 test with Yates cor-rection was applied to the samples positive at the dual test samples; itsvalue is 2.286 which is not significant. The HCV seroprevalence rateof 0.89% in this study is comparable with rates in France, Italy andLibya [1] and is lower than in Japan [1], Saudi Arabia [2] and Qatar [3].However, the seroprevalence was much higher amongst the replace-ment donors, when compared with the non-remunerated voluntarygroup. Another important observation is that all the anti-HCV-positivedonors were males, under the age of 40 years.

Letters to the Editor 259

Our findings, though in a small sample, argue in favour of the en-couragement of altruistic, non-paid voluntary donors, as a means ofincreasing the safety of the blood supply when compared with replace-ment donors or paid donors.

References

1 Choo QL, Weiner AJ, Overby LR, Kuo G, Houghton M: Hepatitis C virus:The major causative agent of viral non-A non-B hepatitis. Br Med Bull1990;46:423–441.

2 Saeed AA, Admawi AM, Rasheed AM, Fairclough D, Bacchus R: Hepati-tis C virus infection in Egyptian volunteer blood donors in Riyadh. Lancet1991;338:459.

3 Lema AM, Cox EA: Hepatitis C antibodies among blood donors in Qatar.Vox Sang 1992;63:237.

Dr. Sudarshan Kumar, Assist. Prof.Department of Blood Transfusion and Immunohaematology, PGIMERChandigarh-160012 (India)Tel. +91172 541467-71, Fax +91 0172-540401

Table1. HCV antibodies in blood donors

Category Testedn

HCV-Abpositive

n %

Replacement donors 391 6 1.53Voluntary donors 389 1 0.25

Total 780 7 0.89

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Concomitant Alloanti-Jka in a Patient with anIgA AutoantibodyRobert Zimmermann, Volker Weisbach, Jürgen Zingsem,Anke Glaser, Reinhold EcksteinDepartment of Transfusion Medicine, Hospital of the University Erlangen-Nürnberg, Erlangen, Germany

A 65-year-old-woman, blood group 0 RhD positive, suffered fromautoimmune haemolysis since 1982, complicated by cholecystolithia-sis requiring endoscopic papillotomy (March 7, 1996) and laparoscop-ic cholecystectomy (March 13, 1996). Preoperative evaluations onMarch 6, 1996, revealed a haemolytic anemia with haemoglobin at9.4 g/dl. The standard direct antiglobulin test (DAT) was negative withanti-IgG, anti-IgM and anti-C3d, positive with anti-IgA and weaklypositive with polyspecific antiglobulin serum composed of anti-IgGand anti-complement (BAG, Lich, FRG). Screening for alloantibodieswas negative using low-ionic-strength solution (LISS) in the indirectantiglobulin test (IAT). The gel centrifugation technique revealed apanagglutinating autoantibody with broad specificity without varia-tions in the strength of reactions demonstrable in the patient’s serumand in the red cell eluate. On March 12, 1996, she received two units ofpacked red cells because her hemoglobin level had dropped to 6.8 g/dl.

Eight days later, the hemoglobin level decreased once more to 4.8 g/dl.An IgG alloanti-Jka was now detectable in the LISS-IAT. The titer was1/2 and rose to 1/8 on March 28, 1996. After transfusion of two units ofJKa-negative RBCs, the haemolysis declined without further transfu-sion requirement.

To our knowledge, this is the first case of an IgG alloanti-Jka in apatient with a warm-reactive IgA autoantibody. The weak reaction ofthe patient’s RBC with a polyspecific antiglobulin serum might be dueto IgG or C3d which were not detectable with monospecific antiglobu-lin reagents. However, it is known that some examples of IgA autoanti-bodies may activate complement [1, 2]. Some other examples of al-loanti-Jk have been reported in patients with autoimmune haemolysis[3, 4]. However, these reports did not differentiate between IgG andIgA autoantibodies. The alloanti-Jka could be detected without ab-sorption studies using the LISS-IAT. This finding is in agreement withIssitt et al. [5] who demonstrated the central role of the LISS-IAT insearching for alloantibodies in sera of patients with red cell autoanti-bodies.

References

1 Sokol RJ, Booker DJ, Stamps R, Booth JR: Autoimmune hemolytic anemiadue to IgA class autoantibodies. Immunohematology 1996;12:14–19.

2 Sokol RJ, Booker DJ, Stamps R, Booth JR, Hook V: IgA red cell autoantibo-dies and autoimmune hemolysis. Transfusion 1997;37:175–181.

3 Sokol RJ, Hewitt S, Booker DJ, Morris BM: Patients with red cell autoanti-bodies: Selection of blood for transfusion. Clin Lab Haematol 1988;10:257–264.

4 Wallhermfechtel MA, Pohl BA, Chaplin H: Alloimmunization in patientswith warm autoantibodies. Transfusion 1984;24:482–485.

5 Issitt PD, Combs MR, Bumgartner DJ, Allen J, Kirkland A, Melroy-Cara-wan H: Studies of antibodies in the sera of patients who have made red cellautoantibodies. Transfusion 1996;36:481–486.

Robert Zimmermann, MDAbteilung für Transfusionsmedizinin der Chirurgischen KlinikFriedrich-Alexander-Universität Erlangen-NürnbergKrankenhausstrasse 12D–91054 ErlangenTel. +49 9131 856972, Fax +49 9131 856987