K. Koerner

Mini-pool screening by nucleic acid testing for hepatitis B virus, hepatitis C virus, and HIV: preliminary results

BACKGROUND: The purpose of this study was to evaluate the feasibility of nucleic acid testing (NAT) of mini‐pools as a blood donation screening test.

In vitro Platelet Function during Storage in Three Different Additive Solutions

Three different synthetic media without glucose were studied for platelet storage. The first medium contained acetate and gluconate. The second contained acetate, gluconate and citrate. Finally the third contained phosphate and mannitol. The purpose of the study was to investigate whether there were differences among the various media in terms of preservation of platelet quality. Pools of platelet concentrates were prepared from buffy coats. In vitro function and metabolic parameters were measured during 5 days of storage in these additive solutions as well as in plasma. Platelet aggregation, hypotonic shock response and release of β‐thromboglobulin, platelet factor 4 and lactate dehydrogenase of the cytosol were equivalent in the media containing acetate compared to plasma storage. In vitro platelet functions and pH in these two media were better preserved compared to the medium with phosphate and mannitol. In addition bacteriological studies using platelets suspended in additive solutions or in plasma were carried out. Carryover of 20% of plasma to the synthetic media necessary for successful platelet storage in these additive solutions allows bacteriological growth. As shown, inoculation of 1 colony/ml Staphylococcus epidermidis leads to 106–107 organisms/ml after 5 days of storage.

Platelet Function of Room Temperature Platelet Concentrates Stored in a New Plastic Material with High Gas Permeability

Abstract. In vitro platelet function during 7 days of storage at room temperature was studied in a conventional polyvinylchloride plastic bag F 76 and in a new plastic bag F 702 which contained as plasticizer a phtalateester analogue. This new material has increased permeability to oxygen and carbon dioxide, and therefore a pH decrease does not occur during 7 days of platelet storage. The decrease of plasma glucose concentration and the increase of plasma lactate in the new bag is less than in the standard plastic currently in use. In vitro platelet function measured as hypotonic shock reaction, aggregation response to ADP and collagen and 14C‐serotonine uptake was better than that found with the standard material. The data indicate that the use of the new platelet storage container F702 will permit satisfactory storage for at least 5 days at 22 °C. It is suggested that it will even improve the quality, as measured by in vitro tests, of platelets stored up to 72 h compared to the standard plastic.

Prevalence of HCV-RNA-positive blood donors and correlation to ELISA and RIBA status

SummaryIn Germany, transmission of hepatitis C virus by blood transfusion is prevented by screening the donations for anti-HCV and ALT. The specificity of the anti-HCV screening in low seroprevalence populations has been questioned. In order to evaluate this screening policy we wanted to estimate the prevalence of viremic and potentially infectious donors by the HCV-RNA polymerase chain reaction (PCR) in our donor population of southern Germany. Donors (n=301) were divided into four subgroups according to anti-HCV status and ALT levels. HCV sequences were detected by nested PCR, using primers for the most conserved region of the viral genome. The recombinant immunoblot assay (RIBA-4) was applied to the same samples. PCR detected 4.2% HCV-RNA carriers in the subgroup anti-HCV−/ALT−; 3% in the subgroup anti-HCV−/ALT+; 19.4% in the subgroup anti-HCV+/ALT−; and 59.4% in the subgroup anti-HCV+/ALT+. It was concluded that, on the one hand, the lack of specificity of the anti-HCV ELISA gives rise to many false-positive results; on the other hand, a minority of infected donations will not be detected by the screening procedure. ALT in conjunction with anti-HCV improves the quality of screening for potentially infectious donors.

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314–330 (EP3) covering the central conserved sequence of this domain. Anti‐hepatitis C virus‐positive blood donors were screened for anti‐EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA‐confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti‐EP3‐producing heterohybridoma cell lines: U1/F30 and U1/F31 produced IgM‐κ, whereas U1/F32 and U1/F33 secreted the isotypes IgG1‐λ and IgG1‐κ, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies U1/F32 and U1/F33 have dissociation constants to the peptide of ∼ 10−9 M, binding to recombinant E1 protein expressed in COS‐7 cells was different. Only U1/F33 detected envelope protein of ∼ 24–35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.

In vitro effect on stored red blood cells and platelets after a 15-hour delayed refrigeration of whole blood prior to component preparation in CPD-AD

Abstract. We extended the time of keeping whole blood at 20–24 °C to 15 h (overnight) after phlebotomy for preparing platelet concentrates. We have evaluated the in vitro characteristics of platelets and blood cells prepared from whole blood drawn into CPD‐AD, an anticoagulant containing 0.4 mM adenine and 1.5 times more dextrose than CPD. We studied in vitro red cell and platelet function of blood cooled either within 4 h after collection or after a 15‐hour delay. In vitro platelet function measured as hypotonic shock reaction, aggregation response to ADP and collagen and 14C‐serotonin uptake were not significantly different after preparation and after a 5‐day storage period. Units held at room temperature for 15 h after blood collection exhibited a level of 2,3‐DPG that was 45% of that exhibited by red cells held for 15 h at 1–6 °C. All other in vitro parameters of red cell concentrates measured during 35 days of storage were not significantly different. Based on these in vitro data blood drawn into CPD‐AD might be kept up to 15 h at room temperature prior to refrigeration in order to prepare platelet concentrates.

Hepatitis C virus stability: the issue!

One of the major challenges concerning the implementation of nucleic acid testing (NAT) as a blood donor screening method is the question of how to store blood samples in order to get reliable results. Many reports have been published which quote the hepatitis C virus (HCV) as very unstable in serum; one of them even condemned the storage of clotted blood for longer than 2 h [1–3]. These findings suggest that for the appropriate NAT screening of blood donations the current sampling and storage conditions of blood samples have to be completely changed. However, sophisticated storage conditions might not always be practicable for the blood transfusion services with mobile blood collection units. Observations made in the German Red Cross Blood Transfusion Service of Baden-Württemberg (DRK-BSD BaWü) have always suggested that HCV is stable in serum samples which are kept at 4°C, even without separation of serum from the clot [4]. The study reported here was designed to evaluate variables such as HCV stability in serum versus plasma, individual stability of different sera and HCV-RNA titer in different blood components. Moreover, the current sampling, transport and storage conditions of blood samples collected by mobile units of the DRKBSD BaWü were taken into account when investigating these parameters. Rapid Communication

PCR Screening in the Routine of Blood Banking of the German Red Cross Blood Transfusion Service of Baden-Würtemberg

© 1998 S. Karger GmbH, Freiburg Fax (07 61) 4 52 07 14 www.karger.com Received: October 29, 1997 Accepted: March 9, 1998 Dr. Marcia da Silva Cardoso DRK-Blutspendedienst und Abteilung für Transfusionsmedizin, Universität Ulm Helmholtzstraße 10, D-89081 Ulm (Germany) Tel. +49 731 150-119, Fax -175 E-mail marcia.cardoso@medizin.uni-ulm.de This article is also accessible online at: http://BioMedNet.com/karger PCR Screening in the Routine of Blood Banking of the German Red Cross Blood Transfusion Service of Baden-Württemberg M. da Silva Cardoso K. Koerner B. Kubanek

Quality of pooled platelet concentrates prepared from buffy coats and stored in an additive solution after filtration

Platelet concentrates prepared from buffy coat were pooled and stored for 6 days after removal of leukocytes by filtration. The platelets were stored in plasma or in an additive solution, Plasmalyte-A. In vitro platelet function was better preserved using Plasmalyte-A than plasma with regard to osmotic reversal and aggregation. No significant differences for the release of platelet markersΒ-thromboglobulin, platelet factor 4, or lactate dehydrogenase pre- and post-filtration and storage in plasma or Plasmalyte-A was observed. Expression of the surface membrane glycoproteins Ib, Ia/ IIa, Ilb/IIIa, and IV measured by flow cytometry after binding of monoclonal antibodies did not change during storage. The expression of activation-dependentα- granula glycoprotein GMP140, the thrombospondin, and the glycoprotein 53 from the lysosomal granules was not different between platelet pools stored in plasma or in Plasmalyte-A. The in vitro quality of platelets stored as pools is comparable for plasma and the additive solution Plasmalyte-A.

The serology of hepatitis C virus (HCV) infection: antibody crossreaction in the hypervariable region 1

SummaryWe determined the NS1/E2 N-terminal sequence including the hypervariable region 1 (HVR1) from five individuals chronically infected with HCV: two from the Czech Republic and three from Germany. From each sequence, six 12-mer overlapping peptides were synthesized and used in a peptide scan to evaluate seroreactivity of each of those patients, as well as three anti-HCV positive blood donors to the different isolates. We could show the general presence of antibodies to multiple HVR1 specific sequences reflecting the existence of multiple variants in infected persons. Finally, we observed the persistance of HCV infections in all individuals despite an active humoral response directed against the virus.