Marcia da Silva Cardoso

Sensitivity of HCV RNA and HIV RNA blood screening assays

BACKGROUND: The FDA requirement for sensitivity of viral NAT methods used in blood screening is a 95‐percent detection limit of 100 copies per mL, whereas the NAT screening system should have a sensitivity of at least 5000 copies per mL per individual donation. According to the Common Technical Specifications of the European Directive 98/79/EC for in vitro diagnostics, viral standard dilutions (calibrated against the WHO standard) should be tested at least 24 times for a statistically valid assessment of the 95‐percent detection limit.

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.

Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

The isolation and characterization of human monoclonal antibodies (humAbs) against the hepatitis C Virus (HCV) glycoproteins E1 and E2 are described. B‐cells from blood donors with anti‐HCV were transformed with Epstein‐Barr virus. The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus RMPA95 expressing the envelope proteins E1 and E2 of an HCV genotype 1a virus (H strain). Positive clones were fused to the heteromyeloma cell line K6H6/B5. Fifteen heterohybridoma cell lines have been established. The specificity of the isolated humAbs was determined both by ELISA and Western blot assays. Several recombinant extracts expressing either the E1 or E2 protein or truncated forms were used in an attempt to map the epitopes on the viral glycoproteins. Some of the humAbs were used successfully for immunofluorescence investigation of transfected cells. Seven specific anti‐E2 humAbs, which react with the envelope protein 2 of genotype 1a and 1b isolates, were characterized. J. Med. Virol. 55:28–34, 1998.

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

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.

Autologous platelet transfusion in alloimmunized patients with acute leukemia.

Seventy-eight transfusions of autologous platelets were given to eight alloimmunized patients receiving curative chemotherapy for acute leukemia. Platelets were collected at regeneration of hematopoiesis after a chemotherapy cycle, cryopreserved with 5% dimethylsulfoxide in liquid nitrogen, and retransfused during bone marrow aplasia following the next treatment cycle. The in vitro platelet recovery after freezing, thawing, and washing was 85 ±4%. The in vivo corrected count increment 1 h after autologous platelet transfusions was 11±5×109/l. With the exception of moderate urticaria and slight nausea each after one transfusion, no immediate or chronic side effects occurred. The bleeding time was shortened and hemorrhage during bone marrow aplasia was prevented in all alloimmunized patients by autologous platelet transfusions.

Hemovigilance survey of pathogen-reduced blood components in the Warsaw Region in the 2009 to 2013 period.

In 2009 the Mirasol Pathogen Reduction Technology (PRT) was introduced to the routine blood component production of the Regional Blood Transfusion Center in Warsaw (RBTCW). The goal of this study was to investigate the safety of Mirasol‐treated blood components.

The first case of HCV seroconversion after 3 years of HCV NAT screening in Baden‐Württemberg

The German Red Cross Blood Transfusion Service of BadenWürttemberg introduced HCV NAT screeening of blood donations in March 1997. This screening was applied to minipools of 96 samples, which were pipetted with a robot (Genesis, TECAN, Crailsheim, Germany).1 Since then, improvements in the original protocol were carried out, so that the sensitivity of the assay increased from 750 IU per mL to 30 IU per mL and the rate of false-positive results fell from 0.5 percent to less than 0.1 percent.2 During this time, over 1,300,000 blood donations were screened by HCV NAT with anti-HCV screening performed simultaneously. Of those, 1 preseroconversion (windowperiod) donation was identified. This donation was from a 22-year-old female donor whose sexual partner, a drug abuser, had been released from prison some 3 months before. The partner was on medical surveillance after having had an episode of hepatitis. The couple also performed mutual tattooing with unsterilized instruments. In Table 1, one can see the results of all tests performed on the sequential samples taken from the donor. To certify that such a viremic donation could be picked up even with the least sensitive method that was used at the beginning of NAT screening, a minipool of 96 negative samples was spiked with 100 μL of this preseroconversion donation. This pool was then treated as at the time of introduction of NAT screening; that is, the nucleic acid isolation was performed manually on 200 μL of pool material. Fifty μL out of the 200-μL eluate was used for amplification. The pool tested positive, as it did when tested with the most sensitive protocol using a kit (NucliSens Extractor, Organon Teknika, Boxtel, the Netherlands) with 2000 μL of pool material and taking 25 μL out of a 50-μL eluate for further amplification. Given that the NAT screening was able to detect all anti-HCV-negative/HCV RNA-positive (preseroconversion) donations, as indeed is the case for the anti-HCV/HCV RNA-positive donations collected by the German Red Cross Blood Transfusion Service of Baden-Württemberg since the introduction of NAT in 1997, we determined a frequency of 1 HCV-seronegative donation in 1,300,000.2 Using the mathematical model from Kleinman et al.,3 the 70-day window period for antibody detection by the third-generation screening test, and the incidence rate of seroconversion of our blood donor population (1.6/100,000), we came to a residual risk estimation of 1 in 300,000 (1/80,000 to 1/ 1,200,000 with a 95% CI). On the other hand, with an HCV RNA window period of 40 days, a value of 1 in 560,000 (1/ 170,000 to 1/3,700,000 with a 95% CI) will be reached. Considering that there was no reported case of HCV transmission through transfusion of blood components from our Blood Transfusion Service in the past 3 years, we believe that the last calculation is the most correct way of describing our blood donor population with respect to HCV residual risk. Marcia da Silva Cardoso, PhD Klaus Koerner, PhD Bernhard Kubanek, MD German Red Cross Blood Transfusion Service of Baden-Württemberg and the Department of Transfusion Medicine University of Ulm Helmholtzstrasse 10 89081 Ulm, Germany e-mail: marcia.cardoso@medizin.uni-ulm.de

Safety of Blood Products Derived from Plasma Pools: The Positive Impact of Anti‐HCV Screening on the Quality of Such Products

Anti‐HCV screening of volunteer blood donors was introduced by the Red Cross Blood Service of Baden‐Württemberg (over 400,000 donations/year) in June 1990. At that time, donors were tested with a first‐generation screening test, which was able to detect antibodies to a single recombinant HCV antigen: c100‐3. Test performance was neither very sensitive nor specific [ 1 ]. The anti‐HCV prevalence observed then was 0.53%. The second generation of screening tests relied on the detection of antibodies to three HCV recombinant antigens c100‐3, c22‐3 and c33c showing improved sensitivity and specificity [2]. It was implemented in Baden‐Württemberg in November of 1991. Anti‐HCV prevalence reached 0.47% after the implementation of this test. Finally, since June 1994 all blood donations are screened with the third generation of antibody tests, which includes the recombinant antigen NS5 and some biochemical modifications on the c33c antigen. The present anti‐HCV prevalence in this population is 0.23%.