Harald Mohr

A novel approach to pathogen reduction in platelet concentrates using short-wave ultraviolet light.

BACKGROUND: Transfusion of platelet concentrates (PCs) is the basic treatment for severe platelet disorders. PCs carry the risk of pathogen transmission, especially bacteria. Pathogen reduction (PR) by addition of photochemical reagents and irradiation with visible or ultraviolet (UV) light can significantly reduce this risk. We present a novel approach for PR in PCs employing UVC light alone.

BLOOD COMPONENTS: A novel approach to pathogen reduction in platelet concentrates using short‐wave ultraviolet light

BACKGROUND: Transfusion of platelet concentrates (PCs) is the basic treatment for severe platelet disorders. PCs carry the risk of pathogen transmission, especially bacteria. Pathogen reduction (PR) by addition of photochemical reagents and irradiation with visible or ultraviolet (UV) light can significantly reduce this risk. We present a novel approach for PR in PCs employing UVC light alone.

Virus Inactivation of Blood Products by Phenothiazine Dyes and Light

Methylene blue (MB) and its derivatives azure A, B, C and thionine are photoactive and, in principle, are suitable for photodynamic virus inactivation of blood and blood products, such as therapeutic plasma. Methylene blue was selected for plasma decontamination because it is being clinically used and because of its known toxicological and other properties. The standard procedure for photodynamic treatment of single units of fresh plasma involves illumination with visible light at an MB concentration of 1 microM. Polymerase chain reaction analysis revealed that, in addition to model viruses, the bloodborne viruses hepatitis B virus, hepatitis C virus, human immune deficiency virus-1 and probably also the nonenveloped parvovirus B19 are sensitive to MB/light treatment. The procedure is further improved when the fluorescent tubes routinely used for illumination are replaced by more intense light sources, e.g. light-emitting diodes or low-pressure sodium lamps. Surprisingly, the improved virus kill is accompanied by reduced damage to plasma proteins.

A fluorescence-based assay for quantitation of lymphokine-activated killer cell activity.

A fluorescence assay for the quantitation of tumor cell lysis by activated and non-activated killer (LAK) cells is described. The target cells are labelled with a europium chelate (Eu-diethylenetriaminopentaacetate) and after cytolysis caused by the LAK cells the Eu3+ complex is released into the culture supernatant. The addition of beta-naphthoyltrifluoroacetone to culture supernatant aliquots leads to the formation of a highly fluorescent chelate which can be measured with a time-resolved fluorometer. The influence of various assay parameters has been evaluated including incubation time, effector-to-target cell ratio, the target cell line and different concentrations of interleukin-2 during cell culture. The optimized time-resolved fluorometric assay was found to be as simple and sensitive as the commonly used cytotoxicity assay in which the release of 51Cr from the labelled target cells is measured. In addition the assay is much faster and safer since the label is not radioactive.

Basics of flow cytometry–based sterility testing of platelet concentrates

BACKGROUND:  Flow cytometry (FACS) is a common technique in blood banking. It is used, for example, for the enumeration of residual white blood cells in plasma and in cellular blood products. It was investigated whether it can also be applied for sterility testing of buffy coat–derived platelet concentrates (PCs).

Sterilization of platelet concentrates at production scale by irradiation with short-wave ultraviolet light

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) is recognized as a serious threat to transfusion safety. We developed a simple method for sterilization of PCs with short‐wave ultraviolet light (UVC). The effects of treatment on the sterility of contaminated PCs and in vitro platelet (PLT) variables were evaluated.

Elimination and multiplication of bacteria during preparation and storage of buffy coat-derived platelet concentrates.

BACKGROUND:  The prevalence of bacterial contamination of random‐donor platelet concentrates (PCs) is considerably lower than that of blood donations. Which key steps of the preparation procedure contribute to the elimination of bacteria was investigated.

West Nile virus in plasma is highly sensitive to methylene blue–light treatment

BACKGROUND:  The epidemic of West Nile virus (WNV) in the US resulted in cases of transfusion‐transmitted WNV. Effective pathogen reduction methods could have removed this infectious agent from the blood supply We have evaluated the efficacy of photodynamic treatment of fresh frozen plasma (FFP) with methylene blue (MB), a decontamination method applied in several European countries.

Methods for the detection of bacterial contamination in blood products

Abstract Culture-based and molecular assays have been developed for the screening of platelet concentrates and other blood components for bacterial contaminations. In this review, the principles of the assays are outlined. The focus of this review is the assessment of the analytical qualities of the methods. Spiking studies by adding defined levels of a wide range of bacteria to the complex biological matrix provide the first basis to evaluate and compare the qualities of methods for bacterial detection. The sensitivity acceptable for reliable screening for bacteria critically depends on the timing of either early sampling (within a period of up to 24 h after preparation of the blood component) or late sampling (a few hours before issuing the blood component). Large screening studies are essential to confirm both adequate sensitivity and specificity of the testing. In the ideal setting, these studies are prospectively planned and include systematic surveillance of adverse events in response to the administration of the screened products. The findings from sterility testing (predominantly with automated systems for detection of bacteria based on CO2 generation) of more than 550,000 platelet concentrates in 13 studies are summarised. The limitations of the early sampling and the “negative-to-date” strategy to issue platelet concentrates are addressed. A few reported cases of probable transmission of bacteria by platelet transfusion despite negative screening tests emphasise the need to further develop optimised methods for testing of bacteria blood components. Clin Chem Lab Med 2008;46:933–46.

Hepatitis C and human immunodeficiency virus RNA degradation by methylene blue/light treatment of human plasma

Treatment of human plasma with methylene blue in combination with visible light (MB/light) inactivates several bloodborne viruses such as retro viruses and herpes viruses. The viral nucleic acid is thought to be a critical target for the inactivation procedure. We investigated the effects of photodynamic treatment on the RNA of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV‐1) using Amplicor reverse transcriptase polymerase chain reaction (RT‐PCR), which detects and quantifies a small fragment of the viral RNA. The detectable HCV RNA load (5‐nontranslated region) in infected human plasma declined by 94–97 % within 10 min of illumination in small‐scale experiments (1–2 ml vol.). Since the same effect was observed in both anti‐HCV positive and negative (“window”) samples, it can be concluded that HCV antibodies do not influence virus inactivation by photodynamic treatment. The effect of treatment on RT‐PCR signals of HIV‐1, which is known to be inactivated rapidly by MB/light treatment, was examined. Plasma was infected with HIV‐1 and subjected to RT‐PCR, which detected a part of the gag gene. The extent and kinetics of PCR signal reduction induced by MB/light treatment were similar to those observed for HCV. Experiments at production scale where single plasma units (300 ml) were infected with HCV showed reduction rates of PCR signals consistent with those measured in the small‐scale experiments. The data support the view that MB/light treatment affects the viral nucleic acids and suggest that HCV is susceptible to the procedure. J. Med. Virol. 56:239–245, 1998.