Bernd Lambrecht

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.

No evidence for neoantigens in human plasma after photochemical virus inactivation

SummaryPhotodynamic virus inactivation of human fresh plasma mediated by visible light in the presence of the phenothiazine dyes methylene blue or toluidine blue was investigated to determine whether it influences functional, structural, and immunological properties of plasma proteins. The activities of the coagulation factors I, VIII, IX, X, and XI were affected to a certain degree, while those of most other plasma proteins were not. The elution profiles obtained by ion exchange chromatography of untreated and photodynamically treated plasma were almost identical. Using a number of antisera against human plasma and single plasma proteins, different immunochemical techniques revealed identical patterns for untreated and treated plasma. Thus, there was no indication that the photodynamic virus inactivation procedure applied considerably influences the properties of plasma proteins.

Establishment of the first International Repository for Transfusion‐Relevant Bacteria Reference Strains: ISBT Working Party Transfusion‐Transmitted Infectious Diseases (WP‐TTID), Subgroup on Bacteria

Background  Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion‐Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion‐Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods.

Evaluation of the tolerability and immunogenicity of ultraviolet C-irradiated autologous platelets in a dog model

BACKGROUND: The THERAFLEX ultraviolet (UV) platelets (PLTs) pathogen reduction system for PLT concentrates (PCs) operates using ultraviolet C (UVC) light at a wavelength of 254 nm. UVC treatment can potentially alter proteins, which may affect drug tolerance in humans and influence the immunogenicity of blood products. This preclinical study in beagle dogs was designed to evaluate the safety pharmacology of UVC‐irradiated PCs after intravenous administration and to determine whether they are capable of eliciting humoral responses to PLTs and plasma proteins.

In vitro variables of buffy coat–derived platelet concentrates with residual plasma of down to 10% are stably maintained in new-generation platelet additive solutions

Platelet additive solutions (PASs) facilitate plasma recovery and may reduce the risk of plasma‐associated adverse transfusion reactions. Whereas current apheresis techniques are able to produce platelet concentrates (PCs) with reduced residual plasma volumes, it is still technically challenging to prepare PCs with plasma levels less than 20% from whole blood. This study aimed to evaluate the feasibility of producing buffy coat (BC)‐derived platelets (PLTs) with as little as 10% residual plasma and to test the ability of PASs to preserve PLT quality under these conditions.

Blood Donations Indeterminate in HIV‐1 Western Blot Analysed by IgM Immunoblot and Polymerase Chain Reaction

The presence of IgM antibodies to human immunodeficiency virus 1 (HIV‐1) was investigated in blood donor sera which were indeterminate in anti‐HIV‐1 IgG Western blot testing. In 7 of 173 instances out of approximately 1,000,000 blood donation sera with an isolated anti‐p24 IgG produced an anti‐gp41‐45 IgM immunoblot reaction. Applying polymerase chain reaction (PCR) to 29 indeterminate samples out of approximately 125,000 blood donations it was found that 2 of them were IgM‐positive and also contained HIV‐1‐specific DNA sequences. Eleven months later 1 of these 2 donors was retested and found IgM and PCR negative.

Durch Methylenblau/Licht-Behandlung virusinaktiviertes Humanplasma: Herstellung und bisherige klinische Erfahrungen

Bei der Anwendung eines neuen photodynamischen Verfahrens zur Virusinaktivierung von Frischplasma fur den therapeutischen Einsatz werden einzelne Plasmaein-heiten in ihren Plastikbehaltnissen in Gegenwart des Phenothiazinfarbstoffes Methylenblau mit sichtbarem Licht bestrahlt. Dabei lassen sich alle auf dem Markt erhaltlichen Beutelsysteme einsetzen. Die photodynamische Behandlung beeintrachtigt die Aktivitaten von Plasmaproteinen nur wenig. Einer der sensibelsten Parameter ist die Thrombinzeit, die sich in Abhangigkeit von der Belichtungszeit und der Farbstoffkonzentration verlangert: unter den gewahlten Bedingungen (1 h Belichtungszeit bei etwa 50000 lx, 1 μM Methylenblau) um ungefahr 25%. Eine Anwendungsbeobachtungs-Studie zeigte, daβ photodynamisch behandeltes Plasma ebensogut vertragen wird wie konventionelles gefrorenes Frischplasma. Zwischen Februar und Ende Juli 1992 wurden etwa 31000 Einheiten des virusinaktivierten Praparates an niedersachsische Kliniken ausgeliefert.

HIV-2 antibody testing of blood donors with doubtful immunoblot results for HIV-1

SummaryAntibodies against human immunodeficiency virus type-1 (HIV-1) in samples from blood donors are commonly detected by various enzyme-linked immunosorbent assays (ELISA) and by confirmatory tests, e.g., “Western blot” or immunofluorescence tests. Immunoblot reactivity, which is directed only towards the HIV-1 core proteins p 18, p 24 and p 55, may represent false-positive reactions. Out of 125,000 blood donations, 140 were repeatably HIV-1 antibody reactive by ELISA; of these, 20 were doubtful positive sera with isolated p 18 and/or p24 bands in the HIV-1 confirmatory assay. Antibodies to HIV-2 are known to cross-react with these HIV-1 core proteins. We therefore assayed the 20 sera by immunofluorescence and immunoblotting for the presence of antibodies to HIV-2. None of these doubtful HIV-1 antibody positive blood donor sera was found to have antibodies to HIV-2.

Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains

Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion‐Relevant Bacterial Reference Strains (the ‘repository’), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion.

An Electrophoretic Study and Amino Acid Analysis of Blood Proteins before and after Photodynamic Treatment of Human Plasma

Absicht der Studie war die elektrophoretische Untersuchung von Proteinen in Humanplasma, vor und nach der photodynamischen Virusinaktivierung mit dem Phenothiazinfarbstoff Methylenblau (MB). Die Ergebnisse der zweidimensionalen (2D-)Immunelektrophorese von unbehandeltem und photodynamisch behandel-tem Plasma zeigen, daβ die photodynamische Behandlung unter Routinebedingun-gen (1 µM MB und 1 h Belichtung) die elektrophoretische Beweglichkeit der gepruf-ten Plasmaproteine nicht beeinfluβt. Unter harteren Bedingungen (25 µM MB und 2 h Belichtung) kommt es dagegen teilweise zu Alterationen im Albumin und Transferrin, wie die 2D-Immunelektrophorese zeigt. Mit Hilfe der 2D-Immunoblottechnik konnten im photodynamisch behandelten und unbehandelten Humanplasma keine Unterschiede in Albumin, Transferrin, Fibrinogen und Histidin-reichem Glykoprotein nachgewiesen werden. Die Aminosaureanalyse demonstriert, daβ es keine signifikanten Alterationen in den Aminosauren der Plasmaproteine von unbehandeltem und unter Routinebedingungen behandeltem Plasma gibt. Lysin ist die einzige Aminosaure von Plasmaproteinen, die unter den oben genannten extremen Bedingungen alteriert wird.