VALIDATION TEST FOR PATHOGEN INACTIVATION USING THE PCR

TRANSFUSION-ASSOCIATED TRANSMISSIBLE DISEASES SUCH AS HEPATITIS AND AIDS REMAIN A SIGNIFICANT PROBLEM DESPITE EXTENSIVE DONOR SCREENING AND IMPROVED TESTING OF BLOOD UNITS. THE DEMAND FOR BLOOD PRODUCTS FOR SUPPORT OF IMMUNOSUPPRESSED PATIENTS UNDERGOING CHEMOTHERAPY AND ORGAN TRANSPLANTATION INCREASES THE RISK OF TRANSFUSION ASSOCIATED VIRAL DISEASE. THE LONG TERM OBJECTIVE OF THIS PROJECT IS TO DEVELOP A SYSTEM TO INACTIVATE PATHOGENS IN BLOOD PRODUCTS. A PHOTOCHEMICAL DECONTAMINATION (PCD) PROCEDURE HAS BEEN DESCRIBED WHICH UTILIZES 8-METHOXYPSORALEN (8-MOP) AND LONG WAVELENGTH ULTRAVIOLET (UVA) LIGHT TO INACTIVATE PATHOGENS IN PLATELET CONCENTRATES (PC) WHILE MAINTAINING ACCEPTABLE PLATELET FUNCTION (LIN ET AL., BLOOD 74"517, 1989). WE SEEK TO DEVELOP A SIMPLE MEANS TO VALIDATE INACTIVATION OF HUMAN PATHOGENS AFTER TREATMENT BY PCD. WE WILL USE INHIBITION OF THE POLYMERASE CHAIN REACTION (PCR) AS A MEASURE OF THE EXTENT OF THE PHOTOREACTION BETWEEN NUCLEIC ACID AND PSORALEN IN A PCD TREATED PLATELET CONCENTRATE. THE INITIAL DEMONSTRATION OF THIS TECHNIQUE WILL BE A CORRELATION OF THE INHIBITION OF PCR AMPLIFICATION OF CELLULAR DNA FROM A PCD TREATED PC WITH THE KINETICS OF INACTIVATION OF HIV-1 AND THE LEVEL OF PHOTOMODIFICATION OF THE CELLULAR DNA. THIS OBJECTIVE WILL BE ACHIEVED THROUGH THE FOLLOWING INDIVIDUAL SPECIFIC AIMS. 1. HIV-1 INFECTED H9 CELLS WILL BE SEEDED INTO PLATELET CONCENTRATES. THE CONCENTRATES WILL THEN BE TREATED WITH 8-MOP PLUS UVA LIGHT FOR VARIOUS TIMES. THE BIOLOGICAL ACTIVITY OF HIV-1 WILL BE MONITORED BY A RAPID MICROPLAQUE ASSAY (HANSON ET AL., J. CLIN. MICRO. 28: 1990). THE INACTIVATION WILL UTILIZE HIGH INTENSITY UVA LAMPS TO DECREASE THE IRRADIATION TIME BY A FACTOR OF 10. 2. THE EXTENT OF PHOTOMODIFICATION OF CELLULAR DNA WILL BE DETERMINED USING 3H-8-MOP. 3. THE INHIBITION OF PCR WILL BE CORRELATED WITH THE LEVEL OF 8-MOP MODIFICATION OF CELLULAR DNA AND WITH THE LOSS OF BIOLOGICAL TITER OF HIV-1. 4. THE EFFECTS OF PHOTODECONTAMINATION TREATMENT ON THE PLATELETS WILL BE ASSESSED BY MEASURING: PLATELET YIELD, EXTRACELLULAR PH, PO2, PCO2, DENSE BODY (ATP) SECRETION, EXTRACELLULAR LACTATE DEHYDROGENASE AND ALPHA-GRANULE SECRETION. SPECIFIC AIMS 2, 3 AND 4 WILL UTILIZE PLATELET CONCENTRATES SEEDED WITH UNINFECTED H9 CELLS TO ELIMINATE ANY HIV-1, BIOHAZARD IN HANDLING THESE SAMPLES.