No abstract

Photochemical treatment of platelet concentrates offers the potential for reducing transfusion-related viral and bacterial diseases.

A photochemical treatment (PCT) process using a novel psoralen and long wavelength ultraviolet light (UVA, 320-400 nm) has been developed to inactivate bacteria and viruses in platelet concentrates. This study evaluated the efficacy of PCT for inactivation of leukocytes that contaminate platelet preparations. Three psoralens, 8-methoxypsoralen (8-MOP), 4′-aminomethyl 4,5′,8-trimethylpsoralen (AMT), and the novel psoralen S-59, were compared using the following four independent but complementary biological and molecular assays. (1) T-cell viability: Treatment with 150 μmol/L S-59 and 1.0 to 3.0 Joules/cm2 UVA inactivated >5.4 ± 0.3 log10 of T cells in full-sized single-donor plateletpheresis units. Using 1.0 Joule/cm2 UVA, the lowest dose of S-59, AMT and 8-MOP required to reduce the number of T cells to the limit of detection was 0.05 μmol/L, 1.0 μmol/L, and 10.0 μmol/L, respectively. (2) Cytokine synthesis: Treatment with 1.9 Joules/cm2 UVA and 150 μmol/L S-59 or AMT completely inhibited synthesis of the cytokine IL-8 by contaminating leukocytes during 5 days of platelet storage. After treatment with 75 μmol/L 8-MOP and 1.9 Joules/cm2 UVA, only low levels of IL-8 were detected. (3) Psoralen-DNA adduct formation: The combination of 1.9 Joules/cm2 UVA and 150 μmol/L S-59, AMT, or 8-MOP induced 12.0 ± 3.0, 6.0 ± 0.9, and 0.7 psoralen adducts per 1,000 bp DNA, respectively. (4) Replication competence: Polymerase chain reaction (PCR) amplification of small genomic DNA sequences (242-439 bp) after PCT was inhibited. The degree of PCR amplification inhibition correlated with the level of adduct formation (S-59 > AMT > 8-MOP). In contrast, 2,500 cGy gamma radiation, a dose that inactivates >5 log10 of T cells in blood products, had minimal effect on cytokine synthesis and did not induce sufficient DNA strand breaks to inhibit PCR amplification of the same small DNA sequences. These results demonstrate that leukocytes are sensitive to PCT with psoralens and among the psoralens tested S-59 is the most effective. Therefore, PCT has the potential to reduce the incidence of leukocyte-mediated adverse immune reactions associated with platelet transfusion.

We report an action spectrum for the photoreversal of a psoralen cross-link joining two self-complementary DNA oligonucleotides. The cross-link was formed between two thymines (T) on opposite strands of the DNA oligomers and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT). For comparison, we also present an action spectrum for the photoreversal of the isolated diadduct T-HMT-T. The wavelength dependence for the diadduct photoreversal parallels its absorption spectrum. Both the diadduct and the cross-linked DNA can be photoreversed by exposure to light with wavelengths between 240 and 313 nm. We did not observe photoreversal at 334 nm or above. At least two distinct absorption bands appear to contribute to photoreversal. We measured a quantum yield of 0.16 for photoreversal of the isolated diadduct at wavelengths between 240 and 266 nm. For wavelengths above 280 nm, the quantum yield is 0.30. We also observed a preferential photoreversal at the furan end of the psoralen in the T-HMT-T diadduct. In contrast, the cross-linked DNA oligonucleotides preferentially photoreversed at the pyrone end of the psoralen adduct. The rate constant for photoreversal of the cross-linked DNA is larger than that for the isolated diadduct at wavelengths below 300 nm.

Viability of 5 day-old PCT platelets was less than for control platelets. However, both were within ranges reported for 5 day-old platelets.