Inactivation of Leukocytes in Platelet Concentrates by Photochemical Treatment With Psoralen Plus UVA

Abstract

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.

Elimination of cytokine production in stored platelet concentrate aliquots by photochemical treatment with psoralen plus ultraviolet A light

“…In addition, the in vivo recovery and survival of platelets treated with S‐59 and UVA light have been evaluated in primates 13 . Inactivation of passenger WBCs in PCs has also been shown 14 . This report describes the potential additional benefit of preventing cytokine synthesis by passenger WBCs.…”

“…An estimate of levels of DNA strand breaks after γ‐radiation based on published data indicates approximately 0.03 breaks per 1000 bp (2 breaks/10 8 Da/10,000 cGy) 25 . The high level of DNA modification in PC samples treated with 150 μ M S‐59 and UVA light suggests that PCT should also be effective in preventing graft‐versus‐host disease 14 …”

“…This report suggests that PCT with S‐59 also results in the inhibition of cytokine synthesis by WBCs. Additional data that demonstrate WBC inactivation are presented elsewhere 14 …”

Inhibition of murine GVHD by PEN110 treatment

“…A variety of approaches have been developed to deplete or inactivate the WBCs that are present in blood components to minimize the role of the WBCs in triggering immune responses. Depletion is accomplished by WBC reduction and inactivation by exposure to gamma irradiation, UV‐B light, or psoralen plus UV‐A light 4‐7 . PEN110 (Inactine, V. I.…”

Evaluation of processing characteristics of photochemically treated pooled platelets: target requirements for the INTERCEPT Blood System comply with routine use after process optimization

“…The INTERCEPT Blood System provides an effective ex vivo method for inactivating blood‐borne pathogens and leucocytes (Lin et al ., 1993, 1998; Grass et al ., 1998) in platelet concentrates stored in a mixture of approximately 35% autologous plasma and 65% PAS III (InterSol®). Besides high efficiency against blood‐borne pathogens, such techniques should be safe for platelets.…”