Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

BACKGROUND: Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. METHODS: PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. RESULTS: PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic medium was irrelevant as compared to PL and PI-PL. CONCLUSION: The replacement of animal additives with human supplements is a basic issue in MSC ex vivo production. PI-PL represents a standardized, plasma-poor, human preparation which appears as a safe and good candidate to stimulate MSC growth in clinical-scale cultures.

Residual risk of HIV-1 transmission: the case of a seroconverter.

In the present report we describe the case of a repeat blood donor infected with HIV-1. In January 2000 the donor was found to be repeatedly reactive to HIV1/2 antibodies and HIV-1 RNA screening tests. The donation was confirmed to be HIV-1 positive by Western blot. During the post-test counselling session, the donor reported a risk sexual behaviour denied during the pre-donation interview, and he recalled that in May 1998 he had undergone a check-up including the test for the detection of HIV1/2 antibodies, which was negative. This check-up was dated four months the next to the donor's previous donation in January 1998, which had been found HIV1/2 antibody negative, too. Serum and plasma specimens, properly stored at -80 degrees C, were available at the hospital where the donor had undergone the HIV antibody test in May 1998. Thus, the specimens dated May 1998 and the specimen of the last donation in January 2000 were investigated again by using the most sensitive tests currently available in the setting of donation screening. On the whole, the results suggest that in May 1998 the donor was in the pre-seroconversion period for HIV-1 infection. The case reported here stresses that a residual risk for HIV transmission through blood products still relies on the possibility that an individual may be accepted as blood donor during the asymptomatic pre-seroconversion window period of HIV-1 infection. Actually, this phase of the infection cannot be detected by the routine antibody/antigen-based HIV1/2 screening tests but only by using more sensitive techniques such as genomic screening.

Identification of low HBV-DNA levels by nucleic acid amplification test (NAT) in blood donors.

OBJECTIVE: To evaluate the presence of HBV-DNA in 22,765 consecutive blood donors, who donated blood in the period from January 2006 to August 2007 at a transfusion centre in Lazio, a region in central Italy with low HBV endemicity. METHODS: Each donation was individually tested using immunoenzymatic assays and nucleic acid amplification technologies (NAT). Samples that were reactive to generic NAT, Procleix Ultrio Assay were tested for HBV-DNA, HCV-RNA and HIV1-RNA by Discriminatory Procleix Ultrio NAT Assay. In samples that were reactive to generic NAT and negative for HBsAg, HCV-RNA and HIV1-RNA, HBV-DNA was further tested using Cobas TaqMan and an in-house nested PCR following an ultracentrifugation step. Sequence analysis confirmed HBV-DNA positivity. RESULTS: Generic NAT identified 31 (0.13%) reactive sera. HBV-DNA discriminatory NAT identified 15 positive sera; HBsAg was positive in 12 sera. Of the 5 generic NAT-reactive/discriminatory NAT-negative/HBsAg-negative sera and of the 3 HBsAg-negative/HBV-DNA discriminatory NAT-positive sera, 7 were positive to Cobas TaqMan or the in-house PCR after ultracentrifugation. The overall HBV-DNA positivity was 0.083% [19 of 22,765 donors: 12 HBsAg-positive (HBV-DNA range 10(2)-10(4) IU/mL), 7 HBsAg-negative/anti-HBc positive (HBV-DNA< 6 IU/mL)]. CONCLUSIONS: For blood transfusion safety, the significance of the finding of very low HBV-DNA levels should be further investigated. Our data indicate that in areas with a low HBV endemicity, single NAT assays may not always identify blood donations with very low HBV-DNA levels.

Characterization of occult hepatitis B virus from blood donors carrying genotype A2 or genotype D strains.

BACKGROUND/AIMS: Nucleic acid testing (NAT) for hepatitis B virus (HBV) DNA in blood donations identified occult HBV infection (OBI) as a potential threat to blood safety. METHODS: A collaborative study was undertaken to explore the molecular basis of OBIs prevalent in Europe in relation to clinical and serological data. RESULTS: Ninety-one percent of 77 donor samples of European origin HBV DNA positive but HBV surface antigen (HBsAg) negative were confirmed. Viral load ranged between unquantifiable and 5640 IU/mL (median 25 IU/mL). Fifty-two strains were genotyped (14 HBV(A2) and 38 HBV(D)). Compared to HBsAg+ samples, genotype D was significantly more frequent than genotype A2 in OBIs from Poland or Italy (P<0.04). Amino acid substitutions were concentrated in the immunologically active parts of the Pre-S/S proteins (P<0.0001) affecting both cellular CD8 T-cell epitopes and B-cell neutralizing Major Hydrophilic Region epitopes. Substitutions were more frequent in OBIs than in HBsAg+ strains of both genotype D (P<0.001) and A2 (P<0.01), in OBIs of genotype D than A2 in the 'a' region (P<0.001) but not cellular epitopes, and in anti-HBs+ than anti-HBs- OBIs (P<0.001). CONCLUSIONS: Results support the hypothesis that humoral and cellular immune pressure on the HBV envelope proteins are major mechanisms generating OBI.