Paolo Rebulla

In addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel. Cord blood units collected at public banks with total nucleated cell counts <1.5×10(9), platelet count >150×10(9)/L and volume >50 mL, ...

... Reasons for discard of umbilical cord blood units before cryopreservation. Lucilla Lecchi ScD 1 ,; Ilaria Ratti ScD 2 ,; Lorenza Lazzari ScD 2 ,; Paolo Rebulla MD 2 ,; Girolamo Sirchia MD, FRCP Edin 2. Article first published online: 24 APR 2002. ...

1. Transfus Med Rev. 1999 Jul;13(3):205-26. Practical placental blood banking. Rebulla P, Lecchi L, Porretti L, Poli F, Ratti I, Mozzi F, Sirchia G. Milano Cord Blood Bank, Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore, Italy. ...

To the Editor: In a recent editorial,’ Vogelsang discussed the role of blood components from donors homozygous for an HLA haplotype that appeared to be shared with the recipient in the pathophysiology of posttransfusion graft-versus-host disease (PTGVHD) in immunocompetent hosts. This phenomenon has recently been documented after surgery in patients transfused with blood components from the recipients’ family In a retrospective analysis of two cases of suspected PTGVHD occurring in our institution during the last 10 years, we noticed that both patients had received platelet concentrates from their fathers during the course of chemotherapy for non-Hodgkin’s lymphoma. This led us to postulate that, a similar mechanism, that is, partial HLA compatibility, could be involved in PTGVHD in immunocompromised recipients, in whom this complication is more often seen. To test this hypothesis, we reviewed the literature4 and found 26 case reports of PTGVHD in patients receiving chemotherapy for hematologic or solid neoplasms, who had received blood transfusions. Seven (28%) of these 26 patients had received blood components from familial donors, mainly parents, children, or siblings, which strongly suggests a role for HLA-A,B,DR haploidentical transfusions. Moreover, in 3 of these 7 patients and in 6 of the 19 who received blood only from unrelated donors, the supposed association was further supported by HLA typing and/or cytogenetic studies. Analysis of these data provided evidence for a role for partially HLA class Iand/or class 11-matched blood products in 5 patients.2 Among the remaining 4 patients, an HLA-A,Bmismatched transfusion was involved in 2 (for whom class I1 typing was not available), and laboratory data were inconclusive in the others. Thus, PTGVHD in cancer patients receiving conventional chemotherapy also seems to be closely related to the transfusion of partially HLA-matched blood components. It appears probable that the greater the degree of immunosuppression, the lower the degree of donor HLA compatibility required to enable immunocompetent allogeneic cell engraftment. Transfusions of such “unrejectable” blood products are generally random events, which probably explains the previously unsuccessful search for predictive risk factors in this rare transfusion complication in immunocompromised patients and its higher frequency in countries with a relatively homogeneous immunogenetic background, such as Japan. In the light of these data, we agree with Vogelsang that directed blood donations from the patients’ family members should be avoided, particularly as no scientific evidence of their lower risk of transfusiontransmitted infections has been reported.’ When unavoidable, irradiation of such fresh or stored blood components might be preferable. We also postulate that unrelated, partially HLA class I-matched fresh platelet concentrates, frequently employed in HLA-immunized thrombocytopenic patients, clearly increase the risk of PTGVHD. Such “non-random” singledonor platelet concentrates should be irradiated as well, regardless of the immunologic status of the recipient. FRANCOIS CHARPENTIER, MD CHRISTINE BRACQ, BSc Gustavc Roussy Institute 1.

A number of ex vivo expansion systems have recently been developed to extend the clinical use of cord blood HPCs to adult recipients. In previous studies, we identified a cytokine cocktail including thrombopoietin (TPO; 10 ng/ mL), Flt-3 ligand (FL; 50 ng/mL), IL-6 (10 ng/mL), and IL11 (10 ng/mL) in serum-free medium (StemPro 34 SFM, Life Technologies, GIBCO BRL, Grand Island, NY), which was able to induce significant and sustained ex vivo expansion of primitive HPCs from cord blood for up to 10 weeks.1 Our experiments had shown that expansion could be slightly improved by the use of serum, but we ultimately chose to use a serum-free medium to reduce variability in cell manipulation and, most of all, to identify good manufacturing practice (GMP) conditions suitable for clinical use. In view of clinical applications, our in vitro results were recently corroborated by the in vivo capacity of cord blood cells expanded with our protocol to reconstitute the bone marrow of NOD/SCID mice after transplantation.2 Because several serum-free media have in the meantime become commercially available for this purpose, in this study, we compared the performance of the StemPro medium with two other serum-free media (CellGro SCGM, CellGenix, Freiburg, Germany; and X-Vivo 10, BioWhittaker, Verviers, Belgium). These serum-free media, whose composition is not disclosed in detail, have been developed for optimal growth support of human HPCs and all the formulations include only human-derived or human recombinant proteins, without antibiotics and cytokines. To this aim, we purified CD34+ cells from cord blood (n = 6) by using a positive selection system (Miltenyi Biotec, Bergisch Gladbach, Germany). The analysis of the CD34+ cell-enriched fraction, performed by flow cytometry with a CD34 MoAb (HPCA-2, Becton Dickinson [BD], San Jose, CA), revealed a purity of 79 to 90 percent. The CD34+ cells purified from each of the 6 units were split into three subsamples, which were then cultured in stroma-free liquid cultures at a concentration of 5 × 103 cells per mL in 24well plates for 2 weeks at 37°C in 5-percent CO2, in the presence of the three commercial media (Medium A: StemPro 34; B: CellGro SCGM; C: X-Vivo 10). All cultures were supplemented with the same cytokine cocktail described above (TPO+FL+IL-6+IL-11). At the onset of culture and after 2 weeks, we determined the nucleated cell, CD34+, CD34+/ 38–/33–, CD34+/38+, and CD34+/33+ cell counts and cell viability (using 7-amino-actinomycin D) by flow cytometry.3 We also determined the CFU–GM content by short-term cultures. The median and range of expansions and the cell viability with different serum-free media are shown in Table 1. Our results indicate that there was significantly less expansion of nucleated, CD34+, CD34+/38–/33–, CD34+/38+, and CD34+/33+ cells and CFU–GM in the presence of Medium C than with the other media. This suggests that that medium, which is often used for gene-marking protocols,4,5 may not provide optimal support to self-renewal of early HPCs and may not sustain ex vivo expansion of CD34+ cells in our ex vivo expansion protocol. Although cell viability with Medium C was lower than that with Media A and B, the difference was not significant. Comparisons of Media A and B also found no statistically significant differences. In conclusion, our data confirm the possibility of using commercial serum-free media in ex vivo expansion protocols. The use of a carefully selected commercial product instead of serum may facilitate the comparison of results between different laboratories and the standardization of the protocols of cell manipulation, which in turn would facilitate the clinical use of ex vivo expanded cells. Lorenza Lazzari, DSc Simona Lucchi, DSc Laura Porretti, DSc Tiziana Montemurro, DSc Rosaria Giordano, MD Raffaella Lopa, DSc Paolo Rebulla, MD Girolamo Sirchia, MD, FRCP Edin Centro Trasfusionale e di Immunologia dei Trapianti

Cell-based therapies promise important developments for regenerative medicine purposes. Adipose tissue and the adipogenic process has become central to an increasing number of translational efforts in addition to plastic and reconstructive surgical applications. In recent experimental clinical trials, human mesenchymal stem cells (MSC) have been proven to be well tolerated because of their low immunoreactivity. MSC are multipotent cells found among mature cells in different tissues and organs with the potentiality to differentiate in many cell types, including osteocytes, chondrocytes and adipocytes, thus being a suitable cell source for tissue engineering strategies. We compared the adipogenic potential of MSC originated from two adult sources as fat pads and bone marrow, and from four foetal sources as umbilical cord blood, Wharton's jelly, amniotic fluid and preterm umbilical cord perivascular cells. Surprisingly, adult MSC displayed higher differentiation capacities confirmed by gene expression analysis on a selected panel of adipogenesis-related genes. Further, an in-depth molecular analysis highlighted the early and vigorous activation of the PPARγ transcription factor-cascade in adipose-derived MSC that resulted to be both delayed and reduced in foetal MSC accounting for their lack of adipogenic potential. Thus, MSC show a different degree of phenotypic plasticity depending on the source tissue, that should be taken into consideration for the selection of the most appropriate MSC type for specific tissue regeneration purposes.

The Oct-4 transcription factor, a member of the POU family that is also known as Oct-3 and Oct3/4, is expressed in totipotent embryonic stem cells (ES) and germ cells, and it has a unique role in development and in the determination of pluripotency. ES may have their postnatal counterpart in the adult stem cells, recently described in various mammalian tissues, and Oct-4 expression in putative stem cells purified from adult tissues has been considered a real marker of stemness. In this context, normal mature adult cells would not be expected to show Oct-4 expression. On the contrary, we demonstrated, using reverse transcription-polymerase chain reaction (PCR) (total RNA, Poly A+), real-time PCR, immunoprecipitation, Western blotting, band shift, and immunofluorescence, that human peripheral blood mononuclear cells, genetically stable and mainly terminally differentiated cells with well defined functions and a limited lifespan, express Oct-4. These observations raise the question as to whether the role of Oct-4 as a marker of pluripotency should be challenged. Our findings suggest that the presence of Oct-4 is not sufficient to define a cell as pluripotent, and that additional measures should be used to avoid misleading results in the case of an embryonic-specific gene with a large number of pseudogenes that may contribute to false identification of Oct-4 in adult stem cells. These unexpected findings may provide new insights into the role of Oct-4 in fully differentiated cells. Disclosure of potential conflicts of interest is found at the end of this article.

In 1999, we implemented an automated platelet cross-matching (XM) programme to select compatible platelets from the local inventory for patients refractory to random donor platelets. In this study, we evaluated platelet count increments in 40 consecutive refractory patients (8.3% of 480 consecutive platelet recipients) given 569 cross-match-negative platelets between April 1999 and December 2001. XM was performed automatically with a commercially available immunoadherence assay. Pre-, 1- and 24-h post-transfusion platelet counts (mean +/- SD) for the 569 XM-negative platelet transfusions containing 302 +/- 71 x 109 platelets were 7.7 +/- 5.5, 32.0 +/- 21.0 and 16.8 +/- 15.5 x 109/l respectively. Increments were significantly higher (P < 0.05, t-test) than those observed in the same patients given 303 random platelet pools (dose = 318 +/- 52 x 109 platelets) during the month before refractoriness was detected, when pre-, 1- and 24-h post-transfusion counts were 7.0 +/- 8.6, 15.9 +/- 16.1 and 9.6 +/- 12.8 x 109/l respectively. The cost of the platelet XM disposable kit per transfusion to produce 1-h post-transfusion platelet count increments >10 x 109/l was euro 447. This programme enabled the rapid selection of effective platelets for refractory patients, from the local inventory.

Platelet concentrates (PC) can be prepared from whole blood donations using two different approaches. An alternative to the classical method of preparing PC from platelet rich plasma (PRP) consists of centrifuging whole blood at high speed so as to concentrate the platelets in the buffy coat (BC), followed by a second centrifugation of the BC (or BC pools) at low speed and collection of the PC in the supernatant.