Paola Bergamaschi

Extracorporeal photochemotherapy for treatment of acute and chronic GVHD in childhood.

BACKGROUND: Extracorporeal photochemotherapy (EPC) has recently been proposed for the treatment of adults with either acute or chronic GVHD. However, data on children given this therapy are scarce. A Phase I‐II study was carried out on EPC in children experiencing GVHD after allogeneic transplantation of HPCs.

Effect of HLA-Matching Recipients to Donor Noninherited Maternal Antigens on Outcomes after Mismatched Umbilical Cord Blood Transplantation for Hematologic Malignancy

Transplantation-related mortality (TRM) is high after HLA-mismatched umbilical cord blood (UCB) transplantation (UCBT). In utero, exposure to noninherited maternal antigen (NIMA) is recognized by the fetus, which induces T regulator cells to that haplotype. It is plausible that UCBTs in which recipients are matched to donor NIMAs may alleviate some of the excess mortality associated with this treatment. To explore this concept, we used marginal matched-pair Cox regression analysis to compare outcomes in 48 NIMA-matched UCBTs (ie, the NIMA of the donor UCB unit matched to the patient) and in 116 non-NIMA-matched UCBTs. All patients had a hematologic malignancy and received a single UCB unit. Cases and controls were matched on age, disease, disease status, transplantation-conditioning regimen, HLA match, and infused cell dose. TRM was lower after NIMA-matched UCBTs compared with NIMA-mismatched UCBTs (relative risk, 0.48; P = .05; 18% versus 32% at 5 years posttransplantation). Consequently, overall survival was higher after NIMA-matched UCBT. The 5-year probability of overall survival was 55% after NIMA-matched UCBTs versus 38% after NIMA-mismatched UCBTs (P = .04). When faced with the choice of multiple HLA-mismatched UCB units containing adequate cell doses, selecting an NIMA-matched UCB unit may improve survival after mismatched UCBT.

A new automated cell washer device for thawed cord blood units.

BACKGROUND:  The current available techniques to wash out DMSO from thawed umbilical cord blood (UCB) units are based essentially on standard centrifugation in an open system with various degrees of cell loss.

HLA haplotypes and birth weight variation: Is your future going to be light or heavy?

Birth weight is known to be a direct indicator of perinatal mortality and a clear predictor of adult pathologies too. It has been correlated with several causes of mortality in adulthood: low birth weight with diabetes, nephropathy and cardiovascular diseases and high birth weight with autoimmune diseases and cancer. In genome-wide studies, an extended human leucocyte antigen (HLA) region has been linked to birth weight variation. We focused our attention on the HLA haplotypes marked by HLA-A, HLA-B and HLA-DRB1 polymorphisms in 1206 healthy Caucasian newborns belonging to the Cord Blood Bank of Pavia (Italy) and their mothers, aiming to investigate the association between this restricted HLA region and birth weight variation. In our study, the HLA-B*38;DRB1*13 haplotype showed an ascending trend among centiles addressing to the high foetal weight. The HLA-A*02;B*15 haplotype showed a descending trend among centiles addressing to the low foetal weight. Besides the acknowledged correlation between the HLA-A*02 and HLA-B*15 alleles (as well as low birth weight) and type I diabetes and between the HLA-B*38 and HLA-DRB1*13 alleles (as well as high birth weight) and several autoimmune diseases, we cannot predict if our babies, healthy at birth, will suffer from these pathologies during life. Nevertheless, our data point to the HLA telomeric end for markers linked to the low birth weight and to the HLA centromeric end for markers linked to the high birth weight, thus limiting the region involved in birth weight variation, which still represents a useful predictor of disease risk in adulthood.

Peripheral blood progenitor cell mobilization and collection in 42 patients with primary systemic amyloidosis

BACKGROUND: High‐dose chemotherapy followed by an inoculum of autologous peripheral blood progenitor cells (PBPCs) can improve survival in patients affected with primary systemic amyloidosis (AL). It has been documented, however, that the morbidity and mortality of PBPC mobilization and collection in this setting are higher than in patients with other diseases. To minimize the mobilization and collection‐related risks, we developed a multidisciplinary approach involving different specialists to manage AL patients with predominant heart and renal involvement.

Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood.

BACKGROUND 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. MATERIALS AND METHODS 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, underwent soft centrifugation within 48 hours of collection. Platelet-rich plasma was centrifuged at high speed to obtain a CBPC with target platelet concentration of 800-1,200×10(9)/L, which was cryopreserved, without cryoprotectant, below -40 °C. RESULTS During 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×10(9)/L. Total platelet count per CBPC was 11.3±4.9×10(9). Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC. DISCUSSION This study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC.

The plasma levels of soluble HLA-G molecules correlate directly with CD34+ cell concentration and HLA-G 14bp insertion/insertion polymorphism in cord blood donors.

BACKGROUND Cord blood provides haematopoietic stem cells for allogeneic transplantation and, thanks to the naivety of its immune system, has several advantages over other sources of stem cells. In the transplantation setting, the presence of immunosuppressive human leucocyte antigen (HLA)-G molecules has been advocated to prevent both rejection and Graft-versus-Host disease. HLA-G is physiologically expressed throughout pregnancy and is contained in cord blood at birth. Moreover, it has recently been reported that not only cord blood mesenchymal cells, but also CD34+ cell progenies produce soluble HLA-G (sHLA-G). We tried to identify the largest producer of sHLA-G among 85 healthy cord blood donors at Pavia Cord Blood Bank, correlating the sHLA-G concentration with the HLA-G 14bp insertion/deletion (INS/DEL) genotype and CD34+ cell concentration. MATERIALS AND METHODS We measured sHLA-G levels in 36 cord blood plasma stored at -20 °C for 2 months and 49 cord blood plasma stored at -196 °C for 4-6 years, by enzyme-linked immunosorbent assay. All cord blood donors were genotyped for the HLA-G 14bp INS/DEL polymorphism by polymerase chain reaction. For each cord blood unit, we measured the cell concentration by flow cytometry. RESULTS We did not find differences in sHLA-G levels between cord blood plasma aliquots stored for 4-6 years at -196 °C and cord blood plasma aliquots stored for 2 months at -20 °C. We observed a higher sHLA-G concentration in cord blood plasma donors who carried the HLA-G 14bp INS/INS genotype and had higher CD34+ cell concentrations (P=0.006). DISCUSSION This is the first report showing that the best cord blood stem cell donor is also the best sHLA-G producer, particularly if genetically characterized by the HLA-G 14bp INS/INS genotype. If the therapeutic role of sHLA-G molecules were to be finally established in the transplantation setting, our data suggest that cord blood plasma donors can provide a safe source of allogeneic sHLA-G immunosuppressive molecules ready for transfusion.

Impact of CTLA4 genotype and other immune response gene polymorphisms on outcomes after single umbilical cord blood transplantation

We evaluated the impact of recipient and cord blood unit (CBU) genetic polymorphisms related to immune response on outcomes after unrelated cord blood transplantations (CBTs). Pretransplant DNA samples from 696 CBUs with malignant diseases were genotyped for NLRP1, NLRP2, NLRP3, TIRAP/Mal, IL10, REL, TNFRSF1B, and CTLA4. HLA compatibility was 6 of 6 in 10%, 5 of 6 in 39%, and ≥4 of 6 in 51% of transplants. Myeloablative conditioning was used in 80%, and in vivo T-cell depletion in 81%, of cases. The median number of total nucleated cells infused was 3.4 × 107/kg. In multivariable analysis, patients receiving CBUs with GG-CTLA4 genotype had poorer neutrophil recovery (hazard ratio [HR], 1.33; P = .02), increased nonrelapse mortality (NRM) (HR, 1.50; P < .01), and inferior disease-free survival (HR, 1.41; P = .02). We performed the same analysis in a more homogeneous subset of cohort 1 (cohort 2, n = 305) of patients who received transplants for acute leukemia, all given a myeloablative conditioning regimen, and with available allele HLA typing (HLA-A, -B, -C, and -DRB1). In this more homogeneous but smaller cohort, we were able to demonstrate that GG-CTLA4-CBU was associated with increased NRM (HR, 1.85; P = .01). Use of GG-CTLA4-CBU was associated with higher mortality after CBT, which may be a useful criterion for CBU selection, when multiple CBUs are available.

Intragenomic conflict of maternal HLA haplotypes: A potential link between vigorous intrauterine growth and risk of autoimmunity in adulthood

The haplotype segregation rate ofMendelian heredity is ordinarily taken for granted. Nevertheless, there are numerous examples of outsider genes that preferentially perpetuate themselves in the population by biasing the Mendelian process; this mechanism is known as intragenomic conflict. A clear mechanism of intragenomic conflict operates in the human leukocyte antigen (HLA) locus, HLA-DR, favoring the transmission of HLADR3 and HLA-DR4 alleles, even though both predispose an individual to diabetes (Noble and Valdes, 2011). In diabetic families, an unexpectedly high proportion (>50%) of newborns carry HLA-DR3 or HLA-DR4 allele, and conversely a disproportionate number of fetuses with an allele other than HLA-DR3 or HLA-DR4 appear to have died in utero. Thus, the inheritance of HLA-DR3 and HLA-DR4 alleles is undoubtedly favorable for intrauterine growth, despite predisposing the carriers to developing diabetes during their lifetime. We investigated the presence of intragenomic conflict in HLA maternal transmission by examining the HLA two-point and three-point haplotypes (marked by HLA-A, HLA-B, and HLA-DRB1 polymorphisms) of 1,206 infant cord blood donors and their corresponding mothers enrolled to the Pavia Cord Blood Bank, following the FACT Netcord international standards (Suppement 1). HLA-A, HLA-B, and HLA-DRB1 polymorphisms were analyzed by PCR techniques, and each genotyping was unambiguously assessed following the European Federation of Immunogenetics recommendations. Fetal HLAhaplotypeswere assignedbymaternal segregation. We found several two-point and three-point, preferentially inherited (PI) and non-inherited (PnI) HLA haplotypes from the maternal side (Table 1). As our sample includes only healthy neonates born from physiological pregnancies (Suppement 1), we conclude that the HLA PI haplotypes are favorable for intrauterine growth, whereas the HLA PnI haplotypes are disadvantageous. In the literature, the majority of the HLA PI haplotypes correlate with adult autoimmune conditions, whereas only two HLA PnI haplotypes correlate with infant autoimmune conditions (Table 1). Further, wepreviously observed a correlation between fetal growth and specificHLA haplotypes connected to the risk of developing autoimmune conditions: the Ancestral Haplotype AH8.1 (HLA-A*01;B*08;DRB1*03) is overrepresented in high-weighted babies (Capittini et al., 2009) despite its association with a wide range of immune-mediated pathologies in adulthood (Price et al., 1999). Theantagonistic pleiotropy theory suggests that reproductionhasacost in longevity: a genetic variation that favors higher fitness will be advantaged in future generations, even if this variation has possible deleterious effects during lifetime. Our observations support the hypothesis that certain HLA haplotypes with initial beneficial effects (improve fetal survival in uterus) prevail, despite their negative effects on adult health (increase the risk of developing autoimmune diseases). In particular, our healthy cord blood donors have preferentially inherited a typology of maternal HLA haplotypes associated with adult autoimmune diseases (Table 1) that present beyond the fertile age, and thus have no influence on fecundity. The preferential inheritance of specific HLA haplotypes (like HLA-DR3 and HLA-DR4 alleles in diabetic families) represents a clear instance of an evolutionary-balanced risk for fetuses, that is a reduced risk of miscarriage in return for a higher risk of developing an autoimmune disease. This concept of fetal protection-at-a-price may help us decipher an evolutionary context to autoimmune diseases. Yet, wemust still understand the immune mechanisms balancing vigorous intrauterine growth (where the mother tolerates the fetal allograft) with adult autoimmunity (where the immune system lost the self-recognition).

On the benefit of blood group molecular genotyping in cord blood banking

Daniels et al. reported on the 3rd International Workshop on Molecular Blood Group Genotyping as a basis for the forthcoming requirement of an inter-Laboratory quality control system for those dealing with erythrogenomics [1]. DNA-based methods for blood group genotyping are spreading worldwide because of their accuracy compared with traditional serologic techniques [2]. Typing for red-blood-cell (RBC) polymorphisms at the DNA level is advantageous in several situations, such as the management of multiply transfused recipients, the prevention of anti-D alloimmunization due to weak RhD variants in blood donors, and the typing of foetuses using amniocytes or cell-free DNA from maternal plasma [3]. We believe molecular blood grouping could also be useful in cord blood (CB) banking. In this setting, serologic blood grouping is usually carried out on fresh CB samples, but is inaccurate after thawing because of haemolysis. The blood group of the newborn itself is not an acceptable substitute. Therefore, if serologic blood grouping has not been carried out on a fresh sample, it is unavailable. And as the CB blood group is a requirement for validation, according to FACT-Netcord international standards [4], CB units lacking it cannot be offered for transplantation, thus wasting human resources. Based on the availability of umbilical cord DNA, we used molecular RBC genotyping in seven out of 3395 units in our CB Bank that were potentially suitable for validation except for lack of blood grouping. ABO and Rh-CDE genotypes were determined by PCR-SSP using the commercial kit READY-GENE ABO and READY-GENE CDE (INNO-TRAIN Diagnostik GmbH, Kronberg ⁄ Taunus, Germany). Genomic DNA was obtained after CB storage times ranging from 1 to 12 years, and from various sources, as shown in Table 1. Molecular genotyping was possible on all the samples, with ABO and Rh polymorphisms as detailed in the table. With minimal increase in initial costs, DNA-based ABO and Rh typing enhanced our CB Bank by validating units otherwise unsuitable, and recovering the cost of their characterization and maintenance. As CB Banks are often in transfusion services, or have their testing done there, we believe this is one more reason to encourage the use of DNA-based methods in transfusion practice. In this setting, the immunogenetics laboratory may reasonably share methods and equipment with an erythrogenomics unit.