Rafael Fores

Unrelated umbilical cord blood transplants in adults: Early recoveryof neutrophils by supportive co-transplantation of a low number of highlypurified peripheral blood CD34+ cells from an HLA-haploidentical donor

UNLABELLED OBJECTIVE, METHODS, AND RESULTS: To reduce the period of posttransplant neutropenia and related early morbidity and mortality of cord blood (CB) transplants, we assessed the feasibility of co-infusion of a low number of highly purified peripheral blood CD34+ cells from a related haploidentical donor with a CB graft. Between March 1999 and May 2002, 11 patients with high-risk hematologic malignancies were transplanted using this strategy. The seven patients who received a haploidentical peripheral blood graft and a CB graft from a sibling (6) or the father (1) had prompt recovery (9-17 days, median 10) of the absolute neutrophil count (ANC) to greater than 0.5 x 10(9)/L. Analysis of DNA polymorphisms showed initial predominance of the haploidentical genotype both in granulocytes and in mononuclear cells, and subsequent progressive replacement by cells of CB genotype until final complete CB chimerism was achieved by patients who survived for sufficient periods of time. The four patients who received maternal haploidentical cells had no significant contribution of these to blood leukocytes, although complete CB chimerism was achieved by three of them and two reached engraftment of the CB on days +20 and +36. Morbidity due to early bacterial or fungal infections was remarkably low in patients with prompt ANC recovery. CONCLUSION Our data show that co-infusion of a CB unit and a low number of haploidentical CD34+ cells may result in a shortened period of posttransplant neutropenia. This is likely the result of prompt and transient engraftment of the haploidentical hematopoietic stem cells that may provide the patient antimicrobial protection until the later engraftment of the CB hematopoietic stem cells.

Cord blood transplants supported by co-infusion of mobilized hematopoietic stem cells from a third-party donor

This open label clinical study provides updated evaluation of the strategy of single unit cord blood transplants (CBTs) with co-infusion of third-party donor (TPD) mobilized hematopoietic stem cells (MHSC). Fifty-five adults with high-risk hematological malignancies, median age 34 years (16–60 years) and weight 70 kg (43–95 kg), received CBTs (median 2.39 × 107 total nucleated cell (TNC) per kg and 0.11 × 106 CD34+ per kg) and TPD-MHSC (median 2.4 × 106 CD34+ per kg and 3.2 × 103 CD3+ per kg). Median time to ANC and to CB-ANC >0.5 × 109/l as well as to full CB-chimerism was 10, 21 and 44 days, with maximum cumulative incidences (MCI) of 0.96, 0.95 and 0.91. Median time to unsupported platelets >20 × 109/l was 32 days (MCI 0.78). MCI for grades I–IV and III–IV acute GVHD (aGVHD) were 0.62 and 0.11; 12 of 41 patients (29%) who are at risk developed chronic GVHD, becoming severely extensive in three patients. Relapses occurred in seven patients (MCI=0.17). The main causes of morbi-mortality were post-engraftment infections. CMV reactivations were the most frequent, their incidence declining after the fourth month. Five-year overall survival and disease-free survival (Kaplan–Meier) were 56 % and 47% (63% and 54% for patients ⩽40 years). In conclusion, CBT with single units of relatively low cell content and 0–3 HLA mismatches is feasible as a first choice option for adult patients who lack a readily available adequate adult donor.

Cord blood transplants: early recovery of neutrophils from co-transplanted sibling haploidentical progenitor cells and lack of engraftment of cultured cord blood cells, as ascertained by analysis of DNA polymorphisms

The number of infused cells is a very important factor in cord blood transplant (CBT) engraftment. Prior ex vivo expansion of aliquots of transplanted cord blood (CB) units is being investigated as a procedure to increase engraftment potential, but results are difficult to evaluate due to a lack of markers for assessing the contribution of expanded cells. We transplanted five patients, infusing the best available CB unit and cells from a second donor simultaneously. In two patients, these cells were obtained from another frozen CB unit by CD34+positive selection and culture expansion; the other three patients received uncultured highly purified haploidentical CD34+ cells. The first two patients had DNA from the culture expanded CB cells detected only for a few days around day +11 when the absolute neutrophil count (ANC) was >200/μl; thereafter and when the ANC was <500/μl, only donor DNA from the uncultured CB was detected. For the other three patients, DNA analysis showed early and transient granulocyte engraftment of haploidentical cells, progressively replaced by the CB-derived granulocytes. We concluded that: (1) simultaneous infusion of lymphocyte-depleted HLA highly mismatched haematopoietic progenitor cells has not produced unfavourable effects for CBT; (2) the double transplant model is suitable for evaluating the engraftment potential of ex vivocultured CB cells in the clinical setting; (3) the culture conditions used did not result in early recovery of ANC; and (4) co-transplantation of purified uncultured HLA haploidentical CD34+ cells may reduce the time of neutropenia following CBT.Bone Marrow Transplantation (2001) 28, 355–363.

Umbilical cord blood banking for unrelated transplantation: Evaluation of cell separation and storage methods

Cost-efficient umbilical cord blood (UCB) banking requires well-standardized methods of volume reduction and storage. To compare UCB fractionation using a technique of hydroxyethyl starch (HES) sedimentation with the Ficoll (double) and Percoll methods, 50 whole units was allocated randomly to each procedure. HES resulted in a significantly better recovery of mononuclear cells (87.5%), granulocyte/macrophage colony-forming units (CFU-GM) (88.4%), and CD34- cells (87.4%) and lesser volume reduction (85.5%). HES was the least laborious, time consuming, and expensive of the three procedures, costing 3.4- and 4.4-fold less than the Ficoll and Percoll methods, respectively. Five units processed by each method was frozen in 4.5-mL cryotubes under optimal conditions. After thawing, the greatest degree of recovery of viable nucleated cells and number of CFU-GM per unit were obtained using the HES procedure. Using 4.5-mL cryotubes, the calculated number of units that could be stored in 600-L containers was 3.8- and 2.2-fold higher for Ficoll- and Percoll-separated than for HES-separated units, respectively. Nevertheless, the higher direct costs of the density gradient separation procedures outweighed their lower storage cost. For long-term cryopreservation, we assessed the freezing of HES-processed units in 50-mL cryobags and their specifically designed canisters. We found cell recoveries similar to those obtained with cryotubes, but storage capacity was decreased. Special racks designed for these canisters resulted in a 5-fold increase over the number of units stored in standard cryobags. This system also is feasible for Percoll- and Ficoll-separated units, resulting in comparable storage costs for the three separation methods. We conclude that this HES procedure and the 50-mL cryobags constitute a cost-efficient system for large-scale UCB banking.

Epstein-Barr virus associated B-cell lymphoma after autologous bone marrow transplantation for T-cell acute lymphoblastic leukaemia

Epstein‐Barr virus associated lymphoproliferative disease after autologous bone marrow transplantation (ABMT) has rarely been reported. We report a case of B‐cell lymphoma following ABMT for T‐acute lymphoblastic leukaemia; bone marrow was purged in vitro with monoclonal antibodies to remove T cells. Immunoglobulin and T‐cell receptor gene rearrangement studies were used to demonstrate clonality and to show that this patient developed a second neoplasm after ABMT. EBV proteins and genome (type A) were present in post‐transplantation lymphoma, suggesting a causative role in its development.

Diagnosis of transfusion‐associated graft‐versus‐host disease by polymerase chain reaction in fludarabine‐treated B‐chronic lymphocytic leukaemia

Summary. Transfusion‐associated graft‐versus‐host disease (TA‐GVHD), has rarely been reported associated with B‐chronic lymphocytic leukaemia (B‐CLL). We report a patient diagnosed with B‐CLL, previously treated with fludarabine, who developed TA‐GVHD after being transfused during surgery for splenectomy. Diagnosis was confirmed by polymerase chain reaction (PCR) detection of donor DNA in the patient, by amplification of Y‐chromosome sequence and analysis of minisatellite polymorphisms. B‐CLL patients treated with fludarabine appear to be at risk for TA‐GVHD and should be regarded as candidates for transfusions with irradiated blood products. This case illustrates that PCR is a rapid technique for the early diagnosis of TA‐GVHD.

Infusion of lymphocytes obtained from a donor immunised with the paraprotein idiotype as a treatment in a relapsed myeloma

A 48-year-old patient with IgA k multiple myeloma received a BMT from his HLA-matched sibling. After transplantation, the disease relapsed. Melphalan therapy followed by reinfusion of haemopoietic blood stem cells collected from the patient led to the improvement of the clinical status, although mixed chimerism and an elevated serum IgA persisted. Successful donor immunisation against an immunogenic preparation of the recipient monoclonal protein was performed before the infusion of donor T lymphocytes (DLI) into the patient. Ten weeks after the lymphocyte infusions, no monoclonal band was evidenced and donor complete chimerism was detected. The patient did not develop GVHD. Once complete remission was achieved, the idiotype vaccine was administered to the patient. Nineteen months after DLI, the patient remains in remission. Bone Marrow Transplantation (2000) 25, 1105–1108.

Management of thrombotic microangiopathy following allogeneic transplantation: what is the role of plasma exchange?

Thrombotic microangiopathy (TMA) is an infrequent but serious complication of allogeneic transplantation. The success rate of plasma exchange (PE) reported in the treatment of this entity is a controversial subject. We report the outcome of 10 patients with TMA post-allogeneic transplantation after treatment with PE. Two out of the 10 patients have not responded, five had a partial response, but died of acute GVHD or interstitial pneumonitis, and three have responded and recovered. Our study suggests that there are different degrees of TMA severity. Only mild multifactorial cases with no severe hemolysis (LDH activity <1000 u/l) may be fully resolved with pe.

Detection of maternal DNA in umbilical cord blood by polymerase chain reaction amplification of minisatellite sequences

One of the concerns about the use of cord blood as a source of hematopoietic stem cells for allogeneic transplantation is the possibility of contamination by maternal cells which could cause life-threatening GVHD. We have assessed cord blood contamination using PCR analysis of several minisatellite regions to detect maternal DNA. Eighty mother–cord pairs were obtained for this study. In one case there were no specific maternal alleles at any loci and, therefore, cord blood could not be evaluated. Thus, there was a total of 79 informative cases for the detection of maternal cells in the fetal circulation. In most cases, the level of detection was between 0.5 and 1%. We detected maternal DNA in the cord blood sample in only one case (1.26%), and the analysis of dilution experiments led to an estimate of 0.5–1% maternal cells. In conclusion, using PCR amplification of hypervariable regions, maternal DNA is very rarely detected in the cord blood collected at birth, although this approach has a relatively low level of sensitivity.

Immune reconstitution after cord blood transplants supported by coinfusion of mobilized hematopoietic stem cells from a third party donor.

Low severity of GVHD, substantial graft vs tumor (GVT) and slow development of protective immunity are well-documented features of cord blood transplants (CBT). We have evaluated the immune reconstitution of adult recipients of single-unit CBT supported by the coinfusion of third party donor (TPD) mobilized hematopoietic stem cells (MHSC), a procedure—‘dual CB/TPD-MHSC transplant’—that results in early recovery of circulating granulocytes, high rates of CB engraftment and full chimerism. Cumulative recovery of natural killer and B cells at or above the median values of normal controls were 1.0 and 0.76 by the sixth and ninth months. Recovery of T cells was much slower, naive cells lagging behind those of memory and effector (committed) immunophenotypes. Serial analyses of signal joint TCR excision circles showed a general pattern of very low levels by the third month after CBT, followed by recovery to levels persistently similar or higher than those observed before transplantation and in normal controls. Our results are consistent with the clinical observations of substantial GVT effect together with low incidence of serious GVHD and slow development of protective immunity and suggest that thymic function contributes substantially to the recovery of T-cell populations in adults receiving dual CB/TPD-MHSC transplants.