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S Querol

Publications and source records attributed to S Querol.

At least 19 recordsLinked to original sources
PubMed2006

Ex vivo expansion of umbilical cord blood CD34 cells in a closed system: a multicentric study.

BACKGROUND AND OBJECTIVES: The Dideco 'Pluricell System' is a CE-marked medical device allowing haematopoietic stem cell (HSC) expansion. It comprises a kit of cGMP cytokines and reagents, a closed-cell expansion chamber and a cell-washing set. We tested the system in a multicentric study by expanding CD34(+) cells from eight fresh umbilical cord blood (UCB) samples. MATERIALS AND METHODS: During culture, the mean nucleated cell (NC) count, the mean CD34(+) cell count, fold expansion, viability and apoptosis were measured. Clonogenic assays and immunophenotypical characterization were performed on days 0, 7 and 12. On the expanded cellular product, in three cases cell genotyping, endotoxin level and mycoplasma detection (by polymerase chain reaction) were performed. RESULTS: The mean CD34(+) cell expansion on days 7 and 12 was sevenfold and 12-fold respectively and the mean NC expansion was 69-fold and 180-fold. The mean NC viability on day 12 was 96.9% (94.4-99.1). After 12 days, granulocyte-macrophage colony-forming units (GM-CFU) showed a 20-fold increase: a slight increase in CD34(+) cell apoptosis was observed during culture. In all of three cases neither chromosomal alterations nor mycoplasma contamination was detected. No significant endotoxin levels were detected after expansion. CONCLUSIONS: The device allows the ex vivo expansion of NC and CD34(+) cells in a closed system. The expanded cellular product is a mixture of progenitors (CD34(+) cells) and differentiated (mainly myeloid and megakaryocytic) cells. To reduce cell apoptosis, more frequent cell feeding during culture should be tested.

Antigens, CD34↗
PubMed2004

Washing of cord blood grafts after thawing: high cell recovery using an automated and closed system.

BACKGROUND AND OBJECTIVES: Cord blood (CB) progenitor cells are an alternative source of haematopoietic stem cells for bone marrow reconstitution. The critical importance of cell dose in the clinical outcome has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. This aim of this study was to evaluate an automated CB washing protocol of thawed cord blood units using the Sepax device. MATERIALS AND METHODS: After an initial 1:1 dilution using a dextran/albumin-containing buffer, the cells were washed in order to obtain a final product ready for transplantation. The automatic method was compared with the conventional manual washing procedure. Blood samples were taken after thawing and after washing. The processing time, viability and mean recovery of nucleated cells (TNC) and progenitors were determined. RESULTS: The automatic procedure resulted in a median recovery of 93% CD34+ cells and 89% TNC; no significant differences were observed between methods. In addition, median viability, as assessed by annexin V and 7-aminoactinomycin D (7-AAD), was 98% and 94%, respectively, within CD34+ cells. CONCLUSIONS: The automatic washing method described is as effective as the manual method in terms of viability and progenitor cells recovery, but faster and easier for the operators to perform. Overall, our data suggest that the automatic method is safe and suitable for the routine washing of thawed CB grafts in the clinic.

Apoptosis↗
PubMed2003

DRB1*1446 allele variations in the group-specific annealing sites of common amplification primers.

A cord blood unit from the Umbilical Cord Bank of Barcelona was initially typed by sequence-based typing (SBT) as DRB1* 0701. However, the subsequent confirmation by sequence-specific oligonucleotide probes (SSOP) revealed an unusual hybridization pattern that did not fit any of the known HLA-DRB1 alleles or allelic combinations. Molecular cloning and sequencing with primers located at introns 1 and 2 provided the definitive identification of a novel DRB1*1446 allele. It was similar to DRB1*1402 at exon 2 except for nucleotide substitutions at codons 10, 11 and 12 (from YST to LRK) that coincide with the 5'-end group specific site for the annealing of common amplification primers used by SBT and sequence-specific primers.

Alleles↗
PubMed2002

Sequence of the HLA-B*1573 allele.

We report here the sequence of an HLA B*15 allele, B*1573. Initially, a cord blood unit from the Umbilical Cord Bank of Barcelona was typed by sequence-specific oligonucleotide hybridization and revealed an unusual hybridization pattern. After the cloning, the sequence-based typing assigned two different alleles: B*07021, and a second allele identical to B*1558 in exons 2 and 3, except for a single nucleotide substitution in exon 3, which changed codon 140 from Phe to Leu (TTX-->TTA).

Alleles↗
PubMed2002

Molecular analysis of HLA-DRB1 polymorphism in north-east Spain.

The DRB1* polymorphism in 941 randomly selected individuals from the Umbilical Cord Blood Bank of Barcelona (92.75% of Spanish origin) was determined by sequence-based typing. The HLA profile was similar to that of other Mediterranean populations, with DRB1*0701 and *0301 being the most frequent alleles. This may be a consequence of the mixture of alleles as a result of migration from contiguous geographical areas.

Alleles↗
PubMed2002

Combined isolation of CD34+ progenitor cells and reduction of B cells from peripheral blood by use of immunomagnetic methods.

BACKGROUND: Malignant cells may contribute to relapse after autologous hematopoietic cell transplantation The effectiveness of a double immunomagnetic purging strategy combining CD34-positive with B-negative cell selection to purge peripheral blood progenitor cells (PBPCs) from patients with chronic lymphoproliferative disorders has been analyzed. STUDY DESIGN AND METHODS: Twenty-two CD34+ cell selections from patients with follicular lymphoma (n = 14), chronic lymphocytic leukemia (n = 6), mantle cell lymphoma (n = 1), and splenic marginal zone lymphoma (n = 1) were performed by use of a magnetic cell selector followed by a negative cell selection step with anti-CD19 monoclonal antibody bound to immunomagnetic beads. RESULTS: The PBPC components contained median CD34+ cells of 1.24 percent (range, 0.38-3.92%) and CD19+ cells of 1.83 percent (range, 0.06-69.7%). After positive selection (n = 22), 49 percent (range, 16-72%) of CD34+ cells were recovered with a purity of 93 percent (range, 24-99%). The double-positive and -negative selections (n = 20) yielded 57.5 percent of CD34+ cells (range, 33.4-79.4%) with a purity of 95 percent (range, 63-99%). Logarithms of B-cell reduction in the CD34+-cell-enriched B-cell-depleted component had a median value of 3.63 (range, 2.74-4.84 log) and CD19+ and CD5+ cells for chronic lymphocytic leukemia patients with more than 4.56 log (>3.6-5.6 log). Of 13 PBPC components that had a tumor-specific clonal signal, 10 became PCR negative after the double-selection procedure. CONCLUSION: Combined positive and negative magnetic cell selection achieves a high grade of tumor cell reduction with up to 77 percent of the grafts being negative for tumor-specific clonal signal by PCR analysis. This technique preserves an adequate recovery of progenitor cells able to engraft.

Adult↗
PubMed2001

Identification and sequencing of the HLA-B*4106 allele.

We report a novel HLA-B*41 allele (HLA-B*4106) initially detected by an unusual sequence-specific oligonucleotide (SSO) hybridization pattern and identified by sequence-based HLA typing. Molecular cloning and sequencing determined that the new HLA-B allele was identical to the HLA-B*4101 in exon 2 and 3 except for a single nucleotide substitution in the exon 3 changing codon 204 from Glu to Gln (GAG-->CAG). In addition, intron 2 was identical to the published HLA-B*4104 intron 2, except for the base 509 (C-->G).

Alleles↗
PubMed2001

Combined positive and negative cell selection from allogeneic peripheral blood progenitor cells (PBPC) by use of immunomagnetic methods.

Twenty-four mobilized peripheral blood products from healthy donors for allogeneic transplantation were positively selected for CD34(+) cells and depleted of CD4(+) and CD8(+) cells (+/- selection) by combining clinical grade immunomagnetic methods. A sequential, "two-step" strategy combining positive selection of CD34(+) cells by use of the Isolex 300i (versions 1 and 2) device and T cell depletion (TCD) using the MaxSep device and a simultaneous, "one-step" method of CD34(+)cell selection and TCD using the Isolex 300i (software versions 1 and 2) have been investigated. Using these magnetic bead separation systems, two groups of sequential +/- selection (Isolex 300i version 1/MaxSep and Isolex 300i version 2/MaxSep) and two groups of simultaneous +/- selection (Isolex 300i versions 1 and 2) were analysed. In the sequential +/- selection, logarithms of TCD (CD3(+) cell depletion) obtained by the positive selection step had median values of 3.7 with the version 1 (n = 5) and 4.5 with version 2 software of the Isolex 300i (n = 5) (P = 0.07). Version 2 also gave a higher CD34(+) cell purity and yield than did version 1 (92% vs77%, P < 0.05 and 55% vs 34%, P = 0.3, respectively). Additional TCD obtained in the second step with the MaxSep device for the two groups had a median value of 0.9 log and 7% CD34(+)cell losses. In the simultaneous +/- selection, the Isolex 300i version 2 (n = 10) gave a median TCD of 5.1 log and version 1 (n = 4) of 4 log (P < 0.005). Higher CD34(+)cell purity and yield were also obtained with version 2 than with version 1 (97% and 76%, P < 0.005 and 57% and 39%, P = 0.07, respectively). These data indicate that simultaneous, "one-step" +/- selection in the Isolex 300i version 2 achieves a high TCD with a high CD34(+) cell purity and an acceptable CD34(+) cell yield.

Algorithms↗
PubMed2001

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/microl; thereafter and when the ANC was <500/microl, 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.

Acute Disease↗
PubMed2000

Genomic typing of minor histocompatibility antigen HA-1 by reference strand mediated conformation analysis (RSCA).

Disparities in minor histocompatibility antigen (mHAg) HA-1 are involved in the development of acute graft-versus-host disease (GvHD) in adult recipient after HLA-identical sibling donor hematopoietic stem cell transplantation. The mHAg HA-1 is an HLA-A*0201-restricted nonapeptide, which derives from the cleavage of a protein encoded at chromosome 19. The sequence analysis of HA-1 cDNA identified two alleles, termed HA-1H and HA-1R, which differ in only two nucleotides at 3' end of exon A, at positions 500 and 504. DNA-based methods for HA-1 typing were developed in 1998, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and restriction fragment length polymorphism (PCR-RFLP). Here, we report the usefulness of reference strand mediated conformation analysis (RSCA), which was developed for mutation detection and typing of polymorphic loci, to discriminate between the two HA-1 alleles. We performed genomic typing of HA-1 locus in 203 HLA-A*0201-positive samples using RSCA and we confirmed these results by PCR-SSP. The results demonstrate the high reproducibility of this method and their strong correlation with the results obtained by PCR-SSP (99%). Only two samples showed disparity between the RSCA typing and the PCR-SSP. Direct sequencing of these samples confirmed that the correct allele assignment was that obtained by the RSCA typing. Furthermore, HA-1- RSCA-based typing provides additional information about the intronic structure of both alleles. With this approach, we describe the almost constant presence (99.2%) of a 5-bp deletion at intronic position 214-218 associated to the HA-1H allele, previously unidentified. We conclude that HA-1 genomic typing by RSCA is easy to perform and that could be used as a routine typing method.

Alleles↗
PubMed2000

HLA-B44 subtyping in the Catalan population using reference strand mediated conformation analysis. Implications for the selection of unrelated bone marrow donors.

Cytotoxic T lymphocytes (CTLs) reactive against the disparity between HLA-B*4402 and HLA-B*4403 have been reported after unrelated donor bone marrow transplantation. These CTLs have been associated with acute graft-versus-host disease and graft rejection. This study describes the HLA-B44-subtyping in the Catalan population using reference-strand mediated conformation analysis. It has been performed on 297 unrelated HLA-B44+ cord blood units from the Barcelona Cord Blood Bank (Barcelona, Spain). We have found a predominance of HLA-B*4403 (66.04%) over HLA-B*4402 (33.02%), whereas the predominant HLA-B44 allele in Northern Europe and the United States is HLA-B*4402. This inverted proportion between HLA-B44 subtypes in Mediterranean populations compared with other Caucasian populations suggests that HLA-B44 subtyping should be performed when an HLA-B44+ unrelated donor marrow is identified.

Alleles↗
PubMed2000

Evaluation of engraftment of ex vivo expanded cord blood cells in humans.

Cord blood transplants (CBT) result in high rates of engraftment in patients transplanted because of inherited diseases even across marked HLA disparities, mostly in children, with less severe manifestations of GVHD than BM and PBSC transplants. Evaluation of engraftment potential of CBT based on early progenitor content is difficult due to their inaccurate quantification. Instead, post-thaw nucleated cell counts (Pt-NCC) are commonly used for this purpose. We have analyzed engraftment as a function of pre-freeze nucleated cell counts (Pf-NCC) in patients receiving CBT because of inherited diseases. We have observed median times to engraftment of 26 days or less, shortest times ranging 8 to 13 days, late engraftment or graft failures tending to be associated with age >15 years and infusions of <3.7 x 10(7)/Pf-NCC/kg. These data may be appropriate references to evaluate engraftment of CBT performed with previously ex vivo expanded cells. CBT performed with units of which one aliquot has been previously culture-expanded have resulted in times to engraftment similar to the ones observed in the above-mentioned analysis. In these trials it is not possible to trace the actual origin of the early engrafting cells because the pre-cultured cells lack differentiating markers. To better evaluate the engraftment dynamics of culture-expanded CB cells in humans, we have used a model of simultaneously transplanting cells from two different donors to the same patient. Preliminary results of patients that have simultaneously received one uncultured CB unit and culture-expanded purified CB CD34+ cells obtained from a second one show no significant contribution of cultured cells to early engraftment, and no prohibitive unfavorable immunological problems have been observed.

Adolescent↗
PubMed2000

Isolation of CD34+ progenitor cells from peripheral blood by use of an automated immunomagnetic selection system: factors affecting the results.

BACKGROUND: The isolation of CD34+ cells from mobilized peripheral blood is being increasingly used in the setting of allogeneic or autologous hematopoietic cell transplantation. Investigation of variables that may influence the effectiveness of CD34+ cell selection is of interest. STUDY DESIGN AND METHODS: Fifty-one CD34+ cell selections from peripheral blood progenitor cells (PBPCs) (39 allogeneic and 12 autologous) were performed using a magnetic cell separator (Isolex 300i, Baxter), including version 2.0 software. The results obtained were analyzed for different processing variables. The feasibility of transplanting these isolated CD34+ cells was also analyzed. RESULTS: The isolated CD34+ cell fraction had a median purity of 88.9 percent (range, 47.8-98.3). The median recovery of CD34+ cells was 45.1 percent (13.8-76.2), and the median colony-forming unit- granulocyte-macrophage (CFU-GM) content was 17. 2 percent (0.8-58.6). Logarithms of T- and B-cell depletion had median values of 3.7 and 2.8, respectively. The version 2.0 software of the Isolex 300i gave a higher CD34+ cell recovery in the enriched cell fraction (median 57.8%) than did version 1.11 (39.4%) or 1.12 (44.4%) (p = 0.01). The use of recombinant human deoxyribonuclease I during cell processing yielded more CD34+ cells (53% vs. 41%, p = 0. 01) and higher purity (92.8% vs. 87%, p = 0.03). There was a correlation between the percentage of CD34+ cells labeled with the monoclonal antibody 8G12 clone and the percentage of CD34+ cells labeled with the monoclonal antibody used during the processing technique (9C5 clone) in the initial, enriched, and depleted CD34+ cell fractions (R(2) = 0.95; 0.92; 0.78, p< 0.005, respectively). Median times for recovering >0.5 x 10(9) per L of granulocytes and >20 x 10(9) per L of platelets were 13 and 16 days in the allograft patients and 13 and 14 days in the autograft patients. CONCLUSION: CD34+ cells can be highly and effectively isolated from allogeneic and autologous grafts by use of this automated technique, with a high grade of T- and B-cell depletion. These purified CD34+ cell components can engraft normally.

Adult↗
PubMed2000

Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols.

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched fraction were cultured for 6 days in serum-free medium supplemented with rHu-SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/- 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colonies.

Antigens, CD34↗
PubMed2000

Leukapheresis components may be cryopreserved at high cell concentrations without additional loss of HPC function.

BACKGROUND: The aim of this study was to assess the feasibility of freezing mobilized peripheral blood progenitor cell (PBPC) components at higher cell concentrations than are classically recommended for bone marrow. This approach might have potential benefits, such as lower cost of processing and storage and less risk of the complications associated with the transfusion of large component volumes and large quantities of DMSO. STUDY DESIGN AND METHODS: In the first phase, small aliquots of 19 apheresis components were cryopreserved at standard and higher cell concentrations (Aliquots A and B, respectively). In the second phase, 21 apheresis components were split into two bags each and frozen at standard (Bag A) and high (Bag B) cell concentrations. The differences in viability, cloning efficiency, and nucleated cell recovery in Bags A and B were examined. Finally, the hematologic recovery of 10 patients who underwent autologous transplantation with PBPC components frozen at high cell concentrations was analyzed. RESULTS: The median cell concentration at freezing was 94 (57-100) x 10(6) per mL and 291 (220-467) x 10(6) per mL for Aliquots A and B, respectively, and 90.9 (45.4-92) x 10(6) per mL and 332 (171-582) x 10(6) per mL for Bags A and B, respectively. The viability was significantly lower in samples frozen at higher cell concentrations: 92 versus 83 percent (p = 0.001) and 87 versus 77 percent (p<0.001) for Aliquots and Bags A and B, respectively. Significant differences were not observed in the recovery of total nucleated cells (102 vs. 101% and 98 vs. 105%) or the cloning efficiency after thawing (13 vs. 16% and 27 vs. 23%) for Aliquots and Bags A and B, respectively. The time to granulocyte engraftment >0.5 x 10(9) per L and platelet engraftment >20 x 10(9) per L was 9 (8-11) and 10.5 (7-21) days, respectively. CONCLUSION: The cryopreservation of PBPC components at standard concentrations and 3.3 (1.8-6.2)-fold cell concentrations has no adverse effect on the function of HPCs after thawing.

Blood Preservation↗
PubMed2000

Follow-up of chimerism status after allogeneic HLA-mismatched stem cell transplantation by detection of non-shared HLA alleles.

BACKGROUND AND OBJECTIVES: Chimerism studies after allogeneic transplantation are usually performed using cytogenetic analysis, PCR-VNTR or PCR-STR. Here, we report an alternative method for following the chimerism status after an HLA-mismatched stem cell transplantation (SCT), detecting the presence of non-shared HLA alleles by reference-strand mediated conformation analysis (RSCA). DESIGN AND METHODS: We tested this new approach on allogeneic related haploidentical SCT, unrelated cord blood transplantation, and HLA-mismatched unrelated donor SCT. The quantification of the chimerism was performed by laser detection of fluorescent-labeled primers on an automated DNA sequencer. RESULTS: In all cases this technique was able to detect mixed chimeras. The technique detected above 5% of residual cells when the analysis was based on HLA-class I and above 3% for HLA-class II. This sensitivity is similar to that of the PCR-VNTR analysis. INTERPRETATION AND CONCLUSIONS: This method avoids the need to search for an informative locus (which is essential for PCR-VNTR or -STR). Moreover, we did not find the phenomenon of preferential amplification that is observed with most VNTR, thus avoiding the need for construction of standard curves to quantify mixed chimeras. We conclude that the detection of the non-shared HLA alleles by RSCA is a useful approach for chimerism follow-up after HLA-mismatched SCT.

Alleles↗