HLA-DRB1: genetic susceptibility and disability progression in a Spanish multiple sclerosis population.

BACKGROUND AND OBJECTIVE: The association of HLA-DRB1*15 with susceptibility to multiple sclerosis (MS) has been consistently reported although its effect on the clinical phenotype is still controversial. The objectives of this study are to investigate the influence of the HLA-DRB1 alleles on the genetic susceptibility to MS and to study their impact on disability progression in a Spanish population. METHODS: HLA-DRB1 typing was performed by PCR-SSP in 380 patients with sporadic MS and 1088 unrelated healthy controls. Allelic frequencies were compared between groups. We studied the correlation between the different alleles and the progression of MS. RESULTS: The HLA-DRB1*15 allele in patients with MS had a statistically significant higher frequency when compared with controls (18.9% in patients vs. 10.1% in controls, Odds ratio (OR)=2.07, 95% CI=1.64-2.60, P<0.001). In the univariate analysis, the DRB1*01 and DRB1*04 alleles were associated with a worse prognosis when considering the time to reach an EDSS of 6, whereas the DRB1*03 was correlated with a better outcome. In the multivariate analysis, the alleles*01 and *04 were demonstrated to be independent factors to have a worse prognosis. CONCLUSIONS: HLA-DRB1*15 is associated with MS when comparing patients with unrelated healthy controls in a Spanish population. The HLA-DRB1*01 and HLA-DRB1*04 alleles are related to a worse prognosis when considering the time taken to reach severe disability.

Quality rather than quantity: the cord blood bank dilemma.

Growing inventories of cord blood units have facilitated access to umbilical cord cell transplantation for many patients lacking conventional stem cell donors. They are in principle 'off-the-shelf', 'fit-for-use', as well as safe and effective therapy products. Cellular enumeration is used as a surrogate of graft potency, and users rely on the rigorous assessment carried out in banks to avoid poor engraftment after thawing (loss of cells or poor function), when the patient's situation is critical. However, in practice, when units are selected, initially on the basis of HLA matching and cell dose assessment, their absolute quality remains uncertain. Unfortunately, quality-related issues (particularly related to viability) are not uncommon in cord blood transplantation. The reasons for potency failures are diverse, but a lack of thorough validation during critical steps of the process and of appropriate use of quality-control tools for timely detection of problematic units are significant contributors. Moreover, incongruence between different sets of standards and regulations, and lack of common quality systems between banks result in a highly heterogeneous international inventory. Therefore, this complicates the matter for the end user of the product. To ameliorate this situation, it is essential to improve quality at each of the critical manufacturing steps wherein potency can be threatened, thereby creating homogeneous inventories of units with excellent quality and quantity.

Enhanced HPC recruitment in children using LVL and a new automated apheresis system.

BACKGROUND: A new automated apheresis system has recently been reported as useful in improving peripheral blood HPC collection in adults. The aim of this study has been to verify the utility of this system (AutoPBSC, COBE BCT) for standard leukapheresis and for LVL in the pediatric setting. STUDY DESIGN AND METHODS: A prospective study was set up in 29 leukapheresis procedures carried out in 26 children with malignant diseases and body weight under 40 kg who had undergone mobilization with G-CSF or with G-CSF and chemotherapy. Leukapheresis procedures were performed under two protocols, depending on the total blood volume processed: standard leukapheresis (< or=3) and LVL (>3). The need to prime the tubing set with blood was determined, and the inlet flow rate, collection time, recruitment of CD34+ cells, CD34+ cell collection efficiency, component volume, leukapheresis cell composition, and preapheresis and postapheresis peripheral blood counts were measured. Paired t test, Spearman's correlation coefficient, and the Mann-Whitney U test were employed for statistical analysis. RESULTS: Because of the low extracorporeal volume (167 mL) of the tubing set of the automated blood processor, priming was necessary in only 2 of 26 patients, both weighing under 10 kg. LVL showed better CD34+ cell yield (7.5 vs. 2.3 x 10(6)/kg; p = 0.047), higher recruitment (2.1 vs. 0.9; p = 0.002), and greater collection efficiency (50% vs. 33%; p = 0.005) than standard leukapheresis. No significant differences were found between groups in collection time. In LVL procedures, CD34+ cell collection efficiency and recruitment were not significantly influenced by the inlet flow rate. CONCLUSION: The AutoPBSC is a reliable system for peripheral blood HPC collection in children mainly when used in combination with LVL. The major advantage of this software is a reduced need for priming. LVL allows better CD34+ cell collection efficiency, enhanced recruitment, and improved CD34+ cell yield.

Administration of recombinant human granulocyte colony-stimulating factor to normal donors: results of the Spanish National Donor Registry. Spanish Group of Allo-PBT.

A Spanish National PBPC Donor Registry has recently been established for short- and long-term safety data collection in normal donors receiving rhG-CSF. To date, 466 donors have been included in the Registry. Median (range) dose and duration of rhG-CSF administration was 10 microg/kg/day (4-20) and 5 days (4-8), respectively. Donors underwent a median of two aphereses (range, 1-5). Adverse effects consisted mainly of bone pain (90.2%), headache (16.9%) and fever (6. 1%), but no donor discontinued rhG-CSF prematurely due to toxicity. Side-effects were more frequent in donors receiving >10 microg/kg/day than in those with lower doses (82.8% vs 61.8%; P = 0. 004). A significant decrease between baseline and post-apheresis platelet counts was the most important analytical finding (229 x 10(9)/l vs 140 x 10(9)/l; P < 0.0001), with a progressive reduction in platelet count with each apheresis procedure. One donor developed pneumothorax that required hospitalization due to central venous line placement. The mean CD34+ cell dose collected was 6.9 x 10(6)/kg (range, 1.3-36), with only 14 donors (2.9%) not achieving a minimum target of CD34+ cells of 2 x 10(6)/kg. No definitive information about potential long-term side effects is yet available. However, we hope this National Registry will serve as a useful basis for better monitoring of the efficiency and side-effects of cytokine administration in healthy people.

Controlled-rate versus uncontrolled-rate cryopreservation of peripheral blood progenitor cells: a prospective multicenter study. Group for Cryobiology and Biology of Bone Marrow Transplantation (CBTMO), Spain.

BACKGROUND AND OBJECTIVE: Cryopreservation of hemopoietic progenitors for transplantation has been traditionally performed by the use of a controlled-rate freezer. Several groups have reported successful cryopreservation of progenitor cells at -80 degrees C without a controlled-rate freezer. In an attempt to elucidate whether both methods are equally efficient, we compared controlled-rate versus uncontrolled cryopreservation of peripheral blood progenitor cells (PBPC) in a prospective, multicenter study. DESIGN AND METHODS: Apheresis products from patients undergoing PBPC mobilization were split into two aliquots, and cryopreserved simultaneously by both methods, in autologous plasma plus 10% dimethylsulfoxide. Controlled-rate samples were placed into a programmable freezer with a cooling rate of 1-2 degrees C/min. Uncontrolled-rate samples were directly introduced into a -80 degrees C mechanical freezer. After thawing, cell counts, assays for viability, clonogenic cultures and CD34+ cell enumeration were performed. RESULTS: A total of 105 cases were included. No significant differences were found in viability (mean 88.8 +/- 13% in the controlled-rate group vs. 89.7 +/- 12% in the uncontrolled-rate group), nucleated cell loss (23.5 +/- 23% vs. 23 +/- 22%), mononuclear cell loss (19 +/- 23% vs. 19.1 +/- 22%), and loss of CD34+ cells (34.3 +/- 33% vs. 28.6 +/- 34%). On the other hand, recovery of granulomonocytic colony-forming units (CFU-GM), was significantly better with the controlled-rate technique, than with the non-controlled-rate method (104.3 +/- 95 vs. 86.5 +/- 80, respectively; p = 0.048). INTERPRETATION AND CONCLUSIONS: Our results indicate that both techniques are suitable for cryopreservation of PBPC, although a better recovery of committed progenitors is achieved by the controlled-rate method. Therefore, the use of controlled-rate freezer should probably be recommended.

Allogeneic transplantation of selected CD34+ cells from peripheral blood: experience of 62 cases using immunoadsorption or immunomagnetic technique. Spanish Group of Allo-PBT.

The objective of this study was to analyze CD34+ cell recovery and T cell depletion (TCD) achieved in CD34+ cell grafts using either immunoadsorption or immunomagnetic methods applied to leukapheresis products from healthy donors. We also wanted to determine the kinetics of engraftment and incidence and severity of graft-versus-host disease (GVHD) after allogeneic transplantation of selected CD34+ cells. HLA-identical sibling donors received G-CSF. After leukapheresis, peripheral blood progenitor cells were selected using immunoadsorption (Ceprate SC) (n = 38) or immunomagnetic (Isolex 300) (n = 24) methods. Sixty-two patients, with a median age of 42 years (range 17-60) diagnosed with hematological malignancies were conditioned with either cyclophosphamide and total body irradiation (n = 43) or busulphan and cyclophosphamide (n = 19). GVHD prophylaxis consisted of cyclosporin A (CsA) and prednisone (n = 48), CsA alone (n = 11) and CsA and methotrexate (n = 3). The median yield and purity of CD34+ cells after the procedure was 65 and 66% with immunoadsorption, and 48 and 86% with immunomagnetic method, respectively. The median number (range) of CD34+ cells infused into the patients was 3.5 x 10(6)/kg (1-9.6). The median number (range) of CD3+ cells administered was 0.4 x 10(6)/kg (0.01-2) using immunoadsorption and 0.14 x 10(6)/kg (0.03-2.5) using immunomagnetic methods. Neutrophil recovery >500 and >1000/microl was achieved at a median (range) of 13 days (8-22) and 14 days (9-31), respectively. Platelets recovered to >20000 and >50000/microl at a median (range) of 13 days (0-128) and 18 days (0-180), respectively. Two patients developed graft failure. Acute GVHD in patients at risk was clinical grade 0 (n = 43), I (n = 8), II (n = 4) and III (n = 1). No patient developed acute GVHD grade IV. Chronic GVHD was limited in two cases and extensive in four cases. The actuarial probability of acute GVHD II-IV was 10% (95% CI, 1-19%), and of extensive chronic GVHD was 12% (95% CI, 11-13%). The cumulative incidence of transplant-related mortality was 12.6%, and this figure was 9% at 6 months. In conclusion, with the immunomagnetic procedure, a lower recovery and higher purity of CD34+ cells, and stronger TCD is obtained as compared to immunoadsorption (P = 0.008, P < 0.0001 and P = 0.0002, respectively). Our results also indicate that allogeneic transplantation of selected CD34+ cells is associated with a very rapid engraftment and with a low incidence of severe GVHD.

Allogeneic transplantation of purified CD34+ cells from peripheral blood: Spanish experience of 62 cases. Spanish Group of allo-PBT.

This report summarizes the Spanish experience of 62 cases of allogeneic transplantation of purified CD34+ cells from peripheral blood. HLA-identical sibling donors received G-CSF. After leukapheresis, peripheral blood progenitor cells were purified using one of two methods: Ceprate (n = 38), or Isolex 300 (n = 24). Sixty-two patients median age 42 years (range 17-60) diagnosed with hematological malignancies were conditioned with either cyclophosphamide and total body irradiation (n = 43) or busulphan and cyclophosphamide (n = 19). GVHD prophylaxis consisted of cyclosporin A (CsA) and prednisone (n = 48), CsA alone (n = 11), and CsA and methotrexate (n = 3). The median yield and purity of CD34+ cells after the procedure was 65% and 66% with Ceprate, and 48% and 86% with Isolex, respectively. The median number of CD34+ cells infused into the patients was 3.5 x 10(6)/kg (range 1-9.6). The median number of CD3+ cells administered was 0.4 x 10(6)/kg (range 0.01-2) using Ceprate and 0.14 x 10(6)/kg (range 0.03-2.5) using Isolex. Neutrophil recovery >500 and >1000/microl was achieved at a median of 13 days (range 8-22) and 14 days (range 9-31), respectively. Platelets recovered to >20,000 and >50,000/microl at a median of 13 days (range 0-128) and 18 days (range 0-180), respectively. The actuarial probability of acute GVHD II-IV was 10% (95% CI, 1-19%), and of extensive chronic GVHD 12% (95% CI, 11-13%).

Umbilical cord blood as a good alternative to bone marrow for allogeneic cell transplantation in children with hemoblastosis.

Umbilical cord blood (UCB) is an alternative source of hematopoietic progenitors and has potential advantages over bone marrow. We present our experience with UCB transplants performed between July 1994 and June 1997 in seven children with hemoblastosis. Two patients underwent transplantation from an HLA-identical sibling donor and five from an unrelated donor. Engraftment was obtained in all but one patient. Acute GVHD was absent in patients with related donors and present in all patients with unrelated donors. No patient showed chronic GVHD. Two patients died from transplant-related complications and a further two relapsed. Four patients were alive with a follow-up of 14, 15, 21 and 39 months post-transplant, respectively. Overall survival was 57% (s.e. 0.19). We conclude that cord blood is a good alternative source of hematopoietic stem cells for children with malignant hematologic diseases.

Isochromosome 17q as a sole anomaly: a distinct myelodysplastic syndrome entity?

We report four patients with myelodysplastic syndrome (MDS) with isochromosome i(17q) as the sole chromosomal anomaly. One patient was classified as refractory anemia (RA) and three as refractory anemia with excess of blasts (RAEB). All four patients shared several features such as male sex, advanced age, severe anemia, as well as a bone marrow with myeloproliferative characteristics: hypercellularity, prominent baso- and eosinophilia, and marked increase of micromegakaryocytes. We suggest that patients with i(17q) as the sole chromosomal anomaly may identify a distinct MDS with characteristics between MDS and chronic myeloproliferative disorders (CMPD).

[Cardiac and hematological involvement in idiopathic hypereosinophilic syndrome. Study of 12 cases]. / Afección cardíaca y hematológica en el síndrome hipereosinofílico idiopático. Estudio de 12 casos.

BACKGROUND: The idiopathic hypereosinophilic syndrome (IHS) is a rare entity of unknown etiology. Hematological and cardiac involvement is predominant. A series of 12 patients with this syndrome, initiated in 1982, is described. METHODS: Cardiological study by repeated echocardiograms and hematological study in peripheral blood and bone marrow upon initiation of the disease were performed. RESULTS: Median follow up was of 48 +/- 31 months. Males predominated (75%) with mean age being 55 +/- 15 years. The principal organs or systems involved were the heart (50%) and the nervous system (41%). Of the 6 cases with cardiac involvement only 2 had clinical manifestations. The remaining 4 patients were diagnosed from echocardiographic changes with the principal alterations observed being: atypical occupation of the ventricles, endocardial thickening and mitral and tricuspid subvalvular cumulus. Echocardiographic follow-up only showed changes in one case. Hematological involvement was characterized by moderate leukocytosis with hypereosinophilia formed by mature eosinophils, conservation of other hematopoietic series, absence of blasts in peripheral blood, finding suggestive of diseosinophilopoiesis and appearance of myelofibrosis and cytogenetic alterations. Survival at four years was 58%. CONCLUSIONS: In the series studied cardiac involvement is frequent, being principally diagnosed in a subclinical phase and with a very slow echocardiographic evolution. At a hematological level changes typical in myelodysplastic syndromes and myeloid leukemia were observed.

Abnormal chromatin clumping in leucocytes: a clue to a new subtype of myelodysplastic syndrome.

We report 6 patients with myelodysplastic syndrome, all of whom showed a bizarre nuclear anomaly within the neutrophils that was characterized by extensive clumping of chromatin into large blocks separated by clear zones, generally associated with a lack of segmentation. Anaemia, thrombocytopenia, variable leucocyte counts with leucoerythroblastic picture, marrow hypercellularity with granulocytic hyperplasia and moderate dysplastic changes in erythroblastic and megakaryocytic lines were present at diagnosis. 2 patients had normal karyotypes and a 3 showed a deletion of chromosome 14. 5 out of 6 patients had pneumonia at diagnosis. The median survival was short (5 months) and haemorrhagic complications were the cause of death in 4 patients. The clinical features and the evolution of these and other reported cases suggest that the presence of abnormal chromatin clumping in leucocytes might be a clue to a new subtype of myelodysplastic syndrome.

Bone marrow biopsy in myelodysplastic syndromes: morphological characteristics and contribution to the study of prognostic factors.

Ten characteristics of bone marrow (BM) biopsies in paraffin sections, obtained at diagnosis from patients with myelodysplastic syndromes (MDS) classified according to the FAB criteria, were analysed to identify both the most relevant morphologic data and any possible influence on survival. Agreement between two observers was obtained for 94% of the data. BM cellularity was increased in 63% of the cases and was higher in refractory anaemia with excess of blasts (RAEB). RAEB in transformation (RAEB-t) and chronic myelomonocytic leukaemia (CMML) (P = 0.001). Dysmegakaryopoiesis and dyserythropoiesis were present respectively in 83% and 72% of the cases, with slight differences among the FAB subtypes. Abnormal localization of immature precursors (ALIP) was found in more than half of the cases and somewhat more frequently seen in the RAEB + RAEB-t + CMML group (P = 0.07). Eosinophilia, plasmacytosis and reticulin fibrosis were evident in 26%, 18% and 47% of the cases respectively. Cellularity (P = 0.006), eosinophilia (P = 0.009) and, to some extent, dysmegakaryopoiesis (P = 0.07) bore a certain relationship with survival on univariate analysis. The presence of ALIP was not seen to affect the outcome. Multivariate analysis showed that the cellularity and presence of dysmegakaryopoiesis, in BM biopsy, added significant independent prognostic information to that achieved with age, platelet count and proportion of blast cells in BM aspirate, three variables with proven prognostic value in MDS patients. Using a regression model including these five characteristics we have stratified the patients into low, intermediate and high-risk groups with different survivals (P = 0.00001). The present findings show that BM biopsy is able to provide both morphological characteristics and information about the prognosis of survival, and should thus be included in the initial evaluation of MDS.

[Autoimmune hemolytic anemia and myelodysplastic syndromes]. / Anemia hemolítica autoinmune y síndromes mielodisplásicos.

The association between autoimmune haemolytic anaemia (AHA) and myelodysplastic syndromes (MDS) was found in seven out of 156 patients with SMD who received several transfusions as supportive therapy. Three patients were diagnosed of refractory anaemia (RA), three more of refractory anaemia with excess of blasts (RAEB) and one of refractory anaemia with ring sideroblasts (RARS). All patients showed a positive direct antiglobulin test (DAT) and the presence of anti-red blood cell IgG type antibodies, both in serum and eluate. Clinically, three patients showed signs of low grade haemolysis. It is suggested that in the reported patients, who seem to be immunologically predisposed, red blood cell transfusions could trigger the autoantibodies and the AHA development.

Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients.

Therapy planning in patients with myelodysplastic syndromes (MDSs) is complicated by its high prognostic heterogeneity. Forty-one patient and disease characteristics at onset of 370 patients with MDS were analyzed to identify significant prognostic factors for survival and transformation to acute myeloblastic leukemia (AML), and to develop and validate a regression model for predicting survival. Multivariate regression analysis showed that the total bone marrow percentage of blast cells, age, platelet count, WBC count, and hemoglobin level were the characteristics more significantly associated with survival in the overall series. The bone marrow percentage of type I blast cells was the most important factor predicting transformation into AML. Proportional hazards regression analysis in a randomly selected training sample of 240 patients demonstrated that the combination of total bone marrow percentage of blast cells, platelet count, and age had the strongest predictive relation to survival length. The resulting regression models, continuous and categorized, were validated in the remaining test sample of 130 patients by demonstrating its capability of segregating patients into low-, intermediate-, and high-risk groups, with distinctively different survival curves (P less than .0001). A scoring system derived from the categorized model also had a great prognostic value (P less than .0001). These regression models and the simpler scoring system may be accurately used for decision-making regarding therapy in MDS patients.

Chronic myelomonocytic leukemia--clinicobiological characteristics: a multivariate analysis in a series of 70 cases.

In a series of 70 patients diagnosed according to the FAB criteria, 42 clinical and biological disease characteristics were analyzed in order to identify significant prognostic factors by means of univariate and multivariate analysis. The univariate analysis identified ten parameters associated with poor prognosis: Symptoms of anemia, WBC over 10 x 10(9)/l, presence of blast cells, myeloid precursors or erythroblasts in peripheral blood (PB), high bone marrow (BM) cellularity, severe dysthrombopoiesis, percent of blast cells in BM and high serum levels of bilirubin and LDH. The Cox proportional hazards regression method revealed that the combination of high leukocyte counts and BM percentage of blast cells had the strongest predictive relation to survival length (p = 0.002 and p = 0.060 respectively). A new multivariate analysis treating the presence of myeloid and erythroid precursors in PB as a single variable selected only this as the most significant prognostic factor (p = 0.001). Both regression models allowed us to discriminate two significantly different risk groups of patients.