A real-life overview of a hematopoietic cell transplant program throughout a four-year period, including prospective registry, exclusion causes and final donor selection.

Traceability of patients who are candidates for Hematopoietic cell transplant (HCT) is crucial to ensure HCT program quality. Continuous knowledge of both a detailed registry from a HCT program and final exclusion causes can contribute to promoting a real-life vision and optimizing patient and donor selection. We analyzed epidemiological data reported in a 4 year-monocentric prospective registry, which included all patients presented as candidates for autologous (Auto) and/or allogeneic (Allo) HCT. A total of 543 patients were considered for HCT: 252 (42.4%) for Allo and 291 (57.6%) for Auto. A total of 98 (38.9%) patients were excluded from AlloHCT due to basal disease progression more commonly (18.2%). Seventy-six (30.2%) patients had an HLA identical sibling, whereas 147 (58.3%) patients had only Haplo. UD research was performed in 106 (42%) cases, significantly more often in myeloid than lymphoid malignancies (57% vs 28.7%, p < 0.001) but 61.3% were finally canceled, due to donor or disease causes in 72.4%. With respect to Auto candidates, a total of 60 (20.6%) patients were finally excluded; progression was the most common cause (12%). Currently, Haplo is the most frequent donor type. The high cancellation rate of UD research should be revised to optimize further donor algorithms.

Analysis of residual disease in chronic lymphocytic leukemia by flow cytometry.

We have investigated the value of both conventional and quantitative flow cytometry to detect minimal residual disease in 21 CLL patients in remission including bone marrow histology: eight in complete remission (CR), 11 in nodular partial remission (nPR) and two in PR. The techniques used were double immunostaining with CD5 and CD19 and quantitative estimation of the number of both antigens with standard microbeads. Reference values were established on normal peripheral blood and bone marrow controls. Patients were considered in 'immunological' remission when the percentage of CD5+ CD19+/total CD19+ cells was <25% in PB and <15% in BM. In six of the eight patients in CR, CLL cells were still detectable by flow cytometry. Only two patients, that underwent allogeneic bone marrow transplant, achieved immunological remission. CLL samples showed significantly higher CD5 and lower CD19 antigen density than normal controls (P < 0.001). Persistence of residual disease was a predictor of time to progression. None of the two patients in immunological remission relapsed within a period of 13 and 33 months, whilst two of the six patients in CR with positive flow cytometry relapsed 3 and 6 months after achieving CR. This study demonstrates that flow cytometry contributes to increase the sensitivity of the clinicohematological criteria to detect residual malignant cells in CLL patients and may be useful to monitor disease status following treatment.

Mobilization kinetics of peripheral blood progenitor cells after IAPVP-16 salvage chemotherapy plus G-CSF in lymphoproliferative disorders.

We have explored the efficacy of salvage chemotherapy combination, IAPVP-16 (ifosfamide 5 g/m2 on day 1; VP-16 100 mg/m2 on days 1-3; ara-C 1.2 g/m2/12 h on days 1 and 2; methylprednisolone 80 mg/m2 on days 1-5) plus G-CSF for PBPC mobilization. This protocol was used in 45 patients with relapsed or refractory lymphoproliferative diseases who underwent 85 leukaphereses. In 41 patients > 2 x 106/kg CD34+ cells were obtained after a median of two procedures. The median number of CD34+ cells harvested was 3.2 x 106/kg per apheresis and 8.4 x 106/kg per patient. Seven of 10 patients who had failed previous mobilization attempts achieved more than 2 x 106 CD34+ cells/kg in a maximum of three aphereses. A history of previous mobilization failure and a low platelet count (<150 x 109/l) negatively influenced the CD34+ cell yield in univariate and multivariate analyses. A good correlation was found between the circulating CD34+ cells/microl and the CD34+ cells and CFU-GM in the leukaphereses products (r = 0.93 and r = 0.73, P < 0.001), and > or =17 CD34+ cells/microl predicted the achievement of > 2 x 106/kg CD34+ cells in a single leukapheresis in more than 90% of cases. IAPVP-16 plus G-CSF may be specially indicated in tandem transplantations or CD34+ selection and in patients who have failed previous mobilization attempts.

Native valve endocarditis due to Candida parapsilosis: a late complication after bone marrow transplantation-related fungemia.

A case of Candida parapsilosis endocarditis observed 16 months after BMT is reported. The patient, a 35-year-old female with CML, suffered from Candida parapsilosis fungemia on day +22 after BMT. In spite of treatment with amphotericin B, fluconazole and catheter withdrawal, the same yeast was isolated > 1 year later from a vegetation on an old rheumatic mitral valve. Although the patient remained in complete cytogenetical and hematological remission, in vitro tests showed reduced phagocytic and chemotactic capacity of neutrophils and monocytes. This case stresses the need of prolonged therapy for patients with candidemia after BMT.

Interstitial del(12)(q15q22) in myelodysplastic syndromes.

A patient with a myelodysplastic syndrome and a 12q deletion was studied and followed-up. After 10 years and several cytogenetic studies, it is suggested that this abnormality can be the sole chromosomal change in myelodysplastic syndromes.

Cunninghamella bertholletiae infection (mucormycosis) in a patient with acute T-cell lymphoblastic leukemia.

Cunninghamella spp. are unusual opportunistic pathogens that have been identified with increased frequency in immunocompromised patients. Clinical infection by this fungus is almost always devastating and usually fatal. Infections with this group of organisms have been seen most frequently in patients with hematological malignancy. Here we report the case of a patient with acute leukemia who developed multiorganic failure as a consequence of hematological dissemination by Cunninghamella bertholletiae. The case highlights the mortality associated with this fungal infection in immunocompromised patients, confirms the risk factors associated with non-candida fungal infections and shows a clinical presentation mimicking myocardial infarct and cerebrovascular stroke.

Juvenile chronic lymphocytic leukemia with unusual intracisternal inclusions: an ultrastructural study.

The electron microscopic analysis of intracisternal inclusions in lymphocytes of the bone marrow and peripheral blood in a case of juvenile chronic lymphocytic leukemia is described. These inclusions consist of well-ordered microtubules attached to a central axis. The contribution of electron microscopic analysis in establishing the substructural pattern of these inclusions is discussed.

[Pentoxifylline is not useful in the prevention of toxicity associated with bone marrow transplantation]. / La pentoxifilina no es útil en la prevención de la toxicidad asociada al trasplante de médula ósea.

BACKGROUND: To evaluate the possible beneficial effect of pentoxifylline (PTX) on both the decrease of toxicity related to bone marrow transplantation (BMT) and the acceleration of the hematopoietic graft. METHODS: Twenty consecutive patients treated with BMT received pentoxifylline (400 mg/6 hours, orally) up to day +50 to prevent toxicity derived from BMT. A previous group of 29 consecutive patients transplanted in the same center were used as controls. The different clinical toxicities (mucositis, kidney failure, hepatic venocclusive disease, graft versus host disease, number of days with fever, day of hospital discharge and survival at day +50), the time elapsed until the hematopoietic graft and the levels of tumoral necrosis factor alpha were evaluated. RESULTS: No significant differences were observed in any of the parameters studied in the two groups of patients. CONCLUSIONS: Treatment with pentoxifylline does not prevent the toxicity derived from BMT or accelerate the hematopoietic grafting.

Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+ cells.

BACKGROUND: This study assesses the feasibility of a new volumetric cytometry system for the enumeration of CD34+ cells in apheresis components, peripheral blood, and cord blood samples in routine laboratory work. This system is compared with the following flow cytometry protocols: Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count fluorospheres. STUDY DESIGN AND METHODS: Correlation, linearity, and reproducibility studies were performed for the various methods. Clonogenic cultures were performed, as an external control, to assess the correlation between the number of CD34+ cells per microL and the number of colony-forming units per microL. RESULTS: The linear regression analysis demonstrated that the five methods were comparable (R2 ranged from 0.86 to 0.96 and slopes were close to 1). The CD34+ assay and the flow-count methods showed poor linearity for CD34+ cell counts below 10 cells per microL (R2 = 0.46 and 0.47). The reproducibility assay for a CD34+ count of 10 cells per microL showed a CV of 12 percent and 25 percent for the Milan and CD34+ assay methods, respectively. The mean CV among all five methods for the 46 evaluated samples was 20 percent. There was a strong correlation between the number of CD34+ cells per microL and colony-forming units per microL in cord blood and apheresis samples (r = 0.71-0.81). CONCLUSION: The CD34+ assay is useful in CD34 enumeration in cord blood, leukapheresis samples, and peripheral blood samples and provides comparable results to the Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count methods. Nevertheless, peripheral blood samples with low CD34 absolute counts (below 10 cells/microL) should be analyzed by alternative flow cytometry protocols. Even though the same operator performed the study in a single laboratory, the high inter-method CV suggests that differences in sample preparation and gating strategy are factors that increase variability. Protocols with fewer intermediate steps or fully automated protocols such as the CD34+ assay are expected to reduced intra- and inter-laboratory variability.

Quantitative analysis of CD79b, CD5 and CD19 in mature B-cell lymphoproliferative disorders.

BACKGROUND AND OBJECTIVE: Distinction between B-cell chronic leukemias can be difficult due to overlap in cell morphology and immunologic features. We investigated, by quantitative flow cytometry, the expression of CD79b, CD5 and CD19 in cells from a variety of B-cell disorders to see whether this analysis adds further information useful to the diagnosis and characterization of these diseases. DESIGN AND METHODS: Peripheral blood cells from 6 normal individuals were used as reference controls. The diseases of the 63 patients investigated comprised: 29 chronic lymphocytic leukemia (CLL), six of them with atypical morphology, 6 B-cell prolymphocytic leukemia (PLL), 12 splenic lymphoma with villous lymphocytes (SLVL) and 16 mantle-cell (Mc) lymphoma in leukemic phase. The study was carried out by triple immunostaining with directly conjugated monoclonal antibodies (MoAb) against CD79b, CD5 and CD19 and quantitative estimation of the antigens per cell assessed with standard microbeads (Quantum Simply Cellular). RESULTS: Compared to normal B-cells, the number of CD19 molecules was significantly lower in cells from all of the B-cell disorders except PLL. The intensity of CD5 in leukemic B-cells was significantly higher in CLL cells, including atypical cases, and Mc lymphoma than in normal B-cells, whilst PLL and SLVL had values similar to those of normal B-lymphocytes. CD79b was expressed at lower levels in all types of leukemic cells compared to normal B-lymphocytes but differences were statistically significant in CLL, Mc lymphoma and SLVL. The number of CD79b molecules per cell was significantly lower in typical CLL than in the remaining B-cell diseases whilst the comparison of CD5 and CD19 intensity between CLL and non-CLL samples failed to show any statistically significant difference. INTERPRETATION AND CONCLUSIONS: Distinct antigen density patterns for the various conditions emerged from this analysis: Typical CLL was characterized by moderate CD5 and weak or negative CD79b expression. Mc lymphoma showed an homogeneous pattern, characterized by similar expression of CD5 than CLL but significantly stronger expression of CD79b whilst PLL and SLVL had weak CD5 and moderate CD79b expression. Atypical CLL had an intermediate pattern of CD79b antigen expression ranging from weak to moderate with bright CD5. Unlike CD5 and CD79b, CD19 did not discriminate the various B-cell disorders but only between normal and leukemic cells.

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.

Predictive factors for a successful mobilization of peripheral blood CD34+ cells in multiple myeloma.

We analyzed the prognostic factors for a successful mobilization and peripheral blood stem cell collection in a series of 57 consecutive patients with multiple myeloma (MM); a new scoring system to predict an adequate mobilization in this subset of patients was also constructed. A total of 221 aphereses were performed in 57 patients with MM. The median time from diagnosis to mobilization was 12 months (range 4-120). Only one line of chemotherapy was administered before mobilization to 36 patients and two or more to 21. The median number of alkylating chemotherapy cycles was 6 (2-33). Two patients were mobilized in complete remission, 32 in partial response, and 23 in stable/progressive disease. Significant adverse prognostic factors for collecting 2.5 x 10(6) CD34+cells/kg or more were: a period of at least 12 months from diagnosis, at least six cycles of alkylating agents, and a plasma cell infiltration of 20% or more prior to mobilization. Patients with three risk factors had a probability of only 0.38 (95% CI 0.3-0.9) for adequate mobilization. Ten patients failed to mobilize; a period from diagnosis of 12 months or more and female sex were unfavorable factors. Patients with two risk factors had a probability of 0.50 (95% CI 0.2-0.8) for failing the mobilization procedure. These findings indicate that MM patients must be mobilized early in the course of the disease, with minimal disease burden before severe hematopoietic progenitor cell injury due to cumulative therapy.