A closed system for the clinical banking of umbilical cord blood.

Umbilical cord blood (UCB) is a source of hematopoietic progenitor cells and is used as an alternative to the bone marrow or peripheral blood for treatment of several onco-hematological diseases. Because of the limited number of CD34+ hematopoietic stem cells present in UCB units and of the elevated costs of cryopreservation, it is of paramount importance to select the UCB units that are clinically useful before storage and optimize banking efficiency by designing reliable procedures to process and freeze the selected units. Among the different parameters characterizing UCB, nucleated cell (NC) and CD34+ cell content provides useful criteria to select UCB units since clinical data documented that the infused cell load (both NC and CD34+ cells) plays an important role in the successful outcome of transplants. By evaluating volume, CD34+ cell content, NC total amount, and NC density of 117 UCB units, we found a significant association between CD34+ cell content and NC density and total amount, indicating these parameters as useful to decide UCB clinical utility. Furthermore, we set up a fast procedure to process UCB units for storage. A system for NC separation and volume reduction of UCB samples in a dedicated, germ-free, closed circuit was developed, where plasma and red blood cells (RBC) depletion was obtained by sedimentation in the presence of a 3.5% Polygeline solution. By this separation system, both RBC depletion and high NC and CD34+ cell recoveries were achieved in 60 min, and the yield was comparable to the one obtained by other separation methods. Since Polygeline has been clinically used as a plasma expander and no toxic effects on patients were reported, the protocol can be applied in the large-scale banking of UCB.

Qualitative multiplex RT-PCR for simultaneous detection of hepatitis C virus and human immunodeficiency virus in plasma samples.

This report describes the development of a one-tube multiplex reverse transcriptase (RT)-PCR assay for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) in plasma samples. The assay was evaluated with two panels of HCV- and HIV-1-positive samples, as well as negative plasma specimens. Extraction and amplification of HCV and HIV-1 RNA from plasma samples were performed in a single reaction, and amplified genomes were detected with specific probes. Serial dilutions of the HCV and HIV-1 first World Health Organization International Standards were used to evaluate the sensitivity of the method. Two RNA controls were constructed to determine inter-assay variations and the sensitivity of the amplification step. The assay had good specificity and detected all the genotypes and subtypes tested. The analytical sensitivity of the entire assay was 100 IU/mL for HCV and 200 IU/mL for HIV-1, while the amplification step detected ten copies/reaction for HCV and 20 copies/reaction for HIV-1. The multiplex assay allowed the simultaneous extraction, amplification and detection of two virus genomes, thereby providing an important practical advantage and an efficient approach for analysing individual and pooled plasma donations.

A novel protocol that allows short-term stem cell expansion of both committed and pluripotent hematopoietic progenitor cells suitable for clinical use.

To obtain long-term engraftment and hematopoiesis in myeloablated patients, the cell population used for hematopoietic reconstitution should include a sufficient number of early pluripotent hematopoietic stem cells (HSCs), along with committed cells from the various lineages. For this purpose, the small subset of CD34+ cells purified from different sources must be expanded ex vivo. Since cytokines may induce both proliferation and differentiation, expansion would provide a cell population comprising committed as well as uncommitted cells. Optimization of HSC expansion methods could be obtained by a combination of cytokines able to sustain renewal of pluripotent cells yet endowed with poor differentiation potential. We used variations of the combinations of cytokines described by Brugger et al. [W. Brugger, S. Heimfels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 333, 283-287] and Piacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Aglietta (1997) Blood 89, 2644-2653] to expand UCB CD34+ cells and monitored proliferation rate and phenotype after 14 days of culture. Several hematopoietic lineage-associated surface antigens were evaluated. Our data show that flt3L and thrombopoietin in combination with IL-3, while sustaining a high CD34+ proliferation rate, provide a relatively low enrichment in very early uncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the number of CD34+/CD38- cells. A combination of the above protocols, applied simultaneously to aliquots of the same sample, would allow expansion of both committed and pluripotent HSC. This strategy may represent a significant improvement for clinical applications.

Phenotypic characterization of immunomagnetically purified umbilical cord blood CD34+ cells.

This study describes the multilineage differentiation pattern of purified CD34+ stem cells obtained from human umbilical cord blood. CD34+ cells were collected from 49 umbilical cord blood samples. Following immunomagnetic purification, cells were double stained with anti CD34 and CD71, CD61, CD7, CD19, CD33, CD36 and triple stained with anti CD34, CD38 and HLA-DR. Analysis were performed using a FACScan flow cytometer. After purification, the mean CD34+ cells' purity was 85.49 +/- 7.08%. Several subpopulations of umbilical cord blood CD34+ cells were identified indicating different lineage commitment. The majority of CD34+ cells expressed both CD38 and HLA-DR (91.74 +/- 3.76%), while those lacking CD38 were 3.43 +/- 2.12% (CD38-DR+) and 1.81 +/- 1.54% (CD38-DR-). These data were compared to the expression of lineage commitment markers on purified CD34+ cells from 5 mobilized peripheral blood samples. The percentage of peripheral blood CD34+CD38-DR+) and CD34+CD38-DR- cells was significantly lower than umbilical cord blood, 0.24 +/- 0.18% and 0.04 +/- 0.03% respectively. The knowledge and standardized of umbilical cord blood CD34+ cells phenotype is critical since umbilical cord blood volume is limited. The homogeneity of CD34+ subpopulation phenotype suggests that monitoring of lineage differentiation antigens may not be relevant for clinical use of umbilical cord blood samples. However, the observed higher percentage of pluripotent CD34+38- stem cells in umbilical cord blood compared to peripheral blood, that might explain the successful clinical use of umbilical cord blood even when low number of cells are used, candidates these antigens as the predictive parameter for clinical use of umbilical cord blood samples.

Immunoreactivity to putative B-cell epitopes of hepatitis G virus polyprotein in viremic and nonviremic subjects.

The hepatitis G virus (HGV) polyprotein was scanned by computer-aided prediction of antigenicity to search for B-cell epitopes. Four polypeptide sequences, V37D (amino acids [aa] 1685 to 1721), V36S (aa 2102 to 2137), P37R (aa 2156 to 2192), and C40P (aa 2280 to 2319), were identified and synthesized for use in immunoassays. Antibodies to these peptides were searched for in a panel of 239 serum samples, which were also tested for anti-E2 antibodies and HGV RNA. Furthermore, the course of HGV markers was studied prospectively in four patients who had been transfused with HGV RNA-positive blood. There was a negative association between immunoreactivity to V37D and P37R and presence of HGV RNA (2 of 53 and 1 of 53, respectively; P < 0.05); none of the subjects with dual antibody positivity was HGV RNA positive. Anti-V37D and anti-P37R antibodies compared favorably with anti-E2 antibodies as markers of recovery from HGV infection. These results might be useful for the development of new, more sensitive diagnostic assays.

[Role of autologous stem cell transplantation in the treatment of Hodgkin's lymphoma]. / Il ruolo del trapianto di cellule staminali autologhe nella terapia del linfoma di Hodgkin.

Among lymphomas, treatment of Hodgkin's disease (HD) allows the highest cure-rate. Radiotherapy (RT) represents first choice therapy in early stages, providing complete remission (CR) rate even superior to 90%. Chemotherapy (CHT) or, when indicated, the combined modality treatment (CHT + RT) is successful, in terms of long overall survival (> 10 yrs) in more than 60% of patients with advanced stage disease at onset. Considering all stages of disease at onset, about 75% of patients can be cured. However, the remaining 25% results resistant to the conventional approach (CHT +/- RT) or, mainly, relapses after first CR. For these poor prognosis patients, it has been assessed the possibility of inducing (or reinducing) a CR by using high dose CHT with stem cells rescue. Autologous stem cell transplantation (ASCT) consists in the administration of antiblastic drugs at so high dosages to require the consequent reinfusion of stem cells, preventively harvested and cryopreserved, thus dramatically decreasing the risk of a prolonged bone marrow aplasia. This procedure is currently performed as intensification treatment in selected cases of patients with advanced stage at onset, once in CR after first-line therapy. Therefore, the development of prognostic models aimed to define with higher sensibility and specificity patients at high risk of relapse and to be submitted to ASCT as consolidation therapy, is becoming of increasing interest.

Peripheral blood stem cell collection from G-CSF-stimulated unrelated donors for second transplant.

Peripheral blood stem cells (PBSCs) collected following stimulation with cytokines are commonly used for autologous haematopoietic transplants. Currently, PBSCs are being used for syngeneic or allogeneic transplants from matched or haploidentical donors. However, many issues are still unanswered regarding the early or late side-effects cytokines have on recipients and on healthy donors. The aims of this paper were to evaluate the experience acquired worldwide in this field, to define the acceptability of stem cell donation by G-CSF-stimulated apheresis from unrelated donors after the failure of a first donation, and to assess side-effects of G-CSF on unrelated donors. The use of PBSCs has increased tremendously over the last few years and in the near future PBSCs will probably become the most relevant source of stem cells. Studies conducted so far have definitely concluded that G-CSF is safe and well tolerated. Results observed in transplants utilizing marrow stem cells compared with results obtained in transplants utilizing PBSCs have shown that patients undergoing this latter procedure recover earlier, require a lower number of transfusions and spend fewer days in hospital with a consequent decrease in costs. We concluded that a second transplant by G-CSF-stimulated apheresis from an unrelated donor is definitely acceptable and we designed a prospective study to better define all controversial aspects. Donors will be given 10 microg/kg/day of G-CSF subcutaneously for 5 days. One or two PBSC collection procedures will be performed: the first on day 5 and the second, if necessary, on day 6. Donors will be surveyed and blood counts monitored in a standardized manner during the process.

Autologous stem cell transplantation for multiple myeloma: a single centre experience.

Thirty-two patients with multiple myeloma (MM) were autografted in our Centre over a 3-year period. Twenty-three patients had a newly diagnosed MM submitted to one induction regimen and 9 had a refractory or relapsing disease treated with at least two different chemotherapy lines: 15 out of 32 patients were sensitive to conventional treatment. In 2 patients BM was harvested while in the majority PBSC were collected after administration of 7 g/m2 Cyclophosphamide plus G-CSF (in 25 patients) or G-CSF alone at the dose of 16 microg/Kg/daily for 5-7 days (in 5 patients). Conditioning regimen was busulfan 16 mg/Kg plus melphalan 120 mg/m2. One patient died of cerebral hemorrhage after reinfusion of PBSC. Out of 31 evaluable patients, 24 (77%) had a response which was complete in 6 patients (19%) and partial in 18 patients (58%), 5 cases (17%) had no response, and 2 (6%) showed myeloma progression. There was a statistical difference in the outcome between newly diagnosed and pretreated patients (p = 0.003). At a median follow-up of 9 months (range 5-37), two patients had died for progression and 3 out of the 29 alive, relapsed after 17, 18 and 36 months respectively. Although median overall survival was not reached, there was a significant survival benefit for autografted patients in comparison with a matched control group conventionally treated in our Centre before 1994 (p = 0.02).

Treatment intensification of malignant lymphomas with autologous bone marrow transplantation and granulocyte colony stimulating factor.

Treatment intensification with autologous bone marrow transplantation (ABMT) was administered to 37 cases of Hodgkin's and non-Hodgkin's lymphoma (HL and NHL) who were in complete or partial remission (CR or PR) after chemotherapy (MOPP/ABVD or F-MACHOP respectively) and to 12 cases of HL and NHL who were in relapse. ABMT treatment was BAVC for NHL and BEAM for HL. Marrow cells were harvested from the marrow and cryopreserved. The number of mononuclear marrow cells that was reinfused ranged between 0.19 and 0.80 x 108/Kg b.w. (median 0.39). All the patients were treated with granulocyte colony stimulating factor (G-CSF, Filgrastim) at a dose of 5 mg/Kg b.w. from day +4 until the absolute neutrophil count exceeded 1 x109/L for 3 consecutive days. Engraftment was observed in all cases, and no transplant-related deaths occurred. The patients with NHL and HL received a median of 12 (range 2-19) and 14.5 (range 9-27) doses of G-CSF respectively. Median time to 20 x 109/L platelet count was 14 to 17 days. Median time to an absolute neutrophil count 0.5x109/L was 13 days. A febrile episode during the period of post-transplant aplasia was documented in 35 patients (71%). Fever was associated with Gram+ bacteraemia in 31% of the cases and with Gram- bacteraemia in 11% of cases. Herpes Simplex infection was documented in two cases. No fungal infections were recorded. Median hospitalisation time from reinfusion ranged between 19.5 days (NHL) and 23 days (HL). Thirty-four of 37 cases (92%) who were transplanted in CR or in PR are currently alive and in continuous CR with a median follow-up time of 37 months after ABMT. Three of 12 cases (25%) who were transplanted in relapse are alive and in CR. Our data point out that ABMT followed by G-CSF is a safe and a very effective procedure for high risk malignant lymphomas, when ABMT is planned and is performed not as a rescue procedure but when it is integrated in the treatment strategy from the very beginning.

Primary systemic CD30 (Ki-1)-positive anaplastic large cell lymphoma of the adult: sequential intensive treatment with the F-MACHOP regimen (+/- radiotherapy) and autologous bone marrow transplantation.

Few series of adult patients with primary systemic CD30 (Ki-1)-positive anaplastic large cell lymphoma (ALCL) are reported in the literature; most of them have been treated with combination chemotherapy (CHT), with only an occasional patient being autotransplanted, mainly after relapsing. The remission rate ranges from 60% to 90%, but relapses are frequent (up to 60%) and precocious (mainly in the first 24 months). The aim of our study was to analyze the outcome of a series of adult patients affected by primary systemic ALCL that were treated at our institution with a sequential intensive therapeutic program including CHT, radiotherapy (RT), and autologous bone marrow transplantation (ABMT). Sixteen consecutive, unselected patients with ALCL were identified. All of them were treated with the 5-fluorouracil, methotrexate, cytosine arabinoside, cyclophosphamide, doxorubicin, vincristine, and prednisone (F-MACHOP) regimen; 9 of 16 (56.2%) reached a complete remission (CR). In six cases with residual mediastinal disease, involved-field RT was performed, allowing three additional patients to become free of disease. All 16 were then autotransplanted with bone marrow stem cells after conditioning with the cytosine arabinoside, etoposide, cyclophasphamide, and carmustine (BAVC) regimen. A present, 16 of 16 patients are alive and in CR. The actuarial overall survival is 100% at a median of 45.5 months, and the actuarial disease-free survival is 100% at a median of 33.5 months. These data suggest that ALCL can be successfully managed with a sequential intensive treatment (CHT +/- RT + ABMT) that prevents early relapses and projects these patients as long-term survivors.

Effect of marrow processing on hematopoietic cell recovery in bone marrow harvests: focus on filtration.

Bone marrow processing requires a first step of filtration to remove small clots, bone fragments, fat cells and fibrin followed by centrifugation to separate mononuclear cells (MNC). These procedures cause a significant loss of cells potentially including hematopoietic stem cells (HSC). We therefore analyzed the cell recovery and phenotype of various fractions (whole marrow; filtered marrow; MNC collected after centrifugation; bone marrow fragments trapped by filtration) of bone marrow harvests (BMH) from patients with different hematological malignancies undergoing autologous bone marrow transplantation. Analysis of 25 BMH showed that the mean percentage of WBC and MNC recovered after filtration was respectively 92.28 +/- 7.42% and 92.3 +/- 9.05% of the original BMH while after centrifugation the percentage was 20.23 +/- 6.47% and 75.7 +/- 12.81%. The percentage of cells present in the tissue fragments trapped in the filters obtained from five BMH was only 3.93 +/- 1.25% (WBC) and 5.65 +/- 2.2% (MNC) of those originally present in the harvest. Phenotypic analysis performed on the same samples showed that there is no selective loss of MNC or CD34+ cells in the filtration process. Our data indicate that processing of BMH, in particular filtration of tissue fragments, does not affect the recovery of HSC.

Autologous bone marrow transplantation in non-Hodgkin's lymphomas: prediction of mononuclear cell yield in bone marrow harvests.

We retrospectively analyzed the factors influencing the mononuclear cell (MNC) yield on bone marrow (BM) harvests in a cohort of 15 non-Hodgkin's lymphoma patients undergoing autologous bone marrow transplantation. All the patients were treated with the F-MACHOP regimen and four of them also received radiotherapy on bulky disease. Before harvesting, the patients underwent complete peripheral blood (PB) count, BM biopsy and aspirate. WBC and MNC/microL were determined on the pre-harvest PB and BM aspirate samples using an automated counter. The following variables were examined in univariate and multivariate regression analysis for possible influence on the MNC yield in the harvests: age, sex, number of cycles of CT, previous radiotherapy, state of the disease at the time of harvest, interval between the end of therapy and BM harvest, cellularity of the BM biopsy, absolute WBC and MNC counts in the PB before harvesting, absolute WBC and MNC counts in the BM aspirate performed before harvesting. The amount of marrow harvested was constant for all patients: 21.2 +/- 0.24 microL/kg B.W. Among the factors analyzed, two correlated strongly with the MNC yield/kg B.W. in the harvests: the MNC count in pre-harvest PB (r = 0.823; p = 0.0001) and the MNC count in pre-harvest BM aspirate (r = 0.802; p = 0.0003). A regression equation was generated that allows calculation of the MNC/kg yield prior to harvesting.

A multicentric seroepidemiological survey of HTLV-I/II in Italy.

BACKGROUND: Several studies carried out in the USA and in Europe have shown the presence of HTLV-I/II antibodies in subjects belonging to high-risk groups for HIV infection as well as blood donors. Concern about the presence of HTLV-I/II markers in the normal population, as well as the efficient transmission of HTLV-I/II by whole blood or infected blood cells have led several countries to include screening for anti-HTLV-I/II among the mandatory serological testing of blood donors. OBJECTIVE: In order to assess the risk of HTLV-I/II infection related to blood transfusions, a multicentric survey for antibodies against HTLV-I and HTLV-II was carried out involving 10 Italian sites during the spring of 1991. STUDY DESIGN: Serum specimens were collected from 14,598 blood donors, 1,411 injecting drug users, 1,015 thalassemics, 142 hemophiliacs and 138 hemodialysis patients. HTLV antibodies were detected by a screening EIA which combines a viral lysate with a recombinant HTLV-I env protein (p21e). The serological confirmation was performed by a semi-automated dot-blot immunoassay that detects gag p19 and p24 and env p21e specific antibodies, while the discrimination of HTLV-I and HTLV-II reactivities was carried out by EIAs employing synthetic peptides of the ENV region specific for each virus. RESULTS: The seroprevalence of confirmed positives was 0.034% among blood donors and 3.61% among IDUs, while no sample of the other categories could be confirmed, although several were indeterminate and one thalassemic reacted against HTLV-I on peptide testing. HTLV-I reactivity was observed in one blood donor, while all 38 of the 51 confirmed seropositive IDU's reacted only to the HTLV-II synthetic peptide. CONCLUSIONS: These data confirm a high prevalence of HTLV-II among Italian IDUs and show an HTLV-I/II seroprevalence among blood donors very similar to that which was found in the USA volunteer blood donors. A surveillance program among blood donors seems advisable in order to establish the possible need of a mandatory screening for HTLV-I/II.

Prevalence of hepatitis B markers among teen-agers in Friuli.

In 1989, the prevalence of hepatitis B virus (HBV) markers was studied by Elisa in 421 healthy teen-agers, 17-19 year old, in Udine, Friuli. The prevalence of any HBV marker was 2.4%, with a male predominance (3.8% vs. 0.5%). The prevalence of hepatitis B surface antigen (HBsAg) was 0.5% (2/421); both subjects were anti-Hbc IgM negative, with AST/ALT values in the normal levels, and males. The prevalence of any HBV marker was not associated with largest family size, nor with lowest father's years of schooling. These findings suggest a very low exposure to HBV infection in such area.

[Platelet aggregation test. When is it useful?]. / Il test di aggregazione piastrinica. Quando è utile?

PIP: The platelet aggregation test is not useful for cases of coagulation disorders, and this study attempts to identify the situations in which it cannot provide useful information. This platelet test using the Born aggregometer can be used to identify various pathologies of adhesion, aggregation and ADP, as well as anomalous bleeding. This is not the case for thrombic diseases, because platelets are involved only in arterial thrombosis, and because it is impossible to tell whether or not the platelet anomaly indicated by a positive test was caused by the thrombosis itself. Once activated, platelets become refractory to in vitro stimulation with aggregating agents. The test is thus not specific for hyperaggregation syndromes, and should not be considered as an aid in their study. Data gathered in the Udine territory showed that, in most cases, the platelet aggregation test is required in situations in which it serves no real purpose, especially for women treated with oral contraceptives. In the absence of risk factors such as tobacco smoke, age, diabetes and obesity, oral contraceptives should not be denied based on a positive test, which does not prove that a platelet anomaly exists because of the pill.^ieng