Optimizing umbilical cord blood collection: impact of obstetric factors versus quality of cord blood units.

INTRODUCTION: The main limitation factor for wide use of umbilical cord blood units (UCBs) as a source of hematopoietic progenitors for transplantation is cell dose. International standard guidelines recommend 2 x 10(7)/kg as the minimal nucleated cell dose for UCB transplantation for adults and 3.7 x 10(7)/kg for children. Therefore it is important to the optimize donor selection and the collection method so as to achieve high cell doses. In this study our main purpose was to determine whether obstetric factors influence UCBs collected. STUDY DESIGN: The study involved 304 UCBs collected from January to December 2004. The UCBs were collected after donor selection based on international criteria for cord blood banking. We analyzed UCB biological features such as collected volume, total nucleated cells (TNC), and CD34-positive cells, and obstetric factors. RESULTS: First, our study showed by multivariate analysis that infant weight was the main factor that influenced biologic features of UCB collected such as total volume (P = .000), TNC (P = .000), CD34 total count (P = .003), and CFU-GM (P = .004). Placental weight > 600 g produced a better volume (P = .007) and increased TNC (P = .056). Gestational age > 39 weeks enhanced CD34% (P = .016). Regarding route of delivery, we found that cesarean section produced higher volume and reduced WBC count compared to vaginal delivery, regarding cord length, it increased TNC (P = .037). And last, we noticed that female infants increased WBC (P = .013) and CD34(+) total count (P = .019) more than male ones. CONCLUSIONS: Our results confirm that volume and TNC are influenced by several obstetric factors, such as greater infant and placental weight, predicting a better collection.

Prognostic value of cell marker analysis in de novo acute myeloid leukemia.

Clinical and cytologic characteristics were correlated to immunologic markers in 154 patients with newly diagnosed acute myeloid leukemia (AML). The panel of monoclonal antibodies (MoAbs) was selected to identify differentiation-associated antigens of both the myeloid and the lymphoid lineages (CD13, CD33, CD14, CD15, CD7, CD34, CD10, HLA-DR, CD19, CD2, CD5, TdT). The expression of multidrug resistance P-glycoprotein (P-170) was also evaluated in 117 patients. Differences in antigenic expression was observed among the various French-American-British (FAB) subgroups. HLA-DR was poorly expressed on the blasts of acute promyelocytic leukemia (M3), and was always found in FAB M5. CD34 was detectable in all M0 cases and only in one M3 (p < 0.001). Lymphoid-associated antigens were positive in 74 cases (48.1%). In particular, CD7 was found in 49 patients (31.8%), and TdT in 30 (21.3%), 15 samples displaying coexpression of these two antigens. The incidence of CD7+ cases was particularly elevated in M0 and M5 AML (p = 0.005). It significantly correlated with the expression of CD34, HLA-DR, P-170 (p < 0.001, p = 0.018 and p = 0.034 respectively), and with a leukocyte count > 50 x 10(9)/l (p = 0.038). Sixty-nine (59%) samples demonstrated P-170 positivity. Again, this phenotype was particularly expressed in the poorly differentiated forms (M5, M0 and M1) and showed significant correlation with the immaturity markers CD34, CD7 and HLA-DR (p = 0.013, p = 0.022 and p = 0.001, respectively). Expression of individual antigens correlated with prognosis. Refractoriness to first line therapy was associated with CD7 expression (p = 0.002) and P-170 (p = 0.001). The CD7 marker was also significantly associated with a very low overall survival (p < 0.001) and continuous complete remission (p < 0.001). CD14 expression also significantly predicted lower survival rates (p = 0.033). The combination (CD7+ CD14+) identified a subset of patients with a particularly adverse outcome. The prognostic value of CD7 expression, alone or in combination with other markers, was confirmed in multivariate analysis.

P-glycoprotein and terminal transferase expression identify prognostic subsets within cytogenetic risk classes in acute myeloid leukemia.

Clinical and biological features were assessed in 204 consecutive de novo adult acute myeloid leukemia (AML) patients who received intensive chemotherapy regimens. Multiparameter flow cytometric assays both of the multidrug resistance (MDR-1)-associated P-glycoprotein (PGP) using the UIC2 monoclonal antibody (MoAb), and of terminal transferase (TdT) were performed. Cytogenetic findings were obtained from 196 patients with high resolution banding. At onset, UIC2 and TdT positivities were detected in 58.5% and 24% of cases, respectively. There were strict correlations either between UIC2 negativity and FAB M3 or between TdT and FAB M0-M1 (P = 0.001 and < 0.0001, respectively). On the other hand, age was significantly associated with cytogenetic risk classes (P < 0.0001). CD34 positivity was highly correlated with TdT expression (P < 0.0001). Moreover, CD7 and CD11b were significantly represented in UIC2+ subset (P < 0.0001). Rhodamine 123 (Rh 123) efflux was significantly higher in 75 UIC2 positive patients compared to 65 UIC2 negative ones (P < 0.001). As regards to cytogenetics, TdT positivity was strongly related either to t(9;22) or single/associated anomalies of chromosome 7; on the other hand, most or all cases with t(8;21) or t(15;17) were UIC2 or TdT negative, respectively. The rate of first complete remission (CR) differed both between UIC2+ and UIC2- cases and between TdT+ and TdT- ones (40% versus 72%, P < 0.001; and 36% versus 61%, P = 0.001, respectively). The survival rates (Kaplan-Meier method) were significantly shorter either in UIC2+ or in TdT+ patients (P = 0.005 and = 0.011, respectively). UIC2 and TdT negative cases showed longer remission duration (P = 0.03 and = 0.22, respectively). The additional effect of UIC2 and TdT on prognosis allowed us to identify two subsets of patients, the first [UIC2- TdT-] at better and the second [UIC2+ TdT+] at worse clinical outcome compared to single UIC2 and TdT cases, concerning CR (P < 0.001), survival (P < 0.0001) and CR duration (P = 0.007). The combinations [UIC2+ TdT-] and [UIC2- TdT+] showed an intermediate clinical course. A strong difference was found between poor risk and intermediate/favorable risk cytogenetic classes with regard to CR rate (P < 0.0001), overall survival and CR duration (P < 0.001). Nevertheless, within the poor risk class, UIC2 positivity was able to identify patients at worst prognosis with regard to CR (P = 0.005), survival (P = 0.02) and CR duration (P = 0.015). On the other hand, UIC2 and TdT negativity allowed us to distinguish patients with longer survival (P = 0.012 and = 0.04, respectively) and CR duration (P = 0.04 and = 0.025, respectively) within the intermediate/favorable risk class. The independent prognostic value of UIC2, TdT and cytogenetic risk classes was confirmed in multivariate analysis. These results suggest that PGP and TdT expressions, together with cytogenetic findings, may represent a basic predictor of chemotherapeutic failure in AML.

Enumeration of CD34+ hematopoietic progenitor cells for clinical transplantation: comparison of three different methods.

Three different methods for determination of CD34+ cells in G-CSF-mobilized peripheral blood were compared. The methods were: the Milan/Mulhouse protocol, the ISHAGE guidelines for CD34+ cells enumeration and our own protocol. The procedure we have adopted is essentially a Milan/Mulhouse protocol-derived methodology combined with a multiparametric approach using the PAINT-A-GATE software analysis program. The samples were collected from 70 patients affected by acute leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, myeloma and breast cancer who were scheduled to receive autologous PBSC transplantation. PBSC collection was performed following mobilization with subcutaneous G-CSF at 5-10 microg/kg/day. A minimum target of 2 x 10(6)/kg CD34+ cells was considered an acceptable harvest to ensure a safe transplant. On average, three aphereses per patient were performed and a total of 204 apheresis samples were analyzed. Regression analysis of the percentage and absolute number of CD34+ cells, as calculated with each method, achieved an excellent correlation in spite of methodological differences. In fact, both CD34+dim and CD34+CD45- events were included in our gating strategy. In the setting of a triple staining associating CD34, CD38 and CD45, we identified a variable fraction of CD34+CD38+CD45- cells which would be otherwise undetected due to its CD45 negativity. To this end, we used a new technology referred to as laser-scanning cytometry (LSC) which allowed the isolation and morphological identification of CD34+CD45- cells. By comparing CD34+CD45+ and CD34+CD45- cells, we found that they share a common morphology, thus confirming the hypothesis that the latter are to be considered for CD34+ cell calculation. The median number of CD34+ cells/kg, as calculated by the three methods, was: 4.79 x 10(6)/kg (range 1-570) for the Milan/Mulhouse protocol, 3.9 x 10(6)/kg (range 0.8-498) for the ISHAGE one, and 5.17 x 10(6)/kg (range 2-599) for our protocol. The median time to ANC and PLT engraftment was 11 (range 9-24) and 20 (range 10-70) days, respectively. Our protocol achieved the best correlation between CD34+ cells/kg and time to ANC/PLT recovery according to the Spearman's rank test (r = -40 and P < 0. 015 for ANC, r= -46 and P = 0.005 for PLT). We conclude that (1) CD45 does not appear the ideal partner of HPCA-2 for determination of hematopoietic progenitors in mobilized peripheral blood; and (2) for clinical application, a single staining with 8G12 appears simple, reliable and feasible when rigorous procedures for sample preparation and acquisition are followed and an adequate software for multiparametric analysis is available.

P-glycoprotein expression in de novo acute myeloid leukemia.

Detection of the multidrug resistance P-glycoprotein (PGP) phenotype was performed at the time of diagnosis in 223 patients with acute myeloid leukemia (AML) by flow cytometry using C219 Monoclonal Antibody (MoAb). On the other hand, JSB1 MoAb was tested in 173 of these samples. At onset, PGP was detected in 57.4% of cases with C219 and 75.9% of cases with JSB1. There was no correlation between PGP expression and sex, age, marrow blast percentage or extramedullary disease. On the contrary, strict correlations were noted either between C219 negativity and FAB M3 subtype or between C219 positivity and FAB M5 group (P = 0.003). Significant correlation was found between PGP phenotype and CD7, as 143 of 223 samples had similar patterns of staining with C219 (P < 0.0001). Finally, there was a close relationship between C219 and JSB1 positivity: all the C219+ cases were positive for JSB1 (P < 0.0001). Concerning the karyotype, most patients with monosomy or del (7) were MDR positive; on the other hand, most patients with t(8;21) or t(15;17) were MDR negative. Rh123 accumulation studies showed a significant decrease of mean fluorescence intensities both in C219 and in JSB1 positive cases in comparison with PGP negative ones (P < 0.001). A significant decrease of remission induction rates (CR) was highlighted both between C219+ and C219- and between JSB1+ and JSB1- cases (32.1% v 62.1% and 32.6% v 73.8%, respectively, with P < 0.0001). The overall survival and the remission duration (CCR) were significantly shorter both in C219+ and in JSB1+ patients with no relationship to age. Furthermore, a higher rate of early relapses was noted among MDR+ when compared with MDR- patients both for C219+ and JSB1+ cases. The combination (C219- JSB1+) identified a subset of patients with an intermediate prognosis. On multivariate analysis, C219 and JSB1 were confirmed to be independent prognostic factors for achievement of CR, overall survival and CCR. In conclusion, the assessment of MDR phenotype by flow cytometry is a crucial prognostic factor of treatment outcome in AML.

Lineage identification of acute leukemias: relevance of immunologic and ultrastructural techniques.

This study assesses the value of immunologic and ultrastructural methods in disclosing the lineage commitment of cells from acute leukemias (ALs). Two hundred and fifty-one ALs were characterized morphologically, cytochemically, and immunologically. Myeloperoxidase (MPO) positivity in > 3% of blasts was regarded as evidence of the myeloid origin of leukemic cells, cytoplasmic CD22 (cCD22) expression was taken as an indication for B-lineage acute lymphoblastic leukemia (ALL), and CD3+ (membrane or cytoplasmic) cases were classified as T-ALL. Diagnosis of minimally differentiated acute myeloid leukemia (AML-M0) was made when blast cells had undifferentiated features by light microscopy, reacted with at least one of the antibodies to myeloid-specific antigens (CD13, CD33, MPO), and lacked CD19, cCD22, and c/mCD3. Megakaryoblastic differentiation was demonstrated by the expression of CD41 and/or CD61. Following these criteria, 209 cases were classified as acute myeloid leukemia (AML) and 39 as ALL. Expression of lymphoid antigens was detected in 45% of AML cases and 30% of ALLs showed myeloid antigens. One case was regarded as a true biphenotypic leukemia because of the combined expression of MPO and CD33 for the myeloid lineage, and cCD3, CD2, and CD5 for the T-cell lineage. Two cases lacked signs of myeloid or lymphoid differentiation and were studied by electron microscopy methods. One displayed platelet peroxidase (PPO) activity and was classified as a megakaryoblastic variant, one other reacted with anti-CD33 and was considered AML-M0. We conclude that light microscopy and standard immunologic methods can accurately demonstrate the lineage orientation in greater than 99% of ALs. Integration with ultrastructural analysis can define the cell nature of virtually all cases of AL.

Diagnosis of acute myeloid leukemia and system Coulter VCS.

BACKGROUND: The aim of this study was to evaluate the possible contribution VCS could make in a hematological laboratory for the diagnosis of acute myeloid leukemia (AML). MATERIALS AND METHODS: Peripheral blood samples from 42 AML patients and 58 normal donors were analyzed by flow cytometry with the VCS. Normal and leukemic peripheral blood samples were tested to establish a correlation between VCS data and the reference manual method. We evaluated the sensitivity threshold of the VCS for blast cell detection in progressively diluted samples. We looked at a correlation between different scatterplots and flags and the FAB classification of acute myeloid leukemia in order to identify a characteristic VCS image for each subtype. Thirty-four bone marrow samples (18 normal donors and 16 leukemic patients) were analyzed by the VCS system to demonstrate a characteristic scattergram distribution. Further, we tried to compare scatterplots to the flags of leukemic bone marrow samples and, finally, we compared VCS scatterplots with aberrant antigen expression in AML cases. RESULTS AND CONCLUSIONS: Overall VCS specificity was 93.1% (54/58) in peripheral blood samples; sensitivity was 100% (42/42) and VCS efficiency was 96%. In AML the characteristic X6 flag was observed in 95.23% of the cases (40/42). In peripheral samples discrimination was made between AML M1 with agranular blasts > 50% of the non erythroid cells (NEC), M4, M5 on the one hand, and AML M1 with granular blasts > 50% of NEC, M2, M3 on the other: the X5 flag was often present in the second group because of the different localization of the cells (p = 0.001). In all normal bone marrow samples we observed granuloblasts in different maturation stages in the neutrophil region of the DF1 VCS scatterplot corresponding to the X5X6 flags or, rarely, to the X5X6X1, because of the presence of immature erythroid cells. This association X5X6 was never observed alone in patients affected by AML. In our study, it was difficult to identify peculiar scatterplots and alarms for each FAB class of AML. Nevertheless, we observed that in all M4 and M5 FAB cases the blastic cells both in the peripheral blood and in the bone marrow samples were located in the monocyte region, with the frequent presence of the X3 flag often associated with the X6 flag. Eight out of the 16 AML bone marrow samples (1 FAB M0, 1 M2, 1 M3, 2 M4, 3 M5) showed the X2 flag and partial localization of blasts in the lymphoid region. In all these cases the presence of some small blastic cells with agranular cytoplasm was confirmed by morphological observation and cytochemical stainings.

Clinical significance of CD38 expression in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) follows heterogeneous clinical courses, and several biological parameters need to be added to the current clinical staging systems to predict which patients will experience an indolent or an aggressive outcome. This study analyzed CD38 expression by flow cytometry and soluble APO1/Fas (sAPO1/Fas), Bcl-2 (sBcl-2), and CD23 (sCD23) proteins by immunoenzymatic methods to evaluate their effect on the clinical course of 168 unselected B-CLL patients. Intermediate/high risk modified Rai stages were characterized by a higher CD38(+) B-cell number (P =.0002) and higher sCD23 levels (P <.0001). Moreover, CD38(+) B-cell percentages were significantly and directly associated both with beta(2)-microglobulin and sCD23 concentrations (P <.0001 and P =.002, respectively). Both a higher tumor burden (lymphadenopathy/splenomegaly) and a lymphocyte doubling time less than 12 months were significantly associated with higher CD38(+) percentages (P <.0001 and P =.0001, respectively). With regard to clinical outcome, progression-free survival was significantly longer (75% versus 37% at 5 years; P =.00006) in patients with lower CD38(+) B-cell percentages. Furthermore, the risk of partial or no response to fludarabine increased with increasing CD38 expression (P =.003), and a shorter overall survival (50% versus 92% at 8 years; P <.00001) characterized patients with more than 30% CD38(+) B-cell number. The predictive value of CD38 expression was maintained among the patients within the Rai intermediate risk group and was confirmed in multivariate analysis. Thus, the percentage of CD38(+) B cells appears to be an accurate predictor of clinical outcome and therefore could be used to indicate when more novel chemotherapeutic approaches are needed.

Quantitative analysis of Fas and bcl-2 expression in hematopoietic precursors.

BACKGROUND AND OBJECTIVES: We investigated the expression of bcl-2 and CD95 (Apo1-/Fas) on CD34+ cells obtained from bone marrow (BM), mobilized peripheral blood (MPB), and umbilical cord blood (UCB) samples. The expression of bcl-2 and Fas was then compared with that of other markers usually associated with immaturity; functional tests using the agonistic antibody anti- Fas CH11 were also carried out. DESIGN AND METHODS: The analysis was performed by flow cytometry on purified CD34+ cells in a three (CD95 PE, CD34 APC and CD71 FITC) and in a four (CD38 PE, HLA-DR PerCP, CD34 APC and bcl-2 FITC) fluorescence assay. RESULTS: The results were expressed as mean fluorescence index (MFI); bcl-2 expression was significantly higher (p < 0.001) in BM (3.73 +/- 0.63) than in MPB (2.47 +/- 0.39) and UCB (2.38 +/- 0.58); Fas was significantly less expressed (p < 0.001) in UCB (1.27 +/- 0.78) than in MBP (3.63 +/- 2.19) and BM (4.56 +/- 1.69). CD34 expression was significantly (p < 0.001) brighter in UCB compared to in MBP and BM, while CD38 and CD71 were significantly (p = 0.005 and p < 0.001, respectively) more expressed in BM than in MPB and UCB. Fas values were directly correlated to CD38; both Fas and bcl-2 were directly related to CD71 and inversely to CD34. Culture assays showed that hematopoietic precursor cells from BM, MPB and UCB had a low susceptibility to undergo Fas-mediated apoptosis. INTERPRETATION AND CONCLUSIONS: In conclusion, bcl-2 and Fas are less expressed in UCB than in MPB and BM; early hematopoietic precursor cells are relatively resistant to CD95-triggered apoptosis; the observed correlation between Fas/bcl-2 and markers of immaturity suggests that they may be determinants of commitment in early hematopoietic precursors.

Minimally differentiated acute myeloid leukemia (AML-M0): comparison of 25 cases with other French-American-British subtypes.

We compared the immunophenotypic and karyotypic features of 25 cases of minimally differentiated acute myeloid leukemia (AML-M0) with those of 247 cases comprising all AML French-American-British (FAB) classification. Myeloperoxidase (MPO) was detectable with a specific monoclonal antibody in all cases of AML-M0, whereas CD13 and CD33 were both negative in 4 of the 25 cases. Thus, anti-MPO reliably detects minimal myeloid differentiation in AML-M0. CD34 and terminal deoxynucleotidyl transferase (TdT) were more frequently expressed in AML-M0 (96% and 68% of the cases, respectively) than in the other FAB subsets (P < .001 for both). By contrast, GP-170 and CD7 were less frequently expressed in AML-M0 than in FAB classes such as M1, M4, and M5 (P = .02 and .003, respectively). A total of 80% of AML-M0 cases carried lymphoid markers (including TdT), and 48% showed a coordinate positivity for two or more of them. CD2, CD5, CD10, and CD19 were expressed in a similar fashion among the different FAB groups, whereas CD4 expression was significantly more frequent in AML-M0, AML-M4, and AML-M5 (P = .014). AML-M0 was characterized by a more frequent occurrence of complex karyotypes. In addition, approximately 20% of cases had TdT positivity, complex karyotypes, and anomalies of chromosome 5 and/or 7, a pattern not observed in the other FAB subsets. Finally, 80% of anomalies of chromosome 5 and/or 7 in AML-M0 were comprised within complex karyotypes, whereas only 13% of the remaining FAB cases carried this feature. In summary, AML-M0 frequently expresses immunophenotypic and karyotypic aspects that are likely to identify a "stem cell" pattern.