Alberto Orfao

Summary. Depending on their stage of maturation and other factors, mast cell (MC) subsets differ from each other in terms of the expression of complement‐associated antigens. This study analysed the expression of various complement‐related cell surface antigens (CD11b/CR3, CD11c/CR4, CD35/CR1, CD55/DAF, CD59/MIRL, CD88/C5aR) on bone marrow mast cells (BMMC) in patients suffering from systemic mastocytosis (SM), other haematological diseases and non‐haematological disorders (control groups). Expression of complement‐associated cell surface antigens was analysed by flow cytometry. There were clear immunophenotypic differences between BMMC obtained from patients with SM and those from the control subjects: the percentage of patients expressing surface CD11c, CD35 and CD88 was significantly higher in patients with SM (76%, 100%, 54%) than in the control subjects (58%, 11%, 18%) (P < 0·05). In addition, the levels of CD11c, CD35 and CD88 expressed per MC (sites per cell) were significantly higher (P < 0·05) in SM than in the control group. Expression of the complement regulatory molecules CD55 and CD59 was detected in BMMC in all patients analysed. However, the levels of CD59 per BMMC were higher in patients with SM as compared with the control subjects, which could help to explain the formation of BMMC aggregates in the former group of individuals. Together, our results showed that BMMC in systemic mastocytosis overexpressed the cell surface membrane receptors involved in binding of complement components and complement‐mediated cell activation. Whether this pathological expression of complement receptors is of pathophysiological significance remains to be determined.

BACKGROUND: Monoclonal gammopathy of undetermined significance (MGUS) is a clonal plasma cell (PC) disorder usually characterized by a benign clinical course. However, in approximately 25% of patients, the disorder has been found to evolve into a multiple myeloma (MM). The mechanism leading to the evolution of MGUS remains unknown. The aim of the current study was, first, to assess by interphase fluorescence in situ hybridization (FISH) the incidence of numerical abnormalities of chromosomes 6, 9, 13, and 17 in MGUS patients and to compare it with that found in MM and PC leukemia (PCL) patients and, second, to explore the potential heterogeneity of the pathologic PC in MGUS as a way to identify unique cytogenetic patterns different from those frequently observed in MM and PCL.METHODS: Numerical abnormalities of chromosomes 6, 9, 13, and 17 were investigated by dual‐ and triple‐color FISH in bone marrow PC from 208 patients corresponding to MGUS (n = 30), MM (n = 158), and PCL (n = 20) cases. In MGUS and MM patients with < 10% PC, both normal and phenotypically aberrant PC were discriminated by multiparameter flow cytometry, the latter subset being specifically sorted for FISH analysis with a purity of 93% ± 6%.RESULTS: Overall, 57% of the MGUS patients displayed abnormalities for at least 1 of the 4 chromosomes analyzed compared with 75% of both MM and PCL cases. The most common single chromosome abnormalities detected in MGUS were gains of chromosomes 9 (23%) and/or 6 (21%) and loss of chromosomes 13 (21%) and/or 17 (17%). Compared with MM patients, MGUS patients were found to have both a lower incidence of gains of chromosome 9 (23% vs. 54%, P = 0.002) and monosomy 13/13q^‐ deletions (21% vs. 38%, P = 0.07); with respect to PCL cases, MGUS patients were found to have a lower incidence of monosomy 13/13q^‐ deletions (21% vs. 75%, P < 0.001) together with a slightly higher frequency of gains of both chromosomes 6 (21% vs. 0%, P = 0.05) and 9 (23% vs. 7%, P = 0.1). The simultaneous use of two or three different chromosome probes showed that within the purified compartment of phenotypically aberrant PC from most MGUS patients (67%), more than 1 PC clone could be identified. In contrast, the incidence of 2 or more PC clones was much lower in MM (19%, P < 0.001) and PCL (15%, P = 0.003). Interestingly, although some FISH patterns were shared by both groups of diseases (i.e., monosomy 13/13q^‐ deletions alone, gains of chromosome 9 alone or together with trisomy 6), others were found almost exclusively in either MGUS (i.e., a clone with monosomy 6 and/or 17 together with nuclei displaying a normal chromosome number) or in MM (i.e., monosomy 13/13q^‐ deletions together with gains of chromosome 6 and/or 9).CONCLUSIONS: In summary, the results of the current study showed that MGUS patients displayed a high incidence of numerical alterations, which are usually associated with the presence of more than one tumor cell clone. It is interesting to note that the cytogenetic patterns observed in the aneuploid PC clones from MGUS patients were frequently different from those observed in both MM and PCL. Cancer 2003;97:601–9. © 2003 American Cancer Society.DOI 10.1002/cncr.11100

Flow cytometry-based immunophenotypic techniques for the analysis of CD55 and CD59 expression on the major cell populations present in blood are the preferred method for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH). In the present study, we comparatively analyze the effects of stain-lyse-and-then-wash techniques and lyse-wash-and-then-stain procedures on the detection of both CD55 and CD59 expression on the major peripheral blood (PB) leucocyte subsets, as analyzed by flow cytometry. Our major goal was to establish the minimum amounts of anti-CD55 and anti-CD59 reagents required to be added to a minimum volume of blood, which would allow an optimal staining for both antigens on red cells, platelets, and leucocytes present in a single tube. Our results show that upon comparing stain-lyse-and-then-wash techniques with lyse-wash-and-then-stain protocols, the presence of important amounts of red cells at the time peripheral blood leucocytes are stained for CD55 and CD59 is associated with a significantly (P < 0.01) lower and more heterogeneous pattern of antigen expression on almost all major PB leucocyte subsets, supporting the need to use red cell lysing procedures prior to the staining of leucocytes. Identical, optimal patterns of antigen staining for CD55 and CD59 were obtained upon incubating 3 microL of blood with 10 microL of each of these monoclonal antibody (mAb) reagents (protein concentration of 0.05 microg/microL and 0.2 microg/microL respectively) for 30 min (room temperature [RT]) using a non-lyse-non-wash sample preparation procedure. This latter procedure allowed for the simultaneous analysis of CD55 and CD59 expression on red cells, platelets, neutrophils, monocytes, and lymphocytes present in the sample through the combined staining of CD55 and CD59 with CD64-fluorescein isothiocyante (FITC) plus CD61-peridinin chlorophyll protein (PerCP) and CD45-PerCP. In summary, our results show that the sample preparation protocol has a significant impact on the quality of the staining obtained for the CD55 and CD59 antigens on the major PB leucocyte subsets; additionally, we propose a simple and reliable stain-non-lyse-non-wash method for the simultaneous analysis of CD55 and CD59 expression on PB red cells, platelets, neutrophils, monocytes, and lymphocytes, which could be reached through the use of two triple stainings.

Objective: Although information on the cytogenetic characteristics of meningioma tumors has accumulated progressively over the past few decades, information on the genetic heterogeneity of meningiomas is still scanty. The aim of the present study was to analyze by interphase fluorescence in situ hybridization (FISH) the incidence of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y in a group of 70 consecutive meningioma tumors. Another goal was to establish the potential associations among the altered chromosomes, as a way to assess both intertumoral and intratumoral heterogeneity. Methods: For the purpose of the study, 70 patients diagnosed with meningioma were analyzed. Interphase FISH for the detection of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y was applied to fresh tumor samples from each of the patients studied. Results: The overall incidence of numerical abnormalities was 76%. Chromosome Y in males and chromosome 22 in the whole series were the most common abnormalities (46% and 61%, respectively). Despite the finding that monosomy of chromosome 22/22q^‐ deletions are the most frequent individual abnormality (53%), we have observed that chromosome gains are significantly more common than chromosome losses (60% versus 40%). Chromosome gains corresponded to abnormalities of chromosomes 1 (27%), 9 (25%), 10 (23%), 11 (22%), 14 (33%), 15 (22%), 17 (23%), and X in females (35%) and males (23%) whereas chromosome losses apart from chromosome 22 frequently involved chromosomes 14 (19%), X in males (23%), and Y in males (32%). Although an association was found among most gained chromosomes on one side and chromosome losses on the other side, different association patterns were observed. Furthermore, in the latter group, monosomy 22/22q^‐ was associated with monosomy X in females and monosomy 14/14q^‐ was associated with nulisomy Y in males. In addition, chromosome losses usually involved a large proportion of the tumor cells whereas chromosome gains were restricted to small tumor cell clones, including tetraploid cells. Conclusions: Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, as assessed by interphase FISH. Cytometry (Clin. Cytometry) 50:153–159, 2002.

Mitochondria play an essential role in the generation of the energy needed for eukaryotic cell life and in the release of molecules involved in initiation of cell death. Here we review the changes in isolated mitochondrial fluorescent populations as distinguished by flow cytometry during postnatal development and their regulation by thyroid hormones and catecholamines. The use of flow cytometry in the study of mitochondrial changes occurring under hypothyroidism, alcohol abuse and aging is also reviewed.

BACKGROUND: Almost all automated hematology cell analyzers use methods based on either the impedance (PLTi) or the optical (PLTo) properties of the cells for performing platelet counts. To improve the accuracy of platelet counts in peripheral blood (PB), the use of CD61 (GPIIIa) MoAbs (ImmunoPLT method) has recently been introduced in an automated hematology blood‐analyzer system (Cell‐Dyn 4000, Abbott Diagnostics). STUDY DESIGN AND METHODS: A comparative evaluation was made of the accuracy and precision of the three methods currently available in the Cell‐Dyn 4000 automated hematology cell analyzer for counting the number of platelets per microliter of PB in a total of 47 patients with chemotherapy‐induced thrombocytopenia. A flow cytometric PB platelet count was also performed in parallel and used as an external reference. RESULTS: PB platelet counts showed a good correlation among the PLTo, CD61‐ImmunoPLT, and flow cytometric methods. In contrast, the PLTi procedure usually provided an overestimation of the number of platelets per microliter. Although a good correlation was observed between the flow cytometric reference method and both the ImmunoPLT and PLTo methods, the highest degree of agreement was found for the ImmunoPLT techniques (94% vs. 67%). A comparative analysis of the PLTo and CD61‐ImmunoPLT methods with regard to their value for predicting platelet transfusion needs on the basis of specific flow cytometric platelet count thresholds showed a good correlation when the cutoff level of 10,000 platelets per μL was used. In contrast, at the threshold of 20,000 platelets per μL, slight differences were observed between the PLTo and CD61‐ImmunoPLT procedures for predicting transfusion needs. CONCLUSION: Such results indicate that, if the CD61‐ImmunoPLT method is used in the platelet transfusion decision‐making process, unnecessary platelet transfusions could be avoided in up to 17.5 percent of persons with a PLTo count of <20,000 platelets per μL.

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II⁺/lineage⁻ were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33^strong+ and CD123^dim+ (0.16 ± 0.06% of the PB nucleated cells and 55.9 ± 11.9% of all PB DC) and the other, CD33^dim+ and CD123^strong+ (0.12 ± 0.04% of PB nucleated cells and 44.53 ± 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor α-chain (CD126) and for CD38. In contrast, the CD123^strong+/CD33^dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33^strong+/CD123^dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1β (97 ± 5% of positive cells), IL-6 (96 ± 1.1% of positive cells), IL-12 (81.5 ± 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-α) (84 ± 22.1% of positive cells) as well as chemokines such as IL-8 (99 ± 1% of positive cells), the functional ability of the CD123^strong+/CD33^dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.

At present, immunophenotyping of hematological malignancies represents one of the most relevant clinical applications of flow cytometry. In recent years, its use has extended from clinical research to diagnostic laboratories. The aim of this report is to critically review the type of information provided by the flow cytometric immunophenotyping of hematological malignancies and its clinical impact as well as to highlight its potential future applications. The currently available information, including that provided by different international consensus groups on the phenotypic characterization of hematologic malignancies, was reviewed. Additionally, recent reports on the immunophenotypic analysis of hematological malignancies published in hematology, oncology, pathology, immunology, and cell biology journals were also analyzed. A careful review of the literature showed that in spite of the well-established utility of immunophenotyping for the diagnosis, classification, prognostic stratification, and monitoring of hematological malignancies, only a small part of the information on the immunophenotypic characteristics of pathological hemopoietic cells has been used routinely. Specific and sensitive identification of neoplastic cells and their accurate enumeration and phenotypic characterization represent the major aims of these procedures. Similarities between leukemic and healthy cells allow the establishment of the lineage and maturation stage of the pathologic cells, this information being of great utility for the diagnosis, classification, and prognostic evaluation of different subtypes of hematological malignancies. On the other hand, the phenotypic aberrations displayed by leukemic cells could allow the selection of cases carrying specific genetic abnormalities in which further confirmatory molecular studies will be performed. The information provided by the flow cytometric immunophenotyping of hematological malignancies is of great clinical utility, with a major challenge for the near future being the standardization of technical procedures, data interpretation, and reporting.

Rapid identification of AML patients carrying the t(15;17) translocation for treatment decision-making is currently made on the basis of morphologic screening. However, the existence of both false positives and negatives highlights the need for more objective methods of screening AML cases and further molecular confirmation of the t(15;17) translocation. In the present study we analyzed a total of 111 AML cases in order to investigate whether immunophenotyping based on the assessment of multiple-stainings analyzed at flow cytometry could improve the sensitivity and specificity of morphologic identification of acute promyelocytic leukemia (APL) carrying the t(15;17) translocation. FISH analysis was used as a complementary technique for cases in which morphology and molecular biology yielded discrepant results. Concordant results between morphology and RT-PCR were found in 102/111 (91.8%) cases: 34 patients had M3/PML-RARalpha+ and 68 non-M3/PML-RARalpha- disease. Nine cases showed discrepants results. Multivariate analysis showed that the best combination of immunologic markers for discriminating between M3/PML-RARalpha+ and non-M3/PML-RARalpha- cases was that of the presence of heterogeneous expression of CD13, the existence of a single major blast cell population, and a characteristic CD34/CD15 phenotypic pattern (p<0.02). A score system based on these parameters was designed, and the 34 M3/PML-RARalpha+ cases showed a score of 3 (presence of the 3 phenotypic characteristics). In contrast, only 1 out of the 68 (1.3%) non-M3/PML-RARalpha- cases had this score, most o these latter cases (53/68, 78%) scoring either 0 or 1. Therefore, among these cases, immunophenotyping showed a sensitivity of 100% and a specificity of 99% for predicting PML/RARalpha gene rearrangements. Of the 9 cases in which morphology and molecular biology results were discrepant, four cases displayed M3 morphology without PML/RARalpha rearrangements by RT-PCR. In only one of these 4 cases did the immunophenotype score 3, this being the only FISH positive case. From the remaining five discrepant cases (non-M3 morphology while positive for PML/RARalpha) two cases had a phenotypic score of 3 and were FISH positive while the other three were negative by FISH. Upon repeating RT-PCR studies, two of these latter three cases became negative. Our results show that immunophenotyping may be of great value for quick screening of APL with PML/RARalpha rearrangements.