Low viral load post-transplant lymphoproliferative disease localized within the tongue.

Post-transplant lymphoproliferative disease (PTLD) can occur in different sites, such as lymph nodes, allograft, and central nervous system. We report a 6-year-old girl with end-stage renal disease secondary to hypoplastic-dysplastic kidneys, who received a kidney transplant. Thirty months post transplant, she developed PTLD in the tongue, an area of muscular tissue only. At that time her peripheral blood Epstein-Barr viral (EBV) load was only 40 copies/10(5) lymphocytes, though the tumor was EB early RNA (EBER) positive. Immunosuppression was reduced with initial improvement in her symptoms. One month later, she returned with abdominal complaints and a contained cecal abscess. The excised cecal tissue revealed CD20 and EBER-positive lymphoid cells. At the same time, her peripheral blood EBV copy number rose to 400 copies/10(5) lymphocytes. She was successfully treated for the progressive PTLD by complete cessation of immunosuppression and a modified reduced-dose chemotherapy protocol plus rituximab. Partial immunosuppression was eventually re-introduced with sirolimus and prednisone. She remains in remission 60 months post transplant, and 30 months post PTLD, with serum creatinine value maintained at 1.3 mg/dL. Unusual localization of PTLD to areas in non-lymphoid tissue without regional lymphoid involvement may result in misleading low peripheral blood EBV viral loads.

2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: medical indications.

The clinical indications for diagnostic flow cytometry studies are an evolving consensus, as the knowledge of antigenic definition of hematolymphoid malignancies and the prognostic significance of antigen expression evolves. Additionally the standard of care is not routinely communicated to practicing clinicians and diagnostic services, especially as may relate to new technologies. Accordingly there is often uncertainty on the part of clinicians, payers of medical services, diagnostic physicians and scientists as to the appropriate use of diagnostic flow cytometry. In an attempt to communicate contemporary diagnostic utility of immunophenotypic flow cytometry in the diagnosis and follow-up of patients with hematolymphoid malignancies, the Clinical Cytometry Society organized a two day meeting of international experts in this area to reach a consensus as to this diagnostic tool. This report summarizes the appropriate use of diagnostic flow cytometry as determined by unanimous approval of these experienced practitioners.

Treatment of refractory acute leukemia with timed sequential chemotherapy using topotecan followed by etoposide + mitoxantrone (T-EM) and correlation with topoisomerase II levels.

A phase I/II clinical study evaluated 17 patients with refractory/recurrent acute leukemia treated with 1.5 mg/m2/day topotecan on days 1-3 followed by etoposide (100 mg/m2/day)+mitoxantrone (10 mg/m2/day) on days 4, 5 and 9, 10. Timed sequential chemotherapy using the topoisomerase I-inhibitor topotecan before the topoisomerase II-inhibitors, etoposide+mitoxantrone (T-EM) treatment is proposed to induce topoisomerase II protein levels and potentiate the cytotoxic activity of the topoisomerase II-directed drugs. Fourteen patients had refractory and three had recurrent acute leukemia. The majority of patients were heavily pre-treated with greater than three re-induction chemotherapy regimens. Ten patients responded to T-EM treatment (59%). Four of seventeen (24%) had a complete remission and one had a partial remission. Four additional patients (24%) who scored complete leukemia clearance had no evidence of disease with complete white and red blood cell recovery but with platelet counts less than 100,000. The lack of platelet recovery in one patient having a partial response was scored as a partial leukemia clearance. The toxicity profile included major non-hematological toxicity including grade 3 mucositis (29%) and neutropenic fever (65%). Paired measurements of intracellular levels of topoisomerase II isoforms alpha and beta in leukemia blast cells (bone marrow) collected before (day 0) and after topotecan treatment (day 4) showed that a relative increase of topoisomerase IIalpha (Topo IIalpha) > or = 40% strongly correlated with response after T-EM treatment. Increased Topo IIalpha levels also corresponded to increased DNA fragmentation. Two patients who had an increase of Topo IIalpha of 20-25% had either a PR or PLC while patients with a < 10% increase showed no response to T-EM treatment. We conclude that timed sequential chemotherapy using topotecan followed by etoposide+mitoxantrone is an effective regimen for patients with refractory acute leukemia, and demonstrate Topo IIalpha protein level increases after topotecan treatment.

Progress toward analysis of D8/17 binding to B cells in children with obsessive compulsive disorder and/or chronic tic disorder.

BACKGROUND: Previous research has suggested that a subgroup of children with obsessive compulsive disorder (OCD) have neuropsychiatric sequelae of streptococcal pharyngitis, similar to that seen in the neurological manifestation of rheumatic fever (RF). Monoclonal antibody D8/17 demonstrates increased binding to B cells in patients with RF and in patients with neuropsychiatric disorders using immunofluorescent microscopy. OBJECTIVE: The aim of this study was to determine if an earlier immunofluorescent microscopy study of monoclonal antibody D8/17 in childhood-onset OCD and/or chronic tic disorder (CTD) could be replicated using the more objective method of flow cytometric analysis. METHOD: D8/17 binding to B cells was determined in patients with OCD and or CTD (N=32), and healthy controls (N=12) by flow cytometric analysis. RESULTS: Subjects with OCD/CTD showed increased mean cell binding (26.0%) of monoclonal antibody compared with healthy controls (9.1%) (p<0.001). When using the threshold of greater than 19% binding (95% upper confidence interval) as a measure of positivity, 65.6% of patients compared with 8.3% of controls showed increased antibody binding to B cells (p=0.01). CONCLUSIONS: Although this study reports positive results, many methodological issues will need to be addressed before generalized use of assay for diagnostic purposes.

Early phase of intestinal mantle cell lymphoma: a report of two cases associated with advanced colonic adenocarcinoma.

Intestinal mantle cell lymphoma characteristically produces multiple polyps, a finding reported as multiple lymphomatous polyposis. The early stages of intestinal mantle cell lymphoma before polyp formation and the pattern of initial lymph node invasion, however, have not been described. We recently encountered two cases of intestinal mantle cell lymphoma in their early development found incidentally associated with advanced colonic adenocarcinoma. We present herein the clinical, histopathological, immunohistochemical, and molecular genetic features of these two cases. In one case, a single polypoid mass was found with invasion limited to mucosa and submucosa of the terminal ileum and without lymph node compromise. In the second case, there were multiple mucosal aggregates of neoplastic cells without formation of polyps. Regional lymph nodes in the latter case showed either partial or complete involvement by lymphoma. In both cases, immunohistochemistry (CD20+, CD5+, cyclin D1+, CD10-, and CD23-), and demonstration of clonal immunoglobulin heavy chain and bcl-1 gene rearrangements by PCR analysis confirmed the diagnosis of mantle cell lymphoma.

The diagnosis of hairy cell leukemia can be established by flow cytometric analysis of peripheral blood, even in patients with low levels of circulating malignant cells.

The diagnosis of hairy cell leukemia (HCL) has traditionally been based on microscopic means. Immunophenotypic analysis of peripheral blood by flow cytometry is not widely recognized as a method for diagnosing HCL, perhaps due to the expectation of low yield of neoplastic cells in patients who are characteristically leukopenic. The abnormal coexpression of CD103, CD25, and intense CD11c and CD20 on monotypic, slightly large B-lymphocytes has previously been shown to be highly characteristic of HCL. We wished to determine if this pattern was valuable in the diagnosis of HCL in leukopenic patients with low levels of neoplastic cells in the peripheral blood. The abnormal immunophenotype above was observed in 25 peripheral blood specimens from patients with unexplained cytopenias or suspected lymphoproliferative processes. Ten of the 25 blood samples exhibited this abnormal phenotype in less than 5% of circulating leukocytes (ranging from <1% to 4%). All 10 patients had other manifestations of HCL, including cytopenias (mean white blood cell count, 1.8 x 10(3)/mm(3); hemoglobin, 11.0 gm/dl; platelets, 74 x 10(3)/mm(3)), splenomegaly, and typical bone marrow morphologic changes. Eight of the 10 patients achieved an excellent response to one course of 2-CDA, with significant improvement of cytopenias (mean white blood cell count: 5.3 x 10(3)/mm(3); hemoglobin: 14.4 g/dl; platelets: 181 x 10(3)/mm(3)) and regression of splenomegaly. One patient had a partial response to alpha interferon and a subsequent complete response to 2-CDA, and one died during treatment. In conclusion, flow cytometric immunophenotyping of peripheral blood is capable of detecting low levels of circulating malignant cells in HCL, even in leukopenic patients. As such, it can be a very useful, non-invasive tool in the diagnosis of this disorder.

Flow cytometric analysis of lymphomas and lymphoproliferative disorders.

Flow cytometry has become an important tool in the diagnosis and characterization of hematologic and lymphoid neoplasia. This technology serves as an excellent complement to microscope-based traditional diagnostic methods and adds distinctive capabilities that are unmatched by any other diagnostic methods. Flow cytometry is ideal for fluids where cells are naturally suspended but is also useful in lymphoid tissues, from which single-cell suspensions can be easily obtained. The advantages of flow cytometry are largely based on its ability to analyze very rapidly, even in small samples, multiple cell properties simultaneously, including size, granularity, surface and intracellular antigens, and DNA content. The quantitative nature of the data produced, both with regard to cell population distributions and to expression of individual cell antigens, offers objective criteria for interpretation of results. Examples of applications include the detection of clonal cells in B-cell lymphoma, the recognition of antigenic expression anomalies in B- or T-cell malignancies, the identification of malignant plasma cells, and the rapid measurement of cell cycle fractions. The unique attributes of flow cytometry allow for increased sensitivity in the detection of neoplastic cells and should contribute to improving accuracy and precision in the diagnosis and classification of lymphomas and lymphoproliferative disorders. Semin Hematol 38:111-123. This is a US government work. There no restrictions on its use.

Optimal number of reagents required to evaluate hematolymphoid neoplasias: results of an international consensus meeting.

At the ISAC 2000 Congress, the Clinical Cytometry Society organized a meeting of international experts to reach consensus on the minimum number of antibodies required for a full evaluation of hematologic and lymphoid neoplasias. A questionnaire was distributed prior to the meeting to numerous experts from US and European institutions and 13 responses were received. At the meeting, 25 individuals, including most of those who returned responses, participated in the discussions and voted on the issues presented. In chronic lymphoproliferative disorders (CLD), 9 antibodies (anti-CD5, CD19, kappa, lambda, CD3, CD20, CD23, CD10, and CD45) were deemed essential for initial evaluation by 75% of the participants. There was near unanimity that additional markers (selected from CD22, FMC7, CD11c, CD103, CD38, CD25, CD79b and heavy chains for B-cell disorders, and CD4, CD7, CD8, CD2, CD56, CD16, TCRa/b, and TCRg/d for T-cell disorders) would be needed to fully characterize CLD, although not every marker would be useful in all cases. Tissue lymphomas were believed to be similar to CLD, needing a minimum of 12--16 markers. However, for some cases, CD30, bcl-2, TdT, CD71, CD1a, and CD34 were cited as useful by the participants. Markers mentioned for plasma cell disorders included kappa, lambda, CD38, CD45, CD56, CD19, CD20, CD138, and heavy chains. Of 17 voting participants, 16 agreed that between 5 to 8 markers would be essential reagents for plasma cell disorders. For acute leukemia (AL), 10 markers (CD10, CD19, CD13, CD33, CD34, CD45, CD7, CD14, CD3, and HLADR) were considered essential by 75% of participants for initial characterization of the leukemia lineage. Most (>75%) agreed that at least one more B (CD20, CD22, CD79a, IgM), T (CD1a, CD2, CD4, CD5, CD8), myeloid (CD11b, CD15, CD64, CD117, myeloperoxidase), erythroid (CD36, CD71, glycophorin A), and megakaryocytic (CD41, CD61) reagents should be included in the essential panel. However, there was no agreement as to which was optimal. Thus, approximately 13--15 of those reagents would be considered essential in all cases of AL, whereas others (CD16, CD56, CDw65, TdT, and cytoplasmic CD3) were mentioned as useful in some cases. Almost all voting participants believed that the appropriate number of markers for complete characterization of AL would average 20--24. The majority of the responders (11 of 13) indicated that fewer reagents could be used in monitoring or staging patients with previously characterized disease, but not all ventured a specific number of reagents. From the above results, we conclude that the phenotypic analysis of hematologic and lymphoid neoplasia requires a rather extensive panel of reagents. Supplementary reagents might even be necessary if they prove to become relevant for diagnostic purposes. Reducing the number of antibodies could significantly compromise the diagnostic accuracy, appropriate monitoring, or therapy of these disorders.

Langerhans cell granulomatosis manifested as pigmented villonodular synovitis.

We report an unusual case of Langerhans cell granulomatosis (LCG) manifested as a villous synovial proliferation in a 38-year-old female jogger. One year after the onset of joint symptoms, she had a classical LCG presentation with skin and visceral lymph node involvement. Review of the literature revealed only one case of synovial shoulder joint tenosynovitis associated with LCG in a middle-aged woman. Ours is the first reported case presenting clinically in the synovium of the hip joint as pigmented villonodular synovitis. Histiocytic/dendritic proliferations involving the synovial tissues are not uncommon. These lesions as well as the rare multicentric reticulohistiocytosis (MRH), a systemic monocytoid/histiocytic disorder with multinucleated giant cells, polyarthritis, and papulonodular skin lesions, should be considered in the differential diagnosis. Clinical and pathologic features will distinguish LCG from MRH.

Flow cytometric analysis of hematologic neoplasia.

Flow cytometry (FCM) is a powerful technology that allows the rapid analysis of cellular components such as surface and intracellular antigens, or DNA content. The measurements are fast and are based on optical signals emitted by cells labeled with fluorochromes as they flow suspended in a liquid medium through an intense laser beam. The signals include scattered light, which provides information on cell size and granularity, and fluorescence derived from dyes or fluorochrome-labeled antibodies bound to specific cell components.

Follicular lymphoma can be distinguished from benign follicular hyperplasia by flow cytometry using simultaneous staining of cytoplasmic bcl-2 and cell surface CD20.

The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (FL) is sometimes problematic. We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of FL, using surface CD20 expression as a discriminator of the various lymphoid compartments. Lymph node cell suspensions from 12 cases of FH and 17 cases of FL were analyzed by flow cytometry using a combined surface CD20 and intracellular bcl-2 staining. CD20- T cells in FH demonstrated the same bcl-2 expression as the CD20+ mantle cells, but the bright CD20+ germinal center cells showed near absence of bcl-2 expression. In contrast, the neoplastic cells of FL showed greater bcl-2 expression than the T cells of the same tumors and all cell populations of FH. This difference was particularly significant between the neoplastic B cells of FL and the germinal center cells of FH. The combined analysis of CD20 and bcl-2 should be useful for the differential diagnosis between FH and FL and particularly applicable to limited samples or when B-cell clonality is in question. Whether the quantitation of bcl-2 expression can be of further discriminatory value in malignant lymphomas remains to be determined.

Lymphohistiocytic anaplastic large cell lymphoma stage I: long-term survival after resection alone.

A 17-year-old female presented with axillary lymphoadenopathy, which, on biopsy, demonstrated an anaplastic large cell lymphoma of the lymphohistiocytic type (ALCL-LH). The tumor cells expressed the CD30 antigen and reacted with the ALK1 antibody, suggesting the presence of the nucleophosmin-anaplastic large cell lymphoma kinase (NPM/ALK) fusion protein. No other adenopathy was found. Following a wide excision of the lymph node and without postoperative treatment, the patient remains free of disease 5 years later. This case demonstrates the potential curability of patients with early stages of ALCL by local treatment.

Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: current status and future directions.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Medulloblastoma: time-dose relationship based on a 30-year review.

PURPOSE: Time-dose relationships have proven important in many cancer sites. This study evaluates the time factors involved in the successful postoperative radiotherapy of medulloblastoma, based on a 30-year experience in a single institution. METHODS AND MATERIALS: Fifty-three patients with medulloblastoma received postoperative craniospinal radiotherapy with curative intent between 1963 and 1993. Seven patients (13%) underwent biopsy alone, 28 patients (53%) had subtotal excision, and 18 patients (34%) had gross total excision. Eleven patients received adjuvant chemotherapy. The mean posterior fossa dose was 53.1 Gy; most patients received 54.0 Gy (range, 34.3 to 69.6 Gy). For 41 patients receiving once-a-day therapy, the mean dose was 50.6 Gy (range, 34.3 to 56.0 Gy). For 12 patients receiving twice-a-day therapy, the mean dose was 61.8 Gy (range, 52.6 to 69.6 Gy). Minimum follow-up was 2 years, and median follow-up was 10.7 years. Survival, freedom from relapse, and disease control in the posterior fossa were calculated using the Kaplan-Meier method, and multivariate analysis was performed for prognostic factors. Variables related to radiotherapy were examined, including dose to the craniospinal axis, dose to the posterior fossa, fractionation (once-a-day vs. twice-a-day), use of adjuvant chemotherapy, risk group [high (> or =T3b or > or =M1) or low (< or =T3a and M0-MX)], interval between surgery and radiotherapy (excluding patients receiving chemotherapy before radiotherapy), and duration of radiotherapy. RESULTS: At 5 and 10 years, overall survival rates were 68 and 64%, respectively, and freedom-from-relapse rates were 61 and 52%, respectively. Rates of disease control in the posterior fossa at 5 and 10 years were 79 and 68%, respectively. At 5 years, absolute survival rates after biopsy alone, subtotal excision, and gross total excision were 43, 67, and 78%, respectively (p=0.04), and posterior fossa control rates were 27, 89, and 83%, respectively (p=0.004). Duration of the treatment course was the only radiotherapy-related variable with a significant impact on freedom from relapse and posterior fossa control. For patients whose radiation treatment duration was < or =45 days, posterior fossa control was 89% at 5 years, compared with 68% for those treated for >45 days (p=0.01). Duration of treatment also affected freedom from relapse at 5 years: < or =45 days (76%) compared with >45 days (43%), p=0.004. CONCLUSION: Our study demonstrates that if adequate doses are used, then radiotherapy treatment duration will significantly affect the outcome in terms of control of disease in the posterior fossa and freedom from relapse. Fractions of at least 1.75 Gy given once a day, or a twice-a-day regimen should yield optimal local control results.

Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes.

Conventional flow cytometric methods for CD34+ cell counting may be affected by the high number of nucleated red blood cells or nonviable cells in cord blood and its products. We developed a simple flow cytometric no-wash procedure that avoids these shortcomings because it provides absolute CD34+ cell counts and assesses cell viability. Samples were incubated with phycoerythrin (PE)-labeled anti-CD34 (Becton Dickinson Immunocytometry Systems [BD], San Jose, CA) and peridinin chlorophyll protein (PerCP)-labeled anti-CD45 (BD) in bead-containing TRUCOUNT tubes (BD). After red cell lysis with a fixative-free reagent, the impermeant nucleic acid dye YO-PRO-1 (Molecular Probes, Eugene, OR) was added and samples were analyzed on a single-laser FACSCalibur (BD). A comparison with the ProCOUNT progenitor cell assay (BD) in 57 samples revealed excellent correlation of results (r = 0.98, intercept -0.2 cells/microl, slope 1.01). Precision studies conveyed coefficients of variation of 6.4 and 8.9% at concentrations of 35 and 16 CD34+ cells/microl, respectively. In untreated and leukocyte-enriched cord blood 4.5+/-3.8% of CD34+ cells were stained by YO-PRO-1, representing apoptotic or necrotic cells. In post-thawing cryopreserved samples this number increased to 10.4+/-5.5%. Isotype controls showed very low blank values of viable cells (0.1+/-0.4 cells/microl, maximum 2.4) and seemed unnecessary. We found no washing-related alteration of results in 35 samples, indicating that the method may also be performed with cell washing. Replacing YO-PRO-1 with TO-PRO-3 facilitated four-color analysis of subpopulations of viable CD34+ cells on a FACSCalibur equipped with a second (diode) laser. We found the proposed method to be a rapid, efficient, and flexible procedure that improved validity of CD34+ cell counts.

Multiparametric flow cytometry in the diagnosis and characterization of low-grade pulmonary mucosa-associated lymphoid tissue lymphomas.

Primary mucosa associated lymphoid tissue (MALT) lymphomas are rare neoplasms that seem to have a better prognosis than nodal lymphomas. Morphologic diagnosis of these lesions may be difficult because of features that overlap with those of benign lymphoid infiltrates. In this study, we assessed the contribution of multi-parametric flow cytometry in demonstrating clonality and further characterizing pulmonary MALT lymphomas. Based on a clinical or pathologic suspicion of MALT-lymphoma, 3 transbronchial biopsies, 4 fine needle aspirates, 1 core needle biopsy, and 13 wedge excisions of lung were submitted fresh (unfixed) to our laboratory for evaluation. Among the 13 cases diagnosed as MALT lymphomas, B-cell monoclonality was established by identifying expression of a single immunoglobulin light chain on CD20 or CD19-positive cells in 12 cases. One case lacked expression of both light chains on B-cells. Of 11 lymphoma cases in which CD5 and CD10 surface antigens were assessed, no cases expressed CD10, and 1 case demonstrated weak CD5 expression. Nine of 10 cases studied were diploid and 1 case was hyperdiploid. All of the lymphomas displayed low (< or = 3%) S-phase fractions consistent with low grade processes. In 10 patients with short follow-up, none died of their disease and the majority had no evidence of lymphoma dissemination. In seven of the remaining eight cases, B-cells were polyclonal consistent with reactive processes. In one morphologically reactive case, flow cytometric analysis was unsuccessful because of poor cell viability. The pulmonary MALT lymphomas in this study represent a group of B-cell tumors with distinctive morphologic, immunophenotypic, and cell kinetic characteristics. Multi-parametric flow cytometry is useful for confirming B-cell monoclonality and illustrating an antigenic profile compatible with this diagnosis. Flow cytometry can be particularly helpful when working with small biopsies and cytologic samples with limited diagnostic material and may abrogate the need for more aggressive surgical procedures.

CD10 expression in follicular lymphoma and large cell lymphoma is different from that of reactive lymph node follicles.

BACKGROUND AND OBJECTIVES: CD10 is a proteolytic enzyme expressed on the surface of germinal center cells and lymphomas derived from these cells. There is a well-known association between CD10 expression and lymphomas of follicular center cell origin. However, the reported frequency of CD10 positivity in follicular lymphomas is widely variable, and no studies have addressed the importance of assessing the intensity of CD10 expression in the diagnosis of these tumors. In this study, we utilized flow cytometry to determine differences in CD10 expression in lymphomas and follicular hyperplasias. METHODS: Cell suspensions from 61 follicular lymphomas, 43 diffuse large B-cell lymphomas, and 44 lymph nodes with follicular hyperplasia were analyzed simultaneously with phycoerythrin-labeled anti-CD20 and fluorescein isothiocyanate-labeled anti-CD10. RESULTS: CD10 expression was mainly observed on B cells and was detected in 89% of follicular lymphomas, 56% of diffuse large B-cell lymphomas, and 55% of lymph nodes showing follicular hyperplasia. In follicular hyperplasia, two subpopulations of B cells displaying dim and bright CD20 expression were recognized. CD10 expression was restricted to the bright CD20-positive cells, which accounted for an average of 16% of B cells. In CD10-positive follicular and diffuse large B-cell lymphoma cases, a significantly higher proportion of B cells (73%) coexpressed CD10. Furthermore, the intensity of CD10 expression in follicular lymphoma and diffuse large B-cell lymphoma was much higher than that of follicular hyperplasia. CONCLUSIONS: Quantitative flow cytometry can detect significant differences in CD10 expression between normal follicular cells and follicular or diffuse large B-cell lymphoma. The use of CD10 intensity of expression as well as the fraction of CD10-expressing B cells should help distinguish reactive from neoplastic B-cell processes.

Hematopoiesis in the liver and marrow of human fetuses at 5 to 16 weeks postconception: quantitative assessment of macrophage and neutrophil populations.

During human development, the liver and marrow both function as hematopoietic organs, but little is known about differences in the production of macrophages and neutrophils by these two organs. We used immunohistochemical stains to quantify the ratio of neutrophils to macrophages within the liver and the marrow of 16 fetuses from 5 to 16 wk postconception. At 5 wk the liver had a ratio of one granulocyte [myeloperoxidase (MPO)-positive cell] to every 9 +/- 5 (X +/- SD) macrophages (KP-1-positive cells). Between 5 and 16 wk, the granulocyte to macrophage ratio in the liver was constant, whereas it changed markedly in the marrow. Before 8 wk no MPO-positive or KP-1-positive cells were observed in bones. At 10 wk, bones still had no MPO-positive cells, but KP-1-positive cells were abundant. At 11-12 wk the granulocyte to macrophage ratio was 1 to 1 +/- 1, but by 13-16 wk it had increased to 8 +/- 3 MPO-positive cells to one KP-1-positive cell. We hypothesized that at 13-16 wk the abundance of MPO-positive cells in the marrow and their scarcity in the liver was the result of production of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSF-R) in the marrow and their absence in the liver. However, by reverse transcriptase-PCR mRNAs for G-CSF and G-CSF-R were positive in both organs at all gestations, and G-CSF and G-CSF-R proteins (by immunohistochemistry) were also abundant in all liver and marrow specimens. We then hypothesized that progenitors in the fetal liver were intrinsically different from those in the marrow, and were unable to generate clones of neutrophils. However, progenitors from the liver produced neutrophils abundantly in culture. Thus, the explanation is likely related to as yet undescribed environmental differences between the fetal liver and marrow.

FMC7 antigen expression on normal and malignant B-cells can be predicted by expression of CD20.

Most antibody panels proposed for flow cytometric immunophenotyping of non-Hodgkin's lymphomas and chronic lymphoid leukemias include anti-CD20 and FMC7 antibodies. As in our experience, reactivity of B-cells with these antibodies seemed to be correlated, we evaluated whether the simultaneous use of anti-CD20 and FMC7 antibodies is justified. Using flow cytometry, we measured the binding of these 2 antibodies to the B-cells of 67 bone marrow aspirates, 31 lymph node biopsies, 18 peripheral blood specimens, and 12 tissue samples from other locations. The diagnoses included 50 cases without overt abnormalities, 5 reactive lymphadenopathies, 56 lymphomas and chronic lymphoid neoplasias, and 17 cases with other malignancies. Although CD20 expression was consistently higher, we observed a significant and strong correlation between CD20 and FMC7 antigen expression on B-lymphocytes, irrespective of the nature of the sample or disease (r=0.910; P < 0.001). Moreover, FMC7 antigen expression on B-cells could be predicted by CD20 expression with a sensitivity of 96%, a specificity of 94% and an efficiency of 96%. Our results show that although differing in intensity, expression of CD20 on B-cells closely parallels that of FMC7 antigen. We, therefore, conclude that little additional information is revealed by using FMC7 in immunophenotyping of non-Hodgkin's lymphomas or chronic lymphoid leukemias if intensity of CD20 expression is taken into consideration.