The use of unfixed bone marrow trephines for multicolour flow cytometry.

An adequate bone marrow aspirate is essential for a rapid diagnosis of acute leukaemia by multicolour flow cytometry enabling the simultaneous assessment of multiple antigens on the cell surface as well as intracellular or nuclear ones. In the context of acute leukaemia, it is important to have a diagnosis of the blasts lineage as soon as possible to decide the appropriate treatment. This is sometimes delayed due to difficulties in obtaining a bone marrow aspirate due to a "dry tap". In this study we evaluated retrospectively cell markers results by flow cytometry of unfixed bone marrow trephines of 65 patients with leukaemia at diagnosis and including a few after treatment. Our aims were: 1) To compare cell markers results between bone marrow trephine (BMT) and bone marrow aspirate (BMA) 24 cases and BMT with peripheral blood (PB) 14 cases in paired samples to establish if they were reproducible with results of the unfixed bone marrow trephine biopsies. 2) To ascertain a precise diagnosis in 27 (42%) of the cases in which only a bone marrow trephine was available. We demonstrated that unfixed bone marrow trephine provides an adequate and representative cell suspension for flow cytometry and it is a powerful tool when no other material (bone marrow aspirate or peripheral blood) is available to make a rapid diagnosis. Furthermore when marrow aspirate or peripheral blood paired samples were available, flow cytometry results obtained were identical across all the sample types. Applicability to the clinical laboratory: We described a method to obtain a cell suspension from core biopsies that can easily be implemented routinely in a laboratory that performs diagnostic flow cytometry immunophenotyping. This method is simple, inexpensive and it doesn't require extra equipment.

Pre-transplant MRD predicts outcome following reduced-intensity and myeloablative allogeneic hemopoietic SCT in AML.

The presence of minimal residual disease (MRD) by multiparametric flow cytometry (MFC) has been associated with adverse outcomes in AML patients treated with chemotherapy alone, but its impact in the setting of allogeneic hematopoietic SCT (HSCT) is less clear. We studied 88 patients who underwent myeloablative (MA) or reduced-intensity conditioned allogeneic HSCT for AML in first or subsequent remission at our center. MRD status was determined using three-color MFC on pre-HSCT BM aspirates, and patients were stratified by MRD status into MRD-negative, low-level MRD-positive (<1%) or high-level MRD-positive groups (1-4.9%). Two-year survival estimates in these groups were 66.8%, 51% and 30%, respectively (P=0.012), and 2-year estimates of relapse were 7.6, 37 and 70% (P<0.001). Pre-HSCT MRD was related to disease characteristics including secondary AML (P=0.002) and primary induction failure (P=0.005), but, despite these strong correlations, MRD remained independently associated with poorer survival in multivariate analysis (hazard ratio, 1.92; P=0.014). Pre-HSCT MRD is associated with adverse clinical outcomes in AML patients undergoing reduced-intensity or MA HSCT in first or subsequent remission and should be integrated into transplant strategies for patients with AML.

Variaciones del ión potasio durante el ayuno del Ramadán. Resultados preliminares en jóvenes musulmanes / Changes of the potassium ion during the fast of Ramadan. Preliminary outcomes

Justificación y objetivos: El precepto islámico del Ramadán (R) impone a las personas que lo practican, importantes modificaciones fisiológicas y psicológicas, debido a las restricciones hídricas y dietéticas a las que se ven sometidos durante las horas diurnas a lo largo de un mes, estableciendo un modelo intermitente de ayuno absoluto, de naturaleza singular, particularmente cuando éste se lleva a cabo en sociedades multiculturales de tipo occidental, en las que no se producen ajustes horarios en las actividades diarias, que sí son habituales en los países de mayoría musulmana. Entre las modificaciones, por esta causa, destacan la activación de mecanismos de adaptación a la restricción hidrosalina, con consecuencias en la homeostasis de agua e iones plasmáticos. Por la relevancia de la cuestión y ante el escaso conocimiento de los efectos del (R) sobre el equilibrio iónico, se plantea como objetivo de este estudio, el análisis del comportamiento del ión potasio durante este mes de ayuno, con el fin de prevenir algunos problemas que pudieran afectar a la salud. Metodología: Se seleccionaron 10 jóvenes musulmanes, varones, sanos y con edades entre 18 y 25 años que realizaron el R y se analizaron parámetros bioquímicos e iones en sangre y orina, así como niveles plasmáticos de Renina y Aldosterona, una semana previa al R, primera y cuarta del periodo de ayuno y una semana después de finalizado éste. Resultados: Durante el mes del R, se produce, durante la mañana, un descenso de la excreción de potasio en orina lo que origina incrementos en la concentración plasmática de potasio; a lo largo de la tarde tiene lugar un aumento de su excreción que resulta más eficaz durante la cuarta semana del R. Discusión: Los cambios experimentados a nivel tubular que afectan a la disponibilidad de Na+, HCO3 - y pH, por efecto del R, parecen estar limitando la excreción del ión, a pesar de que se mantiene estimulado el sistema Renina-Aldosterona a lo largo del ayuno. Conclusión: Estos hallazgos ponen de manifiesto la necesidad de estudios específicos sobre la homeostasis de K+ durante el R, para dilucidar qué factores y mecanismos están determinando los incrementos observados en los niveles plasmáticos del ión (AU)

Background and aims: The Islamic precept of Ramadan (R), imposes on individuals who practice it important physiological and psychological changes due to water and dietary restrictions to which they are subjected during the day, over a month. This fact makes a singular constrat, particularly in multicultural Western societies, where there is no schedule adjustments in daily activities, which are common in predominantly Muslim countries. Among the changes, includes the activation of mechanisms of adaptation to the hydrosalin restriction, with consequences on the homeostasis of water and ion plasma. On the relevance of the issue and the limited knowledge of the effects of (R) on the ion balance, the aim of the present study is to analyze the behaviour of potassium ion for this month, in order to prevent some problems that may affect health. Methodology: We have selected 10 young Muslim healthy men, aged between 18 and 25 years who perform Ramadan. Then we have analyzed biochemical parameters including ions, in blood and urineanalysis, and also plasma levels of renin and aldosterone, one week before R, first and fourth week of R, and one week after the fasting. Results: During the month of R a decrease in potassium urine excretion by the morning is observed. The consequence of this fact is the increase of the levels in plasma concentration of potassium; throughout the afternoon, an increase in its excretion results more effective during the fourth week of R. Discussion: Changes in the availability of Na+, HCO3- and variability of pH, in the distal tubule, appear to be responsible for the potassium limited excretion observed during the fast of Ramadan. Conclusion: These finding put into consideration the need of further studies focussing on potassium homeostasis during Ramadan in order to determine which factors are implicated in the raised levels of K+ observed in plasma (AU)

[Changes of the potassium ion during the fast of Ramadan: preliminary outcomes]. / Variaciones del ión potasio durante el ayuno del Ramadán: resultados preliminares en jóvenes musulmanes.

BACKGROUND AND AIMS: The Islamic precept of Ramadan (R), imposes on individuals who practice it important physiological and psychological changes due to water and dietary restrictions to which they are subjected during the day, over a month. This fact makes a singular constrat, particularly in multicultural Western societies, where there is no schedule adjustments in daily activities, which are common in predominantly Muslim countries. Among the changes, includes the activation of mechanisms of adaptation to the hydrosalin restriction, with consequences on the homeostasis of water and ion plasma. On the relevance of the issue and the limited knowledge of the effects of (R) on the ion balance, the aim of the present study is to analyze the behaviour of potassium ion for this month, in order to prevent some problems that may affect health. METHODOLOGY: We have selected 10 young Muslim healthy men, aged between 18 and 25 years who perform Ramadan. Then we have analyzed biochemical parameters including ions, in blood and urine analysis, and also plasma levels of renin and aldosterone, one week before R, first and fourth week of R, and one week after the fasting. RESULTS: During the month of R a decrease in potassium urine excretion by the morning is observed. The consequence of this fact is the increase of the levels in plasma concentration of potassium; throughout the afternoon, an increase in its excretion results more effective during the fourth week of R. DISCUSSION: Changes in the availability of Na+, HCO3- and variability of pH, in the distal tubule, appear to be responsible for the potassium limited excretion observed during the fast of Ramadan. CONCLUSION: These finding put into consideration the need of further studies focussing on potassium homeostasis during Ramadan in order to determine which factors are implicated in the raised levels of K+ observed in plasma.

Modificaciones dietéticas, en jóvenes musulmanes que practican el ayuno del Ramadán / Dietary modifications, engaged in young muslims of fasting

La práctica del precepto islámico del Ramadán (R), impone a las personas que lo practican importantes modificaciones fisiológicas y psicológicas debido a las restricciones hídricas y dietéticas a las que se ven sometidos, lo que tiene una especial repercusión en el rendimiento físico e intelectual de los estudiantes musulmanes, particularmente en sociedades multiculturales de tipo occidental, en las que no se producen ajustes horarios, en las actividades diarias. Entre las modificaciones introducidas en los hábitos de vida, por esta causa, destacan los cambios dietéticos, tanto cualitativos como cuantitativos. Por la relevancia de la cuestión y ante el escaso conocimiento de los efectos del Ramadán en estudiantes adolescentes que viven en sociedades no islámicas, se plantea como objetivo de este estudio, el análisis de las modificaciones en macronutrientes, ocurridas durante el R, en un grupo de alumnos musulmanes de ambos sexos, con el fin de solucionar y prevenir algunos de los problemas que se plantean en este periodo. Material y método: Se seleccionaron 55 jóvenes musulmanes (24 hombres y 31 mujeres) con edades comprendidas entre 15 y 18 años, que realizan el R y se analizó la dieta mediante inventario de 3 días, una semana antes del ayuno y en la segunda semana durante éste. Resultados: En el R, no hay cambios significativos en la ingesta calórica de los hombres y sí en el de las mujeres. Se incrementa el consumo de carbohidratos y proteínas y desciende el de grasas. Discusión: Los cambios en R conducen a una dieta más ajustada a las recomendaciones nutricionales, y a esto parece haber contribuido el hecho de que los participantes llevaron a cabo sus comidas en el seno doméstico donde, a pesar del corte occidental de la población, se deja sentir una importante influencia de las tradiciones y hábitos magrebíes, por el contacto fronterizo con Marruecos (AU)

The practice of the Islamic precept of Ramadan (R) imposes to those who practice it important physiological and psychological changes due to the water and dietary restrictions they are subjected to, which has a particular impact on the Muslim students' physical and psychologicalperformances, particularly in multicultural Western societies, where there are no schedule adjustments in daily activities. Among the changes in lifestyle, consequently, the dietary changes are stressed, both qualitative and quantitative. Due to the importance of this issue and facing the limited knowledge the Muslim teenagers who live in non-Islamic societies have about the effects of Ramadan, it is suggested as main objective of this study the analysis of changes in nutrients during R in a group of Muslim students of both sexes, in order to solve and prevent some of the problems shown in this period. Materials and methods: 55 Muslim youngsters were selected (24 men and 31 women) aged between 15 and 18, who practise R; and the diet was analysed by inventory over 3 days, one week before fasting and in the second week during it. Results: In R there are no relevant changes in caloric intake. The consumption of carbohydrates and proteins is increased, and that of fat is decreased. Discussion: the changes in R lead to a diet more regulated to nutritional recommendations; and to this seems to have contributed the fact that the participants took their meals at home, where, in spite of the Western cut of the population, it is felt an important influence of North African traditions and habits, due to its border contact with Morocco (AU)

[Dietary modifications, in young Muslims engaged in Ramadan fasting]. / Modificaciones dietéticas, en jóvenes musulmanes que practican el ayuno del Ramadán.

UNLABELLED: The practice of the Islamic precept of Ramadan (R) imposes to those who practice it important physiological and psychological changes due to the water and dietary restrictions they are subjected to, which has a particular impact on the Muslim students' physical and psychological performances, particularly in multicultural Western societies, where there are no schedule adjustments in daily activities. Among the changes in lifestyle, consequently, the dietary changes are stressed, both qualitative and quantitative. Due to the importance of this issue and facing the limited knowledge the Muslim teenagers who live in non-Islamic societies have about the effects of Ramadan, it is suggested as main objective of this study the analysis of changes in nutrients during R in a group of Muslim students of both sexes, in order to solve and prevent some of the problems shown in this period. MATERIALS AND METHODS: 55 Muslim youngsters were selected (24 men and 31 women) aged between 15 and 18, who practise R; and the diet was analysed by inventory over 3 days, one week before fasting and in the second week during it. RESULTS: In R there are no relevant changes in caloric intake. The consumption of carbohydrates and proteins is increased, and that of fat is decreased. DISCUSSION: the changes in R lead to a diet more regulated to nutritional recommendations; and to this seems to have contributed the fact that the participants took their meals at home, where, in spite of the Western cut of the population, it is felt an important influence of North African traditions and habits, due to its border contact with Morocco.

Cytogenetic abnormalities additional to t(11;14) correlate with clinical features in leukaemic presentation of mantle cell lymphoma, and may influence prognosis: a study of 60 cases by FISH.

Mantle cell lymphoma (MCL), characterised by t(11;14)(q13;q32), has a poor prognosis. Many cases have additional cytogenetic abnormalities, and often have a complex karyotype. Fluorescence in situ hybridisation (FISH) was used to study 60 cases with leukaemic presentation of MCL, to determine the frequency, clinical correlations and prognostic impact of a panel of molecular cytogenetic abnormalities: 17p13 (TP53 locus), 13q14, 12 p11.1-q11 (centromere), 6q21 and 11q23. CD38 expression, of prognostic value in chronic lymphocytic leukaemia (CLL), was also studied, and correlations with clinical and cytogenetic abnormalities sought. Eighty per cent of cases had at least one abnormality in addition to t(11;14). Deletions at 17p13 (TP53) and 13q14 were most frequent and involved the majority of the leukaemic clone. Cases with TP53 deletion were more likely to have splenomegaly and marked leucocytosis (>30 x 10(9)/l), and less likely to have lymphadenopathy than those without deletion. Deletions at 11q23 and 6q21 were associated with extranodal disease. 13q14 and 11q23 deletions showed a trend towards worse prognosis by univariate analysis. In multivariate analysis, deletions at 13q14 and 6q21 were independent predictors of poor outcome. Deletion at 17p13 did not show prognostic impact in this series. CD38, positive in two-thirds of cases, was associated with male gender and nodal disease but not with any cytogenetic abnormality, or with survival.

CD52 expression in T-cell large granular lymphocyte leukemia--implications for treatment with alemtuzumab.

Few reports on the successful treatment of T-cell large granular lymphocyte (LGL) leukemia with the humanized anti-CD52 monoclonal antibody alemtuzumab are emerging in the literature. The expression of CD52 by LGLs has not been previously investigated. Using semi-quantitative 2- and 3-color flow cytometry, we documented the expression of CD52 in 100% of abnormal cells in T-cell LGL leukemia (n = 11) and natural killer (NK) cell LGL leukemia (n = 2), and showed no significant difference in CD52 expression between T-cell prolymphocytic leukemia (PLL) and T-cell LGL leukemia. Higher CD52 expression has been noted in responders to alemtuzumab in T-cell PLL and in chronic lymphocytic leukemia (CLL), a B-cell disorder. The strong and consistent expression of CD52 shown here highlights the potential role of alemtuzumab in the treatment of refractory T-cell LGL leukemia and possibly aggressive NK cell leukemia.

The leukemic presentation of mantle-cell lymphoma: disease features and prognostic factors in 58 patients.

Mantle-cell lymphoma (MCL) is a B-cell malignancy with distinct molecular genetics and pathological features. Peripheral blood involvement has been reported with variable frequency, but information on the natural history of cases presenting with leukemia is lacking. This study aimed to determine the clinical and prognostic features of such cases. We studied clinical features, tumor characteristics, prognostic factors and outcome in 58 patients with leukemic presentation of MCL. Diagnosis was based on morphology, immunophenotype, presence of t(11;14), histology and cyclin D1 expression. The median age was 62 years and male:female 2.4:1. Presenting features included splenomegaly (74%), lymphadenopathy (45%), hepatomegaly (17%) and, in a minority, gastro-intestinal involvement or involvement of Waldeyer's ring; 10% had lymphocytosis alone. Six patients developed central nervous system disease. Median lymphocyte count was 58 x 10(9)/l, 55% had anemia and 17% had thrombocytopenia. Morphology of peripheral blood showed small-cell MCL in 15% of cases, typical MCL in 46% and blastoid MCL in 39%. Immunological markers showed a typical phenotype (CD5+ CD23 -) in 68%, and atypical phenotypes, CD5- CD23- in 17% or CD5+ CD23+ in 15%. CLL scores were 0, 1 or 2 in 96%. Median overall survival was 36 months. Good response to first-line treatment (P = 0.0008) and splenomegaly (P = 0.03) were favorable prognostic factors, while other features including morphology and CD38 expression had no impact on survival or treatment response. This analysis demonstrates that except for splenomegaly, survival of MCL patients presenting with leukemia is not significantly influenced by clinical or tumor characteristics. Splenectomy is a useful treatment option in this group of patients.

A comparison of flow cytometry, bone marrow biopsy, and bone marrow aspirates in the detection of lymphoid infiltration in B cell disorders.

AIMS: To evaluate the diagnostic value of bone marrow aspirates, trephine biopsies (BMB), and flow cytometry (FC) in the assessment of bone marrow infiltration in chronic lymphoid disorders. METHODS: Investigations were carried out in 110 diagnostic and follow up specimens from B cell disorders, namely: chronic lymphocytic leukaemia (CLL; 65), non-Hodgkin's lymphoma (NHL; 39), and hairy cell leukaemia (HCL; 6). A selected panel of monoclonal antibodies was used both for FC and immunohistochemistry. RESULTS: In CLL there was agreement between the three investigations in 71% of samples and in 88% when only FC and BMB were compared. In nine of 65 samples, FC and BMB were positive, although the aspirate was reported as negative. Four BMB negative samples had minimal residual disease (MRD) detected by FC, whereas two samples were positive both on BMB and aspirate but showed no evidence of disease on FC. In NHL, there was agreement between the three investigations in 22 of 39 cases, and in 27 of 39 cases there was agreement between FC and BMB. In eight of 39 NHL cases, FC was negative but the BMB was either positive (five) or uncertain (three), whereas in three of 39, FC was positive but BMB was either negative (one) or uncertain (two). In three of five uncertain BMB, no clonal population was detected by the polymerase chain reaction, whereas in the remaining two cases the nodular aggregates disappeared on further sectioning. CONCLUSIONS: Both BMB and FC are better than bone marrow aspirates for the detection of infiltration in B cell disorders. FC might be slightly more sensitive than BMB to detect MRD in CLL, whereas BMB may be slightly better than FC in NHL.

Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry.

AIMS: To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs). METHODS: Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1-120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients. RESULTS: A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC. CONCLUSION: Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay.

Deletions of D13S25, D13S319 and RB-1 mapping to 13q14.3 in T-cell prolymphocytic leukaemia.

Deletions of 13q14.3 are well known in several malignancies and are thought to be associated with tumour suppressor function. The RB-1 gene is a tumour suppressor gene, but other loci including D13S319 and D13S25 telomeric to this within 13q14.3 are deleted in B-cell chronic lymphocytic leukaemia (B-CLL), multiple myeloma and non-Hodgkin's lymphoma, with varying clinical significance. The fluorescence in situ hybridization screening of 22 patients with T-prolymphocytic leukaemia (T-PLL) for deletions of 13q14.3 revealed loss of D13S25 in 17 cases (mean 40% range 13-98%), with 11 patients having at least a 20% deletion. Mapping the deletions for the RB-1, D13S319,and D13S25 loci revealed D13S25 as the most frequently deleted marker. However, patients with only the D13S25 deletion had low percentages of cells with the deletion (12-13%), suggesting that loss of D13S25 on its own may not provide sufficient growth advantage. The use of the YAC 954c12, which maps immediately adjacent to D13S25, defined the telomeric border of the deletion in some of the cases. Inv(14)(q11q32) and t(14;14)(q11;q32) are characteristic of T-PLL, but are also observed in premalignant T-cell clones in patients with ataxia telangiectasia. Transition to overt leukaemia may result from loss of suppressor function. Thus, 13q14.3 deletions could contribute to the development of overt leukaemia in T-PLL, but the involvement of more than one gene in the region cannot be excluded.

The effect of peripheral blood lymphocyte stimulation on zeta chain expression and IL-2 production in Hodgkin's disease.

It has been reported that peripheral blood T cells and NK cells express reduced levels of the T-cell receptor signal-transducing zeta chain in Hodgkin's disease (HD). The zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T and NK-cell activation. We were interested in determining whether the low zeta chain expression in HD could be corrected by anti-CD3, anti-CD3-rIL-2 ex vivo stimulation. Zeta chain expression was analysed by dual immunofluorescence on permeabilized cells before and after 72 hours of culture. The IL-2 concentration in the culture supernatants was measured by ELISA. Zeta chain was significantly reduced on unstimulated CD4+, CD8+ and CD56+ cells from patients in active disease compared with normal subjects. In patients in complete remission, the values were normal except for CD8+ cells, on which zeta expression remained significantly reduced. Stimulation with anti-CD3 did not change zeta expression. Co-stimulation with rIL-2 increased but did not normalize the proportions of CD4(+)/zeta(+), CD8(+)/zeta(+)and CD56(+)/zeta(+)cells and IL-2 production in active disease. Stimulation of cells from patients in clinical remission with anti-CD3(+)rIL-2 increased the proportion of CD8(+)zeta(+)cells and normalized IL-2 production levels. Considering the pivotal role of CD3-zeta in immune response, our data suggest that successful immunotherapy approaches in active HD should consider inclusion of other potent cytokines, as well as genetically engineered tumour vaccines.

Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies.

The translocation (11;14)(q13;q32) and its molecular counterpart the BCL-1 rearrangement are features observed in mantle cell lymphoma (MCL) and less commonly in other B-cell disorders. This rearrangement leads to cyclin D1 overexpression, which may be the main pathogenic event in these tumours and is therefore recognised as a diagnostic marker. We developed a flow cytometry method to detect cyclin D1 overexpression using the monoclonal antibody (MoAb) 5D4, and characterised its frequency in 93 B-cell malignancies. The competitive reverse transcriptase polymerase chain reaction (RT-PCR) for cyclin D1, D2 and D3 was then performed on 40 of these cases to assess the validity of the flow cytometry method. Fluorescence in situ hybridisation (FISH) to detect t(11;14)(q13;q32) was carried out on 31 cases and results were compared with cyclin D1 expression by flow cytometry. Twenty five cases showed cyclin D1 expression using 5D4, including MCL (12/13, 92%), chronic lymphocytic leukaemia (CLL) (4/30), B-prolymphocytic leukaemia (B-PLL) (1/4), splenic lymphoma with villous lymphocytes (SLVL) (4/13), hairy cell leukaemia (HCL) (1/7) and other B-non Hodgkins Lymphoma (B-NHL) (3/15). There was a good correlation between flow cytometry results and RT-PCR in 36/40 cases (90%), and with FISH for t(11;14) in 25/31 cases (80%). We concluded that the detection of cyclin D1 expression by flow cytometry in cell suspensions could be applied routinely to the study of B-lymphoproliferative disorders and may be of value for their diagnosis and management.

Immunophenotype changes and loss of CD52 expression in two patients with relapsed T-cell prolymphocytic leukaemia.

T-cell prolymphocytic leukaemia (T-PLL) is an aggressive disease often resistant to conventional chemotherapy. Long lasting remissions with the monoclonal antibody CAMPATH-1H (anti-CD52) have been documented. We describe two unusual T-PLL patients treated successfully first with CAMPATH-1H in whom, at the time of relapse, the cells underwent a phenotypic switch with loss of CD52 expression. In one of them, cytogenetic analysis demonstrated the same chromosome abnormalities in the cells at diagnosis and relapse. The reasons for the immunophenotypic changes are unknown but it is likely that loss of CD52 antigen expression contributed to the resistance to CAMPATH-1H in one of the patients when re-treated.

Quantitative expression of CD23 and its ligand CD21 in chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: Cells from the great majority of patients with chronic lymphocytic leukemia (CLL) express CD23. A recent histologic study has shown that CD23 is expressed more strongly in the proliferating centers of the lymph nodes, where the large prolymphocytoid cells are located. The aim of our study was to quantify the expression of CD23 and CD21 in small and prolymphocytoid cells from patients with CLL and B-cell lymphomas, and correlate this expression with clinical parameters. DESIGN AND METHODS: Using quantitative flow cyto-metry we analyzed the antigen density of CD23 and CD21 in: 1) 101 cases of chronic lymphocytic leukemia, 84 typical, 14 with increased prolymphocytes (CLL/PL) and 3 atypical, 2) 15 cases of CD23 positive B-cell lymphoma with circulating lymphoma cells and 3) 8 normal subjects. The results were correlated with morphology and clinical staging. RESULTS: Cells from CLL and CLL/PL have a significantly higher number of CD23 molecules than normal and lymphoma B-cells (p<0.001 and p<0.001, respectively). Differences were not significant for CD21. CLL and CLL/PL cases had similar values of CD23 and CD21 molecules, but analysis at a single level showed that prolymphocytes in typical CLL and CLL/PL expressed significantly higher CD23 (p=0.001, p=0.006) and CD21 (p=0.001, p=0.001) than small lymphocytes. There was no correlation between CD23 or CD21 antigen density and clinical stages although there was a trend for a brighter CD23 in stage C patients. INTERPRETATION AND CONCLUSIONS: Since interaction between CD23 and CD21 is important for B-cell activation, proliferation and tumor formation, findings that both molecules are upregulated in prolymphocytes suggest that this is the proliferating cell component in CLL and underline the association between progression and increased prolymphocytes in typical CLL and CLL/PL.

Expression of signal-transducing zeta chain in peripheral blood T cells and natural killer cells in patients with Hodgkin's disease in different phases of the disease.

A number of phenotypic and functional alterations have been described in T cells of cancer patients. These changes are believed to reflect an impaired T-cell mediated immunity, which in turn, may result in a decreased capacity to generate an effective antitumor response. Several mechanisms have been proposed to explain depressed immunity in cancer patients including tumor-derived suppressor factors, abnormal cytokine production, deletion or inactivation of tumor-reactive T-cells. To investigate the mechanism underlying the immunodeficiency in Hodgkin's disease (HD) we studied the expression of T cell receptor zeta chain, which plays a vital role in the cascade of events leading to T and NK cell activation. The expression of the zeta chain of the T cell receptor/CD3 complex was analyzed by dual colour immunofluorescence on peripheral blood T lymphocytes: CD3+, CD4+, CD8+ and NK-cells (CD56+) in patients in different phases of the disease. Zeta chain was significantly reduced on CD3, CD4, CD8, and CD56 positive cells from patients in active phase of the disease compared with normal controls (p=0.05). In patients tested in complete clinical remission the values were normal except for the subpopulation of CD8+ cells in which the expression of zeta chain remained significantly reduced compared with controls. Downregulation of CD3/zeta-chain in PBLs and NK cells in active phase of HD- and to a lesser extent in clinical remission may contribute to immunodeficiency associated with the disease.

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

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