CD8+ chronic lymphocytic leukemia: an extensive characterization of a bizarre hybrid neoplasia.

Atypical chronic lymphocytic leukemia (CLL) expressing the CD8 antigen have a frequency of less than 0.5% of all cases, however, they are not yet been fully characterized. Herein a CD8+ CLL case was extensively studied. Besides the classical CLL antigen expression, an unusual presence of surface markers such as CD11c, CD56, and CD154 was observed. Moreover, gene expression of chemokine receptors belonging to the CCR family were clearly evidenced as well as mRNA for both, Th1 and Th2 cytokines. Likewise, granzyme A, B and perforin gene expression, cytotoxic T cell or NK enzymes were found. The intricate profile of membrane molecules and gene expression suggest that it could be favorable, rather than deletereous, for the maintainance of the neoplastic process.

Flow cytometric analysis of glycosylphosphatidyl-inositol-anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by total or partial deficiency of membrane proteins anchored to the cell surface through a glycosylphosphatidyl-inositol (GPI) moiety. The relationship between the size of the PNH clone, determined by the expression of GPI-anchored proteins (AP; CD14, CD48, CD55, CD59, and CD66b) on erythrocytes, lymphocytes, monocytes, and granulocytes using forward and side scatter analysis, and severity of the disease was evaluated in 19 PNH patients. CD55 antigen expression did not delineate abnormal erythrocytes as well as did anti-CD59. The proportion of monocytes deficient in CD55, CD59, CD48, and CD14 (48-97%) and of granulocytes deficient in CD55, CD59, and CD66b (60-99%) was greater than the proportion of erythrocytes deficient in CD59 (24-95%) and the proportion of lymphocytes deficient in CD55 and CD59 (30-98%). There were no significant correlations among reticulocyte, leukocyte, and platelet counts and GPI-AP-deficient immunophenotypes in red and white blood cells. However, high coefficients of determination were seen between hemoglobin levels and granulocytes deficient in CD59 (r(2) = 0.76), CD55 (r(2) = 0.74), and CD66b (r(2) = 0.74) antigens and between hemoglobin and monocytes deficient in CD55 (r(2) = 0.73), CD59 (r(2) = 0.80), and CD14 (r(2) = 0.75) antigens. These results are interpreted as indicating that the size of PNH clone is better assessed by immunophenotypic analysis of monocytes and granulocytes rather than of lymphocytes and erythrocytes.

Detection of platelet-associated immunoglobulins by flow cytometry for the diagnosis of immune thrombocytopenia: a prospective study and critical review.

BACKGROUND AND OBJECTIVE: Flow cytometry (FC) to identify platelet-associated (PA) immunoglobulin (Ig) is a potentially useful diagnostic test for idiopathic thrombocytopenic purpura (ITP). However, the restricted application of PAIg measurement to thrombocytopenic populations primarily comprised of ITP patients will artificially enhance the test's diagnostic specificity. For this reason, we performed a prospective study in which the results of a sensitive technique for detecting PAIg, as is FC, were correlated to the cause of the thrombocytopenia. DESIGN AND METHODS: A total of 118 patients with platelet counts <100 x 10(9)/L and 30 normal donors with a platelet count >200 x 10(9)/L were studied for PAIg employing a flow cytometer. Forty-two children and 20 adults were diagnosed as having immune thrombocytopenia and 27 children and 29 adults had nonimmune thrombocytopenia of different etiology. RESULTS: Raised levels of PAIg were found in 56/62 patients with immune thrombocytopenia and in 34/56 patients with non-immune thrombocytopenia. Diagnostic values of PAIg for the detection of immune thrombocytopenia were: sensitivity 90.3% and specificity 39. 3%. An enzyme-linked immunoabsorbant assay (ELISA) for the detection of autoantibodies to platelet glycoprotein (GP) complexes was used in adults, 9 with immune-related thrombocytopenia and 16 with non-immune thrombocytopenia, in order to determine the true non-specific nature of the positive PAIg test. By ELISA, 8/9 patients with immune thrombocytopenia and 7/16 with non-immune thrombocytopenic disorders showed autoantibodies to platelet GP complexes. INTERPRETATION AND CONCLUSIONS: PAIg detection by FC constitutes a sensitive but non-specific assay thus making it unnecessary and inappropriate for establishing the diagnosis of ITP.

[Classification of acute leukemias according to the first latin-american consensus conference for the immunophenotyping of leukemias]. / Clasificación de las leucemias agudas de acuerdo con el consenso de la primera conferencia latinoamericana en la tipificación inmunológica de las leucemias.

OBJECTIVE: To evaluate the recommendations of the First Latinamerican Consensus Conference for the Immunophenotyping of Acute Leukemia in untreated patients with de novo disease immunologically classified employing flow cytometry and an extended panel of monoclonal antibodies. MATERIAL AND METHODS: In that conference it was recommended the use of the following antibodies: cytoplasmic CD79a (cCD79a) and CD19 to define B-progenitor acute lymphoblastic leukemia (B-ALL); cCD3 and CD7 for T-cell ALL (T-ALL), and CD13, CD33 and myeloperoxidase (cMPO) for acute myeloblastic leukemia (AML). We analyzed the expression of these cellular antigens in 91 non-consecutive patients classified with the extended panel as: B-ALL 28 cases; T-ALL 7; B-T-ALL 2; AML 47; and mixed-lineage acute leukemia 7 cases. RESULTS: All 28 B-ALL cases were positive with each of the two recommended antibodies cCD79a and CD19, whereas in 24 AML cases (the expression of cCD79a was not assayed in 23 cases) and in 7 T-ALL patients both antigens were absent. cCD3 and CD7 antigens were identified in 71% and 100% of T-ALL, respectively. CD7 antigen was not detected in any of the 28 patients with B-ALL but it was expressed in 6 of 47 AML cases, while none of 75 B-ALL and AML cases were positive to cCD3. Forty nine percent of AML were positive for the three recommended markers: cMPO, CD13 and CD33, and 51% of AML cases reacted with one or two of these three monoclonal antibodies. Six out of 28 cases of B-ALL had aberrant expression of myeloid antigen (CD33 in 3 cases and CD13 in 3 cases). CONCLUSIONS: There was no difference in the definition of AL lineage between employing the extended antibody panel and that recommended by the Latinamerican consensus.

[Laparoscopic splenectomy in autoimmune thrombocytopenic purpura]. / Esplenectomía laparoscópica en púrpura trombocitopénica autoinmune.

OBJECTIVE: To describe the perioperative characteristics, complications and outcome of the first 22 patients who underwent laparoscopic splenectomy for refractary/recurrent ITP in our institution. MATERIAL AND METHODS: Clinical and biochemical characteristics, spleen size, indication for surgery, operative time, blood requirements, complications and outcome of 22 patients who underwent laparoscopic splenectomy between 1994 and 1997 were prospectively recorded. Their mean age was 40 +/- 15 (+/- SD), 15 females and 7 males. RESULTS: The preoperative platelet count was 56 thousand +/- 58.7/uL, average spleen size 10.5 +/- 2 cm. The surgical time averaged 4.5 +/- 1 hours; accessory spleens were removed from two patients. Conversion to the open procedure was necessary in two cases. Complete response was achieved in 59%, partial response in 27%, and no response in 14% (none of these due to missed accessory spleens). Six patients developed complications. One of them died two days after surgery. The mean postoperative stay was 4.7 +/- 2.6 days. CONCLUSIONS: The results of laparoscopic splenectomy were similar to our previous results with the open approach. The laparoscopic technique took longer in our hands but the postoperative stay was shorter.

Circulating lymphocyte subpopulations and activated T and B cells in patients with chagasic and non-chagasic myocardiopathy.

Trypanosoma cruzi causes a profound immune depression in the infected host, and a small proportion of chagasic patients will develop a chronic disease characterized by myocardiopathy. There is evidence suggesting that dilated non-chagasic cardiomyopathy may be mediated by an immunological mechanism. In an attempt to distinguish abnormal immunoregulatory cell patterns in both dilated myocardiopathies, total and activated T and B lymphocyte subpopulations were measured by flow cytometry and double-labeling in whole blood samples from patients with dilated myocardiopathy, 10 with positive serological tests for T. cruzi and 9 with different non-chagasic cardiomyopathies. Several significant differences were found between both groups of patients and 13 sex- and age-matched apparently healthy controls. Chagasic patients besides showing clear decrease in absolute numbers of CD3+/CD71+ and CD8+/CD25+ cell populations also had a significant increase in CD19+, CD10+, and CD19+/HLA-DR+ cell subsets, as well as high helper/ suppressor cell ratio. These findings suggest that concurrently with T cell diminution, which involved activated T lymphocytes displaying suppressor/cytotoxic immunophenotype, chronic chagasic patients with myocardiopathy showed elevated numbers of total and activated B lymphocytes. Patients with dilated non-chagasic myocardiopathy had significantly increased numbers of activated T cells (CD3+/CD25+, CD8+/CD25+, and CD8+/HLA-DR+) and total and activated B lymphocytes (CD10+, CD19+, CD19+HLA-DR+). These data support the notion that dilated myocardiopathies other than the chagasics may be associated with immunological abnormalities.

Immunophenotypic characteristics of the blast crisis in chronic myeloid leukemia.

OBJECTIVE: To characterize the immunophenotype of blast crisis (BC) in Mexican patients with chronic myeloid leukemia (CML). MATERIAL AND METHODS: Mononuclear cells of 17 patients with CML in BC were immunophenotyped employing a panel of 18 monoclonal antibodies: CD5, CD10, CD14, CD22, and anti-HLA-DR used in all patients; CD2, CD15, CD19, CD34, and CD41 in 13 to 16 patients; and CD3, CD7, CD13, CD20, CD21, CD33, CD42b, and CD61 in less than 10 patients. RESULTS: Myeloid was the most frequent type (9/17 cases) followed by lymphoid (6/17) and hybrid or mixed lineage (2/17). Four of the myeloid BC expressed megakaryocyte/platelet associated antigens; 5 of 6 cases with lymphoid BC showed an early precursor B cell immunophenotype (HLA-DR+, CD10+), and the other was an uncommon case of lymphoid B/T transformation (CD19+, CD5+). The CD34 antigen was present in 6 out of 15 cases: 4 patients with lymphoid BC, 1 with myeloid transformation, and 1 with megakaryoblastic BC. CONCLUSIONS: Our findings are comparable to those found in the literature comprising 192 patients. The present study confirms the lineage heterogeneity of CML BC and suggests that extensive immunophenotyping may allow insight for a more precise recognition of normal and leukemic ontogenesis.

[Cellular immunophenotypes in 97 adults with acute leukemia]. / Inmunofenotipos celulares en 97 adultos con leucemia aguda.

OBJECTIVE: To analyze hematopoietic cell surface antigen reactivity in acute leukemia (AL) by flow cytometry and identify acute mixed-lineage leukemias (AMLL) employing the most widely accepted criteria. MATERIAL AND METHODS: Ninety seven patients with de novo AL were studied. Cell surface antigens were investigated with monoclonal antibodies directed to: B lymphoid (CD10, CD19, CD20, CD21, CD22); T lymphoid (CD2, CD3, CD5, CD7); and myeloid (CD13, CD14, CD15, CD33, CD41) cell lineages. Maturation cell-associated antigens (CD34, HLA-DR and TdT) were also studied. RESULTS: Twelve patients unclassified by cytomorphology could be classified by immunophenotype. Using cytomorphologic, cytochemical and immunophenotypic data, 54 cases corresponded to acute lymphoblastic leukemia (ALL) and 43 were acute myeloblastic leukemia (AML). In All there were 63% B lineage, 15% T, 7% T/B, 6% undifferentiated and 9% mixed-lineage (coexpression of two or more myeloid-associated antigens). In AML, myeloid immunophenotype was observed in 86% undifferentiated in 2%, and mixed-lineage in 12% (coexpression of two or more lymphoid-associated antigens). In addition, 26% of ALL cases and 12% of AML cases expressed a single myeloid and lymphoid antigen respectively. The most common aberrant antigens in ALL and AML were CD13 and CD7 respectively. The highest frequency of CD34 antigen expression (90%) was detected in patients with AMLL. CONCLUSIONS: Flow cytometric immunophenotypic analysis allowed to: a) establish diagnosis in cytomorphologically unclassified cases; b) identify AMLL with a frequency similar to that reported in other series; and c) confirm the heterogeneity of AL.

[Response to treatment and survival of adults with acute leukemia classified with immunophenotyping and cytochemistry]. / Respuesta al tratamiento y supervivencia de adultos con leucemia aguda clasificados mediante inmunofenotipo y citoquímica.

We studied the response to treatment and survival of 30 adults with acute lymphocytic leukemia (ALL) and 19 with acute non lymphoid leukemia (ANLL) classified on basis of immunophenotype (monoclonal antibodies) and cytochemistry. For the ALL cases 70% corresponded to common ALL (CALLA positive), 23% to B lymphocytes and 7% to T cells. We had 68% of the ANLL patients classified as myeloid, 21% as hybrid (positive both myeloid and lymphoid markers) and 11% as undifferentiated. We analyzed demographic data (gender and age), basic laboratory values (hemoglobin, leucocytes, platelets and cytomorphology in peripheral blood and bone marrow) using the French-American-British classification, and found no statistically significant differences between ALL and ANLL. Three of four patients (75%) with hybrid ANLL achieved complete remission (CR), while 46% of cases with myeloid ANLL and none of the subjects with undifferentiated ANLL reached CR; these differences were not statistically significant. Patients with common ALL had a median survival (SV) of 499 days, for B cell ALL it was of 212 days, and for T cell ALL of 285 days. Our data suggest that: a) expression of lymphoid markers in patients with ANLL is probably associated with a higher CR ratio, and b) SV in adults with common ALL seems to be longer than in those with B and T cell ALL.

Cellular and humoral immune parameters in women with pathological hyperprolactinemia before and during treatment with bromocriptine.

PROBLEM: Experimental and clinical evidence has suggested an immunostimulatory effect of prolactin and that bromocriptine, an inhibitor of prolactin release, counteracts the actions of prolactin on the immune system. The aim of this study was to determine the impact of elevated serum prolactin levels on the immune system in patients with pathological hyperprolactinemia. METHOD: For this purpose, parameters of the cellular and humoral immune system were studied in six women with prolactinomas and one with idiopathic hyperprolactinemia. Studies were performed when serum prolactin concentrations were high as well as during different phases of the menstrual cycle when prolactin levels had been normalized through treatment with bromocriptine. RESULTS: Hyperprolactinemic subjects, when compared with six age-matched normal women, had significantly higher percentages of total lymphocytes and CD2+ cells. Elevation of CD4+ cells was also observed although to a lesser extent. Bromocriptine-treated patients, when compared with normal women were characterized by increased numbers of total lymphocytes and CD4+ cells, decreased percentage of CD8+ cells, and increased concentrations of serum IgM. These last two findings were also significantly different when compared to those observed in hyperprolactinemia. CONCLUSION: In this study we have described the changes on cellular and immune parameters in patients with hyperprolactinemia before and during bromocriptine therapy, which support the links of communications between the immune and endocrine systems in humans.

Long-term results of high-dose methylprednisolone in aplastic anemia.

Four males and two females, aged 13 to 57 years (median 22 years), with acquired severe aplastic anemia (AA) were treated with intravenous bolus of high doses of 6-methylprednisolone (MPL). Patients received MPL within a 30-day period at a dose of 20 mg/kg/day (3 days), 10 mg/kg/day (4 days), 5 mg/kg/day (4 days), 2 mg/kg/day (9 days), and 1 mg/kg/day (10 days). Within the first 3 months following MPL therapy, a response rate of 83%, assessed by means of increase in reticulocytes, neutrophils or platelets, was recorded in the group: two cases showed partial response and three improvement. The 3-month, and 1-, 2- and 3-year survival of the group was 67%, 50%, 33% and 33%, respectively. Neither the presence of reticulocytopenia or thrombocytopenia prior MPL therapy, nor age, gender, etiology of AA or time between diagnosis and initiation of MPL influenced survival. In contrast, neutrophil counts before MPL treatment had a strong prognostic value. Patients with less than 0.5 x 10(9)/L neutrophils had a median survival of 4.2 months (range 1.2 to 5.2 months) as compared to the 36.1 months median survival (range 12.1 to 36.8 months) of patients whose neutrophil counts were greater than 0.5 x 10(9)/L. Follow-up data suggest that the administration of androgens two months after MPL therapy did not modify survival. It is concluded that high-dose MPL is useful in the treatment of some patients with acquired severe AA, particularly in those with greater than 0.5 x 10(9)/L neutrophils who are not candidates for bone marrow transplantation.

[Splenectomy in idiopathic thrombocytopenic purpura]. / Esplenectomía en púrpura trombocitopénica idiopática.

Between January 1976 and December 1987, 44 patients with idiopathic thrombocytopenic purpura were submitted to splenectomy. This study analyzes the results of treatment and the usefulness of several prognostic markers. There were 38 female patients and six males with a median age of 28.5 years (range 11 to 66 years). Forty two out of 44 patients received preoperative steroids. Seventy five percent achieved a satisfactory response to this treatment but 31 relapsed (in eight recurrence was observed when the steroid dose was tapered). Eleven patients persisted symptomatic notwithstanding medical treatment. In two cases splenectomy was performed as an emergency procedure, and in one patient who was already in remission with previous steroid treatment, splenectomy was performed for other medical reasons. Operative mortality rate was 4 percent, and five patients developed postoperative complications (three had pneumonia, one pulmonary thromboembolism and one would infection). Eighty four percent of the patients were free of disease at one year followup survival at 10 year period was 80 percent. No correlation was found between age, duration of symptoms, previous response to steroids or time-period of medical treatment with the results obtained with splenectomy.

Low doses of high-potency antithymocyte globulin (ATG) in severe aplastic anemia: experience with the Mexican ATG.

Twenty patients with severe aplastic anemia (SAA) were treated with low doses (1-5 mg/kg/day) of a high-potency antithymocyte globulin (ATG) produced in Mexico, shown to have at least a 10-fold potency as compared with other globulins of commercial sources. Patients received ATG within a 10-day period, every other day (5 doses) at a dose of 1 mg/kg/day (4 courses), 2 mg/kg/day (12 courses) or 5 mg/kg/day (8 courses). Four patients received 2 consecutive courses of different doses of ATG. A response rate of 42% was recorded in the group, assessed by means of increases in reticulocytes, granulocytes or platelets. One patient showed a complete remission. The 570-day survival of the group was 51%. It is concluded that the domestically produced ATG is useful in the treatment of some patients with SAA in Mexico.