Paroxysmal nocturnal haemoglobinuria. Experience over a 10 years period.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic, clonal and acquired disorder of the hematopoietic stem cell with a deficiency of all glycophosphatidyl-inositol (GPI) linked proteins. The aim of this retrospective study was to analyse haematological and biochemical data from 152 patients referred to our laboratory for diagnosis of PNH by flow cytometry (FC). METHODS: Patients and healthy donor (152 and 99 respectively) were studied. Ham, sucrose, lactate dehydrogenase (LDH), Iron, haptoglobin (Hp), blood cell morphology and Kaplow cytochemical stain for leukocyte alkaline phosphatase (LAP) were carried out. GPI-proteins anti-CD55 and CD59 in erythrocytes and the former, plus anti CD16b and CD66b on neutrophils were evaluated by FC. RESULTS: Anemia and/or leukopenia and/or thrombocytopenia, increased reticulocyte count and LDH were observed in patients with PNH clone. Some of them had dacriocytes, schistocytes. LAP was low. On average, we detected 50% CD59 (-) erythrocytes and 29, 83, 78% CD55/59 (-), CD16b (-), CD66b (-) neutrophils, respectively. CONCLUSION: Paroxysmal nocturnal hemoglobinuria clone was detected in 20/152 patients. Negative population's percentages were high in patients with classic PNH, Hematimetry, LAP and adequate use of CF contribute to PNH clone detection in the laboratory.

Anti-polymorphonuclear neutrophil antibodies in patients with leukopenia or neutropenia.

INTRODUCTION: Immune humoral neutropenia (Np) could be the consequence of anti-polymorphonuclear neutrophil (PMN) antibodies, circulating immune complexes (CIC) and/or antibodies against myeloid precursors. Granulocyte immunofluorescence test (GIFT) and a leukoagglutination technique (LAGT) assays are recommended for its diagnosis. METHODS: Fifty adult patients with secondary Np were screened for anti-PMN. GIFT by flow cytometry from viable PMN and LAGT were employed. In addition, CIC levels, low expression of CD16(b) (CD16 (b)(low)), PMN phenotype and sera tumor necrosis factor-alpha (TNF-alpha) were also evaluated. RESULTS: Direct IgG-PMN binding (dir-GIFT) was positive in 16% of the patients. Antibodies against autologous PMN were detected in 32% of the samples by indirect (ind)-GIFT and demonstrated in 70% of the sera by both ind-GIFT and/or LAGT. Predominance of human neutrophil alloantigen (HNA)-1b and HNA-2 expression was confirmed. CD16(b)(low) was detected in 16% of the patient's PMN and TNF-alpha in 68% of sera patients. CONCLUSION: Our results suggest that diagnosis of immune Np in the laboratory may be improved by focusing on patient's PMN together with the assessment of cellular markers.

[Evolution of paroxysmal nocturnal hemoglobinuria clone during an hemolytic crisis in a patient with aplastic anemia. Flow cytometry study]. / Evolución del clon de la hemoglobinuria paroxística nocturna en la crisis hemolítica de una anemia aplásica. Estudio por citometría de flujo.

The expansion of paroxysmal nocturnal hemoglobinuria (PHN) clone was evaluated in a patient with aplastic anemia (AA) of 18 years of evolution during an hemolytic crisis. On day 0, Ham and Sucrosa tests were positive and hematological parameters were altered. Low hemoglobin (Hb) levels and erythrocyte and leukocyte counts were found and continued decreasing on days 7 and 24 (last day of study). High LDH levels, indirect bilirubin and reticulocyte counts were detected throughout. We evaluated CD55 and CD59 on erythrocytes by flow cytometry. Our results showed low CD55 expression with respect to the normal pattern. Since day 0, CD59 staining detected two red cell populations: PNH I (48%), cells with positive fluorescence similar to normal and PNH III (52%), negative cells (PNH clone). These negative cells increased, reaching 70% on day 24. Other membrane anchored leukocyte proteins were also absent (CD14) or decreased (CD16). We found a good correlation between clinical observations, evolution of the laboratory values and expansion of the PNH clone.

[Microspherocytosis. Erythroid profile and its relation with different laboratory tests]. / Microesferocitosis. Perfil eritroide y su relación con distintas pruebas de laboratorio.

Hereditary spherocytosis (HS) is a congenital and hemolytic anemia characterized by the presence of microspherocytes on the peripheral blood film and negative Coombs test. Although HS is a heterogeneous syndrome in terms of clinical severity, inherents and underlying molecular defects (deficiency of membrane skeleton proteins), typical HS has a dominant inheritance pattern and presents anemia, jaundice and splenomegaly. The purpose of this study was to present our experience and to establish the relationship between hematological and biochemical parameters and osmotic fragility and autohemolysis, all of them considered as traditionally available tests. In our environment, HS is the second most common inherited anemia after beta thalassemia trait. The diagnosis was based on osmotic fragility measurements, autohemolysis test and microspherocytes in 47 patients (45 of latin, 2 of saxon origin); 12 patients had a negative family history. Mean values: RBC (x 10(12)/L) and Hb (g/dL): children (22): 3.84/10.5; adults (25): female (13): 3.54/10.59; masculine (12): 4.65/13.15. Autohemolysis (average %) was very much increased (15.54) and it was corrected by the addition of glucose (4.07). Median osmotic fragility (average g/dL) was increased in both fresh (0.48) and incubated blood (0.65). 76.5% children and 26.7% adults had absence of haptoglobin. Reticulocytes, indirect bilirubin and LDH were increased (average values 336.35 x 10(9)/L, 36.25 mmol/L and 236.48 UI/L, respectively). Microcytosis, spherocytosis, polycromatophylia, acanthocytosis, basophilic stippling, pincered cells were observed in 43, 41, 41, 12, 11, 1 patients respectively. Morphologic alterations were more marked in children. The laboratory findings seemed more a consequence of microcytosis than of spherocytosis.

Microesferocitosis. Perfil eritroide y su relación con distintas pruebas de laboratorio. / [Microspherocytosis. Erythroid profile and its relation with different laboratory tests]

Hereditary spherocytosis (HS) is a congenital and hemolytic anemia characterized by the presence of microspherocytes on the peripheral blood film and negative Coombs test. Although HS is a heterogeneous syndrome in terms of clinical severity, inherents and underlying molecular defects (deficiency of membrane skeleton proteins), typical HS has a dominant inheritance pattern and presents anemia, jaundice and splenomegaly. The purpose of this study was to present our experience and to establish the relationship between hematological and biochemical parameters and osmotic fragility and autohemolysis, all of them considered as traditionally available tests. In our environment, HS is the second most common inherited anemia after beta thalassemia trait. The diagnosis was based on osmotic fragility measurements, autohemolysis test and microspherocytes in 47 patients (45 of latin, 2 of saxon origin); 12 patients had a negative family history. Mean values: RBC (x 10(12)/L) and Hb (g/dL): children (22): 3.84/10.5; adults (25): female (13): 3.54/10.59; masculine (12): 4.65/13.15. Autohemolysis (average

) was very much increased (15.54) and it was corrected by the addition of glucose (4.07). Median osmotic fragility (average g/dL) was increased in both fresh (0.48) and incubated blood (0.65). 76.5

children and 26.7

adults had absence of haptoglobin. Reticulocytes, indirect bilirubin and LDH were increased (average values 336.35 x 10(9)/L, 36.25 mmol/L and 236.48 UI/L, respectively). Microcytosis, spherocytosis, polycromatophylia, acanthocytosis, basophilic stippling, pincered cells were observed in 43, 41, 41, 12, 11, 1 patients respectively. Morphologic alterations were more marked in children. The laboratory findings seemed more a consequence of microcytosis than of spherocytosis.

Evolución del clon de la hemoglobinuria paroxística nocturna en la crisis hemolítica de una anemia aplásica. Estudio por citometría de flujo. / [Evolution of paroxysmal nocturnal hemoglobinuria clone during an hemolytic crisis in a patient with aplastic anemia. Flow cytometry study]

The expansion of paroxysmal nocturnal hemoglobinuria (PHN) clone was evaluated in a patient with aplastic anemia (AA) of 18 years of evolution during an hemolytic crisis. On day 0, Ham and Sucrosa tests were positive and hematological parameters were altered. Low hemoglobin (Hb) levels and erythrocyte and leukocyte counts were found and continued decreasing on days 7 and 24 (last day of study). High LDH levels, indirect bilirubin and reticulocyte counts were detected throughout. We evaluated CD55 and CD59 on erythrocytes by flow cytometry. Our results showed low CD55 expression with respect to the normal pattern. Since day 0, CD59 staining detected two red cell populations: PNH I (48

), cells with positive fluorescence similar to normal and PNH III (52

), negative cells (PNH clone). These negative cells increased, reaching 70

on day 24. Other membrane anchored leukocyte proteins were also absent (CD14) or decreased (CD16). We found a good correlation between clinical observations, evolution of the laboratory values and expansion of the PNH clone.

[Leucocyte tartrate-resistant acid phosphatase as marker for the transition of monocyte to macrophage]. / Fosfatasa ácida leucocitaria tartrato resistente como marcador de la transformación del monocito en macrófago.

The presence of the 5 degrees isoenzyme of leukocyte tratrate-resistant acid phosphatase (FATRE) was investigated in human peripheral blood monocytes in 32 samples: 26 normal, 4 thrombocytopenia, 1 anemia and 1 hairy cell leukemia. The Cobe Spectra Version 4 cell separador was used for 3 samples while the others were obtained by centrifugation with or without latex particles in order to study macrophages and monocytes, respectively. Using a Sigma Kit for both total acid phosphatase and FATRE reactions, the presence of two monocyte populations was detected, one slightly positive and the other negative for FATRE. Upon the addition of latex particles, the monocytes were transformed into intensely FATRE positive macrophages. It can be concluded that FATRE must play an important role in macrophage function and consequently in human cell immunity.

[Hypocholesterolemia in hematologic neoplasms]. / Hipocolesterolemia en neoplasias hematológicas.

PURPOSE: To evaluate the cholesterol metabolism in oncohaematologic patients and its inclusion as a biochemical marker as well as other parameters in the diagnostic period (leukocytes, deshidrogenase lactate, erithrosedimentation, haptoglobine). Many different epidemiological studies discuss the relation between the high risk for cancer mortality and low blood cholesterol in patients with colon, rectum, ovary and brain cancer as well as in chronic myeloid leukaemia, acute leukaemia and policytemia vera. PATIENTS AND METHODS: Two groups were studied: a normal group (n = 32) was confronted to a group of oncohaematologic patients (n = 37) (15 lymphoma, 4 acute leukaemia, 7 chronic myeloid leukaemia, 1 chronic myelomonocytic leukaemia, 3 policytemia vera, 5 myeloma and 2 chronic lymphoid leukaemia). In all the cases we determinated the total cholesterol, HDL cholesterol, LDL cholesterol, A1 y B apolipoproteins. RESULTS: The means obtained in the normal group were 184.63 mg/dL, 50.34 mg/dL, 116.38 mg/dL, 140.93 mg/dL, 79.57 mg/dL while in the pathologic group were 163.43 mg/dL, 38.19 mg/dL, 106.60 mg/dL, 102.81 mg/dL and 101.23 mg/dL, respectively. There was a significant difference in the total cholesterol, HDL cholesterol and apolipoprotein A1. These values were lower for leukocytes higher than 10.0 x 10(9)/L. Furthermore the lowest total cholesterol value was seen in myeloma patients. CONCLUSION: The cholesterol, its fractions and the apolipoproteins determinations might be considered as a biochemical marker while diagnostic is being made, since they are known to play an important role in the tumoral cell metabolism.

[Concurrence of cytochemical and immune patterns of different cell lines in cases of acute leukemia]. / Concurrencia de marcación citoquímica e inmune de diferentes linajes celulares en casos de leucemias agudas.

Mixed, bilineal, biclonal and hybrid leukemias are synonymous, differing from biphenotypical ones. Mixed acute leukemia is defined by the coincidence of 1) two cytochemical markers of different lineage, or 2) one of them with more than one opposite immunological marker, or 3) more than one immunological marker opposite to another immunological lineage. Seven cases of mixed acute leukemia are presented, two of which showed posttreatment switching. It is concluded that mixed acute leukemias are associated with a poor prognosis, and therapeutic criteria are defined.

[HIV-1 infection in patients with hemophilia. The Argentinian experience from 1983 to 1990]. / Infección por HIV-1 en pacientes con hemofilia. La experiencia Argentina desde 1983 hasta 1990.

We present studies on the evolution of HIV-1 infection in 638 hemophilic patients receiving commercial antihemophilic concentrates (CAH) at the Institute of Hematological Research and the Argentine Foundation of Hemophilia between 1983 and 1990. Positive serology for HIV-1 was detected in 30% of the patients studied. Prevalence of HIV-1 infection was higher (about 70%) in the group with severe hemophilia requiring more CAH, but there were no differences between patients with hemophilia A or B. Sexual transmission was demonstrated in 8/64 women (13%) with stable sexual relationship with HIV-1 + hemophilic patients. Three of them became pregnant, and HIV-1 infection was demonstrated in two of the three children. In general, the clinical evolution, as well as the hematologic and immunologic parameters of infected patients were similar to those described for the hemophilic population in other occidental countries. Opportunistic infections were also those observed elsewhere (with predominance of P. carinii pneumonia and disseminated Candida infections). However, the presence of fatal chagasic encephalitis in two of the patients with AIDS is unusual. Thus, central nervous system localization of T. cruzi (which can be observed during the acute period of T. cruzi infection or in immunosuppressed patients), must be considered as a possible severe complication of HIV-1 disease in T. cruzi infected patients.

Infección por HIV-1 en pacientes con hemofilia. La experiencia Argentina desde 1983 hasta 1990. / [HIV-1 infection in patients with hemophilia. The Argentinian experience from 1983 to 1990]

We present studies on the evolution of HIV-1 infection in 638 hemophilic patients receiving commercial antihemophilic concentrates (CAH) at the Institute of Hematological Research and the Argentine Foundation of Hemophilia between 1983 and 1990. Positive serology for HIV-1 was detected in 30

of the patients studied. Prevalence of HIV-1 infection was higher (about 70

) in the group with severe hemophilia requiring more CAH, but there were no differences between patients with hemophilia A or B. Sexual transmission was demonstrated in 8/64 women (13

) with stable sexual relationship with HIV-1 + hemophilic patients. Three of them became pregnant, and HIV-1 infection was demonstrated in two of the three children. In general, the clinical evolution, as well as the hematologic and immunologic parameters of infected patients were similar to those described for the hemophilic population in other occidental countries. Opportunistic infections were also those observed elsewhere (with predominance of P. carinii pneumonia and disseminated Candida infections). However, the presence of fatal chagasic encephalitis in two of the patients with AIDS is unusual. Thus, central nervous system localization of T. cruzi (which can be observed during the acute period of T. cruzi infection or in immunosuppressed patients), must be considered as a possible severe complication of HIV-1 disease in T. cruzi infected patients.

Infección por HIV-1 en pacientes con hemofilia. La experiencia Argentina desde 1983 hasta 1990. / [HIV-1 infection in patients with hemophilia. The Argentinian experience from 1983 to 1990]

We present studies on the evolution of HIV-1 infection in 638 hemophilic patients receiving commercial antihemophilic concentrates (CAH) at the Institute of Hematological Research and the Argentine Foundation of Hemophilia between 1983 and 1990. Positive serology for HIV-1 was detected in 30

of the patients studied. Prevalence of HIV-1 infection was higher (about 70

) in the group with severe hemophilia requiring more CAH, but there were no differences between patients with hemophilia A or B. Sexual transmission was demonstrated in 8/64 women (13

) with stable sexual relationship with HIV-1 + hemophilic patients. Three of them became pregnant, and HIV-1 infection was demonstrated in two of the three children. In general, the clinical evolution, as well as the hematologic and immunologic parameters of infected patients were similar to those described for the hemophilic population in other occidental countries. Opportunistic infections were also those observed elsewhere (with predominance of P. carinii pneumonia and disseminated Candida infections). However, the presence of fatal chagasic encephalitis in two of the patients with AIDS is unusual. Thus, central nervous system localization of T. cruzi (which can be observed during the acute period of T. cruzi infection or in immunosuppressed patients), must be considered as a possible severe complication of HIV-1 disease in T. cruzi infected patients.

Leukoagglutinins in patients with hemophilia.

Anti-leukocyte antibodies may occur in hemophilic patients as a consequence of replacement therapy with blood derivates. In this report we describe the presence of leukoagglutinins (LA) in serum of HIV-infected hemophiliacs (HIV + He) and their absence in HIV-negative patients (HIV-He). LA activity was recovered in IgG fractions from HIV + He, in the polyethylene glycol (PEG) precipitates from these sera, and in some cases in the supernatant fractions of PEG precipitates. Although the amount of PEG precipitates corresponding to circulating immune complexes (CIC) was higher in HIV + He than in HIV-He and normals, there was no direct relationship between CIC levels and LA. LA reacted both with autologous and with allogeneic polymorphonuclear leukocytes (PMN). In contrast to PMN isolated from HIV-He, HIV + He PMN had membrane associated IgG. In HIV + He, LA activity was more frequently observed in patients with more advanced stages of HIV infection than in asymptomatic individuals. Our results suggest that LA activity could be one of the manifestations of autoreactivity associated with progressing HIV infection in patients with hemophilia.

Hemoglobina inestable adquirida / Acquired unstable hemoglobin

Se describe la aparición de una hemoglobina inestable en una paciente de sexo femenino, de 66 años de edad, con diagnóstico de policitemia vera y diabetes tipo II. La primera se rotuló de acuerdo a los criterios del PVSG cumpliendo con los tres criterios mayores (aumento de masa globular, saturación arterial de oxígeno > ou = 92% y esplenomegalia) y dos criterios menores (leucocitosis y score aumentado de la fosfatasa alcalina leucocitaria). A 11 meses de evolución, durante los cuales se trató con flebotomías, aparecieron alteraciones en la morfología eritroide que orientaron al estudio de hemoglobinopatías. Se realizaron entre otras técnicas: electroforesis de hemoglobina, desnaturalización frente al calor, precipitación con isopropanol, tinción para cuerpos de Heinz. Se confirmó la presencia de una probable hemoglobina inestable. Asimismo se evaluó el carácter adquirido de esa hemoglobina haciendo un estudio paralelo a sus dos hijas. Una fue hematológicamente normal y en la otra se diagnosticó ß-talasemia minor, heredada por rama paterna. Por lo tanto, sería importante el seguimiento de síndromes mieloproliferativos através de la morfología eritroide para detectar, por ejemplo, la aparición de hemoglobinas inestables adquiridas

Hemoglobina inestable adquirida / Acquired unstable hemoglobin

Se describe la aparición de una hemoglobina inestable en una paciente de sexo femenino, de 66 años de edad, con diagnóstico de policitemia vera y diabetes tipo II. La primera se rotuló de acuerdo a los criterios del PVSG cumpliendo con los tres criterios mayores (aumento de masa globular, saturación arterial de oxígeno > ou = 92% y esplenomegalia) y dos criterios menores (leucocitosis y score aumentado de la fosfatasa alcalina leucocitaria). A 11 meses de evolución, durante los cuales se trató con flebotomías, aparecieron alteraciones en la morfología eritroide que orientaron al estudio de hemoglobinopatías. Se realizaron entre otras técnicas: electroforesis de hemoglobina, desnaturalización frente al calor, precipitación con isopropanol, tinción para cuerpos de Heinz. Se confirmó la presencia de una probable hemoglobina inestable. Asimismo se evaluó el carácter adquirido de esa hemoglobina haciendo un estudio paralelo a sus dos hijas. Una fue hematológicamente normal y en la otra se diagnosticó ß-talasemia minor, heredada por rama paterna. Por lo tanto, sería importante el seguimiento de síndromes mieloproliferativos através de la morfología eritroide para detectar, por ejemplo, la aparición de hemoglobinas inestables adquiridas (AU)