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Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease associated with hemolytic anemia, bone marrow failure, thrombosis, and, frequently, poor quality of life. It is caused by defects in the membrane of blood cells, where there is a lack of protein on the cell surface, which inhibits complement activation. We wanted to know the recognition of the disease in Slovenia and the incidence. We prepared the recommendations for discovering of the disease. Patients and methods: We collected data of 68 patients with prospective analysis of one – year period from 1.10.2013 to 30.9.2014 whose blood was sent to the laboratory of immunology and cytology because of suspected presence of PNH clone. The analysis of peripheral blood was performed with multiparametric high specific flow cytometry in a specialized laboratory KO of Haematology University Medical Centre in Ljubljana. Results: PNH clone was positive in 13/68 (19%) patients, 55/68 (81%) patients had a negative PNH clone, most positive samples were sent from the University Medical Centre Ljubljana (7/13). 4/13 positive patients were newly discovered. In average the incidence through 10-years was 1,3 / 1,000,000 population/year. The most common cause of PNH patient referral to a specialist hematologist was unexplained cytopenia - 12/13 (92.3%), the most common symptoms were fatigue and dyspnea (100%), in 2/13 patients was present dark urine with hemoglobinuria, 2/13 patients had transient renal insufficiency. 11/13 patients with positive PNH clone had associated a bone marrow failure (aplastic anemia or myelodysplastic syndrome). The size of PNH clone varied from patient to patient. Conclusions: Early identification of PNH is a key to effective treatment and survival of patients. We recommend determining PNH clone at Coombs negative hemolytic anemia, hemoglobinuria, an unexplained cytopenia, aplastic anemia, myelodysplastic syndrome with laboratory evidence of haemolysis and unexplained thrombosis.

Background. Essential thrombocythemia (ET) is a chronic myeloproliferative disease with a platelet count within the range of 400–2000 × 109/L. Higher percentage of platelets in the circulation of patients with ET express also activation markers on their membranes. Two of such markers are P-selectin and activated fibrinogen on platelet membranes. Because of frequent thrombembolic and also bleeding related complications, treatment of ET is mandatory. Patients whose platelet count is less than 1000 × 109/L and who did not suffer any thrombembolic complication during the course of the disease, are ussually treated with an antiaggregation drug, acetylsalicylic acid 100 mg/daily orally. Clopidogrel is an adenosyn-di-phosphate (ADP) receptor antagonist in platelets. There is no routine clinical data about clopidogrel treatment in the patients with ET and only sporadic case reports can be find in the literature.Patients and methods. In our clinical study we compared antiaggregational effects of acetylsalicylic acid and clopidogrel, by measuring the P-selectin level and activated fibrinogen expression on platelet membranes.There were 35 ET patients included, within the age range between 21 and 78 years and with platelet counts within 451–952 × 109/L. None of the patients did suffer any thrombembolic complication during the course of the disease. During the sequential 14 day periods, patients received acetylsalicylic acid 100 mg/daily orally, followed by clopidogrel 75 mg/daily orally and ultimativelly, together acetylsalicylic acid 100 mg/daily orally plus clopidogrel 75 mg/daily orally. After each fourteen days period the level of P-selectin and activated fibrinogen activated platelets were determined with monoclonal antibodies on flow cytometer. Statistical evaluation was calculated on the difference of average values between the two small, independent pair groups with the t-test.Results. When the patients stopped with acetylsalicylic acid and began with clopidogrel, the percentage of P-selectin exposure on platelet membranes was statistically significant lower (t-value 1.99; p < 0.05). We also noticed statistically significant lower P-selectin exposure in the period, during which patients received acetylsalicylic acid plus clopidogrel, versus the period, during which they received acetylsalicylic acid only (t-value 2.11; p < 0.05). But there was no statistically significant difference in P-selectin exposure within the periods, during which patients received clopidogrel versus acetylsalicylic acid plus clopidogrel (t-value 0.19; p > 0.05). Percentages of activated fibrinogen exposures on platelet membranes were in all clinical study periods comparable and no statistically significant differences between them were noticed (for each pair-relation p > 0.05).Conclusions. In patients with ET clopidogrel statistically significant (p < 0.05) lowers the percentage of P-selectin exposure on platelet membranes. Acetylsalicylic acid has no influence on P-selectin expression and also the combination of acetylsalicylic acid and clopidogrel, does not have higher P-selectin lowering efficacy, than clopidogrel itself. Acetylsalicylic acid and clopidogrel alone, nor in combination don’t have any influence on the expression level of activated fibrinogen on platelet membranes. To conclude, that clopidogrel may be an alternative to acetylsalicylic acid in patients with ET, who can not tolerate acetysalicylic acid because of it’s side-effects.

Background. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal haematopoietic stem cell disorder characterised by intravascular haemolysis, bone marrow failure and increased tendency to thrombosis. It is caused by a somatic mutation in the PIG-A gene, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIG-A mutation results in a clone of blood cells with total or partial deficiency of membrane proteins anchored to the cell surface through GPI anchor. For many years, an increased susceptibility of the PNH red cells to complement lysis in acidified serum (Ham’s test) has been essential for diagnosis of the PNH. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of the specific GPI anchor proteins on blood cells. We evaluated a three-colour flow cytometry method for detection and quantification of CD55/59 negative netrophils.Patients and methods. Nine patients (6 with PNH and 3 with aplastic anaemia) who were previously diagnosed as having PNH or aplastic anaemia were evaluated. In the period of three years the patients were serially evaluated for the extent of PNH clone by quantification of CD55/59 negative neutrophils. Three-colour flow cytometry method was used for quantification of the CD55/CD59 negative neutrophils. We used directly conjugated monoclonal antibodies anti-DC15PC5 for identification of neutrophils and anti-CD59-FITC and anti-CD55-PE for the GPI-linked antigens.Results. Patients with haemolytic PNH had > 50% CD59/CD55 negative granulocytes. The proportion of the PNH granulocytes was higher in the patient with frequent and serious haemolytic attacks. Over the period of three years slow growth of the PNH clone was seen in two cases. Two patients with PNH diagnosed 19 years ago and in remission at the time of flow cytometric analysis was devoid of the PNH clone. Three patients with aplastic anaemia (hypoplastic PNH) had the proportion of CD59/CD55 negative granulocytes < 40%. In one of them PNH clone increased.Conclusions. Three colour flow cytometry of granulocytes using combination of anti-CD15/55/59 provides the accurate technique for detection and quantification of the PNH clone. Monitoring the PNH clone size has clinical and prognostic value. The size of the PNH clone is an important determinant for thrombotic risk. Serial studies allow prediction of remission in some cases or progress from hypoplastic to hemolytic PNH in others.

Background. ALL is a malignant blood disease and lymphoblasts have origin in B or T lymphatic cell line. In 1997 established new World Health Organisation classification (WHO classification) of malignant haematological diseases realizes the importance of cellular immunological markers (immunophenotype) and chromosomal abnormalities (cytogenetics). Based on both findings we may distribute the patients in low, intermediate and high risk groups and the outcome of such distribution is risk adopted ALL treatment strategy. On Clinical department of haematology (CDH) we have decided to overview immunophenotype characteristics of all ALL patients during the January 1, 1995–December 31, 2001 period.Methods and results. During the January 1, 1995–December 31, 2001 period on CDH we have treated 44 patients: 22 males and 22 females. With flow cytometer Coulter Epics XL MCS we have performed first primary antibody panel for acute leukemias antigens (CD2, CD7, CD10, CD19, CD34, cCD3, cCD13, cCD22, MPO, TdT), followed by secondary panel. The later have included antigen CD20, CD23, membrane and/or cytoplasmatic immunoglobulins or their light chains monoclonal antibodies for B-ALL and antigen CD3, CD4, CD5 and CD8 monoclonal antibodies for T-ALL subsets. Besides immunophenotyping we have evaluated all ALL patients also morphologically according to FAB classification (French-AmericanBritish classification), which is an old classification based solely on morphology.32/44 (73%) patients have had immunophenotypic B-ALL, and 12/44 (27%) T-ALL. Subgroups distribution of B-ALL immunophenotype were: pro-B 4/44 (9%), »common«-B 18/44 (41%), pre-B 5/44 (11.5%) and mature B 5744 (11.5%) and for T-ALL immunophenotype were: pro-T 2/44 (4.5%), pre-T 5/44 (11.5%), cortical-T 4/44 (9%) and mature-T 1/44 (2%). Our results are quite comparable with the available data from the literature, despite the fact that newest immunological markers such as CD1a, CD79a and CD22 were not available until last year.ALL morphology does not determine ALL immunophenotype, with the exception of L3 subtype, which usually coincidences with mature B immunophenotype. The commonest morphological variant is L2 subtype, which we have seen in 35/44 (80%) patients and was equally distributed among B and T immunophenotypic subgroups.Conclusions. Determination of ALL immunophenotype is one of the cruical tests for the evaluation of long-term ALL prognosis. Because of the later, every newly diagnosed ALL must be immunophenotyped. Immunophenotype is not the only ALL prognostic factor, but in combination with the others such as cytogenetics, patients age, leukocyte count at diagnosis, we can put together »ALL prognostic mosaic«, followed by an optimal ALL treatment strategy.