Variations of ochratoxin A concentration in the blood of healthy populations in some Croatian cities.

The nephrotoxic mycotoxin ochratoxin A (OTA), a common contaminant of cereals, has been implicated in the etiology of endemic nephropathy. It was also frequently found in low concentrations in blood of healthy populations in countries where endemic nephropathy is not known. However, data on regional and seasonal differences in the frequency and concentration of OTA in human blood are scarce. In June, September and December 1997, and March 1998, about 50 human blood samples were collected randomly from blood donors for blood banks in the Coatian cities of Osijek, Rijeka, Split, VaraZdin and Zagreb. OTA was measured in the total of 983 samples using an HPLC technique with fluorescent detection. The daily intake of OTA was estimated from the mean concentration found in different cities and at different times of year. Samples containing OTA above the detection limit (0.2 ng/ml of plasma) were found in populations from all Croatian cities at all collecting periods. The highest frequency (59%) of samples containing OTA above the detection limit and the highest mean concentration (0.39 ng/ml) were found in June. Both the frequency and the mean concentration were lowest in all samples in December (36% and 0.19 ng OTA/ml, respectively). Osijek was the city with the highest frequency of OTA-positive samples (81%) and the highest mean OTA concentration (0.56 ng/ml). The total mean concentration of OTA in blood of healthy population in Croatia is lower (0.30 ng/ ml) than the mean concentration in European countries as a whole (0.90 ng/ml). The estimated daily intake, calculated from the mean concentration in all blood samples, is 0.40 ng OTA/kg body weight, which is much lower than that proposed by World Health Organization as the tolerable daily intake (16.0 ng/kg body weight). Healthy populations of Croatia are exposed to low, but seasonally and regionally variable amounts of OTA.

[Neonatal alloimmune thrombocytopenic purpura caused by anti-HPA-1a alloantibodies. Case report]. / Neonatalna aloimuna trombocitopenijska purpura (NATP) uzrokovana anti-HPA-1a aloprotutijelima. Prikaz bolesnika.

We report the case of serologically proven HPA-1a NATP. The child was born after uneventful 4th pregnancy. Immediately after birth generalized petechiae and signs of gastrointestinal bleeding were present. Isolated thrombocytopenia with the platelet number of 29 x 10(9)/L was observed. Serological investigation (PSIFT and MAIPA) showed high titre anti-HPA-1a antibody and low titre anti-HLA antibody in mother's sera. Mother's platelets were HPA-1a negative and she was HLA DR 52 positive. Father's platelets were HPA-1a positive. Cross-match between mother's sera and father's platelets was positive. 24 hours after the introduction of corticosteroid therapy platelet number increased to 73 x 10(9)/L and 48 hours later to 155 x 10(9)/L. The child was treated by corticosteroids because the NATP was severe and antigen negative platelets (mother or donor) or IVGG were not available. According to data from the literature the efficiency of corticosteroid therapy in NATP is questionable, but in this case it provided sufficient increase of platelet number with the stop of newborn bleeding.

Transfusion medicine in the new millennium.

Blood is a tissue like all other tissues, however, it has always had a special meaning for man. Blood used to be attributed characteristics of the man whose body it was circulating through. Attempts were made to influence the person's character, properties, or disease, such as strength, old age and illness, by the bloodletting or blood administration. Blood was for centuries considered an elixir of life, and was ascribed various meanings at different times, e.g., mystic, religious, symbolic, racial, patriotic, biological, medical, scientific, industrial, and economic. Nowadays, the meaning of blood is most pronounced in the science, medicine, and economy.

Dynamics of serum hepatitis C virus load and quasispecies complexity during antiviral therapy in patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) infection is a dynamic process during which viral genetic variants continuously develop as a result of the virus adaptation to the host's immune system. The level of viremia and the complexity of the hypervariable region 1 (HVR 1) quasispecies of hepatitis C virus during antiviral therapy reflect the dynamic balance between the viral and host components in response to therapy. OBJECTIVE: The aim of the study was to evaluate the dynamics of HCV viremia and the complexity of the HVR 1 quasispecies during the induction phase of a triple combination therapy regimen in nonresponders to earlier anti-HCV treatment. STUDY DESIGN: Ten patients with chronic hepatitis C undergoing antiviral combination therapy with interferon-alpha, ribavirin, and amantadine were studied. The serum HCV RNA level was monitored by a quantitative RT-PCR assay up to 3 months after start of treatment. The HVR 1 quasispecies complexity was analysed by an "in house" nested RT-PCR mediated single-strand conformation polymorphism (SSCP) assay. RESULTS: Baseline serum HCV RNA levels ranged from 1.94x10(6) to 5.53x10(6) copies/ml. In all patients, HCV subtype 1b was found. At the start of therapy, the SSCP assay revealed a high complexity pattern (at least six SSCP bands) in all patients. None of the patients responded within 4 weeks of treatment, however, the serum HCV RNA level decreased by one to two logs in eight patients. At week 4 after start of treatment, there was a decrease of SSCP bands in five patients. In four patients, SSCP bands remained unchanged and in one patient SSCP bands increased. At month 3 after start of treatment, serum HCV RNA was not detectable in one patient. CONCLUSION: Because of the low number of patients involved in this study, prediction of therapeutical success based on the quasispecies complexity was not possible. Larger studies are urgently needed.

Clinically significant red cell alloantibodies in patients with warm autoimmune hemolytic anemia.

It has been generally accepted that the prevalence of clinically significant red cell alloantibodies is higher in patients with warm autoimmune hemolytic anemia (AIHA) than in other patients. In the present study, immunohematologic testing was performed in 328 polytransfusion patients with internal diseases. The patients were divided into two groups, i.e., without and with clinical signs of warm AIHA. Identification of red cell antibodies was performed in nonadsorbed sera of all patients, and in autoadsorbed and auto- and alloabsorbed sera of patients with warm AIHA. In the AIHA group, antibodies indicating red cell specificity were detected in 38.5% and 24.3% of patients in nonadsorbed and autoadsorbed sera, respectively. Clinically significant red cell alloantibodies were demonstrated in 10.3% nonadsorbed sera of patients free from signs of warm AIHA and in 10.4% auto- and alloadsorbed sera of patients with signs of warm AIHA. Study results showed the prevalence of clinically significant red cell alloantibodies in patients with signs of warm AIHA depend on the method of identification used rather than on the enhanced immune response induced by the autoimmune process.

Hypervariable region 1 of hepatitis C virus genome and response to interferon therapy.

The relationship between the complexity of the hypervariable region 1 (HVR1) quasispecies of hepatitis C virus (HCV) and responsiveness to interferon-alpha (IFN) therapy was studied in patients with chronic hepatitis C. Twelve HCV-RNA-positive patients were treated daily with high dose IFN and ribavirin for 4 weeks, and then with IFN 3 MIU (Million International Units) TIW (three times per week) and ribavirin for 6 months. The HVR1 quasispecies complexity was analyzed by nested polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP). The baseline HCV-RNA levels in the study group ranged from 10(6) to 10(7) copies/ml. All patients exhibited HCV genotype 1 b. Initial SSCP analysis revealed four (33.3%) patients with a low complexity pattern (SSCP bands < or =4) and eight (66.6%) patients with high complexity pattern (SSCP bands >4). After 4 weeks of IFN therapy, one patient became HCV negative, and among those remaining positive, the HCV-RNA levels decreased by 2 to 3 logs and the number of SSCP decreased by 2 to 3 bands per sample. After 6 months of IFN therapy, five (41.7%) patients became HCV-RNA-negative. Seven (58.3%) patients did not respond to IFN therapy with sustained viral load from 10(3) to 10(5) copies/ml, and high complexity SSCP patterns. Our data support the HVR quasispecies complexity to be an independent predictive factor for IFN responsiveness in patients infected with HCV.

Decreasing risk of viral transfusion-transmitted diseases in Croatia.

AIM: To assess the risk of viral transfusion-transmitted infections in Croatia. METHODS: The following parameters were analyzed: frequency of blood donations repeatedly reactive for HBsAg and anti-HCV (1993-1999); blood donations confirmed positive for HBsAg and anti-HCV (1997-1999), anti-HIV1/2, and syphilis reactivity (1993-1999); number of registered patients with hepatitis B and C; transfusion-associated hepatitis B and hepatitis C; and frequency of HBV, HCV and HIV markers in patients with congenital bleeding disorders (1993-1998). RESULTS: The frequency of repeatedly reactive HBsAg and anti HCV markers and confirmed positive HBsAg, anti-HCV, and syphilis markers in donors blood decreased during the study, whereas the frequency of anti-HIV1/2 positivity did not change. The frequency of confirmed positive donors in 1999 was 0.068% for HBsAg, 0.035% for anti HCV, 0.002% for anti HIV1/2, and 0.0056% for syphilis. The number of patients with hepatitis B, hepatitis C, and transfusion-associated hepatitis B and C steadily decreased during the 1993-1998 period. The number of transfusion-associated hepatitis patients leveled off in 1997. From the beginning of the follow-up of AIDS patients in 1987, only 7 (2%) of hemophiliacs have been HIV-infected, all before 1990 and due to non-inactivated coagulation factor concentrates. There were no cases of transfusion-associated HIV2 infection in patients with congenital bleeding disorders or transfusion-associated HIV1 infection through transfusion of labile blood components. CONCLUSION: The safety of transfusion therapy in Croatia has improved, and the present risks of viral transfusion transmitted diseases are very low.

Ochratoxin A in blood of healthy population in Zagreb.

Healthy blood donors from the city of Zagreb were checked for the presence of a nephrotoxic mycotoxin ochratoxin A (OTA) in the plasma. Samples of blood were collected in June, September, and December 1997, and March 1998, totalling 200 or 50 in each round. The concentrations of OTA were measured using high pressure liquid chromatography (HPLC) method (detection limit 0.2 ng OTA/ml of plasma). The frequency of OTA-positive samples (> 0.2 ng/ml of plasma) showed significant seasonal variation (P < 0.001). The frequency of OTA-positive samples was the highest in March (65%) and it gradually decreased towards December (12%). The high frequency of positive samples coincided with seasons favouring growth of moulds and production of toxins. The daily intake of OTA by healthy persons in Zagreb was estimated from the mean concentration of OTA in samples collected during the whole year (0.19 ng OTA/ml plasma). The estimated daily intake was 0.26 ng/kg b.w., that is, substantially below the tolerable daily intake proposed by World Health Organization (16.0 ng/kg b.w.).

Evaluation of the enzyme test for the detection of clinically significant red blood cell antibodies during pregnancy.

In the Croatian transfusion medicine, no general agreement has yet been achieved whether red blood cell (RBC) Rhesus (Rh) antibodies detected during pregnancy only by enzyme tests can cause hemolytic disease of the newborn (HDN). Results of the detection of clinically significant RBC antibodies by low-ionic-strength additive solution antiglobulin test (LISS-IAT) and trypsin enzyme test in 22,947 pregnant women are presented. All pregnant women in whom clinically significant RBC antibodies (RBC-CSA) were detected by LISS-IAT and/or enzyme tests were followed and observed during pregnancy. The women who had enzyme-only anti-D antibodies in their serum were followed up during subsequent pregnancies. Out of 302 positive results obtained by both techniques, irregular clinically significant enzyme-only antibodies (anti-RhD and anti-RhE specificity) were detected in 14 (4.6%) pregnant women. None of 11 RhD positive newborns whose mothers had enzyme-only anti-D antibodies, had signs of HDN after delivery. In these 11 women, anti-D antibodies were detected by LISS-IAT in the first trimenon of subsequent pregnancy. Nine infants born from subsequent pregnancies to women who had previously had enzyme-only anti-D, had clinical signs of HDN. The authors concluded that there is no need for enzyme tests in prenatal testing because enzyme tests are not reliable in the prediction of HDN.

A perception of transfusion safety and risks.

Transfusion therapy is today safer than ever. However, administration of homologous transfusions still has a number of different, even serious potential hazards. Therefore, transfusion therapy should only be given when the reasons for it are clear, its benefits outweigh the hazards, and the patient has accepted them after being well informed about them.

Croatian blood transfusion service in prevention of HIV spread during the war.

Beside the war, an unfavorable epidemiological situation and a large number of foreign peace troops that entered the country without having been previously tested for infectious diseases, the number of AIDS cases in our country remained relatively low. The transfusion service played a considerable part in the prevention of HIV infection spread. Although the blood transfusion service was faced with higher demands for blood and blood products, throughout the period of the war not a single blood unit was imported and no single unit of blood components was transfused without having been previously tested for the presence of viral disease markers. Despite enormous economic difficulties, three new diagnostic tests were then introduced in our transfusion practice as a regular procedure: anti-HCV in 1993, anti-HIV 2 in 1994 and anti-HIV 1/0 in 1995.

[What we can learn from refusal of transfusion therapy from experience with Jehovah's Witnesses]. / Sto mozemo nauciti o odbijanju transfuzijskog lijecenja iz iskustva s Jehovinim svjedocima.

Patients who refuse blood transfusion for personal or religious reasons present complex medical, legal and moral problems. Blood transfusion has been doctrinally forbidden for Jehovah's Witnesses since 1945. Their refusal is based on the strict interpretation of several Biblical passages. A clear understanding of the philosophy of the Jehovah's Witnesses regarding blood transfusion and of the medicolegal and ethical aspects of their care is essential to clinicians who care for such patients. Various ethical principles, including the patient's autonomy, the interest of society in preserving life and the dignity of medical profession can be confronted. There are no clear guidelines which physicians can follow in deciding to treat or not treat in the presence of a patient's refusal. However, most authors agree that a competent adult has an absolute right to decline medical treatment, and that it is not morally or ethically correct to force patient to an unwanted treatment. We wished to present the experiences with the use of alternative methods in the treatment of Jehovah's Witnesses and to discuss ethical and legal aspects of treatment decisions in the presence of blood transfusion refusal.

[The effect of Rh-D immunoprophylaxis with hyperimmune anti-D-immunoglobulin on the occurrence of RhD immunization in pregnancy]. / Utjecaj provodenja RhD-imunoprofilakse hiperimunim anti-D-imunoglobulinom na ucestalost RhD-imunizacija trudnica.

During prenatal immunohaematological examination in the period from January 1, 1991 to December 31, 1995, in the Croatian Institute of Transfusion Medicine we tested sera of 5107 RhD negative women. All of them had pregnancies in their medical history. The frequency of Rh immunization was 4.6% in 1991; 4.1% in 1992; 2.5% in 1993; 2.5% in 1994 and 2.4% in 1995. Rh immunization during the first pregnancy was observed in 0.46% of women, in 1.8% during the second, in 9.4% during the third, in 22.4% during the fourth pregnancy, and 33.8% in women with more than five pregnancies. In women that have no abortions in their medical history, anti-D alloantibodies were found with the frequency of 0.46% at the end of the first pregnancy, 1.2% at the end of the second pregnancy, 5.9% at the end of the third pregnancy, 14.3% at the end of the fourth pregnancy, and 15.3% in women with more than five pregnancies. The frequency of anti-D alloantibodies in women who in their medical history have only abortions is 3.4% after the first abortion, 10.5% after the second, 17.8% after the third and 20.8% after the fourth or more abortions. The frequency of antibodies of anti-D specificities in women who had abortions and births is 17.1% at the end of the third pregnancy, 26.2% at the end of the fourth pregnancy, and 42.7% after more than five pregnancies. The frequency of anti-D alloantibodies in women who were protected from Rh immunization by hyperimmune anti-D globulin is 1%. The obtained results demonstrate that prevention of Rh immunization by hyperimmune anti-D globulin does not comprise all the Rh negative women, and is especially inadequate after abortions and multiple pregnancies.

[Pseudothrombocytopenia caused by ethylenediaminetetraacetic acid (EDTA) (case report)]. / Pseudotrombocitopenija uzrokovana etilendiamintetraoctenom kiselinom (EDTA) (prikaz bolesnice).

Pseudothrombocytopenia is a laboratory artefact that can introduce serious problems in diagnosis and treatment in patients with low platelet count. The most common reason for this artefact is in vitro platelet clumping in blood samples collected into ethilenediaminetetraacetic acid (EDTA) anticoagulant. The clumping activity is greater at temperatures less than 37 degrees C, and the EDTA concentrations required for clumping are 20 times below anticoagulant concentrations. In this article we described the case of a female patient with diagnosed EDTA induced pseudothrombocytopenia. The cause of incorrectly low platelet counts was proved by simultaneous analysis in blood samples collected into EDTA anticoagulant and into heparin as a control sample. Absences of incorrectly low platelet count in heparin sample and rapid decrease of platelet count in EDTA sample were noticed. Decrease in platelet count was accompanied by increase in the number of leukocytes, so called pseudoleukocytosis. Careful examination of blood film is necessary to establish correct diagnosis, promptly after the blood collection and approximately two hours later. It is important to verify formation of clumps two hours after the blood collection and also progressive reduction in the platelet count in EDTA sample. By blood assessment conducted in this concern it is possible to avoid severe misinterpretation in such patients.

[Case report of a female patient with autoimmune hemolytic anemia and cold agglutinin antibodies]. / Prikaz bolesnice s autoimunom hemolitickom anemijom s hladnim protutijelima.

We report a case of acute transient cold agglutinin disease, the etiology of which we could not determine with the available methods. Cold autoagglutinins had anti I specificity, high titers of the autoantibody (> 1:1,000) and the thermal range was relative wide. Our patient had severe haemolysis and immunosuppressive therapy with methylprednisolone and cyclophosphamide was administered. It is a question how much these immunosuppresive agents influenced the recovery, and in what extent it was a self limited disease with spontaneous recovery.

[Evaluation of the direct antiglobulin test]. / Evaluacija direktnog antiglobulinskog testa.

The validity of a direct antiglobulin test (DAT) was evaluated by testing 7645 blood samples from hospitalized patients. DAT was routinely done in 7201 blood samples sent for pretransfusion testing and 444 blood samples specifically sent for the examination of immune hemolysis. Positive DAT was discovered in 0.04% (3/7201) pretransfusion samples and in 3.83% (17/444) samples examined for immune hemolysis. In 16 of the samples with positive DAT, IgG antibodies with or without complements and in 4 samples only components of complements were detected on RBC. The cost of positive DAT in pretransfusion testing is 92 times higher than that of DAT during laboratory investigation of immune hemolysis. Due to a low frequency of positive DAT during pretransfusion testing, its cast and the fact that patients had no clinical signs of immune hemolysis, we advocate no use of a routine DAT during pretransfusion testing.