Sažetak: The outbreak of thrombotic diathesis in younger people requires additional diagnostic ex... more Sažetak: The outbreak of thrombotic diathesis in younger people requires additional diagnostic examination, including genetic testing for possible inherited factors. Incidence of the G20210A mutation in the gene for prothrombin is such patients, as well as frequency ...
Journal of clinical chemistry and clinical biochemistry. Zeitschrift für klinische Chemie und klinische Biochemie, 1987
Blood sampling on filter paper was tested for determination of glycated haemoglobin. The method s... more Blood sampling on filter paper was tested for determination of glycated haemoglobin. The method showed coefficients of variation of 3.4% and 4.1%, and linearity coefficients of 0.978 and 0.91 for the microchromatographic and colorimetric methods respectively. A blood sample on filter paper impregnated with 5% ethylene glycol solution remains stable for 8 days at room temperature. In a group of 30 diabetics educated in the filter paper blood sampling technique, no statistically significant differences were registered among the mean values of their blood glucose profiles, glycated haemoglobin levels determined at the hospital and those obtained at home 14 days after discharge. In conclusion, the use of blood spotted on filter paper seems a cheap and convenient method for collecting, storing and transporting samples for analysis of glycated haemoglobins. It is also a useful alternative for home monitoring of diabetics. Moreover, it could also be useful in epidemiological studies of dia...
Despite the identification of a wide range of inherited and acquired risk factors for arterial is... more Despite the identification of a wide range of inherited and acquired risk factors for arterial ischemic stroke (AIS) in children, genetic risk factors are incompletely characterized and may vary among different populations. We investigated the role of individual and combined inherited prothrombotic and intermediate-risk factors in 73 children with perinatal (n = 35) and childhood AIS (n = 38) and 100 age- and sex-matched controls. Ten polymorphisms in 8 candidate genes encoding coagulation and fibrinolytic proteins (factor V [FV] Leiden, FV HR2, factor II [FII] G20210A, β-fibrinogen [β-FBG]-455G>A, factor XIII [FXIII]-A p.Val34Leu, plasminogen activator inhibitor 1 4G/5G), homocysteine metabolism (methylenetetrahydrofolate reductase [MTHFR] C677T, MTHFR A1298C), and intermediate-risk factors (angiotensin-converting enzyme I/D, apoE ∊2-4) were detected using a multilocus genotyping assay. Allele-specific polymerase chain reaction was used for the determination of human platelet alloantigens (HPA-1, HPA-2, HPA-3, and HPA-5). Factor V Leiden was associated with an increased risk of AIS (odds ratio [OR]: 4.72, 95% confidence interval [CI]: 1.22-18.27) and perinatal AIS (OR: 8.29, 95% CI: 1.95-35.24). Human platelet antigen-3b allele carriers had a 2-fold lower risk of AIS (OR: 0.51, 95% CI: 0.26-0.98) and perinatal AIS (OR: 0.40, 95% CI: 0.18-0.92). A 2.21-fold increased risk of childhood AIS (95% CI: 1.03-4.73) was identified in FXIII-A Leu34 allele carriers. Combined FV Leiden/FV HR2, FV Leiden/MTHFR A1298C, FV Leiden/MTHFR C677T/MTHFR A1298C, and FV Leiden/FV HR2/MTHFR A1298C heterozygosity was identified in children with AIS but not in controls, which revealed a statistically significant difference. This case-control study shows that besides already documented association between FV Leiden and AIS, other previously unreported polymorphisms (FXIII-A p.Val34Leu, HPA-3) and several genotype combinations that always include heterozygous FV Leiden can be related to AIS in Croatian population.
Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardize... more Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently utilize a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that's traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR, and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample in...
European journal of medical research, Jan 21, 2003
The main consequences of human immunodeficiency virus (HIV) infection and AIDS are frequent and p... more The main consequences of human immunodeficiency virus (HIV) infection and AIDS are frequent and persistent opportunistic infections at mucosal surfaces, but data upon impaired oral mucosal response in AIDS patients are still lacking. - The aim of this study was to determine salivary flow rates and peroxidase levels in unstimulated whole saliva in AIDS patients together with comparison to the healthy controls. Salivary peroxidase levels were determined according to Putter and Becker in 20 AIDS patients and 18 HIV-seronegative healthy controls. Statistical analysis was performed using Student t-test. Salivary peroxidase levels were significantly increased in the AIDS group (9.41 +/- 8.50 kU/L; p<0.009) when compared to the healthy controls (3.1 +/- 2.0 kU/L). Salivary flow rates were significantly decreased in AIDS patients (0.17+/-0.11 ml/min, p<0.009) when compared with healthy controls (0.58 +/- 0.19 ml/min). Elevated salivary peroxidase levels indicate increased salivary ant...
Haemophilia : the official journal of the World Federation of Hemophilia, 2014
Diagnosis of haemophilia A is usually made by the measurement of factor VIII (FVIII) activity tha... more Diagnosis of haemophilia A is usually made by the measurement of factor VIII (FVIII) activity that allows categorization of the disease severity. However, tests that assess global haemostasis may better reflect clinical features and give additional clinically relevant information. The aim of this study was to develop a new quantitative activated partial thromboplastin time (aPTT) waveform analysis and compare it with FVIII activities to find out whether waveform parameters are superior determinants of clinical phenotype. A total of 81 haemophilia A patients divided into two groups (37 severe, 44 non-severe) were included in the study. The control group comprised 101 healthy male volunteers. Quantitative aPTT waveform analysis was performed with Actin FS on BCS (Siemens Healthcare Diagnostics, Marburg, Germany) using three parameters (DELTA, RATIO-1, RATIO-2) obtained from a single aPTT measurement with two evaluation modes. FVIII activities were measured by one-stage clotting and tw...
European journal of medical research, Jan 28, 2009
The occurrence of acute promyelocytic leukemia (APL) in HIV-infected patients has been reported i... more The occurrence of acute promyelocytic leukemia (APL) in HIV-infected patients has been reported in only five cases. Due to a very small number of reported HIV/APL patients who have been treated with different therapies with the variable outcome, the prognosis of APL in the setting of the HIV-infection is unclear. Here, we report a case of an HIV-patient who developed APL and upon treatment entered a complete remission. A 25-years old male patient was diagnosed with HIV-infection in 1996, but remained untreated. In 2004, the patient was diagnosed with primary central nervous system lymphoma. We treated the patient with antiretroviral therapy and whole-brain irradiation, resulting in complete remission of the lymphoma. In 2006, prompted by a sudden neutropenia, we carried out a set of diagnostic procedures, revealing APL. Induction therapy consisted of standard treatment with all-trans-retinoic-acid (ATRA) and idarubicin. Subsequent cytological and molecular ana?lysis of bone marrow d...
European journal of medical research, Jan 28, 2001
The aim of our investigation was to evaluate possible connection between burning mouth syndrome a... more The aim of our investigation was to evaluate possible connection between burning mouth syndrome and hematinic deficiencies, a hypothesis previously reported in the literature with contradictory results. Serum levels of iron, vitamin B12, folic acid, calcium and magnesium were determined in 41 (aged 31-87 years, mean 68,7 yrs) patients with burning mouth syndrome and 35 matched controls (35-83, mean 63 yrs). Serum iron levels were determined according to Fairbanks and Klee. Levels of vitamin B12 and folic acid were determined on commercially available kits (Imx12 and Imx folate assay, Abbot Park lab, IL, USA) on Imx analyser. Calcium and magnesium levels were determined using atomic absorption spectrophotometry. No statistically significant differences in serum levels of iron, folic acid, calcium and magnesium were found between patients with burning mouth syndrome and controls. Statistically significant lowered vitamin B12 levels were found in patients with burning mouth syndrome. O...
To determine the prevalences of factor V Leiden and the G20210A mutation in the prothrombin gene ... more To determine the prevalences of factor V Leiden and the G20210A mutation in the prothrombin gene (PT20210A) and the frequency of their association in healthy subjects and in patients with venous thromboembolism (VTE). We studied 160 Croatian patients with at least one episode of VTE and 155 healthy subjects as a control group. Genomic DNA was extracted according to standard procedures and the presence of factor V Leiden and PT20210A were determined by polymerase chain reaction-restriction fragment length polymorphism method. The prevalences of factor V Leiden and PT20210A were in VTE patients 21% and 8% respectively, and 4% in controls for both mutations. Additionally, 4 patients were affected by double heterozygous defects, corresponding to a frequency of 3%, whereas none of the controls were double heterozygotes. The coexistence of the PT20210A in heterozygous carriers of factor V Leiden was 15% in VTE group. The results obtained for different subgroups of VTE patients showed that...
International Journal of Oral-Medical Sciences, 2004
Oral lichen planus (OLP) is a chronic inflammatory disease whose etiology has not yet been identi... more Oral lichen planus (OLP) is a chronic inflammatory disease whose etiology has not yet been identified. It is characterizied by immunoreactivity directed against basal keratinocytes and mediated by T-lymphocytes. In health, salivary peroxidase together with other salivary antimicrobial enzymes maintains the ecological balance in the oral cavity.
ABSTRACT Background: Despite extensive investigations and identification of a wide range of inher... more ABSTRACT Background: Despite extensive investigations and identification of a wide range of inherited and acquired risk factors for childhood stroke in recent years, genetic risk factors for this disease are still incompletely characterized. Taking into consideration that the frequency of genetic factors may vary among different populations, the aim of this study was to investigate the possible role of individual inherited prothrombotic and intermediate risk factors in Croatian children with a confirmed diagnosis of arterial ischemic stroke (AIS) and to check the possible association of investigated risk factors with the disease. Methods: A genotype analysis of 14 polymorphisms in 12 candidate genes encoding proteins of the coagulation and fibrinolysis systems (FV Leiden, FV HR2, FII G20210A, b-FBG −455G&amp;gt;A, FXIII-A Val34Leu, PAI-1 4G/5G), human platelet alloantigens (HPA-1, -2, -3 and -5), homocysteine metabolism (MTHFR C677T, MTHFR A1298C) and intermediate risk factors (ACE I/D and apoE e2–4) was performed in 73 children (48 boys and 25 girls) with AIS aged ≤18 years and compared with 100 age- and sex-matched control subjects from the same geographical region. Investigated polymorphisms were also analyzed in AIS subgroups defined according to the time of stroke onset into perinatal (n = 35, 20 boys and 15 girls) and childhood AIS (n = 38, 28 boys and 10 girls). Results: FV Leiden was found to be significantly associated with approximately 5-fold increased risk for AIS (OR = 4.72; 95% CI 1.22–18.27), and 8-fold increased risk for perinatal AIS (OR = 8.29; 95% CI 1.95–35.24). The presence of at least one HPA-3b allele was associated with approximately 2-fold lower risk for the development of AIS (OR = 0.51; 95% CI 0.26–0.98) and perinatal AIS (OR = 0.40; 95% CI 0.18–0.92). Increased risk for childhood AIS was identified in carriers of at least one FXIII-A Leu34 allele (OR = 2.21; 95% CI 1.03–4.73). Moreover, combined heterozygosity for FV Leiden and FV HR2 was found in 5/73 children with AIS (P = 0.012), in both AIS subgroups (perinatal AIS 2/35, childhood AIS 3/35) whereas this combination was not found in control subjects. Conclusions: This case-control study confirmed the association between FV Leiden and AIS that has also been observed in numerous studies so far, but it also showed that other previously not reported polymorhisms (FXIII-A Val34Leu, HPA-3) as well as the combination of FV Leiden and FV HR2 polymorphisms, can be related to AIS subgroups in Croatian population.
The human platelet antigens (HPA) are genetically defined polymorphisms expressed on platelet mem... more The human platelet antigens (HPA) are genetically defined polymorphisms expressed on platelet membrane glycoproteins. As platelet antigens are very important in several clinical situations and in population genetics, we used the polymerase chain reaction with sequence-specific primers (PCR-SSP) to investigate HPA-1, -2, -3 and -5 allele frequencies in the Croatian population. The HPA frequencies obtained in 219 Croatians were: 1a-0·854, 1b-0·146, 2a-0·890, 2b-0·110, 3a-0·575, 3b-0·425, 5a-0·895 and 5b-0·105. These data are similar to the frequencies reported in most European studies with some significant differences in HPA-2 when compared with the Dutch and German population, in HPA-3 when compared with the Swiss population and in HPA-5 when compared with the Finnish population. The three most common condensed HPA genotypes in the Croatian population were: HPA-1a/a, -2a/a, -3a/b, -5-a/a (0·283), HPA-1a/a, -2a/a, -3a/a, -5-a/a (0·137) and HPA-1a/b, -2a/a, -3a/b, -5-a/a (0·087). Data obtained in this study can be used for better understanding and treatment of immune-mediated platelet disorders in our population.
ABSTRACT Perinatal arterial stroke (PAS), defined as a cerebrovascular event which occurs between... more ABSTRACT Perinatal arterial stroke (PAS), defined as a cerebrovascular event which occurs between 28 weeks of gestation and 28 days of postnatal age with either pathological or radiological evidence of focal arterial infarction, has received increased attention as an important cause of cerebral palsy and other neurologic disabilities, including epilepsy and cognitive impairment. Although sensitive neuroimaging techniques have dramatically improved the detection of PAS in recent years, the cause of PAS is still poorly understood. The increasing evidence that inherited or acquired prothrombotic disorders may be implicated in the pathogenesis of PAS prompted us to investigate the prevalence of genetic polymorphisms that encode proteins associated with thrombosis (factor V G1619A [FVL], factor II G20210A [PT], homocysteine metabolism (methylenetetrahydrofolate reductase C677T [MTHFR]), and human platelet antigens (HPA) in children with PAS. Polymorphisms were investigated in 24 children (11 boys, 13 girls) with PAS confirmed by brain imaging and in 103 children (72 boys, 31 girls) from the same geographical region that represented the control group, with standard laboratory techniques. At least 1 prothrombotic abnormality was identified in 18/24 (75 %) children with PAS: 3 were heterozygous for FVL, 16 were carriers of the MTHFR mutation (13 heterozygotes, 3 homozygotes) and 1 was double heterozygous for FVL and MTHFR ; the presence of PT 20210A was not detected. Similar genotype and allele frequencies of HPA-1, HPA-2 and HPA-5 were observed in both study groups. Although higher frequencies of HPA-3a/a (50%), HPA-3a allele (68.7%) and FVL (12.5%) were observed in children with PAS compared to the control group (28.2%, 53.4% and 1.9% respectively), these differences failed to reach statistical significance. On the contrary, homozygosity for MTHFR (12.5% in patients and 1.9% in controls) was found to be the only statistically significant difference between the studied groups.
Journal of Hematotherapy & Stem Cell Research, 2002
Acute promyelocytic leukemia (APL) M3 is an acute myeloid leukemia (AML) subtype characterized by... more Acute promyelocytic leukemia (APL) M3 is an acute myeloid leukemia (AML) subtype characterized by proliferation of malignant promyelocytes with mature myeloid immunophenotype and the translocation t(15;17)(q22;q11), which results in the fusion of retinoic acid receptor-alpha (RARalpha) gene on chromosome 17 and the gene PML on chromosome 15. There are three M3 morphologic variants: the typical hypergranular form and the microgranular and basophilic variants. Although most leukemic cells in M3 patients express t(15;17), other cytogenetic abnormalities have also been reported. Also, there are three molecular variants of the PML/RARalpha transcript (bcr1, bcr2, bcr3). Blasts had typical hypergranular appearance (13 patients) with a mature myeloid immunophenotype (HLA-DR(-),CD13(+), and/or CD33(+)) (10 patients) in the majority of patients with M3 followed in this study. The typical translocation [t(15;17)(q22;q11)] was detected by cytogenetic analysis in 5 M3 patients, but PML/RARalpha was positive in 13 out of 15 patients, as assessed by RT-PCR (8 patients with bcr1 and 5 with bcr3 subtype). Cytogenetic diversity was found in three patients (1 with t(17;17), 1 with +8, and 1 with add (7)(q22); -7; +8). According to many studies, leukemic cell heterogeneity in APL influences the clinical outcome of disease. The analysis of certain leukemic cell characteristics on the clinical outcome in our study revealed that patients with bcr3 had shorter medians of first remission and survival in comparison to patients with the bcr1 isoform of PML/RARalpha. Also, the clinical relapse of disease in 4 APL patients with reverted PML/RAR alpha positivity is consistent with the view that detection of PML/RARalpha by RT-RCR in patients in remission implies a poor prognosis. On the contrary, lack of detection of PML/RARalpha by RT-PCR at least three times is a sign of long remission and survival.
Journal of Hematotherapy & Stem Cell Research, 1999
Detection of unusual or aberrant cell immunophenotype with flow cytometry is the basis for the im... more Detection of unusual or aberrant cell immunophenotype with flow cytometry is the basis for the immunologic recognition of minimal residual disease (MRD) in patients with acute leukemia (AL). In this study, we have shown that the double immunocytochemical alkaline phosphatase antialkaline phosphatase (APAAP) staining technique also makes possible the detection of leukemic cells with unusual (leukemic) combinations of antigens (ULCA) both at diagnosis and during follow-up of patients with ULCA+ AL. The applicability of double APAAP was analyzed on bone marrow (BM) samples obtained from 12 patients (8 with AML, 3 with ALL, and 1 with undifferentiated acute leukemia [AUL]) randomly chosen from a larger group of 22 ULCA+ patients treated at our center in a 3-year period (22% observed ULCA+ AL frequency). The percentages of ULCA+ BM cells before chemotherapy were in the range of 5%-60%, which dropped to 0%-7% in 10 patients who achieved remission (range 0%-7%, p &lt; 0.01). However, these cells could also be found 60 days after the initiation of therapy, ranging from 0%-2% of all nucleated cells. In 2 of 10 patients who achieved remission, 2% ULCA+ BM cells were found on days 35 and 60 after initiation of chemotherapy, and this finding was followed by relapse on days 110 and 270. However, the other 8 patients remained in remission despite positive finding of ULCA+ BM cells ranging from 0.2%-2% on at least one occasion. In 2 patients with AML FAB-M3 and cytomorphologic remission, the finding of ULCA+ cells by double APAAP correlated with the molecular finding of PML/RARalpha junction. These results indicate that double APAAP staining can identify leukemic cells in samples with a cytomorphologic pattern consistent with remission, but its applicability in detection of MRD awaits additional studies on a larger number of patients with ULCA+ AL.
Several inherited prothrombotic risk factors, particularly the presence of factor V G1691A, facto... more Several inherited prothrombotic risk factors, particularly the presence of factor V G1691A, factor II G20210A, and MTHFR C677T, have been demonstrated to play a role in childhood stroke, but the role of platelets has not been well studied in children.
The External RNA Controls Consortium* Standard controls and best practice guidelines advance acce... more The External RNA Controls Consortium* Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.
In this study we investigated IgH and TCRgamma gene rearrangements, cyclin A1 and HOXA9 gene expr... more In this study we investigated IgH and TCRgamma gene rearrangements, cyclin A1 and HOXA9 gene expression as well as the in vitro growth of biphenotypic acute leukemia (BAL) blasts in relation to acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The aim of the study was to correlate BAL morphology and its biological parameters in order to get information that might be used for additional stratification of BAL. This rare form of AL was identified in a total of 10 patients, comprising 4.3% of adult and 3.0% of pediatric patients with de novo AL referred to our institution during the 1999-2003 period. Our results indicate that IgH and TCRgamma gene rearrangements correlated well with lymphoid BAL morphology, whereas the expression of cyclin A1 correlated with myeloid and undifferentiated BAL morphology. Surprisingly, HOXA9 expression, a marker associated with myeloid cell lineage, showed no strong correlation with BAL morphology. Finally, in vitro growth of blasts during a 7-day culture showed autonomous cell growth in 3/10 AML and 3/8 myeloid BAL samples tested, but not in any of the AL with lymphoid features. Further studies are needed to confirm these findings and to extend research to a broader spectrum of cell markers.
Sažetak: The outbreak of thrombotic diathesis in younger people requires additional diagnostic ex... more Sažetak: The outbreak of thrombotic diathesis in younger people requires additional diagnostic examination, including genetic testing for possible inherited factors. Incidence of the G20210A mutation in the gene for prothrombin is such patients, as well as frequency ...
Journal of clinical chemistry and clinical biochemistry. Zeitschrift für klinische Chemie und klinische Biochemie, 1987
Blood sampling on filter paper was tested for determination of glycated haemoglobin. The method s... more Blood sampling on filter paper was tested for determination of glycated haemoglobin. The method showed coefficients of variation of 3.4% and 4.1%, and linearity coefficients of 0.978 and 0.91 for the microchromatographic and colorimetric methods respectively. A blood sample on filter paper impregnated with 5% ethylene glycol solution remains stable for 8 days at room temperature. In a group of 30 diabetics educated in the filter paper blood sampling technique, no statistically significant differences were registered among the mean values of their blood glucose profiles, glycated haemoglobin levels determined at the hospital and those obtained at home 14 days after discharge. In conclusion, the use of blood spotted on filter paper seems a cheap and convenient method for collecting, storing and transporting samples for analysis of glycated haemoglobins. It is also a useful alternative for home monitoring of diabetics. Moreover, it could also be useful in epidemiological studies of dia...
Despite the identification of a wide range of inherited and acquired risk factors for arterial is... more Despite the identification of a wide range of inherited and acquired risk factors for arterial ischemic stroke (AIS) in children, genetic risk factors are incompletely characterized and may vary among different populations. We investigated the role of individual and combined inherited prothrombotic and intermediate-risk factors in 73 children with perinatal (n = 35) and childhood AIS (n = 38) and 100 age- and sex-matched controls. Ten polymorphisms in 8 candidate genes encoding coagulation and fibrinolytic proteins (factor V [FV] Leiden, FV HR2, factor II [FII] G20210A, β-fibrinogen [β-FBG]-455G&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;A, factor XIII [FXIII]-A p.Val34Leu, plasminogen activator inhibitor 1 4G/5G), homocysteine metabolism (methylenetetrahydrofolate reductase [MTHFR] C677T, MTHFR A1298C), and intermediate-risk factors (angiotensin-converting enzyme I/D, apoE ∊2-4) were detected using a multilocus genotyping assay. Allele-specific polymerase chain reaction was used for the determination of human platelet alloantigens (HPA-1, HPA-2, HPA-3, and HPA-5). Factor V Leiden was associated with an increased risk of AIS (odds ratio [OR]: 4.72, 95% confidence interval [CI]: 1.22-18.27) and perinatal AIS (OR: 8.29, 95% CI: 1.95-35.24). Human platelet antigen-3b allele carriers had a 2-fold lower risk of AIS (OR: 0.51, 95% CI: 0.26-0.98) and perinatal AIS (OR: 0.40, 95% CI: 0.18-0.92). A 2.21-fold increased risk of childhood AIS (95% CI: 1.03-4.73) was identified in FXIII-A Leu34 allele carriers. Combined FV Leiden/FV HR2, FV Leiden/MTHFR A1298C, FV Leiden/MTHFR C677T/MTHFR A1298C, and FV Leiden/FV HR2/MTHFR A1298C heterozygosity was identified in children with AIS but not in controls, which revealed a statistically significant difference. This case-control study shows that besides already documented association between FV Leiden and AIS, other previously unreported polymorphisms (FXIII-A p.Val34Leu, HPA-3) and several genotype combinations that always include heterozygous FV Leiden can be related to AIS in Croatian population.
Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardize... more Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently utilize a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that's traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR, and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample in...
European journal of medical research, Jan 21, 2003
The main consequences of human immunodeficiency virus (HIV) infection and AIDS are frequent and p... more The main consequences of human immunodeficiency virus (HIV) infection and AIDS are frequent and persistent opportunistic infections at mucosal surfaces, but data upon impaired oral mucosal response in AIDS patients are still lacking. - The aim of this study was to determine salivary flow rates and peroxidase levels in unstimulated whole saliva in AIDS patients together with comparison to the healthy controls. Salivary peroxidase levels were determined according to Putter and Becker in 20 AIDS patients and 18 HIV-seronegative healthy controls. Statistical analysis was performed using Student t-test. Salivary peroxidase levels were significantly increased in the AIDS group (9.41 +/- 8.50 kU/L; p<0.009) when compared to the healthy controls (3.1 +/- 2.0 kU/L). Salivary flow rates were significantly decreased in AIDS patients (0.17+/-0.11 ml/min, p<0.009) when compared with healthy controls (0.58 +/- 0.19 ml/min). Elevated salivary peroxidase levels indicate increased salivary ant...
Haemophilia : the official journal of the World Federation of Hemophilia, 2014
Diagnosis of haemophilia A is usually made by the measurement of factor VIII (FVIII) activity tha... more Diagnosis of haemophilia A is usually made by the measurement of factor VIII (FVIII) activity that allows categorization of the disease severity. However, tests that assess global haemostasis may better reflect clinical features and give additional clinically relevant information. The aim of this study was to develop a new quantitative activated partial thromboplastin time (aPTT) waveform analysis and compare it with FVIII activities to find out whether waveform parameters are superior determinants of clinical phenotype. A total of 81 haemophilia A patients divided into two groups (37 severe, 44 non-severe) were included in the study. The control group comprised 101 healthy male volunteers. Quantitative aPTT waveform analysis was performed with Actin FS on BCS (Siemens Healthcare Diagnostics, Marburg, Germany) using three parameters (DELTA, RATIO-1, RATIO-2) obtained from a single aPTT measurement with two evaluation modes. FVIII activities were measured by one-stage clotting and tw...
European journal of medical research, Jan 28, 2009
The occurrence of acute promyelocytic leukemia (APL) in HIV-infected patients has been reported i... more The occurrence of acute promyelocytic leukemia (APL) in HIV-infected patients has been reported in only five cases. Due to a very small number of reported HIV/APL patients who have been treated with different therapies with the variable outcome, the prognosis of APL in the setting of the HIV-infection is unclear. Here, we report a case of an HIV-patient who developed APL and upon treatment entered a complete remission. A 25-years old male patient was diagnosed with HIV-infection in 1996, but remained untreated. In 2004, the patient was diagnosed with primary central nervous system lymphoma. We treated the patient with antiretroviral therapy and whole-brain irradiation, resulting in complete remission of the lymphoma. In 2006, prompted by a sudden neutropenia, we carried out a set of diagnostic procedures, revealing APL. Induction therapy consisted of standard treatment with all-trans-retinoic-acid (ATRA) and idarubicin. Subsequent cytological and molecular ana?lysis of bone marrow d...
European journal of medical research, Jan 28, 2001
The aim of our investigation was to evaluate possible connection between burning mouth syndrome a... more The aim of our investigation was to evaluate possible connection between burning mouth syndrome and hematinic deficiencies, a hypothesis previously reported in the literature with contradictory results. Serum levels of iron, vitamin B12, folic acid, calcium and magnesium were determined in 41 (aged 31-87 years, mean 68,7 yrs) patients with burning mouth syndrome and 35 matched controls (35-83, mean 63 yrs). Serum iron levels were determined according to Fairbanks and Klee. Levels of vitamin B12 and folic acid were determined on commercially available kits (Imx12 and Imx folate assay, Abbot Park lab, IL, USA) on Imx analyser. Calcium and magnesium levels were determined using atomic absorption spectrophotometry. No statistically significant differences in serum levels of iron, folic acid, calcium and magnesium were found between patients with burning mouth syndrome and controls. Statistically significant lowered vitamin B12 levels were found in patients with burning mouth syndrome. O...
To determine the prevalences of factor V Leiden and the G20210A mutation in the prothrombin gene ... more To determine the prevalences of factor V Leiden and the G20210A mutation in the prothrombin gene (PT20210A) and the frequency of their association in healthy subjects and in patients with venous thromboembolism (VTE). We studied 160 Croatian patients with at least one episode of VTE and 155 healthy subjects as a control group. Genomic DNA was extracted according to standard procedures and the presence of factor V Leiden and PT20210A were determined by polymerase chain reaction-restriction fragment length polymorphism method. The prevalences of factor V Leiden and PT20210A were in VTE patients 21% and 8% respectively, and 4% in controls for both mutations. Additionally, 4 patients were affected by double heterozygous defects, corresponding to a frequency of 3%, whereas none of the controls were double heterozygotes. The coexistence of the PT20210A in heterozygous carriers of factor V Leiden was 15% in VTE group. The results obtained for different subgroups of VTE patients showed that...
International Journal of Oral-Medical Sciences, 2004
Oral lichen planus (OLP) is a chronic inflammatory disease whose etiology has not yet been identi... more Oral lichen planus (OLP) is a chronic inflammatory disease whose etiology has not yet been identified. It is characterizied by immunoreactivity directed against basal keratinocytes and mediated by T-lymphocytes. In health, salivary peroxidase together with other salivary antimicrobial enzymes maintains the ecological balance in the oral cavity.
ABSTRACT Background: Despite extensive investigations and identification of a wide range of inher... more ABSTRACT Background: Despite extensive investigations and identification of a wide range of inherited and acquired risk factors for childhood stroke in recent years, genetic risk factors for this disease are still incompletely characterized. Taking into consideration that the frequency of genetic factors may vary among different populations, the aim of this study was to investigate the possible role of individual inherited prothrombotic and intermediate risk factors in Croatian children with a confirmed diagnosis of arterial ischemic stroke (AIS) and to check the possible association of investigated risk factors with the disease. Methods: A genotype analysis of 14 polymorphisms in 12 candidate genes encoding proteins of the coagulation and fibrinolysis systems (FV Leiden, FV HR2, FII G20210A, b-FBG −455G&amp;gt;A, FXIII-A Val34Leu, PAI-1 4G/5G), human platelet alloantigens (HPA-1, -2, -3 and -5), homocysteine metabolism (MTHFR C677T, MTHFR A1298C) and intermediate risk factors (ACE I/D and apoE e2–4) was performed in 73 children (48 boys and 25 girls) with AIS aged ≤18 years and compared with 100 age- and sex-matched control subjects from the same geographical region. Investigated polymorphisms were also analyzed in AIS subgroups defined according to the time of stroke onset into perinatal (n = 35, 20 boys and 15 girls) and childhood AIS (n = 38, 28 boys and 10 girls). Results: FV Leiden was found to be significantly associated with approximately 5-fold increased risk for AIS (OR = 4.72; 95% CI 1.22–18.27), and 8-fold increased risk for perinatal AIS (OR = 8.29; 95% CI 1.95–35.24). The presence of at least one HPA-3b allele was associated with approximately 2-fold lower risk for the development of AIS (OR = 0.51; 95% CI 0.26–0.98) and perinatal AIS (OR = 0.40; 95% CI 0.18–0.92). Increased risk for childhood AIS was identified in carriers of at least one FXIII-A Leu34 allele (OR = 2.21; 95% CI 1.03–4.73). Moreover, combined heterozygosity for FV Leiden and FV HR2 was found in 5/73 children with AIS (P = 0.012), in both AIS subgroups (perinatal AIS 2/35, childhood AIS 3/35) whereas this combination was not found in control subjects. Conclusions: This case-control study confirmed the association between FV Leiden and AIS that has also been observed in numerous studies so far, but it also showed that other previously not reported polymorhisms (FXIII-A Val34Leu, HPA-3) as well as the combination of FV Leiden and FV HR2 polymorphisms, can be related to AIS subgroups in Croatian population.
The human platelet antigens (HPA) are genetically defined polymorphisms expressed on platelet mem... more The human platelet antigens (HPA) are genetically defined polymorphisms expressed on platelet membrane glycoproteins. As platelet antigens are very important in several clinical situations and in population genetics, we used the polymerase chain reaction with sequence-specific primers (PCR-SSP) to investigate HPA-1, -2, -3 and -5 allele frequencies in the Croatian population. The HPA frequencies obtained in 219 Croatians were: 1a-0·854, 1b-0·146, 2a-0·890, 2b-0·110, 3a-0·575, 3b-0·425, 5a-0·895 and 5b-0·105. These data are similar to the frequencies reported in most European studies with some significant differences in HPA-2 when compared with the Dutch and German population, in HPA-3 when compared with the Swiss population and in HPA-5 when compared with the Finnish population. The three most common condensed HPA genotypes in the Croatian population were: HPA-1a/a, -2a/a, -3a/b, -5-a/a (0·283), HPA-1a/a, -2a/a, -3a/a, -5-a/a (0·137) and HPA-1a/b, -2a/a, -3a/b, -5-a/a (0·087). Data obtained in this study can be used for better understanding and treatment of immune-mediated platelet disorders in our population.
ABSTRACT Perinatal arterial stroke (PAS), defined as a cerebrovascular event which occurs between... more ABSTRACT Perinatal arterial stroke (PAS), defined as a cerebrovascular event which occurs between 28 weeks of gestation and 28 days of postnatal age with either pathological or radiological evidence of focal arterial infarction, has received increased attention as an important cause of cerebral palsy and other neurologic disabilities, including epilepsy and cognitive impairment. Although sensitive neuroimaging techniques have dramatically improved the detection of PAS in recent years, the cause of PAS is still poorly understood. The increasing evidence that inherited or acquired prothrombotic disorders may be implicated in the pathogenesis of PAS prompted us to investigate the prevalence of genetic polymorphisms that encode proteins associated with thrombosis (factor V G1619A [FVL], factor II G20210A [PT], homocysteine metabolism (methylenetetrahydrofolate reductase C677T [MTHFR]), and human platelet antigens (HPA) in children with PAS. Polymorphisms were investigated in 24 children (11 boys, 13 girls) with PAS confirmed by brain imaging and in 103 children (72 boys, 31 girls) from the same geographical region that represented the control group, with standard laboratory techniques. At least 1 prothrombotic abnormality was identified in 18/24 (75 %) children with PAS: 3 were heterozygous for FVL, 16 were carriers of the MTHFR mutation (13 heterozygotes, 3 homozygotes) and 1 was double heterozygous for FVL and MTHFR ; the presence of PT 20210A was not detected. Similar genotype and allele frequencies of HPA-1, HPA-2 and HPA-5 were observed in both study groups. Although higher frequencies of HPA-3a/a (50%), HPA-3a allele (68.7%) and FVL (12.5%) were observed in children with PAS compared to the control group (28.2%, 53.4% and 1.9% respectively), these differences failed to reach statistical significance. On the contrary, homozygosity for MTHFR (12.5% in patients and 1.9% in controls) was found to be the only statistically significant difference between the studied groups.
Journal of Hematotherapy & Stem Cell Research, 2002
Acute promyelocytic leukemia (APL) M3 is an acute myeloid leukemia (AML) subtype characterized by... more Acute promyelocytic leukemia (APL) M3 is an acute myeloid leukemia (AML) subtype characterized by proliferation of malignant promyelocytes with mature myeloid immunophenotype and the translocation t(15;17)(q22;q11), which results in the fusion of retinoic acid receptor-alpha (RARalpha) gene on chromosome 17 and the gene PML on chromosome 15. There are three M3 morphologic variants: the typical hypergranular form and the microgranular and basophilic variants. Although most leukemic cells in M3 patients express t(15;17), other cytogenetic abnormalities have also been reported. Also, there are three molecular variants of the PML/RARalpha transcript (bcr1, bcr2, bcr3). Blasts had typical hypergranular appearance (13 patients) with a mature myeloid immunophenotype (HLA-DR(-),CD13(+), and/or CD33(+)) (10 patients) in the majority of patients with M3 followed in this study. The typical translocation [t(15;17)(q22;q11)] was detected by cytogenetic analysis in 5 M3 patients, but PML/RARalpha was positive in 13 out of 15 patients, as assessed by RT-PCR (8 patients with bcr1 and 5 with bcr3 subtype). Cytogenetic diversity was found in three patients (1 with t(17;17), 1 with +8, and 1 with add (7)(q22); -7; +8). According to many studies, leukemic cell heterogeneity in APL influences the clinical outcome of disease. The analysis of certain leukemic cell characteristics on the clinical outcome in our study revealed that patients with bcr3 had shorter medians of first remission and survival in comparison to patients with the bcr1 isoform of PML/RARalpha. Also, the clinical relapse of disease in 4 APL patients with reverted PML/RAR alpha positivity is consistent with the view that detection of PML/RARalpha by RT-RCR in patients in remission implies a poor prognosis. On the contrary, lack of detection of PML/RARalpha by RT-PCR at least three times is a sign of long remission and survival.
Journal of Hematotherapy & Stem Cell Research, 1999
Detection of unusual or aberrant cell immunophenotype with flow cytometry is the basis for the im... more Detection of unusual or aberrant cell immunophenotype with flow cytometry is the basis for the immunologic recognition of minimal residual disease (MRD) in patients with acute leukemia (AL). In this study, we have shown that the double immunocytochemical alkaline phosphatase antialkaline phosphatase (APAAP) staining technique also makes possible the detection of leukemic cells with unusual (leukemic) combinations of antigens (ULCA) both at diagnosis and during follow-up of patients with ULCA+ AL. The applicability of double APAAP was analyzed on bone marrow (BM) samples obtained from 12 patients (8 with AML, 3 with ALL, and 1 with undifferentiated acute leukemia [AUL]) randomly chosen from a larger group of 22 ULCA+ patients treated at our center in a 3-year period (22% observed ULCA+ AL frequency). The percentages of ULCA+ BM cells before chemotherapy were in the range of 5%-60%, which dropped to 0%-7% in 10 patients who achieved remission (range 0%-7%, p &lt; 0.01). However, these cells could also be found 60 days after the initiation of therapy, ranging from 0%-2% of all nucleated cells. In 2 of 10 patients who achieved remission, 2% ULCA+ BM cells were found on days 35 and 60 after initiation of chemotherapy, and this finding was followed by relapse on days 110 and 270. However, the other 8 patients remained in remission despite positive finding of ULCA+ BM cells ranging from 0.2%-2% on at least one occasion. In 2 patients with AML FAB-M3 and cytomorphologic remission, the finding of ULCA+ cells by double APAAP correlated with the molecular finding of PML/RARalpha junction. These results indicate that double APAAP staining can identify leukemic cells in samples with a cytomorphologic pattern consistent with remission, but its applicability in detection of MRD awaits additional studies on a larger number of patients with ULCA+ AL.
Several inherited prothrombotic risk factors, particularly the presence of factor V G1691A, facto... more Several inherited prothrombotic risk factors, particularly the presence of factor V G1691A, factor II G20210A, and MTHFR C677T, have been demonstrated to play a role in childhood stroke, but the role of platelets has not been well studied in children.
The External RNA Controls Consortium* Standard controls and best practice guidelines advance acce... more The External RNA Controls Consortium* Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.
In this study we investigated IgH and TCRgamma gene rearrangements, cyclin A1 and HOXA9 gene expr... more In this study we investigated IgH and TCRgamma gene rearrangements, cyclin A1 and HOXA9 gene expression as well as the in vitro growth of biphenotypic acute leukemia (BAL) blasts in relation to acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The aim of the study was to correlate BAL morphology and its biological parameters in order to get information that might be used for additional stratification of BAL. This rare form of AL was identified in a total of 10 patients, comprising 4.3% of adult and 3.0% of pediatric patients with de novo AL referred to our institution during the 1999-2003 period. Our results indicate that IgH and TCRgamma gene rearrangements correlated well with lymphoid BAL morphology, whereas the expression of cyclin A1 correlated with myeloid and undifferentiated BAL morphology. Surprisingly, HOXA9 expression, a marker associated with myeloid cell lineage, showed no strong correlation with BAL morphology. Finally, in vitro growth of blasts during a 7-day culture showed autonomous cell growth in 3/10 AML and 3/8 myeloid BAL samples tested, but not in any of the AL with lymphoid features. Further studies are needed to confirm these findings and to extend research to a broader spectrum of cell markers.
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Papers by Renata Zadro