Hematological investigations in a case of intravascular hemolysis due to Clostridium perfringens infection.

A patient presented with fever, severe pain and edematous tight due to hip trauma and was scheduled for urgent fasciotomy. Following physical examination, laboratory analyses were requested, and results revealed anemia and severe infection. As the patient's condition was serious, a new set of samples was sent to the laboratory four hours later. Following centrifugation, severely hemolyzed dark-colored serum and plasma samples were obtained and in vitro hemolysis was suspected. The collection of samples was repeated, but a new set of samples was also hemolyzed with a significant decrease in the hemoglobin value. At that point, in vivo hemolysis was suspected, and samples were processed according to standard laboratory procedures for hemolytic samples. Following confirmation of the gas gangrene diagnosis by clinicians, the cause of hemolysis was attributed to the cytotoxic activity of α-toxin produced by the anaerobic gram-positive bacterium Clostridium perfringens. An insight into the laboratory procedure that could help to narrow down the causes of hemolysis and single out C. perfringens as a cause of intravascular hemolysis was given.

Reporting of activated partial thromboplastin time (aPTT): Could we achieve better comparability of the results?

INTRODUCTION: Activated partial thromboplastin time (aPTT) is determined and reported as clotting time in seconds aPTT(s), but it is presumed that reporting results as patient-to-normal clotting time ratio, aPTT(r), could minimize within-laboratory variability. The aim of study was to investigate differences in reporting aPTT results that can affect comparability of the results among Croatian laboratories and suggest further steps for its harmonization. MATERIALS AND METHODS: The questionnaire on aPTT reporting practice was distributed to 83 laboratories through Survey Monkey application in March 2019 as the part of the first regular round of Croatian Centre for Quality Assessment in Laboratory Medicine proficiency testing. RESULTS: The survey response rate was 0.49. Majority of laboratories report aPTT results as both, seconds and ratio. Participants reported use of 23 different aPTT(s) reference intervals along with 17 different combinations of reagent/coagulometer and 25 aPTT(s) denominators of different origin for aPTT(r) calculation. Despite the same aPTT(s) results, the use of different denominators caused a dispersion of aPTT(r) results that can lead to exceeding external quality assessment performance criteria of 7%, particularly when results were compared for the same reagent group only. By applying aPTT(s) reference interval mean as denominator for calculation of aPTT(r) reference interval better concordance to harmonized one was obtained (17 vs. 27; χ2 = 3.972; P = 0.046). CONCLUSION: In order to improve comparability of the results, laboratories are advised to use mean of aPTT(s) reference interval as denominator for aPTT(r) calculation. Type of coagulometer need to be considered when evaluating aPTT proficiency test results and its currently acceptable limit of performance evaluated accordingly.

Association of fibrinogen and plasmin inhibitor, but not coagulation factor XIII gene polymorphisms with coronary artery disease.

BACKGROUND: In the final phase of clot formation, fibrinogen constitutes frame, whereas factor XIII (FXIII) active form is responsible for the covalent cross-linking of fibrin fibres and plasmin inhibitor (PI), thus contributing to clot stability. It could be expected that any change of coagulation factors' structure affects the clot formation and modulates the atherothrombotic risk. The aim was to determine the frequency of four single nucleotide polymorphisms: (i) A > G in codon 312 of the fibrinogen α-chain gene (rs6050, Thr312AlaFGA), (ii) C > T at position 10034 of the 3 - untranslated region in the fibrinogen γ-chain gene (rs2066865, 10034C > T FGG), (iii) C > T in codon 564 of the FXIII-A subunit gene (rs5982, Pro564LeuFXIII-A), and (iv) C > T in codon 6 of the plasmin inhibitor gene (rs2070863, Arg6TrpPI) in Croatian patients and their association with coronary artery disease (CAD). METHODS: We performed the unrelated case-control association study on the consecutive sample of patients 18 years old, who had undergone coronary angiography for investigation of chest pain and suspected CAD. The cases were patients with confirmed CAD (N=201), and the controls were the subjects with no CAD (N=119). Samples were genotyped using PCR-RFLP analysis. RESULTS: Observed frequencies of the rare alleles of Thr312Ala FGA, 10034C > T FGG, Leu564Pro FXIII-A and Arg6Trp PI polymorphisms were 21%, 17%, 14%, 20%, respectively. Patients with 10034C > T FGG CC genotype had 3.5 times (95% CI 1.02-12.03) higher adjusted odds for CAD than patients with 10034C > T FGG TT genotype. Patients with Arg6Trp PI CC genotype had 3.86 times (95% CI 1.23-12.12) higher odds for CAD than patients with Arg6Trp PI TT genotype. It seems that those genotype-related higher odds are also male-gender related. No difference was observed regarding any other investigated polymorphism. CONCLUSIONS: Our finding suggests that 10034C > T FGG and Arg6Trp PI are associated with CAD.

Knowledge and awareness of iodine intake - survey among Croatian women of reproductive age.

INTRODUCTION: Appropriate iodine intake is important for the entire population, especially in fertile women due to decisive role of thyroid hormones in normal foetal brain development. The aim of this study was to investigate knowledge on iodine role among Croatian women of reproductive age. MATERIALS AND METHODS: The survey was conducted among 378 women of reproductive age during May-September 2018. Data on age, education level, salt intake habits, knowledge of the iodine role and possible presence of thyroid disease were collected and results were presented as numbers or percentage of total number of participants. Comparison between groups was performed by Chi square test. RESULTS: Of 378 participants, 178 reported to be familiar with the iodine role in the body (P = 0.115). Significantly higher proportion of the younger woman and woman with lower degree of education weren't familiar with the iodine role (P < 0.001). More woman were introduced to consequences of insufficient than to excessive iodine intake (273 vs 213; P < 0.001). In addition, participants mainly weren't familiar with obligation of salt iodination (P < 0.001). Presence of thyroid disease was recorded in 75 subjects with higher prevalence in subjects 36-49 years (P < 0.001). Presence of thyroid disease was not associated with knowledge on iodine role on health. CONCLUSION: Women of reproductive age are not completely aware of the consequences of excessive iodine intake. Thus, further education focusing on more sensitive groups such as woman of younger age should be considered in order to preserve their and their children's health.

Croatian Society of Medical Biochemistry and Laboratory Medicine: National recommendations for blood collection, processing, performance and reporting of results for coagulation screening assays prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer.

A modern diagnostic laboratory offers wide spectrum of coagulation assays utilized in the diagnosis and management of patients with haemostatic disorders, preoperative screening and anticoagulation therapy monitoring. The recent survey conducted among Croatian medical biochemistry and transfusion laboratories showed the existence of different practice policies in particular phases of laboratory process during coagulation testing and highlighted areas that need improvement. Lack of assay standardization together with non-harmonized test results between different measurement methods, can potentially lead to incorrect decisions in patient's treatment. Consequently, patient safety could be compromised. Therefore, recommended procedures related to preanalytical, analytical and postanalytical phases of prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer testing are provided in this review, aiming to help laboratories to generate accurate and reliable test results.

Effect of cold agglutinins on red blood cell parameters in a trauma patient: a case report.

The presence of cold agglutinins (CAs) in samples intended for complete blood count (CBC) using automated haematology analysers might cause serious preanalytical errors. In this report we describe the case of a 90-year old female patient admitted to the Emergency department following trauma injuries. A blood testing on admission revealed surprisingly low red blood cell count (0.99 x 1012/L), low haematocrit (0.102 L/L) which did not correlate with haemoglobin concentration (100 g/L), and high erythrocytes indices (mean corpuscular haemoglobin, 101 pg; mean corpuscular haemoglobin concentration, 980 g/L). In the second sample, after repeated collection, almost equal results were observed. Blood smear examination under the microscope revealed clusters of erythrocytes. Cold agglutinins presence was suspected and, in order to get valid results, sample was warmed to 37 °C. Correction of CBC was observed. Furthermore, we performed some additional analysis to confirm the presence of CAs in this patient. The aim of this report was to present the laboratory findings in a case of CAs and propose a laboratory procedure for whole blood samples with suspected CAs.

Policies and practices in haemostasis testing among laboratories in Croatia: a survey on behalf of a Working Group for Laboratory Coagulation of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

INTRODUCTION: The objective of this survey was to assess current policies and practice in haemostasis testing among both hospital and outpatient laboratories in Republic of Croatia. MATERIALS AND METHODS: A questionnaire with seventy questions divided into nine sections was created in May 2015. Participants were asked about their practice related to test request form, sample collection, prothrombin time (PT) and activated partial thromboplastin time assays, other individual haemostasis assays, point-of-care testing (POCT), reporting of coagulation tests results and quality assurance of procedures, the personnel and other laboratory resources, as well as on issues related to education and implementation of additional coagulation assays in their laboratory. The survey was administered and data were collected between June and September 2015. RESULTS: A total survey response rate was 104/170 (61.2%). Most respondents were faced with incomplete information on prescribed therapy and diagnosis on the test request or inappropriate samples withdrawn on distant locations, but also do not have protocols for handling samples with high haematocrit values. Reporting of PT-INR and D-dimer results was different between laboratories. Although almost all laboratories developed a critical value reporting system, reporting a value to general practitioners is still a problem. Result on coagulation POCT testing showed that not all devices were supervised by laboratories, which is not in compliance with Croatian Chamber of Medical Biochemistry acts. CONCLUSION: Obtained results highlighted areas that need improvement and different practice patterns in particular field of haemostasis testing among laboratories. A harmonization of the overall process of haemostasis testing at national level should be considered and undertaken.

Multiple presence of prothrombotic risk factors in Croatian children with arterial ischemic stroke and transient ischemic attack.

AIM: To determine the frequency of inherited and acquired prothrombotic risk factors in children with arterial ischemic stroke (AIS) and transient ischemic attacks (TIA) in Croatia. METHODS: We investigated 14 prothrombotic risk factors using blood samples from 124 children with AIS or TIA and 42 healthy children. Prothrombotic risk factors were classified into five groups: natural coagulation inhibitors (antithrombin, protein C, protein S), blood coagulation factors (FV Leiden and FII 20210), homocysteine, lipid and lipoprotein profile (lipoprotein (a), triglycerides, total, high- and low-density lipoprotein), and antiphospholipid antibodies (lupus anticoagulant, anticardiolipin, and antiphosphatidylserine antibodies). RESULTS: The most common prothrombotic risk factor was elevated lipoprotein (a), which was identified in about 31% of patients and in 24% of controls. Natural coagulation inhibitors were decreased in about 19% of patients, but not in controls. Pathological values of homocysteine, blood coagulation factor polymorphisms, and antiphospholipid antibodies were found in similar frequencies in all groups. Fourteen children with AIS and TIA (11.3%) and no children from the control group had three or more investigated risk factors. CONCLUSION: The presence of multiple prothrombotic risk factors in children with cerebrovascular disorder suggests that a combination of risk factors rather than individual risk factors could contribute to cerebrovascular disorders in children.

Contraception-related deep venous thrombosis and pulmonary embolism in a 17-Year-old girl heterozygous for factor V leiden, prothrombin G20210A mutation, MTHFR C677T and homozygous for PAI-1 mutation: report of a family with multiple genetic risk factors and review of the literature.

We present the case of a 17-year-old girl who suddenly woke up with localized pain in the left groin and the inability to twist her leg. After comprehensive physician and laboratory examinations, deep venous thrombosis with consequent pulmonary embolism was ascertained. She had not experienced any recent trauma, but she had started to take oral contraceptives 6 months prior to the onset of the symptoms. Her parents and sisters had been asymptomatic throughout their lives, but the family history revealed a few thromboembolic accidents. Using DNA analysis, heterozygosity for factor V Leiden, prothrombin gene mutation G20210A and methylenetetrahydrofolate reductase C677T, as well as the homozygous 4G/4G genotype in the plasminogen activator inhibitor 1 were identified in our patient. Subsequently, DNA analysis was performed in all living family members, and multiple factors associated with thrombophilia were discovered. Our case confirms the multifactorial cause of thromboembolic events and emphasizes the importance of oral contraceptive use in the onset of venous thrombosis, especially in teenage females. In addition, this case indicates that teenage females with a family history of thrombosis who are making choices about contraception could most likely benefit from advanced thrombophilia testing.

Impact of FXIII-A Val34Leu polymorphism on coronary artery disease in Croatian patients.

Arterial thrombosis is the major reason for severe complications of coronary artery disease (CAD). Recently it has been suggested that the FXIII-A Val34Leu polymorphism, affecting clot stability, provides protection against thrombosis. Results published up to date implicate that there is a significant correlation between geographical area and the Leu34 allele prevalence and that its contribution to arterial thrombosis is different in different populations. The purpose of this study was to determine frequency of Leu34 allele in Croatian subjects as well as to estimate its association with a CAD. FXIII-A Val34Leu genotyping was carried out by real-time PCR method on the LightCycler using melting curve analysis with forward 5'-AACTTCCAGGACCGGCTTT-3' and reverse 5'-ACCCAGAGTGGTGGGGAA-3' primers. The Leu34 allele frequency in studied Croatian subjects was 24.3%. No significant differences were found in the prevalence of FXIII-A Val34Leu genotype or Leu34 allele distribution between studied subjects (P > 0.05). Carriage of the Leu34 allele was not significantly associated with CAD or MI risk reduction (P > 0.05). This is the first report that studies the prevalence of the Leu34 allele frequency in Croatian subjects and our results suggest that possession of the Leu 34 alele does not provide protection against MI.