Inherited Thrombophilia Associated With Ischemic Pediatric Stroke in Parent-Child Pairs.

BACKGROUND: We aimed to examine inherited thrombophilia frequencies by extending genetic profile to previously rarely or not investigated polymorphisms in children with ischemic pediatric stroke (IPS) and their parents. METHODS: The study included 33 children: 23 with perinatal arterial ischemic stroke (PAIS), eight with childhood arterial ischemic stroke (CAIS), and two with sinovenous thrombosis and their parents (33 mother-child, 12 father-child, and 12 mother-father-child pairs). Genotyping of FV-Leiden, FV-H1299R, FII-G20210A, ß-fibrinogen-455G>A, FXIII-A-Val34Leu, PAI-1(4G/5G), HPA-1, MTHFR-C677T, MTHFR-A1298C, ACE(I/D), and APOE(ε2-4) was performed using CVD Strip assay (ViennaLab, Austria). RESULTS: At least one and up to seven simultaneously present polymorphisms were observed in all children with IPS, mothers, and fathers. More than five simultaneously present polymorphisms were identified threefold more frequently in children with IPS (10 of 33; 30%) compared with the child control group (17 of 150; 11%), yielding a statistically significant difference between the two groups (odds ratio [OR] = 3.40; 95% confidence interval [CI] = 1.39 to 8.35; P = 0.012). Stronger association was revealed for PAIS (OR = 4.17; 95% CI = 1.55 to 11.29; P = 0.008) and CAIS subgroups (OR = 7.82; 95% CI = 1.79 to 34.20; P = 0.012). Complete match of polymorphisms was not identified in any parent-child pair. A partial match (one to four mutual polymorphisms) was found in 11 of 12 parent-child pairs where until three mutual polymorphisms was present in 11 of 12 (91.7%) father-child compared with 21 of 33 (63.6%) mother-child pairs. CONCLUSIONS: According to obtained results the simultaneous presence of more than five polymorphisms is associated with a higher risk for IPS occurrence, suggesting the risk enhancement for PAIS in the presence of pregnancy complications or for CAIS in conjunction with maternal comorbidity and positive family history. The presence of up to three mutual polymorphisms more frequently in father-child than mother-child pairs suggests significant paternal contribution of inherited thrombophilia to increased risk of IPS.

SARS-CoV-2 Seroprevalence and Neutralizing Antibody Response after the First and Second COVID-19 Pandemic Wave in Croatia.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus with a pandemic spread. So far, a total of 349,910 SARS-CoV-2 cases and 7687 deaths were reported in Croatia. We analyzed the seroprevalence and neutralizing (NT) antibody response in the Croatian general population after the first (May-July 2020) and second (December 2020-February 2021) pandemic wave. Initial serological testing was performed using a commercial ELISA, with confirmation of reactive samples by a virus neutralization test (VNT). A significant difference in the overall seroprevalence rate was found after the first (ELISA 2.2%, VNT 0.2%) and second waves (ELISA 25.1%, VNT 18.7%). Seropositive individuals were detected in all age groups, with significant differences according to age. The lowest prevalence of NT antibodies was documented in the youngest (<10 years; 16.1%) and the oldest (60-69/70+ years; 16.0% and 12.8%, respectively) age groups. However, these age groups showed the highest median NT titers (32-64). In other groups, seropositivity varied from 19.3% to 21.5%. A significant weak positive correlation between binding antibody level as detected by ELISA and VNT titer (rho = 0.439, p < 0.001) was observed. SARS-CoV-2 NT antibody titers seem to be age-related, with the highest NT activity in children under 10 years and individuals above 50 years.

Third-party verification of total and specific immunoglobulin E on analyzer Immulite® 2000XPi.

BACKGROUND: Although Immulite®2000XPi (Siemens) allergy testing is routinely used in laboratories worldwide; a well-designed comprehensive third-party verification of its analytical performance has not yet been published. Our aim was to verify stability, precision and reference intervals for total and some frequent specific IgEs, test trueness for several most common allergens and assess the comparability of fluorescent- and chemiluminescent-enzyme immunoassay for total IgE. METHODS: Verification was based on EP15-A3, EP9-A3 and EP28-A3c CLSI guidelines. Stability was tested on serum pools stored at 2-8 °C, during 7 days. Manufacturer's reference intervals were verified for children and adults. Acceptance criteria were based on manufacturer specifications and national External Quality Assessment provider data. RESULTS: Samples for total IgE analysis were stable at 2-8 °C for 7 days. Precision was within manufacturer's specifications for most of tested allergens, except for total IgE (92.5 kIU/L, CVs 5.6% and 7.6%) and w1 (9.3 kU/L, CV = 5.9%). Bland-Altman plot revealed statistically significant constant (-32.4 kIU/L) and proportional bias (-20.2%) for total IgE between two methods. Passing Bablok showed statistically but not clinically significant biases between two Immulite analyzers for betula verrucosa (constant and proportional), house dust mite and egg white allergens (proportional). For adults and children, total IgE results were within reference intervals declared by Siemens. CONCLUSIONS: Immulite®2000XPi assay has acceptable precision and trueness. Samples for total IgE are stable even longer than declared by the manufacturer. Declared reference intervals can safely be adopted for both adult and children population in our laboratory. Fluorescent- and chemiluminescent-enzyme immunoassay results of total IgE are not comparable.

External quality assessment of medical laboratories in Croatia: preliminary evaluation of post-analytical laboratory testing.

INTRODUCTION: Proper standardization of laboratory testing requires assessment of performance after the tests are performed, known as the post-analytical phase. A nationwide external quality assessment (EQA) scheme implemented in Croatia in 2014 includes a questionnaire on post-analytical practices, and the present study examined laboratory responses in order to identify current post-analytical phase practices and identify areas for improvement. MATERIALS AND METHODS: In four EQA exercises between September 2014 and December 2015, 145-174 medical laboratories across Croatia were surveyed using the Module 11 questionnaire on the post-analytical phase of testing. Based on their responses, the laboratories were evaluated on four quality indicators: turnaround time (TAT), critical values, interpretative comments and procedures in the event of abnormal results. Results were presented as absolute numbers and percentages. RESULTS: Just over half of laboratories (56.3%) monitored TAT. Laboratories varied substantially in how they dealt with critical values. Most laboratories (65-97%) issued interpretative comments with test results. One third of medical laboratories (30.6-33.3%) issued abnormal test results without confirming them in additional testing. CONCLUSION: Our results suggest that the nationwide post-analytical EQA scheme launched in 2014 in Croatia has yet to be implemented to the full. To close the gaps between existing recommendations and laboratory practice, laboratory professionals should focus on ensuring that TAT is monitored and lists of critical values are established within laboratories. Professional bodies/institutions should focus on clarify and harmonized rules to standardized practices and applied for adding interpretative comments to laboratory test results and for dealing with abnormal test results.

Evaluating performance in sweat testing in medical biochemistry laboratories in Croatia.

INTRODUCTION: Sweat test has a diagnostic role in evaluation of cystic fibrosis. Its performance includes sweat stimulation, collection and analysis. All listed may be sources of inconsistencies in everyday practice. The aim of this study was an evaluation of external quality assessment (EQA) of sweat chloride measurement including sweat test performance in medical biochemistry laboratories in Croatia. MATERIALS AND METHODS: EQA for sweat chloride measurement was provided by Croatian Centre for Quality Assessment in Laboratory Medicine (CROQALM) in five consecutive exercises to medical biochemistry laboratories (MBL) that offered sweat testing. A questionnaire regarding all phases of testing was mailed to involved MBL (N = 10). Survey results were compared to current guidelines for sweat test performance. RESULTS: Reported results of EQA in 2015 exercises showed coefficients of variation (CV) from 28.9%, 29.0% to 35.3%, respectively. An introduction of uniform sweat chloride measurement protocol resulted in CV of 15.5% and 14.7% reported in following two exercises in 2016. All MBL included in this study replied to the questionnaire. Results reported by MBL indicated: lack of patient information policy (7/10), use of unacceptable electrodes (6/9), misuse of minimum of acceptable sweat weight (6/9), lack of internal quality assessment (5/9) and recommended reference ranges (5/9 and 4/9). Agreements to guidelines were found in approach to unsuitable patients (9/10) and sweat collection (8/9). CONCLUSION: Presented results indicate major weak points of current practice in sweat test performance in Croatian MBL and stress the need for its standardization on a national level.

External Quality Assessment in Croatia: problems, challenges, and specific circumstances.

In Croatian medical laboratories (ML), external quality assessment (EQA) has a long tradition of almost half of a century. At national level, EQA is provided by Croatian Centre for Quality Assessment in Laboratory Medicine (CROQALM) which is a part of Croatian Society of Medical Biochemistry and Laboratory Medicine.This case study aimed to summarize the main challenges, which are set to CROQALM and their possible solutions.CROQALM has 10 schemes, covering majority of analysis for which medical biochemistry laboratories in Croatia are authorized for, including pre-analytical and post-analytical phase of laboratory work. Assessment scheme has three exercises per year. One sample per scheme and exercise is distributed to participants depending on their application. All data transfer and evaluation of the results are done using web interface and statistical software for evaluation of quality in laboratory medicine.Since CROQALM has relatively small number of participating laboratories (N = 197) with lot of different manufacturers of instruments used for analysis in all schemes, constant challenges are present in the evaluation of the results (commutability problems, statistical analysis etc.). Further, number of participating medical laboratories is even lower for highly specific parameters, which are in the scope of clinical laboratories only.Despite the obstacles we are faced to, EQA at national level is useful tool regarding standardization and harmonization aspects in total testing process within the country. Furthermore, it gives participating laboratories recognition and proof for meeting expected quality criteria in the community they serve.

Preanalytical external quality assessment of the Croatian Society of Medical Biochemistry and Laboratory Medicine and CROQALM: finding undetected weak spots.

INTRODUCTION: The aim of this paper is to present results of first two years of preanalytical external quality assessment (EQA) in Croatia. MATERIALS AND METHODS: This paper summarizes results from 6 rounds of preanalytical EQA during 2014-2016 in 161-175 Croatian laboratories (number ranged between cycles). EQA was designed as an online survey of the compliance with National recommendations for phlebotomy (NRP). Forty-seven questions in 5 categories are analyzed (materials and equipment, patient identification, patient preparation, sampling and storage). Additionally, preanalytical cases are presented. Overall performance scores (Question score (Qscore) for compliance with NRP and Case score (Cscore) for preanalytical cases) are calculated for each question/case as a proportion of laboratories with satisfactory procedure (x 100). Qscores and Cscores ≥ 70 were classified as acceptable (maximal score = 100). RESULTS: In investigation of compliance with NRP, acceptable Qscores were obtained for 34/47 questions. The lowest scores were observed for the availability of sterile disposable tourniquets (Qscore = 15) and safe-sharp needles (Qscore = 34), obtaining patients address as an identifier (Qscore = 21), using glycolysis inhibitor tubes for glucose concentration measurement (Qscore = 21) and verification of manufacturers declarations on temperature and time of storage (Qscore = 31). There was no statistically significant difference in overall Qscore according to different categories of phlebotomy procedures (P = 0.284). The results of preanalytical cases showed acceptable Cscore values for all cases (89-96). CONCLUSION: First two years of preanalytical EQA showed good compliance with the NRP and excellent expertise in resolving complex preanalytical issues. Major critical spots are lack of availability of safe-sharp needles, disposable tourniquets and glucose inhibitor tubes.

Capillary blood sampling: national recommendations on behalf of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

Capillary blood sampling is a medical procedure aimed at assisting in patient diagnosis, management and treatment, and is increasingly used worldwide, in part because of the increasing availability of point-of-care testing. It is also frequently used to obtain small blood volumes for laboratory testing because it minimizes pain. The capillary blood sampling procedure can influence the quality of the sample as well as the accuracy of test results, highlighting the need for immediate, widespread standardization. A recent nationwide survey of policies and practices related to capillary blood sampling in medical laboratories in Croatia has shown that capillary sampling procedures are not standardized and that only a small proportion of Croatian laboratories comply with guidelines from the Clinical Laboratory Standards Institute (CLSI) or the World Health Organization (WHO). The aim of this document is to provide recommendations for capillary blood sampling. This document has been produced by the Working Group for Capillary Blood Sampling within the Croatian Society of Medical Biochemistry and Laboratory Medicine. Our recommendations are based on existing available standards and recommendations (WHO Best Practices in Phlebotomy, CLSI GP42-A6 and CLSI C46-A2), which have been modified based on local logistical, cultural, legal and regulatory requirements. We hope that these recommendations will be a useful contribution to the standardization of capillary blood sampling in Croatia.

Nationwide survey of policies and practices related to capillary blood sampling in medical laboratories in Croatia.

INTRODUCTION: Capillary sampling is increasingly used to obtain blood for laboratory tests in volumes as small as necessary and as non-invasively as possible. Whether capillary blood sampling is also frequent in Croatia, and whether it is performed according to international laboratory standards is unclear. MATERIALS AND METHODS: All medical laboratories that participate in the Croatian National External Quality Assessment Program (N = 204) were surveyed on-line to collect information about the laboratory's parent institution, patient population, types and frequencies of laboratory tests based on capillary blood samples, choice of reference intervals, and policies and procedures specifically related to capillary sampling. Sampling practices were compared with guidelines from the Clinical and Laboratory Standards Institute (CLSI) and the World Health Organization (WHO). RESULTS: Of the 204 laboratories surveyed, 174 (85%) responded with complete questionnaires. Among the 174 respondents, 155 (89%) reported that they routinely perform capillary sampling, which is carried out by laboratory staff in 118 laboratories (76%). Nearly half of respondent laboratories (48%) do not have a written protocol including order of draw for multiple sampling. A single puncture site is used to provide capillary blood for up to two samples at 43% of laboratories that occasionally or regularly perform such sampling. Most respondents (88%) never perform arterialisation prior to capillary blood sampling. CONCLUSIONS: Capillary blood sampling is highly prevalent in Croatia across different types of clinical facilities and patient populations. Capillary sampling procedures are not standardised in the country, and the rate of laboratory compliance with CLSI and WHO guidelines is low.

Inherited prothrombotic risk factors in children with stroke, transient ischemic attack, or migraine.

OBJECTIVE: The aim of this study was to investigate the prevalence and possible association of inherited prothrombotic risk factors in children with stroke, transient ischemic attack, or migraine. METHODS: We performed genotypic analysis for factor V G1691A, factor II G20210A, methylenetetrahydrofolate reductase C677T, and 4 common platelet glycoprotein polymorphisms (human platelet alloantigen-1, -2, -3, and -5) in 150 children <18 years of age with established diagnoses of stroke, transient ischemic attack, or migraine. Children were classified into 5 groups, namely, childhood arterial ischemic stroke (N = 33), perinatal arterial ischemic stroke (N = 26), hemorrhagic stroke (N = 20), transient ischemic attack (N = 36), and migraine (N = 35). The control group consisted of 112 children < or =18 years of age from the same geographical region who had no history of neurologic or thromboembolic diseases. RESULTS: Heterozygosity for factor V G1691A was associated with approximately sevenfold increased risk for arterial ischemic stroke, perinatal arterial ischemic stroke, and transient ischemic attack. Increased risk for transient ischemic attack was found in carriers of the human platelet alloantigen-2b allele, human platelet alloantigen-5a/b genotype, and combined human platelet alloantigen-2b and human platelet alloantigen-5b genotype. The presence of the human platelet alloantigen-2b allele was associated with a 2.23-fold increased risk for migraine, whereas carriers of the human platelet alloantigen-3b allele had a lower risk for arterial ischemic stroke than did carriers of the human platelet alloantigen-3a allele. CONCLUSIONS: Factor V G1691A has an important role in susceptibility to arterial ischemic stroke, both in the perinatal/neonatal period and in childhood, as well as transient ischemic attacks. A minor impact of human platelet alloantigen polymorphisms suggests that platelet glycoprotein polymorphisms may increase the risk of transient ischemic attacks and migraine, but this should be confirmed in larger studies.