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.

Role of platelet gene polymorphisms in ischemic pediatric stroke subtypes: a case-control study.

AIM: To assess the role of human platelet antigens (HPA), P-selectin gene (SELP) polymorphisms, and HPA and SELP haplotypes with factor V (FV) R506Q in ischemic pediatric stroke (IPS) subtypes: cerebral sinovenous thrombosis (CSVT), perinatal (PAIS), and childhood (CAIS) arterial ischemic stroke. METHODS: This case-control study enrolled 150 children with confirmed IPS and 150 age- and sex-matched controls. FV R506Q and HPA-1 were genotyped with CVD StripAssay®, HPA-2 and HPA-3 with real-time polymerase chain reaction, SELP S290N, V599L, and T715P with high resolution melting analysis, and SELP N562D with sequence-specific polymerase chain reaction. RESULTS: HPA-1b allele (odds ratio [OR] 2.75, 95% confidence interval [CI] 1.02-7.42, P=0.048) and HPA-1a2a3b (OR 5.46, 95% CI 1.51-19.76, P=0.011), HPA-1b2a3a (OR 7.00, 95% CI 1.25-39.13, P=0.028), and HPA-1b2b3a (OR 11.39, 95% CI 1.39-92.95, P=0.024) haplotypes increased the risk for CSVT. HPA-3b allele was significantly associated with 2-fold lower risk for PAIS (OR 0.49, 95% CI 0.26-0.89, P=0.020) and CAIS (OR 0.47, 95% CI 0.26-0.86, P=0.014) and non-significantly associated with increased risk for CSVT (OR 6.43, 95% CI 0.83-50.00, P=0.022). HPA-1a2b3a haplotype was significantly associated with CAIS (OR 6.76, 95% CI 2.13-21.44, P=0.001). The inclusion of FV R506Q in SELP haplotype analysis increased the risk for PAIS 4-fold in QNDVT carriers (OR 8.14, 95% CI 0.93-71.33, P=0.060) compared with NDVT haplotype (OR 2.45, 95% CI 0.98-6.18, P=0.058), but the result was not significant. CONCLUSION: Individual HPAs, and particularly HPA haplotypes, are involved in IPS subtypes pathogenesis. A possible risk-inducing synergistic effect of SELP haplotypes with FV R506Q is restricted to PAIS only.

Development and validation of a rapid method for genotyping three P-selectin gene polymorphisms based on high resolution melting analysis.

BACKGROUND: High resolution melting (HRM) analysis is one of the newer, reliable, and sensitive genotyping techniques, which offers considerable time and cost savings. P-selectin is an adhesion molecule that has a role in the initial phases of leukocyte adhesion to stimulated platelets and endothelial cells in inflammation. Multiple polymorphisms in P-selectin gene (SELP) that affect the protein sequence have been described. The aim of this study was to design, optimize, and validate a simple and rapid in-house HRM-based method for genotyping the NM_003005.3:c.992G>A (c.992G>A), NM_003005.3:c.1918G>T (c.1918G>T), and NM_003005.3:c.2266A>C (c.2266A>C) SELP polymorphisms. METHODS: Initial genotyping of three SELP polymorphisms was performed by applying polymerase chain reaction (PCR) with sequence-specific primers (SSP), which was used as a reference method for determination of analytical sensitivity. PCR-HRM was performed with primers for c.2266A>C reported in the literature. Primers for the remaining two polymorphisms were designed using Primer-BLAST. Precision testing was performed using three samples with different genotypes. For accuracy, analytical sensitivity and specificity testing, 20 wild type, 10 heterozygous, and 10 homozygous samples were chosen per polymorphism. Results were expressed as percentage of concordance with the acceptability criterion ≥95%. RESULTS: Agreement of results was 100% for all validation parameters except for analytical sensitivity for c.1918G>T and c.2266A>C, with agreement of 90%. Repeated analysis using both methods revealed an error in initial genotyping and correct genotyping by PCR-HRM, which was confirmed by Sanger sequencing. CONCLUSION: The validation confirmed PCR-HRM as a precise, accurate, and specific method for genotyping the c.992G>A, c.1918G>T, and c.2266A>C SELP polymorphisms.

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.

Advanced Age, High ß-CTX Levels, and Impaired Renal Function are Independent Risk Factors for All-Cause One-Year Mortality in Hip Fracture Patients.

Risk factors for increased mortality in hip fracture patients include older age, male sex, fracture type, bone mineral density, and pre-existing co-morbidities. The role of biochemical and other anthropometric parameters on hip fracture mortality remains unclear. The aim of this study was to identify the risk factors for one-year mortality in patients with hip fractures. A total of 236 consecutive patients (59 males) with hip fractures were followed over a one-year period. Patient age, gender, type of fracture, type of treatment, time from admission to surgery, type of anesthesia, body mass index, and electrocardiograms were recorded. Complete blood counts, serum electrolytes, urea, creatinine, d-dimers, calcium, phosphate, osteocalcin, and beta-isomerised C-terminal telopeptide of collagen type I (ß-CTX) were measured at admission and estimated glomerular filtration rate (eGFR) was calculated. Multivariate Cox regression models were used to analyze the association of these parameters with survival. One-year mortality rate was 28.4%. Age was independently associated with mortality (HR 1.117, 95% CI 1.062-1.174, P < 0.001). In a multivariable model, mortality was increased in patients with higher ß-CTX (HR 4.63 95% CI 1.87-11.45, P = 0.001) and lower eGFR (HR 0.972, 95% CI 0.956-0.987, P < 0.001). Patients younger than 84 years, with eGFR < 55.4 ml/min had ten times higher mortality rates (3.2 vs. 24.5%, HR 9.73, 95% CI 2.06-45.93) as well as those with ß-CTX > 0.276 g/L (3.5 vs. 25.7%, HR 9.5, 95% CI 2.11-42.76). Advanced age, high ß-CTX levels, and impaired renal function are independent risk factors of mortality in patients with hip fractures.

Platelet satellitism in a trauma patient.

Platelet satellitism (PS) is a rare phenomenon observed in blood smears obtained from blood anticoagulated with EDTA. It is characterised by platelet rosetting around polymorphonuclear neutrophils and in rare cases around other blood cells. PS is a rare cause of pseudothrombocytopenia. References about the phenomenon of PS in medical literature are few. In this report we describe a case of PS fortunately noticed in one trauma patient. Furthermore, we discuss the possible pathophysiological mechanisms of PS proposed in the literature. To our knowledge this is the first case of PS reported in Croatia.

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.