The novel IMAGE001 genotyping array as a valuable alternative for genetic diversity screening in chicken: a demonstration in a local chicken breed in Belgium.

Screening for genetic diversity in livestock species breeds is of utmost importance, especially for local, small populations that are at the risk of extinction. Luckily, recent developments in technology increase access to genotyping, also for numerically small breeds. One of these new technologies is the IMAGE001 single nucleotide polymorphism genotyping array that includes markers for 6 different species (cow, pig, sheep, chicken, horse and goat). For our current study, we studied the Turkey-headed Malines chicken, a local chicken breed in Belgium, for the first time. A total of 110 animals were genotyped, together with 29 samples from 4 supposedly related breeds. The genotypes were used to assess the genetic diversity of this local breed. Our analysis revealed an average inbreeding coefficient of 0.20 through runs of homozygosity analysis, and effective population size estimation based on linkage disequilibrium indicated a low genetic diversity (Ne = 34). Moreover, a principal component analysis and a genetic differentiation study (FST) were performed using these marker data to position the Turkey-headed Malines relative to the 4 other indigenous Belgian chicken breeds. Finally, we discussed the practical implications of the overlap between the IMAGE001 array and other existing chicken genotyping arrays. This study is the first use of the novel IMAGE001 array to evaluate a local chicken breed, and demonstrates it as a viable option for genomic characterization a breed. Moreover, with this research, we are able to provide a good basis for further evaluation of the Belgian chicken heritage.

A Revised Phylogeny of the Mentha spicata Clade Reveals Cryptic Species.

The genus Mentha is taxonomically and phylogenetically challenging due to complex genomes, polyploidization and an extensive historical nomenclature, potentially hiding cryptic taxa. A straightforward interpretation of phylogenetic relationships within the section Mentha is further hindered by dominant but outdated concepts on historically identified hybrid taxa. Mentha spicata is traditionally considered to be of hybrid origin, but the evidence for this is weak. Here, we aim to understand the phylogenetic relationships within the section Mentha using large sample sizes and to revisit the hybrid status and identity of M. spicata. We show that two of three traditional species in the subsection Spicatae are polyphyletic, as is the subsection as a whole, while the real number of cryptic species was underestimated. Compared to previous studies we present a fundamentally different phylogeny, with a basal split between M. spicata s.s. and M. longifolia s.s. Cluster analyses of morphological and genotypic data demonstrate that there is a dissociation between morphologically and genotypically defined groups of samples. We did not find any evidence that M. spicata is of hybrid origin, and we conclude its taxonomic status should be revised. The combination of genetic and phenotypic information is essential when evaluating hyperdiverse taxonomic groups.

Lethal Injection of a Castor Bean Extract: Ricinine Quantification as a Marker for Ricin Exposure Using a Validated LC-MS/MS Method.

Ricin is a highly toxic agent derived from the castor bean plant (Ricinus communis). Poisoning occurs commonly by oral ingestion of the beans. Injection of ricin is believed to be more lethal. Ricin is a large glycosylated protein difficult to detect in clinical samples. Instead, ricinine, a small alkaloid found in the same beans, is used as surrogate marker for ricin exposure. We describe a simple LC-MS/MS method for the detection of ricinine in serum, blood and urine, validated according to EMA guidelines and successfully applied to patient samples of a suicidal death after injection of a castor bean extract. A 26-year-old man self-presented to the emergency department with severe abdominal cramps and nausea after injection of a castor bean extract. Due to rapid deterioration of his hemodynamic function despite early aggressive fluid resuscitation, he was transferred to ICU. Abdominal cramps worsened and a fulminant diarrhea developed, resulting in hypovolemic shock and cardiorespiratory collapse. Despite full supportive therapy, the patient died approximately 10 hours after injection due to multiple organ failure. Ricinine was quantified by LC-MS/MS after LLE with diethyl ether using ricinine-D3 as internal standard. Six hours after injection, ricinine concentrations in serum and blood were 16.5 and 12.9 ng/mL, respectively, which decreased to 12.4 and 10.6 ng/mL, 4 hours later. The urinary concentration was 81.1 ng/mL 7 hours after injection, which amply exceeded the levels previously reported in similar cases with lethal outcome. Concentrations of ricinine, compatible with a lethal exposure to castor beans, were detected in serum, blood and urine. Ricinine was also found in bile and liver tissue.

Gamma-hydroxybutyrate (GHB), an unusual cause of high anion gap metabolic acidosis.

The causes of high anion gap metabolic acidosis (HAGMA) are well described in the literature. However, sometimes more frequent causes of HAGMA cannot explain its occurrence.In the case of HAGMA and severe neurological depression in the absence of other causes of HAGMA, clinicians should consider an intoxication with gamma-hydroxybutyrate (GHB) as a possible cause.GHB is endogenous to the mammalian central nervous system (CNS). Synthetic GHB was initially used as an anesthetic but is now only licensed for medical use in a limited number of indications such as the treatment of narcolepsy. Because of its euphoric effects, it became popular for recreational use under the street names: Liquid Ecstasy, Georgia Home Boy, and Liquid G.We describe the clinical case of a patient who suffered from severe neurological depression and HAGMA.

Performance Evaluation of Serum Free Light Chain Analysis: Nephelometry vs Turbidimetry, Monoclonal vs Polyclonal Reagents.

OBJECTIVES: Free light chain (FLC) measurement gained a lot of interest for diagnostic workup of monoclonal gammopathy. METHODS: We evaluated the performance of turbidimetric polyclonal Freelite (The Binding Site, Birmingham, UK) assays on Cobas 6000 (Roche Diagnostics, Rotkreuz, Switzerland) and nephelometric monoclonal N Latex (Siemens Healthcare Diagnostics, Marburg, Germany) assays on BN ProSpec (Dade Behring, Deerfield, IL) vs established nephelometric Freelite assays on BN ProSpec. RESULTS: Analytical performance was acceptable. Method comparison (n = 118) showed significant proportional FLC differences for N Latex assays. However, good correlation and clinical concordance were shown. Recovery study in the low concentration range demonstrated consistent over- and underrecovery for Freelite reagents, hampering future research on prognostic value of suppressed noninvolved FLC. Antigen excess detection was successful for κ FLC in three-fourths of cases with Freelite reagents and in all cases with N Latex reagents. However, the latter resulted in underestimated κ FLC concentrations. CONCLUSIONS: FLC analysis requires continuous awareness of analytical limitations. Monitoring of disease response requires FLC analysis on the same platform using the same reagents.

Antithrombin heparin binding site deficiency: A challenging diagnosis of a not so benign thrombophilia.

BACKGROUND: Hereditary antithrombin (AT) deficiency is a rare autosomal dominant disorder characterised by decreased AT activity in plasma and predisposition to recurrent venous thromboembolism (VTE). Thrombotic risk is thought to vary according to the subtype of deficiency, with Heparin Binding Site (HBS) deficiencies being the less thrombogenic. OBJECTIVES AND METHODS: The study population consisted of 82 genetically confirmed HBS deficient patients sharing six different mutations. Plasma samples of 35 of them, including one homozygous patient, were used for the evaluation of 4 commercial activity assays in their ability to diagnose HBS deficiency. We assessed mutation-specific prevalence of venous and arterial thrombosis and the contribution of additional thrombophilic risk factors. RESULTS AND CONCLUSIONS: Only one assay showed 100% sensitivity for all HBS mutations. The other ones failed mainly in the cases with p.Pro73Leu and p.Arg79His mutations. Shortening of incubation time resulted in an increase in sensitivity. In one patient, a novel HBS mutation, p.Asn77His, was identified, a quite exceptional and important finding given the restricted number of causal mutations reported so far in AT HBS deficiency. The overall prevalence of VTE in our study population (35%) was higher than previously reported (6-8%) in these patients. The presence of additional thrombophilic risk factors such as Factor V Leiden or prothrombin gene mutation G20210A contributed to a higher risk of VTE. Interestingly, the p.Pro73Leu and p.Arg79His mutations were associated with a high prevalence of arterial thrombosis. Our data suggest that AT HBS deficiencies are probably more prevalent and less benign than previously assumed.

Evaluation of the Sebia CAPILLARYS 2 flex piercing for the measurement of HbA(1c) on venous and capillary blood samples.

OBJECTIVES: We evaluated the Sebia CAPILLARYS 2 Flex Piercing (Cap 2FP; Sebia, Lisses, France) for measurement of hemoglobin A1c (HbA1c) on venous and capillary blood samples. METHODS: We analyzed whole-blood samples and control materials with the Cap 2FP and Tosoh G8 (Tosoh Corporation, Tokyo, Japan). Capillary blood samples were analyzed on the Cap 2FP on different storage conditions and were compared with venous samples. RESULTS: Both instruments achieved total imprecision of less than 2.5% (International Federation of Clinical Chemistry units). Bias was 1 mmol/mol or less and 4 mmol/mol or less for the Cap 2FP and Tosoh G8, respectively. The Cap 2FP was not prone to common interferences. The Tosoh G8 showed significant bias only for carbamylated hemoglobin and did not completely separate hemoglobin D and hemoglobin E. On the Cap 2FP, storage of capillary blood at room temperature showed no significant bias. There was good agreement with venous blood. CONCLUSIONS: The Cap 2FP and Tosoh G8 perform excellently for HbA1c determination. Capillary blood can be analyzed on the Cap 2FP as an acceptable alternative to venous blood and point-of-care testing. Home collection and central analysis of capillary blood could contribute to a reduction of health care costs without reducing quality of HbA1c determination.

Diagnostic potential of CD34+ cell antigen expression in myelodysplastic syndromes.

The World Health Organization introduced flow cytometry as an additional criterion for diagnosis of myelodysplastic syndromes (MDS). Aberrant antigen expression on bone marrow (BM) blasts may identify "low-grade MDS." This study aimed to examine differences in antigen expression on CD34+ BM cells between patients with MDS and those with secondary cytopenia. BM aspirates of 175 patients with cytopenia were classified as MDS or secondary cytopenia. Expression of stem cell antigens (CD34, CD133), myeloid antigens (CD13, CD33), B-cell antigens (CD19, CD10), growth factor receptors (CD117, CD123), and chemokine receptor (CD184) was examined. Thirty-two normal adults and 49 patients with CD34+ acute myeloid leukemia (AML) were also examined. High percentage of CD34+ cells, CD117 and CD123 overexpression, and abnormal CD45 expression on these cells are the best markers for MDS. These phenotypic aberrancies correlate with number of blasts and degree of dysplasia, and were similar to those in CD34+ AML, thus reflecting the relationship between these disorders.

Use of likelihood ratios improves interpretation of laboratory testing for pulmonary sarcoidosis.

Laboratory tests for pulmonary sarcoidosis (percentage lymphocytes and CD4/CD8 ratio in bronchoalveolar lavage fluid and serum angiotensin-converting enzyme activity) lack sensitivity and specificity. In a retrospective study of 153 subjects under suspicion of pulmonary sarcoidosis (36 cases and 117 patients with other diseases [control patients]), we defined likelihood ratios (LRs) for rationally selected result intervals of these tests, which improve clinical interpretation as compared with dichotomous interpretation based on a single cutoff value. By using logistic regression analysis, we further integrated the 3 individual tests into a unified algorithm that could rule out diagnosis in 57 (48.7%) of the 177 control subjects and confirm diagnosis in 12 (33%) of the 36 pulmonary sarcoidosis cases. We conclude that use of LRs improves interpretation of laboratory tests for pulmonary sarcoidosis. In addition, we present a prediction algorithm based on the combination of laboratory tests that helps clinicians confirm or exclude diagnosis in almost half of the study population.