Routine Laboratory Setting Research Articles

Vivax malaria detection using a parasitic red blood cell flag generated by the Sysmex XN-9000 hematology analyzer.

A Sysmex XN-series hematology analyzer (Sysmex), the next generation up from the Sysmex XE-series, can provide information regarding malaria infection in the form of a parasitic red blood cell (pRBC) flag. This study aimed to determine the usefulness of the pRBC flag for early detection and follow-up in patients infected with Plasmodium vivax. A total of 221 patients with fever for whom CBC and malaria microscopy had been requested were analyzed. Sixty-seven individuals were diagnosed with P vivax infection, and 154 were diagnosed with other febrile diseases. The sensitivity and specificity of the pRBC flag for malaria parasite detection and the relationship between parasite density and presence of the pRBC flag were determined. The concordance rate between malaria microscopy and pRBC flag in 147 follow-up cases was calculated. The pRBC flag was detected in 56 of 67 malaria patients (sensitivity, 83.6%; specificity, 100%). The patients with the pRBC flag at initial diagnosis revealed significantly higher parasite density than the patients without the pRBC flag (P<.05). The concordance rate between malaria microscopy and pRBC flag in the follow-up cases was 53.1%. Considering its high sensitivity in malaria-suspicious patients, unexpected vivax malaria cases can be detected with the pRBC flag when CBC is done in a routine laboratory setting. The pRBC flag provided by the Sysmex XN series is a valuable tool for vivax malaria detection.

Rapid typing of carbapenem-resistant Acinetobacter baumannii and Acinetobacter nosocomialis by multiplex Pan- and OXA-PCR assays.

Introduction. Outbreaks of carbapenem-resistant A. baumannii and A. nosocomialis have occurred worldwide in healthcare settings. Rapid and reliable molecular typing of bacterial isolates is vital for the effective surveillance of institutional outbreaks. The Pan-PCR and OXA-PCR assays are two multiplex PCR-based assays for the molecular typing of Acinetobacter species.Gap statement. However, few studies have investigated the discriminatory power of two multiplex PCR assays in in the genotyping of Acinetobacter species.Aim. We aimed to evaluate the efficacies of the Pan-PCR and OXA-PCR assays for molecular typing of A. baumannii and A. nosocomialis.Methodology. A total of 105 carbapenem-resistant A. baumannii isolates (CRABs) and 93 carbapenem-resistant A. nosocomialis isolates (CRANs) obtained from blood cultures were used for molecular typing by the Pan-PCR and OXA-PCR assays and two multilocus sequence typing (MLST) schemes.Results. The isolates were individually divided into 12 and 21 different sequence types via the Pasteur and Oxford MLST schemes, respectively. Additionally, these isolates were distinguished into 18 different types by the Pan-PCR and OXA-PCR assays. The results of the Pan-PCR and OXA-PCR assays distinguished CRABs and CRANs with a sensitivity of 98.13 % and a specificity of 100 %.Conclusion. The Pan-PCR and OXA-PCR assays are promising alternative methods for rapid molecular typing of CRABs and CRANs in a routine laboratory setting.

Evaluation of the Succeeder SF-8200 Fully Automated Coagulation Analyzer.

The SF-8200 is a new coagulation analyzer developed by Beijing Succeeder Technology Inc., China. The SF-8200 is able to perform clotting, chromogenic, and immunoturbidimetric tests. System capability is a throughput of up to 360 tests per hour, 60 sample tubes can be loaded at any time, and its reaction cuvette capacity is 1,000. The analyzer also has an optional cap piercing module to reduce manual sampling time. We aimed to perform an analytical performance comparison study between Succeeder SF-8200 and Stago Compact Max3 because fully automated coagulation analyzers have become one of the most essential components of clinical laboratories. Routine coagulation tests were assessed, which are the most ordered in laboratories such as PT, aPTT, and fibrinogen. Stago Compact Max-3 was accepted as the reference instrument in the comparison study. The assay precisions were assessed using fresh and pooled plasma samples or consumer internal quality controls. Hemolysis, lipemia, and icterus interferences were also verified. The coefficients of variation assessed in the intra and inter-assay precision analyses were below 5% representatively for assessed parameters. The inter-analyser comparison demonstrated good results. Results obtained by the SF-8200 showed high comparability predominantly to used reference analyzers, with correlation coefficients ranging from 0.953 to 0.976. In our routine laboratory setting, SF-8200 reached a sample throughput rate of 360 tests per hour. No considerable influence on tests was found for elevated levels of free hemoglobin, bilirubin, or triglycerides. In conclusion, the SF-8200 was an accurate, precise, and reliable coagulation analyzer in routine testing. According to our study, the results demonstrated excellent technical and analytical performance.

Asymmetric dimethylarginine–A potential cardiac biomarker in horses

Introduction/objectivesAsymmetric dimethylarginine (ADMA) is a cardiac biomarker in humans, symmetric dimethylarginine (SDMA) a renal biomarker in humans, cats, and dogs. The purpose of this prospective study was to investigate if measuring serum ADMA and SDMA concentrations via ELISA allows detection of cardiac disease in horses in a routine laboratory setting. In this context, reference values in horses were established. Animals, materials, and methodsSeventy-eight horses with no known medical history were compared to 23 horses with confirmed structural cardiac disease with/or without arrhythmias. Horses underwent physical examination, electrocardiography, echocardiography and venous blood sampling and were staged based on the severity of cardiac disease from 0 to II. Asymmetric dimethylarginine and SDMA were measured via ELISA and crosschecked using liquid chromatograph triple quadrupole mass spectrometry. Reference intervals with 90th percent confidence intervals were evaluated and standard software was used to test for significant differences in ADMA, SDMA, and the l-arginine/ADMA ratio between groups. ResultsThe reference ranges were 1.7–3.8 μmol/L and 0.3–0.8 μmol/L for ADMA and SDMA, respectively. Serum ADMA was higher in horses with heart disease compared to healthy horses (p < 0.01) and highest in horses with stage II heart disease (p = 0.02). The l-Arginine/ADMA ratio was significantly higher in healthy animals than those with cardiac disease (p = 0.001). ConclusionsReference values for serum ADMA and SDMA using ELISA methods are presented in horses. This study confirms the association between heart disease and increased serum ADMA concentration as well as a decreased l-Arginine/ADMA ratio in horses.

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Anti-neutrophil cytoplasmic antibody testing by indirect immunofluorescence: Computer-aided versus conventional microscopic evaluation of routine diagnostic samples from patients with vasculitis or other inflammatory diseases

BackgroundDetection of anti-neutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence assays (IFA) is of diagnostic importance in vasculitides and some other inflammatory diseases. Automation of IFA may be beneficial in high-throughput clinical laboratories. An analytical appraisal of the EUROPattern (EPa) automated microscope and image analysis system has not been reported in a routine clinical laboratory setting testing samples from both vasculitis and non-vasculitis patients. MethodsResults of EPa and on-screen ANCA pattern recognition of 568 consecutive routine serum samples were compared to those of conventional visual evaluation. ResultsAgreement of discrimination between negative and non-negative samples was 86.1% comparing EPa and conventional reading, and it increased to 96.7% after on-screen user validation. Importantly, from the 334 samples classified as negative by EPa 328 (98.2%) were also negative by conventional evaluation. Pattern recognition showed ‘moderate’ agreement between classical microscopic and EPa analysis (κ = 0.446) and ‘very good’ agreement after user validation (κ = 0.900). Misclassification by EPa was dominantly due to the presence of anti-nuclear/cytoplasmic antibodies (incorrect pattern, 80/568) and the lower fluorescence cut-off of the automated microscope (false positives, 73/568). ConclusionsAutomated ANCA testing by EPa is a reliable alternative of classical microscopic evaluation, though classification of sera needs correction by trained personnel during on-screen validation.

Rapid Molecular Detection for Differentiation of Homozygous HbE and ß0-Thalassemia/HbE in Samples Related With HbE >80% and Variable HbF Levels.

To validate a novel rapid molecular testing method for differentiation of homozygous hemoglobin (Hb)E and HbE/β 0-thalassemia genotypes using multiplex melt curve combined with high-resolution melt (HRM) analysis in a single test tube. All 10 genotypes contained (β N/β N; n = 95), (β N/β 3.5-kb; n = 71), (β N/β 45-kb; n = 28), (β N/β E; n = 10), (β E/β 3.5-kb; n = 6), (β E/β 45-kb; n = 4), (β E/β 41/42; n = 28), (β E/β 17; n = 9), (β E/β IVSI#1; n = 6), and (β E/β E; n = 76) were recruited for validation. A proposed strategy for rapid differentiation of β 0-thalassemia/HbE disease and homozygous Hb E in specimens with HbE greater than 80% and variable HbF levels was demonstrated. In the validation method, all genotypes showed 100% concordance, compared with the conventional reverse dot blot (RDB) and gap-polymerase chain reaction (PCR) methods. Our newly developed method could be useful in routine laboratory settings. The method is rapid, simple, and cost effective; does not require a post-PCR step; and can be applied in routine settings.

System performance evaluation of the cobas t 711 and cobas t 511 coagulation analyzers in routine laboratory settings

Utility of coagulation analyzers in real-world settings depends on characteristics that are often not studied comprehensively. This study aimed to investigate the analytical performance, system functionality, practicability, consistency and throughput of two new automated coagulation analyzers in routine laboratory practice. Real-world settings were simulated in three major European hemostasis laboratories and multiple assays were performed in anonymized plasma samples in parallel with routine clinical practice on the cobas t 711 (high-throughput) and cobas t 511 (mid-throughput) analyzers using activated partial thromboplastin time (aPTT), aPTT Lupus, aPTT Screen, Antithrombin (AT), d-Dimer, Fibrinogen, Prothrombin Time (PT)-derived Fibrinogen, PT Owren, PT Rec (recombinant human thromboplastin reagent) and Thrombin Time assays. Precision was tested in a 21-day experiment and accuracy was compared with reference methods of the same laboratory. A number of experiments simulated challenging real-life situations. Pearson's correlation coefficient was more than 0.98 in all assays. Across assays, coefficients of variation ranged from 0.0 to 1.5% for intermediate precision; 0.2 to 3.0% for repeatability and 0.4 to 3.7% for total precision. Good between-run comparability was seen when testing samples under random conditions. Calculated maximum throughput was 197 and 387–402 tests/h for the cobas t 511 and 711 analyzers, respectively. Practicability met or exceeded user expectations in 98% of cases. In a simulated real-life setting of three major laboratories, the new cobas t 511 and cobas t 711 coagulation analyzers demonstrated a good functionality, practicability and performance and the throughput was high.

Therapeutic drug monitoring of valproic acid using a dried plasma spot sampling device.

Valproic acid (VPA) dosing needs to be individualized for epilepsy patients through therapeutic drug monitoring (TDM). The patients must show up in the clinic at the therapeutic window time to venipuncture sample. Dried plasma spot (DPS) sampling is an alternative way to replace conventional venipuncture sampling. The aim of this study was to develop and validate a DPS-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to monitor VPA in a routine clinical laboratory setting. We compare the DPS with the wet plasma method of clinical samples by LC-MS/MS. The method was linear over the dynamic range of 10-200 μg/ml (covering entire therapeutic range) with a correlation coefficient r2 ≥ 0.995. Both the DPS and wet plasma methods were fully validated for the accuracy, precision, recovery, and matrix effect. The analyte stability was examined under conditions mimicking the sample storage, transport, and analysis procedures. A clinical study with epilepsy patients receiving VPA (n = 35) showed that, after correction for hematocrit (HCT), plasma concentrations can be successfully calculated from the DPS quantification results. Passing-Bablok regression coefficients showed no proportional bias between estimated and measured plasma concentrations. Similar agreement was found by Bland-Altman plots. The dried sample could be mailed to the clinical lab to test by regular mail service. So DPS can be used for drug monitoring with self-sampling strategy at the patient's convenient time and place specially for ambulatory patients not attending a clinic.

Reduction of non-typeable results using a plasmid oriented Lymfogranuloma venereum PCR for typing of Chlamydia trachomatis positive samples.

Typing of Chlamydia trachomatis (CT) is traditionally performed by characterising the ompA gene, resulting in more than a dozen different genovars, A to L. Type L is associated with Lymphogranuloma venereum (LGV) and commonly screened for using PCR, targeting the chromosomal pmpH gene. We aimed to develop and validate a new CT/LGV plasmid-based typing assay targeting the pgp3 gene, to increase sensitivity and thus reduce the number of non-typeable results. The new pgp3 PCR assay using LNA probes to detect point mutations was analytically and prospectively validated in a routine diagnostic laboratory setting. For the analytical tests, quantified nucleotide constructs (gBlocks) were used to perform limit of detection analyses. Quality control panel samples from 2018 and 2019 for CT were also tested. For the clinical study patient samples which were collected in two months in 2018 were tested simultaneously using the pmpH PCR and the pgp3 PCR. Analytically, the assay proved to be 100% specific relative to the previously used LGV typing assay targeting the single copy pmpH gene but it was much more sensitive to detect non-LGV CT. In the quality control panel 2 nonLGV samples and 7 LGV samples were solely positive with the pgp3 PCR and not with the pmpH PCR. None of the samples from analytical specificity panels were positive, indicating 100% specificity. In a prospective panel of 152 clinical samples, 142 (93%) were successfully typed with the pgp3 PCR compared to 78% with the pmpH PCR. The pgp3 PCR was fully concordant with the pmpH PCR to identify all LGV subtypes and detected an increased number of clinical samples of non-LGV subtype. We developed and validated a sensitive and specific plasmid-based typing assay to discriminate LGV from non-LGV CT subtypes. This is useful in a clinical setting to quickly determine the optimal treatment for Chlamydia trachomatis infections.

Diagnosing Herpetic Genital Ulcers in a Resource-Limited Setting- What Works?

Aims: Globally, viral agents, especially herpes simplex virus (HSV), have overtaken the bacterial causes of genital ulcers. Very few laboratories in India, perform culture techniques and polymerase chain reaction (PCR) for diagnosis of genital ulcers. This study aimed to establish the utility of existing tests, which are cheaper and need less technical expertise, when compared to newer tests such as PCR.&#x0D; Study Design: This cross sectional study was carried out to determine the aetiology of genital ulcers, with emphasis on diagnosis of herpetic ulcers, using newer and more accurate methods of diagnosis and evaluating their performance by comparing against viral culture as gold standard test.&#x0D; Place and Duration of the Study: The study was carried out over a period of one year in the Apex Regional Sexually Transmitted Diseases (STD) Centre at Safdarjung Hospital, New Delhi and the Department of Microbiology, AIIMS, New Delhi.&#x0D; Methodology: Fifty three patients with genital ulcers were included in the study. Specimens from ulcers were taken for various tests, including Giemsa stain, ELISA for HSV-1 &amp; 2, PCR and Viral culture for HSV.&#x0D; Results: HSV was identified in 31 of 53 cases (58.5%), including 03 cases of HSV-1, and 28 cases of HSV-2. Sensitivity and specificity of PCR was 90.0% and 84.85%, respectively. Viral culture positivity was 37.7%.&#x0D; Conclusion: Genital herpes is associated with an increased risk of Human Immunodeficiency Virus (HIV) acquisition, and clinical manifestations are diverse; hence a presumptive diagnosis should be confirmed by reliable laboratory tests. Nucleic acid amplification tests (NAAT) are the most sensitive methods for direct detection of HSV. The extensive validation of these tests allows for their application in routine laboratory settings with consistency and greater diagnostic accuracy. When standardised and used, PCR is a highly reproducible, rapid and labour efficient method for HSV detection.

Routine diagnostics for neural antibodies, clinical correlates, treatment and functional outcome

ObjectiveTo determine frequencies, interlaboratory reproducibility, clinical ratings, and prognostic implications of neural antibodies in a routine laboratory setting in patients with suspected neuropsychiatric autoimmune conditions.MethodsEarliest available samples from 10,919 patients were tested for a broad panel of neural antibodies. Sera that reacted with leucine-rich glioma-inactivated protein 1 (LGI1), contactin-associated protein-2 (CASPR2), or the voltage-gated potassium channel (VGKC) complex were retested for LGI1 and CASPR2 antibodies by another laboratory. Physicians in charge of patients with positive antibody results retrospectively reported on clinical, treatment, and outcome parameters.ResultsPositive results were obtained for 576 patients (5.3%). Median disease duration was 6 months (interquartile range 0.6–46 months). In most patients, antibodies were detected both in CSF and serum. However, in 16 (28%) patients with N-methyl-d-aspartate receptor (NMDAR) antibodies, this diagnosis could be made only in cerebrospinal fluid (CSF). The two laboratories agreed largely on LGI1 and CASPR2 antibody diagnoses (κ = 0.95). The clinicians (413 responses, 71.7%) rated two-thirds of the antibody-positive patients as autoimmune. Antibodies against the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), NMDAR (CSF or high serum titer), γ-aminobutyric acid-B receptor (GABABR), and LGI1 had ≥ 90% positive ratings, whereas antibodies against the glycine receptor, VGKC complex, or otherwise unspecified neuropil had ≤ 40% positive ratings. Of the patients with surface antibodies, 64% improved after ≥ 3 months, mostly with ≥ 1 immunotherapy intervention.ConclusionsThis novel approach starting from routine diagnostics in a dedicated laboratory provides reliable and useful results with therapeutic implications. Counseling should consider clinical presentation, demographic features, and antibody titers of the individual patient.

Relation between cell-bound exopolysaccharide production via plasmid-encoded genes and rugose colony morphology in the probiotic Lactobacillus brevis KB290.

The probiotic Lactobacillus brevis KB290 is a natural producer of cell-bound exopolysaccharide (EPS), and the plasmid-encoded glycosyltransferase genes are responsible for this EPS production. KB290 forms unique rugose colonies inside an agar medium; this characteristic is useful for detecting and enumerating KB290 in the gut or feces. However, the genetic elements associated with this morphology remain unclear. Here, we aimed to investigate the relation between the plasmid eps genes and rugose colony morphology in KB290. The plasmid-cured mutants formed smooth colonies, and the rugose colony morphology was restored after complementation with the eps genes. The eps genes were successfully cloned and expressed in other L. brevis and L. plantarum strains. In these transformant strains, the presence of the EPS, consisting of glucose and N-acetylglucosamine, correlated with rugose colonies, indicating that EPS is responsible for rugose colony formation. To the best of our knowledge, this is the first report identifying the genetic factors influencing rugose colonies in Lactobacillus strains. This rugose colony formation may serve as a useful selective marker for KB290 in routine laboratory and research settings and can be used to detect the spontaneous loss of plasmids in this strain.

P513 Performance of a rapid test for adalimumab monitoring vs. conventional ELISA in a routine laboratory setting

P513 Performance of a rapid test for adalimumab monitoring vs. conventional ELISA in a routine laboratory setting

Carbohydrate-deficient transferrin: utility of HPLC in handling atypical samples uninterpretable by capillary electrophoresis

Carbohydrate-deficient transferrin (CDT) is a marker of chronic alcohol abuse. Uninterpretable (atypical) CDT patterns have been detected by both capillary electrophoresis (CE) and HPLC. The aim of this study was to evaluate the performance of HPLC as a second-line test for the interpretation of most frequent atypical CDT profiles detected by CE. CDT was analyzed by CE (Capillarys 2, Sebia) on 9120 consecutive samples in a routine laboratory setting during a 2-year period. A commercial method (ClinRep CDT kit, Recipe) was employed to retest 123 (1.4%) samples with atypical CDT patterns on a Prominence LC-20AT HPLC (Shimadzu). CE-uninterpretable samples were categorized as having low transferrin (Tf) concentration (LT; n = 42, 0.5%), di-trisialotransferrin bridging (D-TB; n = 63, 0.7%) or atypical peak profile (APP; n = 18, 0.2%). CDT was detectable by HPLC in 58 of 123 (47%) samples including 21of 42 (50%) with LT, 27 of 63 (43%) with D-TB and 10 of 18 (56%) with APP. Second-line HPLC testing reduced uninterpretable samples by 47%, with similar rates of improvement regardless of the type of CDT pattern. The usefulness of HPLC as a second-line test for CDT should be evaluated according to cost-benefit considerations in the context of each laboratory.

Advantages of the AdvanSure MDR-TB GenoBlot assay containing disputed rpoB mutation-specific probes in a routine clinical laboratory setting.

The AdvanSure MDR-TB GenoBlot Assay detects isoniazid- and rifampin-resistant tuberculosis using mutation-specific probes, including probes to disputed rpoB mutations. The aim of this study was to evaluate the clinical usefulness of molecular drug susceptibility testing (DST) using the AdvanSure assay with weekly batch testing in routine clinical laboratory settings in a country with an intermediate tuberculosis burden. The AdvanSure assay was evaluated against an absolute concentration (AC) method and the Mycobacterial Growth Indicator Tube (MGIT) 960 System, which are phenotypic DST methods, using 496 Mycobacterium tuberculosis (MTB) isolates. We retrospectively reviewed and compared DST results and turnaround times (TATs), the time intervals from MTB culture identification to final reporting, for these methods. For rifampin, the AdvanSure assay showed 99.2% (492/496) concordance with both the AC and MGIT methods. AdvanSure also detected an rpoB mutation (D516Y) conferring low-level resistance in three isolates categorized as rifampin-susceptible by both phenotypic DST methods. For isoniazid, AdvanSure concordance rates with the AC method and MGIT DST were 96.6% (479/496) and 95.4% (473/496), respectively. The median TAT of AdvanSure in weekly batch testing was 5.8 days, shorter than the times for the phenotypic DST methods, which were 35.1 days for the AC method and 8.9 days for MGIT DST. AdvanSure shows promising clinical usefulness for rapid detection of rifampin- and/or isoniazid-resistant tuberculosis when used as a complementary method to phenotypic DST assays in weekly batch testing. Furthermore, MTB isolates with disputed mutations for rifampin resistance were detectable by the AdvanSure assay.

Diagnosis of feline mesenchymal nasal hamartoma by squash preparation cytology.

Feline Mesenchymal Nasal Hamartoma (MNH) is a rare benign tumor-like lesion of the sinonasal tract affecting young cats. This study aimed to determine the diagnostic significance of osteoblast-like (OB-L) and osteoclast-like cells (OC-L) in squash preparation cytology from endoscopic biopsies. A 5-year database was retrospectively reviewed and included 109 cases of which 24 were diagnosed as MNH by histopathology. Slides were examined by two cytologists (one experienced and one inexperienced in nasal and squash cytology) in a double-blind study. The inexperienced cytologist counted OB-L and OC-L in 500 intact nucleated cells. The experienced cytologist assigned samples to four categories for OB-L (0, 1-5, 6-10, >10/field) and OC-L (0, 1-2, 3-5, >5/field). The presence of OB-L and OC-L was significantly associated (P<0.001) with the histologic diagnosis of MNH. Receiver operating characteristic curves from the counts by the inexperienced cytologist revealed 3/500 OB-L and 2/500 OC-L as the best cut-offs for the diagnosis of MNH. Those of the experienced cytologist evaluation revealed that all the MNHs presented more than 10 OB-L/field and 3 or more OC-L/field. Both cytologists detected each cell type in all MNHs with an overall concordance of 0.93. The presence of OB-L and OC-L is a consistent finding in MNH, and thus, represents a reliable cytologic diagnostic criterion. The described methods are applicable in routine in-clinic laboratory settings and are easy to apply at any expertise level. Further prospective studies are needed to assess the accuracy of the proposed cut-off values.

Easy Employment and Crosstalk-Free Detection of Seven Fluorophores in a Widefield Fluorescence Microscope.

Immunofluorescence staining has become an essential tool in pathology and biomedical sciences to identify rare cells, cell–cell interactions, and submicroscopic cellular components. Many experimental settings, however, suffer from the fact that traditional widefield fluorescence microscopy is usually restricted to imaging three or four fluorophores only. Due to a lack of morphological information and a high detection limit, even flow cytometry—which is capable of staining 20 or more fluorophores at the same time—is limited in its applicability, especially in areas such as rare cell detection. Other advanced imaging approaches, such as confocal laser scanning microscopy and imaging flow cytometry, may be addressing these shortcomings, but in turn require sophisticated downstream data processing and high capital outlay. Here, we describe a new method and filter set-up to routinely employ up to seven fluorophores on a traditional widefield fluorescence microscope equipped with a standard high-pressure mercury light source. Quantification of crosstalk between channels and actual seven-color imaging of cancer cells spiked into leukocytes demonstrate that there is no need for digital compensation correction algorithms. Our set-up thus permits a detailed analysis of rare cell populations, co-localization of antigens, and cell morphology in a standard research or routine laboratory setting.

Next Generation Sequencing in Molecular Diagnosis of Lynch Syndrome - a Pilot Study Using New Stratification Criteria.

The development of the new technologies such as the next-generation sequencing (NGS) makes more accessible the diagnosis of genetically heterogeneous diseases such as Lynch syndrome (LS). LS is one of the most common hereditary form of colorectal cancer. This autosomal dominant inherited disorder is caused by deleterious germline mutations in one of the mismatch repair (MMR) genes - MLH1, MSH2, MSH6 or PMS2, or the deletion in the EPCAM gene. These mutations eventually result in microsatellite instability (MSI), which can be easily tested in tumor tissue. According to the actual recommendations, all patients with CRC that are suspect to have LS, should be offered the MSI testing. When the MSI is positive, these patients should be recommended to genetic counseling. Here we report a pilot study about the application of NGS in the LS diagnosis in patients considered to have sporadic colorectal cancer. The inclusion criteria for the NGS testing were MSI positivity, BRAF V600E and MHL1 methylation negativity. We have used 5 gene amplicon based massive parallel sequencing on MiSeq platform. In one patient, we have identified a new pathogenic mutation in the exon 4 of the MSH6 gene that was previously not described in ClinVar, Human Gene Mutation Database, Ensembl and InSight databases. This mutation was confirmed by the Sanger method. We have shown that the implementation of new criteria for colorectal patients screening are important in clinical praxis and the NGS gene panel testing is suitable for routine laboratory settings.

Carbamazepine, lamotrigine, levetiracetam and valproic acid in dried blood spots with liquid chromatography tandem mass spectrometry; method development and validation.

Monitoring of antiepileptic drugs in children with epilepsy require multiple visits at a clinic for blood collection. Dried blood spot sampling is an alternative way of collection, performed at home by self-collection and can save time and costs for patients and family members. The aim was to develop and validate an LC-MS/MS dried blood spot method for carbamazepine, lamotrigine, levetiracetam and valproic acid with the requirements of using standard equipment and material in a routine laboratory setting. Whatman-903 filter paper was utilized, and discs were punched into a 96 well plate with an automated puncher and barcode reading. Extraction with methanol/water solution including internal standards on an orbital shaker was followed by a vacuum centrifuge step and reconstitution in mobile phase. Bioanalytical validation was performed according to guidelines from European Medicines Agency and additional dried blood spot specific validation. Calibration curves of the four included drugs had R2 values ≥0.994. Therapeutic relevant concentrations were well within measuring ranges. Within and -between run precision had %CV:s of 2.9-10.5%. Accuracy (%bias) was between -16.5% (lower limit of quantification) to +7.4%. Blood spots in a volume range of 15-50μL with hematocrit in expected ranges for this patient group were within precision and accuracy limits. To test the method, concentrations from dried blood spot venous and capillary patient samples (n=50) were compared with plasma concentrations. Good correlations for all four drugs with R2 of >0.92 was shown. In summary, a fast method for dried blood spots based on a 96 well format was developed for four commonly prescribed antiepileptic drugs. This validated method with traceability in sample preparation by bar code reading makes it suitable for the clinical laboratory.