Clinical Diagnostic Laboratories Research Articles

Application Areas of Traditional Molecular Genetic Methods and NGS in relation to Hereditary Urological Cancer Diagnosis.

Next generation sequencing (NGS) is widely used for diagnosing hereditary cancer syndromes. Often, exome sequencing and extended gene panel approaches are the only means that can be used to detect a pathogenic germline mutation in the case of multiple primary tumors, early onset, a family history of cancer, or a lack of specific signs associated with a particular syndrome. Certain germline mutations of oncogenes and tumor suppressor genes that determine specific clinical phenotypes may occur in mutation hot spots. Diagnosis of such cases, which involve hereditary cancer, does not require NGS, but may be made using PCR and Sanger sequencing. Diagnostic criteria and professional community guidelines developed for hereditary cancers of particular organs should be followed when ordering molecular diagnostic tests for a patient. This review focuses on urological oncology associated with germline mutations. Clinical signs and genetic diagnostic laboratory tests for hereditary forms of renal cell cancer, prostate cancer, and bladder cancer are summarized. While exome sequencing, or, conversely, traditional molecular genetic methods are the procedure of choice in some cases, in most situations, sequencing of multigene panels that are specifically aimed at detecting germline mutations in early onset renal cancer, prostate cancer, and bladder cancer seems to be the basic solution for molecular genetic diagnosis of hereditary cancers.

Laboratory monitoring analysis and prospects for using its results in specialized clinical diagnostic laboratory

This article presents a retrospective analysis of the laboratory monitoring orders from the specialized clinical laboratory. It has been established that large amounts of information about rare, poorly understood diseases and special types of pathology are accumulated during the everyday activity of specialized clinical laboratory. This research indicated that the share of laboratory monitoring, with 17 % of all orders, has a tendency to increase, which may be associated with the improvement of laboratory diagnostic methods. The possibility of laboratory monitoring results using in order to facilitate diagnostic process by the means of an optimal examination algorithm compiling, as well as to study regional pathology, is discussed. Besides, the prospects of using information on laboratory monitoring in improving the organization of specialized clinical laboratory’s activity are assessed.

Validation of a next generation sequencing assay for BRCA1, BRCA2, CHEK2 and PALB2 genetic testing

BRCA1, BRCA2, CHEK2 and PALB2 genes are associated with hereditary breast and ovarian cancer syndrome. Genetic testing of these genes is of increasing importance to guide therapeutic and management decisions. In this study, we evaluated the performance of a next generation sequencing (NGS) assay for the complete analysis of BRCA1, BRCA2, CHEK2 and PALB2 genes using Agilent's SureMASTR BRCA Screen that enabled the detection of single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variations (CNVs) in a single-tube PCR based library preparation. The results showed 100% sensitivity and specificity on a set of 52 known samples from de-identified patients and external quality assessment program. A concordance rate of 87.5% was achieved in the comparison of variant classification with the external laboratories. The high accuracy of the assay supports the use of SureMASTR BRCA Screen in clinical diagnostic laboratories (SureMASTR BRCA Screen is for research use only, not for use in diagnostic procedures).

Precision of autoantibody assays in clinical diagnostic laboratories: What is the reality?

BackgroundISO 15189 accreditation remains a challenge for specialized laboratories. In the field of autoimmunity, beside the crucial problem of absence of standardization, laboratories have to manage the analytical performances of the large panel of assays in terms of sensitivity and specificity, but also on their measurement precision for which no reference values are available on biorepositories. MethodsAs an initiative of the French EASI (European Autoimmunity Standardization Initiative) group, French clinical diagnostic laboratories were requested to participate in a survey aiming to analyze the coefficients of variation (CVs) of intra-run and inter-run variability obtained with assays quantifying 14 different autoantibodies. Two performance goals corresponding to the 90th percentile and the 50th percentile (lowest CV values reached by 90% and 50% of laboratories respectively) defined for three levels of concentration were calculated. The impact on the assay performances of the number of measurements, of the nature of the internal quality control (IQC) and the type of immunoassay, was also analyzed. Results414 and 616 values of intra-run and inter-run CVs were collected, respectively. The 50th percentile performance goals were comprised between 1.0% and 8.9% for the intra-run CVs, and between 1.8% and 14.6% for the inter-run CVs. At 90th percentile, the performance goals were comprised between 3.2% and 13.5% for the intra-run CVs, and between 7.3% and 30.8% for the inter-run CVs. CVs calculated from 10 values were similar to those obtained from more values. Higher imprecision was observed when the antibody levels of the IQC was lower than 2 fold the positive threshold. Commercial IQCs gave lower CVs than IQCs derived from patient samples. ConclusionOur results allow proposing some acceptability limits for the precision performances of the autoantibody assays, compatible with the reality of life in diagnostic laboratories and clinical care.

Likelihood Ratios as Value Proposition for Diagnostic Laboratory Tests.

The clinical and health economic value of clinical laboratory diagnostics has been debated increasingly in recent years without resulting in practical recommendations for measuring the effectiveness of diagnostic tests. One way to achieve such a goal could be to enrich the mere data of laboratory test results with additional information about their likelihood ratios for diagnosis. The diagnostic significance of test results can be judged subjectively based on the experience of the treating physician or expressed objectively in the form of likelihood ratios. The provision of likelihood ratios by the laboratory would increase the impact of laboratory diagnostics in healthcare and thus have positive economic value. Consequently, likelihood ratios should be taken into account in reimbursement strategies.

Clinical utility of next generation sequencing based HLA typing for disease association and pharmacogenetic testing

HLA associations have been linked to many diseases and are important for risk assessment of drug hypersensitivity reactions. The increasing number of HLA alleles discovered generated a list of ambiguities that cannot be resolved with the current clinical assays, which commonly include sequence-specific oligonucleotide probe (SSOP) genotyping, and real-time PCR with melting curve analysis. HLA typing by next-generation sequencing (NGS) has recently been adopted by clinical laboratories for transplantation testing, as it provides unambiguous and cost-effective HLA typing. The goal of this study was to evaluate the feasibility of using NGS-based HLA-B and DQ genotyping for clinical HLA disease association testing, and provide direct comparison with the currently used clinical tests, including SSOP genotyping, and real-time PCR with melting curve analysis. While the real-time PCR method is easy and inexpensive to perform, ambiguities are rapidly increasing as more and more HLA alleles are discovered. SSOP genotyping identifies the alleles present but limitations include ambiguities and underreporting less common alleles. Our data show that HLA typing by NGS is superior to the existing clinical methods for identifying HLA alleles associated with disease or drug hypersensitivity, and offers a viable approach for high volume clinical diagnostic laboratories.

AB1058 JOINT HYPERMOBILITY SYNDROME AND PRIMARY OPEN-ANGLE GLAUCOMA

Background:Eye symptoms: myopia, prolapse of the upper eyelid, epiblepharon in the upper eyelid are small diagnostic criteria for joint hypermobility syndrome (JHS).There are few publications in the literature on the relationship between JHS and primary open-angle glaucoma (POAG).It is known that in the development of JHS, the distribution of collagen of types I and III with the predominance of collagen of type III is important, the latter is encoded by the COL3A1 gene. When using POAG in the connective tissue of the middle and deep layers of the sclera by the immunohistochemical method, intense focal accumulation of type I and III collagen was previously revealed, and in the layers of the sclera’s own substance, type III collagen, unusual for it.Objectives:To study articular and extraarticular clinical manifestations, instrumental, laboratory signs, as well as to conduct molecular genetic studies on the carriage of the Col3Al gene in patients with a diagnosis of POAG and compare them.Methods:Nine consecutive patients with an established diagnosis of POAG (burdened heredity by glaucoma) with arthralgia were sent for consultation to the V.A. Nasonova Research Institute of Rheumatology from the Moscow Helmholtz Research Institute of Eye Diseases. All patients are women, the average age is 56.7 ± 10.5 years, the average Beighton score - 4.86 ± 1.7, the mean value of the Westergren ESR - 11,8 ± 5.1 mm/h, CRPhs- 4.9 ± 9.4 mg / 1, all of them were seronegative for rheumatoid factor (RE) and ACCP. All patients responded to the JHS diagnosis according to the 1998 Brighton diagnostic criteria. DNA was isolated from the leukocyte fraction of venous blood using the Wizard DNA Purification Kit (Promega) according to the manufacturer’s instructions. The study of gene polymorphisms was performed by the method of minisequencing with subsequent time-of-flight mass spectrometry of a sample on a matrix (MALDI-TOF) in the clinical diagnostic laboratory of NPF L1TEX LLC using a standard protocol (Wise C.A., 2003).Results:9 patients had arthralgia, 8 - vertebralgia, 3 - myalgia. 2 had a history of wrist joint dislocation, 7 had flat feet (3 of them had Hallucis valgus), 5 had spondylosis and spondylolisthesis (protrusions and disc herniation according to MRI of the spine), and 4 had excessive skin and / or striate atrophy of skin. Extraarticular manifestations: mitral valve prolapse was detected in 3 patients (in 1 of them + atrial septal defect) with ultrasound of the heart, in 3 - descent of the internal organs (nephroptosis, uterine prolapse), in 4 - pronounced varicose veins of the lower extremities. All patients had a carrier state of the A allele identified by marker C.2092G> A and C allele c.2244T> C of the COL3A1 gene, and a family history of glaucoma. Identification of compliance with JHS diagnostic criteria and the presence of genetic factors (COL3A1 gene) in patients with POAG is of great scientific importance, since it confirms not only clinical associations, but also the genetic proximity of these two conditions. It is also difficult to overestimate the practical value, since patients with POAG need the help of a doctor in the treatment of their articular and other non-ophthalmological manifestations of JHS, and establishing a diagnosis of JHS will require a more thorough examination of the eyes in terms of detecting POAG, its treatment or prevention.Conclusion:The association of JHS, POAG and COL3A1 gene necessitates further study of the association of JHS and POAG: POAG as a clinical manifestation of JHS, on the one hand, and the role of JHS as a possible risk factor for the development of POAG - on the other hand.Table 1.Demographic, clinical and laboratory characteristics of patients with Schnitzler’s syndromePtsThe age (y)The age at onset (y)Diagnosis delay (y)ESR (<15 mm/h)СRP (<6g/l)M-gradient (g/l)AnakinraCanakinumabThe treatment duration (y)151404311077,1015253282240295,7000336293140447,80134585331001927,6110,556966249965,1110,5Abbreviations: Pts – patients, y- years, ESR – erythrocyte sedimentation rate, CRP – C-reactive proteinDisclosure of Interests:None declared

Identification of salivary volatile organic compounds as potential markers of stomach and colorectal cancer: A pilot study

ObjectivesThe purpose of the pilot study was to determine the potential diagnostic capabilities for the analysis of oxygen-containing salivary volatile organic compounds (VOCs) in stomach and colorectal cancer. MethodsSaliva samples of 11 patients with stomach cancer, 18 patients with colorectal cancer, and 16 healthy volunteers were analyzed through capillary gas chromatography. The levels of lipid peroxidation products and catalase activity were determined in all samples. To assess saliva diagnostic potential, we constructed a Classification and Regression Tree (CART). ResultsIt was shown that the use of a combination of saliva VOCs (acetaldehyde, acetone, propanol-2, and ethanol) allowed classification into Cancer/Control groups with a sensitivity and specificity of 95.7 and 90.9%, respectively. To clarify the location of the tumor, it was necessary to add a methanol level; in this case, the sensitivity for detecting stomach and colorectal cancer was 80.0% and 92.3%, respectively, while the specificity in both cases was 100%. When the lipid peroxidation product content was added to the VOC indicators, they were selected as the main factors for constructing the decision tree. For classification into Cancer/Control groups, only the triene conjugate and Schiff base content in saliva was sufficient. The combination of VOCs in saliva and lipid peroxidation indices improved the sensitivity and specificity for classification to 100%. ConclusionPreliminary data were obtained on the sensitivity and specificity of the diagnosis of stomach and colorectal cancer, which confirmed the promise of further studies on saliva VOCs for the purpose of clinical laboratory diagnostics.

Phenotypic manifestation of homozygous partial deletion of the chromosome 1 segment spanning <i>CFHR3</i> region

This article presents two clinical cases of patients with a homozygous deletion of segment of chromosome 1, which covers regions of genes associated with complement factor H, in particular CFHR3. Patients underwent in-depth clinical studies, heredity assessment, laboratory, instrumental and genetic diagnostics. The first clinical case describes a clinical case with deleted chromosome 1 segment in a 9-year-old girl who was diagnosed with atypical hemolytic-uremic syndrome. This is a complement-dependent disease that affects both adults and children. It is known that a defect in any proteins included in the alternative complement activation pathway can lead to atypical hemolytic-uremic syndrome. However, this syndrome is most often caused by defects in chromosome 1 region, including gene sequences associated with complement factor H – CFHR1 and CFHR3. Modern treatment of atypical hemolytic uremic syndrome involves targeted pathogenetic treatment, therefore, the genetic diagnosis seems to be a necessary step for differential diagnosis and confirmation. The patient had fairly typical clinical symptoms, including signs of thrombotic microangiopathy, thrombocytopenia, hemolytic anemia and increasing renal failure. It is also known that her mother had congenital hydronephrosis, and the pregnancy proceeded against a background of ureaplasma, mycoplasma, cytomegalovirus infection, chronic pyelonephritis, and preeclampsia.The second clinical case of a deleted chromosome 1 region, involving the CFHR3 gene, is a description of the disease in a boy of 8 years old, while the disease manifested with alopecia at the age of 4. Intermittent alopecia was the main symptom, while there were no signs of renal failure, thrombocytopenic purpura, and other symptoms characteristic of atypical hemolytic-uremic syndrome. The boy also revealed some congenital defects of the urinary system: bladder diverticulum, unilateral ureterohydronephrosis, and bilateral dilatation of the pyelocaliceal system. The detected genetic defect is usually associated with atypical hemolytic uremic syndrome. However, the phenotype, i.e., clinical manifestations, determined a completely different diagnosis – primary immunodeficiency, a group of complement defects, and a deficiency of complement factor H-related protein. After analyzing the given clinical cases, we can conclude that clinical manifestations may vary significantly in carriers of same gene mutations. This suggests that there are additional factors (genetic or environmental) that can influence the formation of various phenotypic manifestations of this pathology.

FACSCanto II and LSRFortessa flow cytometer instruments can be synchronized utilizing single-fluorochrome-conjugated surface-dyed beads for standardized immunophenotyping.

BackgroundMultiparameter flow cytometry is the preferred method to determine immunophenotypic features of cells present in a wide variety of sample types. Standardization is key to avoid inconsistencies and subjectivity of interpretations between clinical diagnostic laboratories. Among these standardization requirements, synchronization between different flow cytometer instruments is indispensable to obtain comparable results. This study aimed to investigate whether two widely used flow cytometers, the FACSCanto II and LSRFortessa, can be effectively synchronized utilizing calibration bead–based synchronization.MethodTwo FACSCanto II and two LSRFortessa flow cytometers were synchronized with both multicolor hard‐dyed and single‐fluorochrome–conjugated surface‐dyed beads according to the manufacturer's instructions. Cell staining was performed on five whole‐blood samples obtained from healthy controls and were analyzed upon synchronization with the respective synchronization protocols.ResultsComparability criteria (defined as <15% deviation from the reference instrument) were met with both bead sets when synchronizing different FACSCanto II or LSRFortessa instruments. However, we observed that the criteria could not be met when synchronizing FACSCanto II with LSRFortessa instruments with multicolor hard‐dyed beads. By utilizing single‐fluorochrome–conjugated surface‐dyed beads to determine and adjust PMT voltages, the accepted comparability criteria were successfully met. The protocol has been validated using five different eight‐parameter stained samples.ConclusionWe show that FACSCanto II and LSRFortessa instruments can effectively be synchronized using single‐fluorochrome–conjugated surface‐dyed beads in case deviation criteria cannot be met using multicolor hard‐dyed beads. Synchronization with single‐fluorochrome–conjugated surface‐dyed beads results in decreased deviations between instruments, allowing comparability criteria to become stricter.

Clinical evaluation and performance of QIAGEN Clinical Insight Interpret (QCI-I) as a reporting tool for a comprehensive cancer panel (TSO 500).

e14048 Background: The rapid adoption of next-generation sequencing (NGS) methods into clinical diagnostic laboratories have revolutionized the field of genetic testing. To interpret the exhaustive NGS data, bioinformatics has become an inevitable component of any laboratory implementing a clinical NGS test. However, there is an unmet need for accurate, efficient and standardized variant interpretation for actionable insights. In this effort, AMP/ASCO/CAP released a joint consensus recommendation proposing a four-tiered system to categorize somatic alterations based on their clinical significance in cancer diagnosis, prognosis, or therapeutics for standardization of interpretation and reporting of results among laboratories. Thus, the aim of the study was to assess and validate QIAGEN Clinical Insight (QCI) Interpret software for clinical laboratory test interpretation. Methods: 67 malignant cases (myeloid neoplasm and lung) were analyzed using the TSO-500 NGS assay and secondary analysis platform (Illumina, San Diego, CA). The data was uploaded and analyzed using QCI Interpret. Patient case draft reports were produced using a TS0-500 customizable workflow with automated variant filtering, and variant classifications. Results: Data analysis on 67 cases resulted in a total of 251 alterations (SNVs and Indels). A total of 77 and 174 alterations were reported as pathogenic/ likely pathogenic into tier based classification by the QCI Interpret for myeloid and lung malignancies, respectively. For myeloid malignancies, the QCI Interpret was able to detect accurately all the clinically relevant variants and listed them into appropriate tier-based classification compared to the expertly reported alterations. For lung malignancies, 16 alteration were classified in tier 1, 96 alteration in tier 2, 61 alteration in tier 3, and 1 alteration in tier 4. The QCI interpret was able to detect 100% of the alterations being validated, and reported them into tier based classification. Conclusions: Systematic evaluation of an automated classification based on AMP/ASCO/CAP using an up to date knowledge base is critical to accurately classify actionable mutations and detect emerging relevant mutations. The QCI Interpret seems to be a critical bioinformatics tool that adheres to the AMP/ASCO/CAP classification system and incorporates TMB and MSI values from the TSO500 assay, which can be used across clinical laboratories for accurate, efficient and standardized variant interpretation for actionable insights.

SUN-720 LC-MS/MS Measurements of Parathyroid Hormone-Related Protein (PTHrP): Association Between Ca, PTH and PTHrP in Clinical Samples

Parathyroid hormone related protein (PTHrP) is involved in intracellular calcium (Ca) regulation and homeostasis. The main clinical utility of PTHrP as a biomarker is to assist with determining the cause of unexplained hypercalcemia (UH). Considering that PTHrP, along with parathyroid hormone (PTH), is involved in Ca homeostasis, we evaluated associations between concentrations of Ca, PTH, PTHrP and age, and the between-sex differences in clinical patient samples. We reviewed historic data on Ca, PTH and PTHrP concentrations measured in routine patient samples (n=2,701) submitted for testing to a large clinical diagnostic laboratory. Among the analysed samples 31.1% were from adults (18-59y), 52.9% from older adults (60-79y) and 16.9% from elderly (>79y). Out of the samples, 60.5% were from women (W) and 39.5% from men (M). PTHrP was measured using a validated LC-MS/MS method; Ca and parathyroid hormone (PTH) were measured using the Cobas 8000 (Roche). The imprecision of all assays was <10%. Reference intervals (RI) for PTHrP were <3.3 pmol/L and <2.2 pmol/L in W and M, respectively; RI for calcium was 8.4–10.2 mg/dL, and for PTH was 15–65 pg/mL. Median (mean) PTHrP concentrations in samples from adults, older adults and elderly were 1.5 (7.9), 2.5 (8.7) and 3.2 (5.3) pmol/L, respectively. Median (mean) concentrations in samples from W and M were 2.4 (6.3) and 2.5 (10.4) pmol/L, respectively. Statistically significant difference in PTHrP concentrations was observed between W and M from adult (p=0.0005) and older adult (p=0.0038) groups; between W from adult and elderly groups (p=0.0113); and between M from older adult and elderly groups (p=0.028). No significant difference among the age groups was observed in concentrations of Ca and PTH. As expected, direct relationship was observed between concentrations of Ca and PTHrP (ρ=0.189, p<0.0001), while inverse relationships were observed between concentrations of Ca and PTH (ρ=-0.393, p<0.0001), and PTH and PTHrP (ρ=-0.226, p<0.0001). Assessment of the distribution of PTHrP concentrations in samples from W (n=472) and M (n=446) with UH (Ca >10.2 mg/dL and PTH< 15 pg/mL), revealed elevated PTHrP concentrations in 35.0% of W and 59.4% of M; with odds ratio for PTHrP as cause of UH of 0.7 and 2.2, in W and M, respectively. Among the study samples, PTHrP concentrations in 29.3% of samples from W and 54.2% of samples form M were above the sex specific RI. The PTHrP positivity rates in adult, older adult and elderly W (M) were 22.1%, 28.2% and 44.2% (41.2%, 58.5% and 70.5%), respectively. We observed higher prevalence of elevated PTHrP concentrations in M than in W, and in older individuals. PTHrP concentrations above the RI were observed in 39.1% of the analyzed samples. Our data suggest that hypercalcemia caused by elevated PTHrP concentrations is more common for pathologic conditions, which have higher prevalence or specific of M.

Nonceliac Gluten Sensitivity—A Masquerading IBS or a Real Phenomenon?

The gluten-free diet has become popular among the public. People who are using this diet have reported symptom relief once gluten has been removed from their diet. Nonceliac gluten sensitivity (NCGS) has emerged as a new diagnosis for those patients who have tested negative for celiac disease. Although there are no diagnostic markers established for NCGS, its symptomatology ranges from gastrointestinal symptoms to neuropsychiatric symptoms. Indeed, some of these symptoms are also seen in patients with irritable bowel syndrome (IBS), such as abdominal pain, bloating, altered bowel movement, diarrhea, and constipation. It is important to add that unlike celiac disease, NGCS has never been associated with any long-term malignancy. We aim to review the recent clinical data available on this topic and address the overlap symptoms between NCGS and IBS. We concluded that despite the overlap symptoms between both diseases, NCGS is a real clinical phenomenon that awaits its own diagnostic clinical criteria and specific laboratory markers. We suggest that patients with gluten sensitivity who are negative for celiac disease should be considered for NCGS.

The new era for diagnostic microbiology laboratories: ‘All things are ready, if our mind be so’

Identification and characterization of micro-organisms that cause infections are crucial for successful management of patients. For several decades, routine clinical microbiological diagnostic laboratories have been equipped with a growing panel of culture-dependent and culture-independent methods to investigate the microbial etiology of infectious diseases. Considering the disadvantages of traditional methods, multiplex PCR techniques have been routinely endorsed in clinical microbiology laboratories as rapid and sensitive diagnostic and prognostic tool including many PCR panels routinely used in microbial diagnostics. The great advance in medical biotechnology has been associated with development of Matrix-Associated Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF MS). The power of MALDI TOF MS correctly identified 93.2% of organisms to the species level and 5.3% to the genus level. Recently, the application of next-generation sequencing (NGS) technology and its various methodological variants makes it possible to detect different types of microorganisms present within a microbial sample simultaneously, using a culture-independent approach and in a single sequencing run. Over the next 5 to 10 years, although it is unlikely to see NGS completely replacing the conventional culture and susceptibility methods, a wealth of NGS applications will be acquired in the vast majority of diagnostic microbiology laboratories worldwide, providing enhanced diagnostic capabilities and improving the quality of patients care. In Egyptian diagnostic microbiology laboratories, we have to ask ourselves; are we ready to subsist this new era? Sure the answer is: ‘All things are ready, if our mind be so’.

Laboratory diagnostics in medicine

The development of clinical laboratory diagnostics is in line with the evidence-based medicine, which requires that clinical decisions have to be based on diagnostic methods with proven informativity. This creates a request for the scientific validity of the use of laboratory researches and application of probabilistic interpretation tools corresponding to the tasks. The concept of indefiniteness (analytical, biological and clinical) is at the heart of interpretation of laboratory results. The inclusion of laboratory research in clinical guidelines, the choice and appointment of this research to the patient should not be made from the position of ideas about increasing or decreasing the laboratory index in the disease, but on the basis of its scientifically proven characteristics as a laboratory biomarker sensitivity, specificity, predictive value, as well as the relationship with certain clinical events, outcomes, risks. These characteristics are probabilistic and can be defined.

Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists.

The indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting. We developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting. One hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials. Based on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

A high performance liquid chromatography fluorescence method for the analysis of both pyridoxal-5-phosphate and thiamine pyrophosphate in whole blood

Background and aimsThe active coenzymes of the water soluble vitamins B1 and B6 (thiamine pyrophosphate (TPP) and pyridoxal-5-phosphate (P5P) respectively) play an important role in numerous bodily functions. The simultaneous analysis of both these analytes is limited to either mass spectrometry based methods or commercial kit suppliers. In this study we developed a novel method for analysis of both TPP and P5P by fluorescence detection. MethodsBriefly, whole blood samples are precipitated by trichloroacetic acid, and P5P and TPP are both derivatised before separation on a C18-PFP column. The new assay’s performance was compared against a recent cycle from an external quality assurance program (RCPAQAP) and the current only existing commercial kit (n = 76). ResultsLinearity for both analytes was above 0.99 (r2) up to a concentration range of: 4000 nmol/L (P5P) and 2000 nmol/L (TPP). Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 6% (coefficient of variation). Recovery of both compounds exceeded 90%. Accuracy of the protocol displayed satisfactory results in proficiency testing and had an acceptable level of agreement with the existing current kit method. ConclusionsOverall, this method provides an economical alternative in routine clinical diagnostic laboratories wishing to perform P5P and TPP analysis.

Competence-oriented exam in clinical laboratory diagnostics for students studying in the specialty «Medical Biochemistry».

In connection with the introduction of annexes and changes to the Federal State Educational Standard of Higher Education in the specialty 30.05.01 «Medical Biochemistry», a need arose to improve the assessment tool of general professional and professional competencies formation in the discipline - clinical laboratory diagnostics. A competence-oriented exam (COE) in the studied discipline is developed and implemented. Admission to the exam is the development of practical skills by students in the discipline modules, portfolio design and posting the results on the "Moodle" platform. COE includes two stages: testing to assess the level of theoretical knowledge deepening and solving a situational problem to assess the skills and abilities, interpret the results of laboratory tests and prepare a clinical and laboratory report. Situational tasks and checklists have been developed that make it possible to conduct an objective and comprehensive assessment of the competencies formed by students during the training process, which allows the graduate completed the program to solve professional problems.

Breast cancer detection by ATR-FTIR spectroscopy of blood serum and multivariate data-analysis

Detection of breast cancer has particular importance for the diagnosis of cancer diseases. This is the most common type of cancer among women. Breast cancer is a malignant tumor of the glandular tissue of the breast. It is proposed to use infrared spectroscopy of blood serum as a simple and quick way to detect breast cancer. The paper presents the results of research using the methods of multivariate processing of IR spectra of human blood serum obtained by ATR-FTIR spectroscopy. The paper presents the results of research using the methods of multivariate processing of IR spectra of human blood serum obtained by ATR-FTIR spectroscopy. A sufficiently large sample of patients and healthy donors was diagnosed. Blood samples are examined from 66 patients who are clinically diagnosed with breast cancer and 80 healthy volunteers. A feature of the applied approach was a combination of the method of principal component analysis (PCA) and principal component regression (PCR) for processing the IR spectra of blood serum. The PCA method allows us to determine the spectral bands referring for the intensity differences between the control group and the patient group. Shown, that the range of 1306–1250cm−1 in the IR spectrum of blood serum is diagnostically significant for breast cancer. This range corresponds to the vibrations of several functional groups of DNA and RNA, which play a key role in discrimination in breast cancer screening using ATR-FTIR spectroscopy. It is shown that the proposed method has advantages in ease of use for clinical diagnosis and gives good results for the identification of breast cancer. The values of sensitivity (92.3%) and specificity (87.1%) obtained using the PCR method are close to those of mammography and ultrasound. This indicates the possibility of using this method in real clinical laboratory diagnostics.

Nanopore metagenomic sequencing to investigate nosocomial transmission of human metapneumovirus from a unique genetic group among haematology patients in the United Kingdom

BackgroundHuman metapneumovirus (HMPV) infection causes a spectrum of respiratory tract disease, and may be a significant pathogen in the context of immunocompromise. Here, we report direct-from-sample metagenomic sequencing of HMPV using Oxford Nanopore Technology. MethodsWe applied this sequencing approach to 25 respiratory samples that had been submitted to a clinical diagnostic laboratory in a UK teaching hospital. These samples represented 13 patients under the care of a haematology unit over a 20-day period in Spring 2019 (two sampled twice), and ten other patients elsewhere in the hospital between 2017–2019. ResultsWe generated HMPV reads from 20/25 samples (sensitivity 80% compared to routine diagnostic testing) and retrieved complete HMPV genomes from 15/20 of these. Consensus sequences from Nanopore data were identical to those generated by Illumina, and represented HMPV genomes from two distinct sublineages, A2b and B2. Sequences from ten haematology patients formed a unique genetic group in the A2b sublineage, not previously reported in the UK. Among these, eight HMPV genomes formed a cluster (differing by ≤3 SNPs), likely to reflect nosocomial transmission, while two others were more distantly related and may represent independent introductions to the haematology unit. ConclusionNanopore metagenomic sequencing can be used to diagnose HMPV infection, although more work is required to optimise sensitivity. Improvements in the use of metagenomic sequencing, particularly for respiratory viruses, could contribute to antimicrobial stewardship. Generation of full genome sequences can be used to support or rule out nosocomial transmission, and contribute to improving infection prevention and control practices.