Clinical Laboratory Practice Research Articles

Ethical and Professional Issues Training for Students and Staff of Laboratory Medicine

Background : Medical Laboratory plays a critical role in maintaining and promoting health. Increasing wide applications of laboratory medicine in clinical practice have highlighted specific topics in the areas of social, professional and ethical issues in the laboratory. Despite the important role of clinical lab in health system, ethical issues have been rarely discussed for clinical labs. Considering the need to provide efficient training program for students, this study was designed and conducted to assess the views of laboratory science educators about offering a professional ethical course in laboratory science curriculum. Methods: The study population was 28 laboratory science educators at Mashhad University of Medical Sciences. A questionnaire was designed to assess instructors' views about the importance, quantity, quality, and the need to provide a professional ethical course for medical laboratory students. Results: The majority of respondents (82.15%) evaluated the importance of offering an ethical course for laboratory medicine to be at a very high level. Most educators (75%) believed that good clinical laboratory practice is extremely related to moral issues. Many lecturers (85.7%) completely agreed with the addition of professional ethical course for laboratory science students. Respondents (89.3%), also, believed that the educating professional ethical issues to medical lab students will enhance service quality and performance of the future laboratories. In addition, most professors suggested offering two credit points to cover such issues for the students (85.7%). Conclusion: We recommend offering a separate course in laboratory science curriculum to cover the clinical ethical issues, such as modality of taking samples, interaction with patients, the way of giving information about genetic tests, viral and malignancies, the way of storing data and access to medical records, the diagnosis of infertility, prenatal diagnosis, legal issues in poisonings, and the way of interacting with the doctor and medical private issues.

Statistical validation of reagent lot change in the clinical chemistry laboratory can confer insights on good clinical laboratory practice

Verification of new lot reagent's suitability is necessary to ensure that results for patients' samples are consistent before and after reagent lot changes. A typical procedure is to measure results of some patients' samples along with quality control (QC) materials. In this study, the results of patients' samples and QC materials in reagent lot changes were analysed. In addition, the opinion regarding QC target range adjustment along with reagent lot changes was proposed. Patients' sample and QC material results of 360 reagent lot change events involving 61 analytes and eight instrument platforms were analysed. The between-lot differences for the patients' samples (ΔP) and the QC materials (ΔQC) were tested by Mann-Whitney U tests. The size of the between-lot differences in the QC data was calculated as multiples of standard deviation (SD). The ΔP and ΔQC values only differed significantly in 7.8% of the reagent lot change events. This frequency was not affected by the assay principle or the QC material source. One SD was proposed for the cutoff for maintaining pre-existing target range after reagent lot change. While non-commutable QC material results were infrequent in the present study, our data confirmed that QC materials have limited usefulness when assessing new reagent lots. Also a 1 SD standard for establishing a new QC target range after reagent lot change event was proposed.

Factor XIII Assays and Associated Problems for Laboratory Diagnosis of Factor XIII Deficiency: An Analysis of International Proficiency Testing Results

We analyzed results from the External quality Control of diagnostic Assays and Tests program to assess current clinical laboratory practice and performance of different methods for factor XIII (FXIII) testing internationally. FXIII proficiency testing data from all eight surveys conducted in 2010 and 2011 were analyzed (1,283 results), comparing the three available methods for detecting FXIII deficiency, thus including clot-solubility qualitative activity, quantitative activity, and antigen. Clot-solubility qualitative assays detected a deficiency in only 16% (11/69) of samples with less than 2% FXIII. Assays using added thrombin detected more deficiencies (33%) than did assays without added thrombin (11%). The most commonly used quantitative activity method tended to produce higher results for low FXIII samples than other quantitative activity methods. Antigen results generally showed good accuracy compared with expected levels. The mean interlaboratory coefficients of variation showed wide variability, especially for samples with less than 10% FXIII activity. Laboratory self-classification of results (as normal vs. abnormal) was good, and was slightly better for specimens with ≤ 25% FXIII than for specimens with 26 to 70% or those with >70% FXIII. We conclude that quantitative activity assays perform better for detecting FXIII deficiency than clot solubility assays, although some quantitative activity assays overestimate low FXIII levels.

Separate Quality-Control Measures Are Necessary for Estimation of RNA and Methylated DNA from Formalin-Fixed, Paraffin-Embedded Specimens by Quantitative PCR

Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in clinical laboratory practice. Excluding specimens with poorly preserved nucleic acids is an important quality-control step for avoiding unreliable results. Because the assays for RNA abundance and DNA methylation have different critical limiting factors, we examined the extent of overlap of excluded specimens for RNA abundance versus methylated DNA. The transcript abundance of three reference genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's κ. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to determine usability in the MethyLight assay. Excluding specimens with mean reference gene CT values exceeding the group mean by >1 SD led to significant improvement of the concordance of ESR1 and ER. Specimens with usable DNA after bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA extracted from the same FFPE block need separate exclusion criteria for qPCR assays of transcript abundance and methylated DNA.

Within the context of good clinical laboratory practices evaluation of measurement uncertainty of Thyroid Stimulating Hormone and Prostate Specific Antigen parameters

Within the context of good clinical laboratory practices evaluation of measurement uncertainty of Thyroid Stimulating Hormone and Prostate Specific Antigen parameters

Utility and Necessity of Repeat Testing of Critical Values in the Clinical Chemistry Laboratory

ContextRoutine repeat testing of critical values is a long-standing practice in many clinical laboratories; however, its usefulness and necessity remain to be empirically established and no regulatory requirements yet exist for verification of the critical value results obtained by repeat analysis.ObjectiveTo determine whether repeat testing of critical values is useful and necessary in a clinical chemistry laboratory.MethodsA total of 601 chemistry critical values (potassium, n = 255; sodium, n = 132; calcium, n = 108; glucose, n = 106) obtained from 72,259 routine clinical chemistry specimens were repeat tested. The absolute value and the percentage of difference between the two testing runs were calculated for each of the four critical values and then compared with the allowable error limit put forth in the College of American Pathologists (CAP).ResultsAmong the repeat data for the 601 critical values, a total of 24 showed large differences between the initial result and the repeated result which exceeded the CAP limits for allowable error. The number and rates (%) of large differences for within and outside the analytical measurement range (AMR) were 12 (2.1%) and 12 (41.4%), respectively. For the 572 critical values within the AMR for each test category, the mean absolute difference (mmol/L) and difference(%) between the two testing runs were: potassium, 0.1 mmol/L (2.7%); sodium, 2.1 mmol/L (1.7%); calcium, 0.05 mmol/L (3.0%); glucose, 0.18 mmol/L (2.6%).ConclusionsWhen the initial chemistry critical values are within the AMR, repeated testing does not improve accuracy and is therefore unnecessary. When the initial chemistry critical values are outside the AMR, however, the benefit of repeated testing justifies its performance and makes it necessary. Performing repeat clinical testing on a case-by-case, rather than routine, basis can improve patient care by delivering critical values more rapidly while providing savings on reagent costs associated with unnecessary repeat testing.

Measurement of ganciclovir concentration in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry

BackgroundGanciclovir/valganciclovir plays an important role in the treatment and prevention of cytomegalovirus disease after organ transplantation. Material and methodsWe developed and validated a simple chromatographic method by ultra-performance liquid chromatography tandem mass spectrometry to measure plasma concentration of ganciclovir in human plasma. Chromatographic separation was achieved using an Acquity® UPLC® BEH™ (2.1×50mm id, 1.7μm) reverse-phase C18 column, with a water/methanol linear gradient containing ammonium acetate/formic acid at a 0.4mL/min flow rate. Ganciclovir and its internal standard (acyclovir) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode. ResultsThe limit of detection and quantification were 0.03 and 0.06mg/L, respectively, and linearity was observed between 0.06 and 30.0mg/L. Intra-day and day-to-day coefficients of variation and relative biases ranged from 3.6 to 5.4%, 4.2 to 6.2%, −2.6 to −1.1% and −4.0 to −2.8%, respectively. Recovery values were greater than 81.9%. Evaluation of the matrix effect showed ion suppression for ganciclovir and acyclovir. No carry-over was observed. ConclusionsThe validated method is useful for both therapeutic drug monitoring and pharmacokinetic studies. It could be applied to the daily clinical laboratory practice to measure the concentration of ganciclovir in human plasma.

Cancer Stem Cells: A Review of Potential Clinical Applications

Cancer stem cells (CSCs) comprise a minor cell population in a tumor; however, they possess self-renewal capacity and are responsible for tumor recurrence and the emerging issue of tumor resistance. Despite recent advances in the study of pathogenesis and mechanisms of CSC-mediated disease recurrence and multidrug resistance, many questions remain unanswered. To provide an overview of CSC theory and to describe major methods of CSC detection and isolation, with the emphasis on those techniques that are potentially relevant in clinical laboratory practice. Particular attention is given to CSC markers, such as cancer testis antigens, which could become promising targets in the development of immunotherapy in settings of minimal residual disease. The review is based on analysis of peer-reviewed literature cited in PubMed, as well as preliminary results of studies conducted in our laboratory. Despite a lack of consensus in the scientific community on research methodology, CSCs have demonstrated significant potential as therapeutic targets in the treatment of cancer. Further research of CSC biology and markers will eventually lead to the development of novel therapeutic approaches for targeting these cells to treat resistant and recurrent tumors and minimal residual disease.

Development and Validation of Multiplex Real-Time PCR Assay for Noninvasive Prenatal Assessment of Fetal RhD Status and Fetal Sex in Maternal Plasma

Objective: Noninvasive prenatal detection of RhD status and fetal sex is becoming part of daily practice in clinical laboratories. We evaluated a high throughput procedure for automated DNA extraction and developed a multiplex real-time PCR (rt-PCR) for the simultaneous detection of three fetal loci in a single reaction to assess fetal sex and RhD status in maternal plasma. Methods: An automated DNA extraction method was evaluated together with a new multiplex rt-PCR assay for the simultaneous detection of exons 5 and 7 of the RHD gene together with the Y chromosome marker DYS14 in maternal plasma. The test was evaluated on 60 samples of known fetal genotype obtained from RhD-negative pregnant women before being applied prospectively on 158 consecutive clinical cases. Results were compared with newborn phenotypes. Results: Automated DNA extraction allowed successful analysis of all samples. DYS14 was detected in 118 cases (male fetuses) and both RHD exon 5 and 7 were detected in 148 samples. In 70 samples neither RHD exon 5 nor RHD exon 7 were detected (RhD-negative fetuses). Absence of all three sequences (female RhD-negative fetuses) was assessed in 33 samples. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. Conclusion: The automated DNA extraction procedure coupled with a novel multiplex rt-PCR assay proved accurate, efficient and reliable allowing rapid and high throughput noninvasive determination of fetal sex and RhD status in clinical samples.

An Automated HIV-1 Env-Pseudotyped Virus Production for Global HIV Vaccine Trials

BackgroundInfections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed.Methodology/Principal FindingsThe automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO2 incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity.ConclusionsAn automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials.

Challenges of non‐commercial multicentre North‐South collaborative clinical trials

The last decade has witnessed a substantial increase of multi-centre, public health-oriented clinical trials in poor countries. However, non-commercial research groups have less staff and financial resources than traditional commercial sponsors, so the trial teams have to be creative to comply with Good Clinical Practices (GCP) requirements. According to the recent experience of a large multicentre trial on antimalarials, major challenges result from the complexity of multiple ethical review, the costs of in-depth monitoring at several sites, setting up an adequate Good Clinical Laboratory Practices (GCLP) framework, lack of insurers in host countries, and lack of adequate non-commercial data management software. Public research funding agencies need to consider these challenges in their funding policies. They also could support common spaces where North-South collaborative research groups may share critical information, such as on research insurance and open-source, GCP-compliant software. WHO should update its GCP guidelines, which date back to 1995, to incorporate the perspectives and needs of non-commercial clinical research.

Assuring the quality of next-generation sequencing in clinical laboratory practice

Assuring the quality of next-generation sequencing in clinical laboratory practice

OMIP‐014: Validated multifunctional characterization of antigen‐specific human T cells by intracellular cytokine staining

OMIP‐014: Validated multifunctional characterization of antigen‐specific human T cells by intracellular cytokine staining

Gene patents: perspectives from the clinical laboratory.

Patents involving human genes, human genetic material, and genotype-phenotype correlations are a reality and are increasingly having a negative effect on the clinical molecular diagnostic laboratory and on patient care. Specifically, gene patents and exclusive licensing of diagnostic testing has a detrimental effect on the quality of laboratory testing, the cost of testing, turnaround time, coordination of care, patient access to testing and the ability to confirm testing at a separate laboratory. In this article, gene patents are discussed from the perspective of a medical director of a molecular diagnostics laboratory, and the effect of such patents on clinical laboratory practice is examined.

Pathology harmony moves on: progress on implementation in haematology

Pathology harmony moves on: progress on implementation in haematology

Variability in laboratory reporting practices for regions of homozygosity indicating parental relatedness as identified by SNP microarray testing

Single-nucleotide polymorphism (SNP) microarrays are capable of detecting regions of homozygosity (ROH) that can suggest parental consanguinity or incest. This study was designed to describe the variable reporting practices of clinical laboratories in the United States regarding ROH found on SNP microarray tests, to discuss the follow-up practices of laboratory personnel when findings of ROH indicate consanguinity or incest, and to highlight the legal and ethical dilemmas faced by workers who have discovered these incidental findings. A 20-question survey was administered to microarray experts at 18 laboratories offering clinical SNP microarray tests. The results are presented using descriptive statistics. There was variability in laboratory SNP microarray reporting practices with respect to information and interpretation of ROH findings. All the laboratories agreed that they have a duty to inform the ordering physician about results suggesting consanguinity or incest, but the follow-through practices varied among laboratories. This study discovered variability in reporting practices and follow-up procedures for microarray results that suggest parental consanguinity or incest. Our findings highlight the need for laboratory guidelines to standardize reporting practices for SNP microarray and other tests that are capable of detecting ROH.

Good Clinical Laboratory Practices Improved Proficiency Testing Performance at Clinical Trials Centers in Ghana and Burkina Faso

BackgroundThe recent drive towards accreditation of clinical laboratories in Africa by the World Health Organization – Regional Office for Africa (WHO-AFRO) and the U.S Government is a historic step to strengthen health systems, provide better results for patients and an improved quality of results for clinical trials. Enrollment in approved proficiency testing (PT) programs and maintenance of satisfactory performance is vital in the process of accreditation. Passing proficiency testing surveys has posed a great challenge to many laboratories across sub-Saharan Africa. Our study was aimed at identifying the causes of unsatisfactory PT results in clinical research laboratories conducting or planning to conduct malaria vaccine trials sponsored by the National Institutes of Health (NIH).MethodologyPT reports for 2009 and 2010 from the College of American Pathologists (CAP) for the laboratories were reviewed as part of the process. Errors accounting for unsatisfactory results were classified into clerical, methodological, technical, problem with PT materials, and random errors. A training program on good clinical laboratory practices (GCLP) was developed for each center to address areas for improvement.ResultsThe major cause of PT failure in the four centers was methodological. The application of GCLP improved the success rate in the PT surveys from 58% in 2009 to 88% in 2010. It also decreased the error rate on PT by 35%.ConclusionA previous report from the CAP- PT participating laboratories indicated that the major causes of error were clerical. These types of errors were predominantly made in laboratories in the US, with much more experience in quality control, and varied significantly from what we found. In our centers in sub-Saharan Africa, methodological errors, and not clerical errors, accounted for the vast majority of errors. A process was started for continuous improvement which has decreased methodological errors by 35%, but more improvement is needed.

Clinical Laboratory Practices for the Isolation and Identification of Campylobacter in Foodborne Diseases Active Surveillance Network (FoodNet) Sites: Baseline Information for Understanding Changes in Surveillance Data

Campylobacter is a leading cause of foodborne illness in the United States. Understanding laboratory practices is essential to interpreting incidence and trends in reported campylobacteriosis over time and provides a baseline for evaluating the increasing use of culture-independent diagnostic methods for Campylobacter infection. The Foodborne Diseases Active Surveillance Network (FoodNet) conducts surveillance for laboratory-confirmed Campylobacter infections. In 2005, FoodNet conducted a survey of clinical laboratories to describe routine practices used for isolation and identification of Campylobacter. A profile was assigned to laboratories based on complete responses to key survey questions that could impact the recovery and isolation of Campylobacter from stool specimens. Of 411 laboratories testing on-site for Campylobacter, 97% used only culture methods. Among those responding to the individual questions, nearly all used transport medium (97%) and incubated at 42°C (94%); however, most deviated from existing guidelines in other areas: 68% held specimens in transport medium at room temperature before plating, 51% used Campy blood agar plate medium, 52% read plates at <72 hours of incubation, and 14% batched plates before placing them in a microaerobic environment. In all, there were 106 testing algorithms among 214 laboratories with a complete profile; only 16 laboratories were fully adherent to existing guidelines. Although most laboratories used culture-based methods, procedures differed widely and most did not adhere to existing guidelines, likely resulting in underdiagnosis. Given the availability of new culture-independent testing methods, these data highlight a clear need to develop best practice recommendations for Campylobacter infection diagnostic testing.

Analysis of Factors Influencing the Generation of Unqualified Clinical Samples and Measures to Prevent this Generation

BackgroundWe investigated the influence of pre-analytical factors on the results of clinical tests and thereby analyzed approaches to improve quality management in clinical laboratories.MethodsUnqualified clinical samples were selected from all the samples received at our clinical laboratory. The data were collected for 2009 and 2010, i.e., the years before and after the establishment of the laboratory quality management system. The rate and causes of generation of unqualified samples were analyzed, and measures to improve the laboratory practices were studied and implemented.ResultsA total of 1,051 unqualified samples were identified from among the 553,158 samples (the overall incidence rate of unqualified samples was 0.19%). The number of unqualified samples substantially varied according to the nature of the sample, and clinical samples collected for routine blood tests or coagulation tests were the predominant unqualified samples. The main causes of generation of unqualified samples were insufficient sample volumes and improper methods of mixing the samples. The rate of generation of unqualified samples decreased significantly after the implementation of improvement measures (0.26% in 2009 vs. 0.13% in 2010, P<0.001).ConclusionsThe number of unqualified samples decreased significantly after the establishment of the laboratory quality management system, which promoted active communication among and training of the clinical staff to reduce the occurrence of pre-analytical errors. Comprehensive control of pre-analytical factors is an important approach in improving the clinical laboratory practices.

Promoting good clinical laboratory practices and laboratory accreditation to support clinical trials in sub-Saharan Africa.

Laboratory capacity in the developing world frequently lacks quality management systems (QMS) such as good clinical laboratory practices, proper safety precautions, and adequate facilities; impacting the ability to conduct biomedical research where it is needed most. As the regulatory climate changes globally, higher quality laboratory support is needed to protect study volunteers and to accurately assess biological parameters. The University of Bamako and its partners have undertaken a comprehensive QMS plan to improve quality and productivity using the Clinical and Laboratory Standards Institute standards and guidelines. The clinical laboratory passed the College of American Pathologists inspection in April 2010, and received full accreditation in June 2010. Our efforts to implement high-quality standards have been valuable for evaluating safety and immunogenicity of malaria vaccine candidates in Mali. Other disease-specific research groups in resource-limited settings may benefit by incorporating similar training initiatives, QMS methods, and continual improvement practices to ensure best practices.