Quality control validation for a veterinary laboratory network of six Sysmex XT-2000iV hematology analyzers.

BACKGROUND: Quality control (QC) validation is an important step in the laboratory harmonization process. This includes the application of statistical QC requirements, procedures, and control rules to identify and maintain ongoing stable analytical performance. This provides confidence in the production of patient results that are suitable for clinical interpretation across a network of veterinary laboratories. OBJECTIVES: To determine that a higher probability of error detection (Ped ) and lower probability of false rejection (Pfr ) using a simple control rule and one level of quality control material (QCM) could be achieved using observed analytical performance than by using the manufacturer's acceptable ranges for QCM on the Sysmex XT-2000iV hematology analyzers for veterinary use. We also determined whether Westgard Sigma Rules could be sufficient to monitor and maintain a sufficiently high level of analytical performance to support harmonization. METHODS: EZRules3 was used to investigate candidate QC rules and determine the Ped and Pfr of manufacturer's acceptable limits and also analyzer-specific observed analytical performance for each of the six Sysmex analyzers within our laboratory system using the American Society of Veterinary Clinical Pathology (ASVCP)-recommended or internal expert opinion quality goals (expressed as total allowable error, TEa ) as the quality requirement. The internal expert quality goals were generated by consensus of the Quality, Education, Planning, and Implementation (QEPI) group comprised of five clinical pathologists and seven laboratory technicians and managers. Sigma metrics, which are a useful monitoring tool and can be used in conjunction with Westgard Sigma Rules, were also calculated. RESULTS: The QC validation using the manufacturer's acceptable limits for analyzer 1 showed only 3/10 measurands reached acceptable Ped for veterinary laboratories (>0.85). For QC validation based on observed analyzer performance, the Ped was >0.94 using a 1-2.5s QC rule for the majority of observations (57/60) across the group of analyzers at the recommended TEa . We found little variation in Pfr between manufacturer acceptable limits and individual analyzer observed performance as this is a characteristic of the rule used, not the analyzer performance. CONCLUSIONS: An improved probability of error detection and probability of false rejection using a 1-2.5s QC rule for individual analyzer QC was achieved compared with the use of the manufacturers' acceptable limits for hematology in veterinary laboratories. A validated QC rule (1-2.5s) in conjunction with sigma metrics (>5.5), desirable bias, and desirable CV based on biologic variation was successful to evaluate stable analytical performance supporting continued harmonization across the network of analyzers.

Comparison of traditional statistical quality control using commercially available control materials and two patient-based quality control procedures for the ADVIA 120 Hematology System.

BACKGROUND: Quality control procedures are an important part of the overall quality assurance for production of accurate and reliable hematologic results. OBJECTIVES: This study aimed to validate a quality control material-based procedure and assess two patient-based quality control procedures (repeat patient testing [RPT] and average of normals [AoN]) with the ADVIA 120 Hematology System. METHODS: Requirements for quality control procedures were obtained with the computerized statistical and quality program, EZRules3. The procedures were evaluated comparing the probability of error detection (Ped), probability of false rejection (Pfr), and sigma metrics. RESULTS: All three of the quality control procedures could be applied with 1-3s control rules, achieving the desired quality requirements. Validation of the quality control materials achieved values for Ped and Pfr of ≥90% and 0%, respectively. Patient-based procedures obtained a ≥85% Ped and a 0% Pfr, except for platelets in the AoN procedure, which achieved a 77% Ped. The RPT achievable total errors were similar to those of the traditional quality control materials and the AoN procedures, except for platelets, which had an achievable total error of 75%. CONCLUSIONS: Patient-based procedures are suitable for veterinary laboratories. The RPT approach may benefit laboratories with limited budgets and low hematology caseloads. The AoN procedure may benefit laboratories with higher hematology caseloads.

Observational Study Design in Veterinary Pathology, Part 2: Methodology.

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.

Evaluation of information presented within soft tissue sarcoma histopathology reports in the United States: 2012-2015.

Despite the existence of the American College of Veterinary Pathology guidelines for tumour biopsy specimens, anecdotally the authors' have seen inconsistency of reporting of information on the pathology report for canine soft tissue sarcomas (STSs). If crucial aspects are not reported this can result in slower or impeded patient care. This retrospective study evaluated 255 STS histopathology reports submitted from across the United States. Reports were evaluated by a single observer to assess for information contained in 5 main categories: patient history and signalment, gross and microscopic description, grading, histologic margins and the comments section. Inclusion criteria for histopathology reports included a final diagnosis of STS, having a microscopic description and resulting from the initial surgical resection. The majority of the reports stated the patient signalment (91.2%) and clinical history (90.8%). However, only 64.8% of the reports had a gross description of the specimen. Histologic margin description was present in 229 reports (91.6%), however, only 149 reports (59.6%) stated an objective measurement of these margins. Histologic classification was stated in 50.0% of the reports, while grade was given on 97.2% of the reports. Variability in histopathologic reporting including histologic margin description for resected canine STS was identified. Given surgical treatment is the mainstay for STS and histopathological assessment plays an important role in determination of whether additional surgery, radiation or chemotherapy is needed. Standardization or checklists like the American College of Pathology utilize may be helpful to ensure histopathologic characteristics are reported that may guide further treatment recommendations.

ASVCP guidelines: Allowable total error hematology.

The purpose of this document is to provide total allowable error (TEa ) recommendations for commonly analyzed hematology measurands for veterinary personnel. These guidelines define relevant terminology and highlight considerations specific to hematology measurands. They also provide reasons and guidelines for using TEa in instrument performance evaluation, including recommendations for when the total observed error exceeds the recommended TEa . Biological variation-based quality specifications are briefly discussed. The appendix describes the derivation of the hematology TEa recommendations and provides resources for external quality assurance/proficiency testing programs and a worksheet for implementation of the guidelines.

Comparison of direct in-house cerebrospinal fluid cytology with commercial pathology results in dogs.

OBJECTIVES: To investigate the correspondence between in-house direct cytological assessment of cerebrospinal fluid and results from a commercial veterinary pathology laboratory. METHODS: Prospective inclusion of samples from dogs that were presented for investigation of suspected neurological disease and had cerebrospinal fluid samples submitted to a commercial pathology laboratory for analysis. A board-certified veterinary pathologist assessed all cerebrospinal fluid samples, and a line smear was assessed in-house by two observers. Nucleated cell count, red blood cell count and differential cell counts were recorded and compared. RESULTS: In-clinic and commercial pathology nucleated cell counts and red blood cell counts were strongly correlated. In-house line smear results were compared with the gold standard of a defined dichotomous rating of 'increased nucleated cell count' provided by the external pathology service. Sensitivity was 93% and specificity 80% for samples with at least two cells per linear field. CLINICAL APPLICATION: Although not a replacement for the assessment of cerebrospinal fluid samples by specialist veterinary pathologists, this method can provide rapid and clinically meaningful information before externally processed sample results are available.

Objective evaluation of analyzer performance based on a retrospective meta-analysis of instrument validation studies: point-of-care hematology analyzers.

BACKGROUND: Information on quality requirements and objective evaluation of performance of veterinary point-of-care analyzers (POCAs) is scarce. OBJECTIVES: The study was aimed at assessing observed total errors (TEobs s) for veterinary hematology POCAs via meta-analysis and comparing TEobs to allowable total error (TEa ) specifications based on experts' opinions. METHODS: The TEobs for POCAs (impedance and laser-based) was calculated based on data from instrument validation studies published between 2006 and 2013 as follows: TEobs = 2 × CV [%] + bias [%]. The CV was taken from published studies; the bias was estimated from the regression equation at 2 different concentration levels of measurands. To fulfill quality requirements, TEobs should be < TEa . Measurands were considered as globally acceptable if > 60% of analyzers showed TEobs < TEa . RESULTS: Six studies evaluating 11 analyzers and 5 studies evaluating 5 analyzers were included for canine and feline hematology variables, respectively. For the CBC, TEobs was < 15% for canine and < 13% for feline measurands, except for HGB and platelet counts. Measurands of the CBC, excluding differential WBC and platelet counts, and HGB concentration were considered globally acceptable. For most of the cell types in the WBC differential count, TEobs was > TEa (data from 3 analyzers). CONCLUSION: This meta-analysis is considered a pilot study. Experts' requirements (TEobs < TEa ) were fulfilled for most measurands except HGB (due to instrument-related bias for the ADVIA 2120) and platelet counts. Available data on the WBC differential count suggest an analytic bias, so nonstatistical quality control is recommended.

Recommendations for designing and conducting veterinary clinical pathology biologic variation studies.

The recent creation of a veterinary clinical pathology biologic variation website has highlighted the need to provide recommendations for future studies of biologic variation in animals in order to help standardize and improve the quality of published information and to facilitate review and selection of publications as standard references. The following recommendations are provided in the format and order commonly found in veterinary publications. A checklist is provided to aid in planning, implementing, and evaluating veterinary studies on biologic variation (Appendix S1). These recommendations provide a valuable resource for clinicians, laboratorians, and researchers interested in conducting studies of biologic variation and in determining the quality of studies of biologic variation in veterinary laboratory testing.

Interobserver Agreement Using Histological Scoring of the Canine Liver.

BACKGROUND: Grading schemes for the assessment of hepatic fibrosis and necroinflammatory activity in humans previously have been applied to dogs with chronic hepatitis. Interobserver agreement is a desirable characteristic for any histological scoring scheme. HYPOTHESIS/OBJECTIVES: To assess interobserver agreement associated with pathologists using a previously published histological scoring scheme to assess hepatic fibrosis and necroinflammatory activity in dogs and to compare fibrosis scores assigned to serial sections stained with hematoxylin & eosin (H&E) and picrosirius red. ANIMALS: Histological sections of liver from 50 dogs with variable degrees of hepatic fibrosis and necroinflammatory activity were selected from institutional tissue archives. METHODS: Six board-certified veterinary anatomic pathologists assigned fibrosis and necroinflammatory activity scores to the histological sections. The multiuser kappa statistic was calculated to assess interobserver agreement. Fibrosis stage assigned to serial sections stained with picrosirius red and H&E was compared using the Wilcoxon signed-rank test. RESULTS: Multiuser kappa statistics for assessment of fibrosis and necroinflammatory activity from H&E-stained sections were 0.35 and 0.16, respectively. There was no difference in median fibrosis scores assigned to serial section stained with H&E and picrosirius red (P = .248). CONCLUSIONS AND CLINICAL IMPORTANCE: There was fair interobserver agreement when pathologists assessed fibrosis and poor agreement when they assessed necroinflammatory activity. This suboptimal agreement must be taken into account by clinicians making decisions based on histology reports of the liver and in the design of studies evaluating these findings. To decrease this variability, ideally >1 pathologist should evaluate each section.

Reproducibility of histopathological findings in experimental pathology of the mouse: a sorry tail.

Reproducibility of in vivo research using the mouse as a model organism depends on many factors, including experimental design, strain or stock, experimental protocols, and methods of data evaluation. Gross and histopathology are often the endpoints of such research and there is increasing concern about the accuracy and reproducibility of diagnoses in the literature. To reproduce histopathological results, the pathology protocol, including necropsy methods and slide preparation, should be followed by interpretation of the slides by a pathologist familiar with reading mouse slides and familiar with the consensus medical nomenclature used in mouse pathology. Likewise, it is important that pathologists are consulted as reviewers of manuscripts where histopathology is a key part of the investigation. The absence of pathology expertise in planning, executing and reviewing in vivo research using mice leads to questionable pathology-based findings and conclusions from studies, even in high-impact journals. We discuss the various aspects of this problem, give some examples from the literature and suggest solutions.

Principles for Assessing Adversity in Toxicologic Clinical Pathology.

There is limited direction in the literature or regulatory guidance on determination of adversity for clinical pathology (CP) biomarkers in preclinical safety studies. Toxicologic clinical pathologists representing the American Society for Veterinary Clinical Pathology-Regulatory Affairs Committee and Society of Toxicologic Pathology-Clinical Pathology Interest Group identified principles, overall approach, and unique considerations for assessing adversity in CP data interpretation to provide a consensus opinion. Emphasized is the need for pathophysiologic context and a weight-of-evidence approach. Most CP biomarkers do not have the potential to be adverse in isolation, regardless of magnitude of change. Rather, they quantify or describe the impact of effects, provide adjunct or supportive information regarding a process or pathogenesis, and provide translational biomarkers of effect. Most often, CP changes are part of a constellation of findings that collectively are adverse. Thus, most CP changes must be interpreted in conjunction with other study findings and require contextual and integrative interpretation. Exceptions include critical CP changes without correlates that indicate a health risk in the tested species. Overall, CP changes should not be interpreted in isolation and their adversity is best addressed with an integrated approach.

ASVCP quality assurance guidelines: external quality assessment and comparative testing for reference and in-clinic laboratories.

The purpose of this document is to educate providers of veterinary laboratory diagnostic testing in any setting about comparative testing. These guidelines will define, explain, and illustrate the importance of a multi-faceted laboratory quality management program which includes comparative testing. The guidelines will provide suggestions for implementation of such testing, including which samples should be tested, frequency of testing, and recommendations for result interpretation. Examples and a list of vendors and manufacturers supplying control materials and services to veterinary laboratories are also included.

The preanalytic phase in veterinary clinical pathology.

This article presents the general causes of preanalytic variability with a few examples showing specialists and practitioners that special and improved care should be given to this too often neglected phase. The preanalytic phase of clinical pathology includes all the steps from specimen collection to analysis. It is the phase where most laboratory errors occur in human, and probably also in veterinary clinical pathology. Numerous causes may affect the validity of the results, including technical factors, such as the choice of anticoagulant, the blood vessel sampled, and the duration and conditions of specimen handling. While the latter factors can be defined, influence of biologic and physiologic factors such as feeding and fasting, stress, and biologic and endocrine rhythms can often not be controlled. Nevertheless, as many factors as possible should at least be documented. The importance of the preanalytic phase is often not given the necessary attention, although the validity of the results and consequent clinical decision making and medical management of animal patients would likely be improved if the quality of specimens submitted to the laboratory was optimized.

How much reproducibility do we need in human and veterinary pathology?

In diagnostic and research reports as well as text-books of human and veterinary pathology repeatability, reproducibility, inter- and intra-observer variation are mentioned rarely as a problem in preparing diagnosis from macroscopic and/or microscopic samples and discussed inconsistently. However, optimal care and restoration of health for a patient are dependent on reliability of diagnosis, therapy, prognosis and prophylaxis. This requires for all tests and procedures a maximal repeatability and reproducibility, a sensitivity and specificity of 85-95% for procedures and methodologies and a comparison of results procedures and methodologies to a gold standard. Looking at the various steps on the road to diagnosis in pathology this is influenced by a series of laboratory steps preparing tissue samples but most importantly reproducibility depends on the handling of visual information in the central nervous system of the individual diagnostician. Thus reproducibility in this context has to be divided into at least three levels: individual (epistemological, organoleptic, inter- and intra-observer variation, and formal/technological- and normative reproducibility). The aim of the present manuscript is to stimulate the reflection among the pathology experts on this most important topic.

Reporting and interpreting red blood cell morphology: is there discordance between clinical pathologists and clinicians?

BACKGROUND: Clinical pathologists (CPs) report RBC morphologic (RBC-M) changes to assist clinicians in prioritizing differential diagnoses. However, reporting is subjective, semiquantitative, and potentially biased. Reporting decisions vary among CPs, and reports may not be interpreted by clinicians as intended. OBJECTIVES: The aims of this study were to survey clinicians and CPs about RBC-M terms and their clinical value, and identify areas of agreement and discordance. METHODS: Online surveys were distributed to small animal clinicians via the Veterinary Information Network and to CPs via the ASVCP listserv. A quiz assessed understanding of RBC-M terms among respondent groups. Descriptive statistics were used to analyze responses to survey questions, and quiz scores were compared among groups. RESULTS: Analyzable responses were obtained from 1662 clinicians and 82 CPs. Both clinicians and CPs considered some terms, e.g., agglutination, useful, whereas only CPs considered other terms, e.g., ghost cells, useful. All groups interpreted certain terms, e.g., Heinz bodies, correctly, whereas some clinicians misinterpreted others, e.g., eccentrocytes. Responses revealed that CPs often do not report RBC-M they consider insignificant, when present in low numbers. Twenty-eight percent of clinicians think CPs review all blood smears while only 19% of CPs report reviewing all smears. CONCLUSIONS: Important differences about the clinical relevance of certain RBC-M terms exist between clinicians and CPs. Inclusion of interpretive comments on CBC reports is the clearest way to ensure that RBC-M changes are interpreted as intended by the CP. Reporting practices should be examined critically to improve communication, transparency, and ultimately medical decisions.