Quality and resilience of clinical laboratories in Rwanda: a need for sustainable strategies.

BACKGROUND: Quality healthcare is a global priority, reliant on robust health systems for evidence-based medicine. Clinical laboratories are the backbone of quality healthcare facilitating diagnostics, treatment, patient monitoring, and disease surveillance. Their effectiveness depends on sustainable delivery of accurate test results. Although the Strengthening Laboratory Management Towards Accreditation (SLMTA) programme has enhanced laboratory quality in low-income countries, the long-term sustainability of this improvement remains uncertain. OBJECTIVE: To explore the sustainability of quality performance in clinical laboratories in Rwanda following the conclusion of SLMTA. METHODS: A quasi-experimental design was adopted, involving 47 laboratories divided into three groups with distinct interventions. While one group received continuous mentorship and annual assessments (group two), interventions for the other groups (groups one and three) ceased following the conclusion of SLMTA. SLMTA experts collected data for 10 years through assessments using WHO's StepwiseLaboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist. Descriptive and t-test analyses were conducted for statistical evaluation. RESULTS: Improvements in quality were noted between baseline and exit assessments across all laboratory groups (mean baseline: 35.3%, exit: 65.8%, p < 0.001). However, groups one and three experienced performance declines following SLMTA phase-out (mean group one: 64.6% in reference to 85.8%, p = 0.01; mean group three: 57.3% in reference to 64.7%, p < 0.001). In contrast, group two continued to enhance performance even years later (mean: 86.6%compared to 70.6%, p = 0.03). CONCLUSION: A coordinated implementation of quality improvement plan that enables regular laboratory assessments to pinpoint and address the quality gaps is essential for sustaining quality services in clinical laboratories.

Main findings: We found that continuous laboratory quality improvement was achieved by laboratories that kept up with regular follow-ups, as opposed to those which phased out these followups prematurely.Added knowledge: This study has affirmed the necessity of maintaining mentorship and conducting regular quality assessments until requisite quality routines are established to sustain laboratory quality services.Global health impact for policy and action: These findings emphasise the significance of instituting a laboratory quality plan, with regular assessments, as policy directives to uphold and enhance quality standards, which benefits both local and global communities, given the pivotal role of laboratories in patient treatment, disease prevention, and surveillance.

Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

Whole blood clotting time: Current practices among Thailand National External Quality Assessment Scheme for blood coagulation member laboratories.

INTRODUCTION: The Thai National Guidelines for Hemostatic Laboratory Testing were established in 2018. The guidelines recommend that the 20-min whole blood clotting time (20WBCT) method be used to diagnose/monitor snake bites. The aim of this study was to survey members of the Thailand National External Quality Assessment Scheme (NEQAS) for Blood Coagulation to investigate the use of 20WBCT testing compared between the 2021 post-guideline and 2007 pre-guideline periods. METHODS: In July 2021, questionnaires were sent from the Faculty of Medicine Siriraj Hospital, Mahidol University to 521 Thailand NEQAS for Blood Coagulation member laboratories to survey their WBCT practices. Current WBCT practices were compared with pre-guideline WBCT practices, and chi-square test (x2) was used to test for differences between groups. RESULTS: Ninety-seven (18.6%) of 521 surveys were returned. Seventy-one laboratories (73.2%) reported knowing about 20WBCT from the Thai national guidelines. The reported average frequency of overall WBCT testing in 2021 was 12.4 times/month. The proportion of laboratories that reported using the 20WBCT test increased from 2.0% in 2007 to 46.4% in 2021 (p < 0.001), and the indications for performing WBCT were virtually unchanged from 2007 to 2021. The proportion of laboratories that reported having problems with WBCT testing decreased from 32.7% in 2007 to 16.5% in 2021. CONCLUSION: Despite our findings that almost three-quarters of respondent laboratories reported knowing about 20WBCT testing from the WBCT guidelines, and that WBCT-specific problems decreased significantly from 2007 to 2021, more work and training is needed to improve WBCT guideline dissemination, understanding, and adherence in Thailand.

A Novel Approach for Routinely Assessing Laboratory Sigma Metrics for a Broad Range of Automated Assays.

BACKGROUND: Sigma metrics have been adapted for the clinical laboratory to incorporate observed accuracy, precision, and total error allowed. The higher the Sigma level for a process, the better performance that process has. A limitation of studies assessing Sigma metrics is that they are performed on a small number of well-controlled systems. METHODS: An algorithm was developed to extract QC data and derive the Sigma metric for 115 analytes from sites connected to the QuidelOrtho E-Connectivity® database. The median of these results was then used to derive the Sigma metric for each assay. RESULTS: In this analysis, 79 out of 115 (68.7%) of the assays assessed achieved 6 Sigma or better and 98 out of 115 (85.2%) achieved 5 Sigma or better. CONCLUSIONS: This study has demonstrated a methodology that can be used to condense Sigma metrics from hundreds of analyzers into a single metric of assay quality. Because these analyzers are running in working laboratories from around the world, this analysis can serve as a baseline for understanding the assay performance achieved in the presence of variabilities such as lab-to-lab, instrument-to-instrument, material handling, environmental conditions, and reagent lot. The significant number of assays demonstrating high Sigma levels did so despite this variation. The ability of the methods reported here to include hundreds of analyzers represents a novel approach for assessing Sigma metrics in clinical laboratories.

Analysis of hematology quality control using six sigma metrics.

INTRODUCTION: Clinical laboratories serve a critical role in increasing the efficiency of patient care. Choosing the right test, getting trustworthy results and appropriate interpretation are of utmost importance in improving the patient's well-being. Quality management strategies should be applied in routine patient care because laboratory errors have a major impact on the quality of patient care. In sigma metrics, errors identified are quantified as percentage errors or defects per million (DPM). It aims at improving the quality control (QC) process by forming an appropriate strategy. AIM AND OBJECTIVES: To analyze the internal quality control (IQC) of hematology analytes using the sigma metrics method and to devise the frequency of IQC by the results of six sigma metric analysis. MATERIALS AND METHODS: This study was conducted in a tertiary care center of western India. Internal quality control (IQC) data sets of five analytes- Red Blood Cell count (RBC), Hemoglobin (Hb), Hematocrit (Hct), White blood cell count (WBC), and Platelet count (PLT) were analyzed retrospectively of six months using Beckman Coulter DXH 800 hematology analyzers. RESULTS: The observed sigma value was >6 for Hb, TLC, and PLT, indicating excellent results and requiring no modification in IQC. The Sigma value was between 3 and 4 for RBC and Hct suggested the need for improvement in quality control (QC) processes. No analytes showed a Sigma value of <3. CONCLUSION: Sigma metrics provide a quantitative framework that helps to assess analytic methodologies and can serve as an important self-assessment tool for quality assurance in the clinical laboratory.

A survey of total IgE reference intervals reported by Scandinavian and British medical laboratories - a need for harmonisation.

OBJECTIVES: There appears to be marked discrepancies between total IgE reference intervals (RIs) in use by many laboratories and those recommended by published studies. The aim of this study was therefore to review total IgE RIs currently reported by Scandinavian and British laboratories and to compare these to published RIs identified by a literature review. METHODS: Relevant laboratories were identified by test directories provided by the national accreditation bodies in Norway, Sweden, Denmark and the UK. Total IgE RIs and their sources were acquired by accessing laboratory user handbooks or by an electronic survey. In addition a literature review of published total IgE RI studies was performed. RESULTS: From 172 accredited laboratories providing total IgE analysis, data was acquired from 122 laboratories. An adult upper reference limit between 81 to 150 kU/L was reported by 89% of these. Denmark and Sweden reported the most harmonised RIs whilst Norway and the UK exhibited the least degree of harmonisation. Published adult (n = 6) and paediatric (n = 6) RI studies reported markedly higher upper limits than those currently in use by the laboratories included in this study. There were also large variations in the number of age strata in use for paediatric RIs. CONCLUSION: This study demonstrates large variations in currently utilised IgE RIs by Scandinavian and British accredited laboratories and most report markedly lower RIs than those recommended by recent RI publications. Many laboratories likely utilise outdated RIs and should consider critically reviewing and updating their RIs.

A performance evaluation of sthemO 301 coagulation analyzer and associated reagents.

AIM: The study objective was to evaluate the performance of sthemO 301 system and to compare it with the analyzer used in our university hospital laboratory (STA R Max® 2), for a selection of hemostasis parameters. METHODS: Method comparison (according to CLSI EP09-A3), carryover (according to CLSI H57-A), APTT sensitivity to heparin (according to CLSI H47-A2), HIL level assessment, and productivity were performed using leftover samples from our laboratory (n > 1000). Commercial quality control materials were used to evaluate precision (according to CLSI EP15-A3) and accuracy. The assays tested on sthemO 301 were: PT, APTT (silica and kaolin activators), fibrinogen (Fib), thrombin time (TT), chromogenic and clotting protein C (PC) activity, and von Willebrand factor antigen (VWF:Ag) levels. RESULTS: All intra-assay and inter-assay precision CVs were below the maximal precision limit proposed by the French Group for Hemostasis and Thrombosis (GFHT). Accuracy was verified with bias below GFHT criteria and most Z-scores were between -2 and +2. No clinically relevant carryover was detected. Silica APTT reagent sensitivity to unfractionated heparin was moderate, as expected. Productivity results were consistent over the 10 repeats performed. The overall agreement between the two systems was excellent for all assays, with Spearman rank correlation coefficient all above 0.9 and slopes of Passing-Bablok correlation near 1 and intercepts close to 0. CONCLUSION: For the methods tested, sthemO 301 system met all the criteria to implement a novel coagulation analyzer in the laboratory and result comparability with STA R Max® 2 was good.

Reported Awareness and Adoption of 2021 Estimated Glomerular Filtration Rate Equations Among US Clinical Laboratories, March 2022.

This study includes clinical laboratories that participated in the first general chemistry proficiency testing survey in 2022 to assess awareness and adoption of new equations from the Chronic Kidney Disease Epidemiology Collaboration for estimated glomerular filtration rate (eGFR) that eliminated race-adjustment factors, including one based on creatinine and one based on creatinine and cystatin C.

Canadian Society of Clinical Chemists Harmonized Clinical Laboratory Lipid Reporting Recommendations on the Basis of the 2021 Canadian Cardiovascular Society Lipid Guidelines.

There is limited guidance on laboratory reporting and interpretation of lipids and lipoproteins used in cardiovascular risk stratification. This contributes to inconsistencies in lipid reporting across clinical laboratories. Recently, the Canadian Cardiovascular Society (CCS) published the 2021 CCS guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult. A subcommittee of the Working Group on Reference Interval Harmonization of the Canadian Society of Clinical Chemists has developed harmonized lipid reporting recommendations that are aligned with the 2021 CCS guidelines, to improve the standardization of lipid assessment and clinical decision-making. The proposed harmonized lipid reporting recommendations were critically reviewed by a broad range of laboratory and clinical experts across Canada. Feedback from approximately 30 expert reviewers was reviewed by the Working Group on Reference Interval Harmonization lipid subcommittee, and consensus decisions were incorporated into the 2021 harmonized lipid reporting recommendations. In this position statement, we provide 6 recommendations for laboratory reporting of lipid parameters. These recommendations include implementing the new National Institutes of Health equation to replace the Friedewald equation for calculating low-density lipoprotein cholesterol, offering lipoprotein (a), either as an in-house or send-out test, and using assays that report lipoprotein (a) in molar units (nmol/L). We also developed a harmonized lipid reporting format with interpretive comments that includes flagging results based on screening patients using treatment decision thresholds in a primary prevention setting. Overall, harmonized lipid reporting will help bridge the gap between clinical guideline recommendations and clinical laboratory reporting and interpretation, and will improve cardiovascular risk assessment across Canada.

Investigation of the quantitative detection of serum Helicobacter pylori antibody in clinical laboratories in China.

BACKGROUND: This study aimed to investigate the implementation and quality control of the quantitative detection of serum Helicobacter pylori (H. pylori) antibody in clinical laboratories in China. METHODS: Online external quality assessment (EQA) questionnaires were distributed to the clinical laboratories by National Center for Clinical Laboratories (NCCL) of China. We collected information on the quantitative detection procedures of serum H. pylori antibody in clinical laboratories, including detection reagents, methods, instruments, calibrators, and internal quality control (IQC). We distributed quality control products to some select laboratories that conducted quantitative detection and analyzed the obtained test data. We evaluated the quantitative detection procedure based on the standard evaluation criteria set at a target value of ±30%. RESULTS: 70.9% (146/206) of the laboratories conducted quantitative detection of H. pylori antibody; 29.1% (60/206) of the laboratories performed qualitative detection. Domestic reagents and matching calibrators accounted for more than 97.1% (200/206) of all reagents. Latex-enhanced immunoturbidimetry was used in 89.7% (131/146) of the laboratories for quantitative determination, while the colloidal gold method was used in 66.7% (40/60) of the laboratories for qualitative determination. A total of 130 laboratories participated in the EQA; 123 completed the assessment, and the pass rate was 75.6% (93/123). CONCLUSION: Clinical quantitative detection of serum H. pylori antibody is performed at a high rate in China. Thus, further studies on the specificity of commercial detection reagents are needed. EQAs are useful to monitor and improve the detection quality of H. pylori antibodies.

Manual de biossegurança e bioprotecção 2ª edição, 2022 / Biosafety and bioprotection 2nd edition, 2022

A Biossegurança e Bioprotecção em Laboratórios de Saúde Pública, Laboratórios de Análises Clínicas e instituções de pesquisa em saúde são áreas do conhecimento relativamente novas, reguladas em vários países por um conjunto de leis, normas, directrizes e procedimentos específicos. Actualmente, a Biossegurança e a Bioprotecção são vistas como disciplinas científicas que requerem um domínio de conhecimentos, habilidades e atitudes (CHA) multi-disciplinares. A aplicação dos princípios de Biossegurança e Bioprotecção é essencial para conter, prevenir, reduzir e eliminar os riscos de exposição por agentes microbiológicos, químicos, físicos, ergonómicos, acidentais e entre outros perigos que podem ou poderiam causar doenças de âmbito ocupacional. A Biossegurança e a Bioprotecção são também importantes para prevenir a exposição a riscos aos familiares de profissionais de saúde, utentes dos serviços de saúde, e para garantir a protecção do meio ambiente e da vida selvagem

A method for measuring the experimental resolution of laboratory assays (clinical biochemical, blood count, immunological, and qPCR) to evaluate analytical performance.

BACKGROUND: The measurement method for experimental resolution and related data to evaluate analytical performance is poorly explored in clinical research. We established a method to measure the experimental resolution of clinical tests, including biochemical tests, automatic hematology analyzer methods, immunoassays, chemical experiments, and qPCR, to evaluate their analytical performance. METHODS: Serially diluted samples in equal proportions were measured, and correlation analysis was performed between the relative concentration and the measured value. Results were accepted for p ≤ 0.01 of the correlation coefficient. The minimum concentration gradient (eg, 10%) was defined as the experimental resolution. For this method, the smaller the value, the higher the experimental resolution and the better the analytical performance. RESULTS: The experimental resolution of the most common biochemical indices reached 10%, with some even reaching 1%. The results of most counting experiments showed experimental resolution up to 10%, whereas the experimental resolution of the classical chemical assays reached 1%. Unexpectedly, the experimental resolution of more sensitive assays, such as immunoassays was only 25% when using the manual method and 10% for qPCR. CONCLUSION: This study established a method for measuring the experimental resolution of laboratory assays and provides a new index for evaluating the reliability of methods in clinical laboratories.

Application of a six sigma model to evaluate the analytical performance of urinary biochemical analytes and design a risk-based statistical quality control strategy for these assays: A multicenter study.

BACKGROUND: The six sigma model has been widely used in clinical laboratory quality management. In this study, we first applied the six sigma model to (a) evaluate the analytical performance of urinary biochemical analytes across five laboratories, (b) design risk-based statistical quality control (SQC) strategies, and (c) formulate improvement measures for each of the analytes when needed. METHODS: Internal quality control (IQC) and external quality assessment (EQA) data for urinary biochemical analytes were collected from five laboratories, and the sigma value of each analyte was calculated based on coefficients of variation, bias, and total allowable error (TEa). Normalized sigma method decision charts for these urinary biochemical analytes were then generated. Risk-based SQC strategies and improvement measures were formulated for each laboratory according to the flowchart of Westgard sigma rules, including run sizes and the quality goal index (QGI). RESULTS: Sigma values of urinary biochemical analytes were significantly different at different quality control levels. Although identical detection platforms with matching reagents were used, differences in these analytes were also observed between laboratories. Risk-based SQC strategies for urinary biochemical analytes were formulated based on the flowchart of Westgard sigma rules, including run size and analytical performance. Appropriate improvement measures were implemented for urinary biochemical analytes with analytical performance lower than six sigma according to the QGI calculation. CONCLUSIONS: In multilocation laboratory systems, a six sigma model is an excellent quality management tool and can quantitatively evaluate analytical performance and guide risk-based SQC strategy development and improvement measure implementation.

The commercial genetic testing landscape for Parkinson's disease.

INTRODUCTION: There have been no specific guidelines regarding which genes should be tested in the clinical setting for Parkinson's disease (PD) or parkinsonism. We evaluated the types of clinical genetic testing offered for PD as the first step of our gene curation. METHODS: The National Institutes of Health (NIH) Genetic Testing Registry (GTR) was queried on 12/7/2020 to identify current commercial PD genetic test offerings by clinical laboratories, internationally. RESULTS: We identified 502 unique clinical genetic tests for PD, from 28 Clinical Laboratory Improvement Amendments (CLIA)-approved clinical laboratories. These included 11 diagnostic PD panels. The panels were notable for their differences in size, ranging from 5 to 62 genes. Five genes for variant query were included in all panels (SNCA, PRKN, PINK-1, PARK7 (DJ1), and LRRK2). Notably, the addition of the VPS35 and GBA genes was variable. Panel size differences stemmed from inclusion of genes linked to atypical parkinsonism and dystonia disorders, and genes in which the link to PD causation is controversial. CONCLUSION: There is an urgent need for expert opinion regarding which genes should be included in a commercial laboratory multi-gene panel for PD.

International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing.

This guidance document has been prepared on behalf of the International Council for Standardization in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for the processing of citrated blood samples for coagulation tests in clinical laboratories in all regions of the world. The following areas are included in this document: Sample transport including use of pneumatic tubes systems; clots in citrated samples; centrifugation; primary tube storage and stability; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport, storage and processing; preanalytical variables for platelet function testing. The following areas are excluded from this document, but are included in an associated ICSH document addressing collection of samples for coagulation tests in clinical laboratories; ordering tests; sample collection tube and anticoagulant; preparation of the patient; sample collection device; venous stasis before sample collection; order of draw when different sample types are collected; sample labelling; blood-to-anticoagulant ratio (tube filling); influence of haematocrit. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Modifications of antifungal sensibility testing as suggested by CLSI document M27-A4: proposal for using different culture medium and buffer.

A common strategy in antifungal susceptibility testing is the utilization of the standardized protocol based on the microbroth dilution assay approach as described by the Clinical Laboratory Standards Institute (CLSI) (M27-A4). One major problem for laboratories in resource-limited countries with this protocol arises from the use of expensive culture media like RPMI-1640 and 3-N-morpholinopropanesulfonic acid (MOPS) buffer. One approach of circumventing this problem in cases of economic need is the evaluation of alternative culture media and buffers. The overall goal of this work was to investigate the influence of modifications in the protocol M27-A4 on diagnostic reliability. We performed univariate analyses evaluating (1) 2 different culture media (YNB and modified SAB); (2) three different buffers (sodium bicarbonate, Tris-HCL, and phosphate), as well as the influence of inoculum concentration (102, 103, 104, 105 cells/mL), the influence of incubation time, and the influence of the assessment mode (visual, biological dye, and spectrophotometer). Our results suggested that (1) RPMI-1640 may be substituted by modified SAB and (2) MOPS buffer may be substituted by Tris-HCl buffer for defined analyses. By comparing the CLSI protocol and the alternative protocol proposed in the present study (modified SAB and Tris-HCl buffer) for the assessment of fluconazole susceptibility of eighteen yeasts (clinical isolates), similar results with both methodologies were recorded. We feel that this study should stimulate a discussion on the feasibility and evolution of the M27-A4 protocol in order to include pragmatic alternatives for resource-limited settings.

A practical guide to chimerism analysis: Review of the literature and testing practices worldwide.

BACKGROUND AND PURPOSE: Currently there are no widely accepted guidelines for chimerism analysis testing in hematopoietic cell transplantation (HCT) patients. The objective of this review is to provide a practical guide to address key aspects of performing and utilizing chimerism testing results. In developing this guide, we conducted a survey of testing practices among laboratories that are accredited for performing engraftment monitoring/chimerism analysis by either the American Society for Histocompatibility & Immunogenetics (ASHI) and/or the European Federation of Immunogenetics (EFI). We interpreted the survey results in the light of pertinent literature as well as the experience in the laboratories of the authors. RECENT DEVELOPMENTS: In recent years there has been significant advances in high throughput molecular methods such as next generation sequencing (NGS) as well as growing access to these technologies in histocompatibility and immunogenetics laboratories. These methods have the potential to improve the performance of chimerism testing in terms of sensitivity, availability of informative genetic markers that distinguish donors from recipients as well as cost. SUMMARY: The results of the survey revealed a great deal of heterogeneity in chimerism testing practices among participating laboratories. The most consistent response indicated monitoring of engraftment within the first 30 days. These responses are reflective of published literature. Additional clinical indications included early detection of impending relapse as well as identification of cases of HLA-loss relapse.

Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study.

BACKGROUND: Emerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is ideally suited to such assessments. Standardization and validation of an end-to-end workflow that supports multisite trials and clinical laboratory processes are vital. Six institutions collaborated to: (1) optimize an automated six-plex assay focused on the PD-1/PD-L1 axis, (2) assess intersite and intrasite reproducibility of staining using a locked down image analysis algorithm to measure tumor cell and immune cell (IC) subset densities, %PD-L1 expression on tumor cells (TCs) and ICs, and PD-1/PD-L1 proximity assessments. METHODS: A six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, and CK) was rigorously optimized as determined by quantitative equivalence to immunohistochemistry (IHC) chromogenic assays. Serial sections from tonsil and breast carcinoma and non-small cell lung cancer (NSCLC) tissue microarrays (TMAs), TSA-Opal fluorescent detection reagents, and antibodies were distributed to the six sites equipped with a Leica Bond Rx autostainer and a Vectra Polaris multispectral imaging platform. Tissue sections were stained and imaged at each site and delivered to a single site for analysis. Intersite and intrasite reproducibility were assessed by linear fits to plots of cell densities, including %PDL1 expression by TCs and ICs in the breast and NSCLC TMAs. RESULTS: Comparison of the percent positive cells for each marker between mIF and IHC revealed that enhanced amplification in the mIF assay was required to detect low-level expression of PD-1, PD-L1, FoxP3 and CD68. Following optimization, an average equivalence of 90% was achieved between mIF and IHC across all six assay markers. Intersite and intrasite cell density assessments showed an average concordance of R2=0.75 (slope=0.92) and R2=0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R2=0.72 (slope=0.86) and R2=0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R2 value of 0.88 and slope of 0.92. Assessments of PD-1/PD-L1 proximity also showed strong concordance (R2=0.82; slope=0.75). CONCLUSIONS: Assay optimization yielded highly sensitive, reproducible mIF characterization of the PD-1/PD-L1 axis across multiple sites. High concordance was observed across sites for measures of density of specific IC subsets, measures of coexpression and proximity with single-cell resolution.