A Consortium for Analytic Standardization in Immunohistochemistry.

CONTEXT.­: The authors announce the launch of the Consortium for Analytic Standardization in Immunohistochemistry, funded with a grant from the National Cancer Institute. As with other laboratory testing, analytic standards are important for many different stakeholders: commercial vendors of instruments and reagents, biopharmaceutical firms, pathologists, scientists, clinical laboratories, external quality assurance organizations, and regulatory bodies. Analytic standards are customarily central to assay development, validation, and method transfer into routine assays and are critical quality assurance tools. OBJECTIVE.­: To improve immunohistochemistry (IHC) test accuracy and reproducibility by integrating analytic standards into routine practice. To accomplish this mission, the consortium has 2 mandates: (1) to experimentally determine analytic sensitivity thresholds (lower and upper limits of detection) for selected IHC assays, and (2) to inform IHC stakeholders of what analytic standards are, why they are important, and how and for what purpose they are used. The consortium will then publish the data and offer analytic sensitivity recommendations where appropriate. These mandates will be conducted in collaboration and coordination with clinical laboratories, external quality assurance programs, and pathology organizations. DATA SOURCES.­: Literature review and published external quality assurance data. CONCLUSIONS.­: Integration of analytic standards is expected to (1) harmonize and standardize IHC assays; (2) improve IHC test accuracy and reproducibility, both within and between laboratories; and (3) dramatically simplify and improve methodology transfer for new IHC protocols from published literature or clinical trials to clinical IHC laboratories.

Quantitative comparison of PD-L1 IHC assays against NIST standard reference material 1934.

Companion diagnostic immunohistochemistry (IHC) tests are developed and performed without incorporating the tools and principles of laboratory metrology. Basic analytic assay parameters such as lower limit of detection (LOD) and dynamic range are unknown to both assay developers and end users. We solved this problem by developing completely new tools for IHC-calibrators with units of measure traceable to National Institute of Standards & Technology (NIST) Standard Reference Material (SRM) 1934. In this study, we demonstrate the clinical impact and opportunity for incorporating these changes into PD-L1 testing. Forty-one laboratories in North America and Europe were surveyed with newly-developed PD-L1 calibrators. The survey sampled a broad representation of commercial and laboratory-developed tests (LDTs). Using the PD-L1 calibrators, we quantified analytic test parameters that were previously only inferred indirectly after large clinical studies. The data show that the four FDA-cleared PD-L1 assays represent three different levels of analytic sensitivity. The new analytic sensitivity data explain why some patients' tissue samples were positive by one assay and negative by another. The outcome depends on the assay's lower LOD. Also, why previous attempts to harmonize certain PD-L1 assays were unsuccessful; the assays' dynamic ranges were too disparate and did not overlap. PD-L1 assay calibration also clarifies the exact performance characteristics of LDTs relative to FDA-cleared commercial assays. Some LDTs' analytic response curves are indistinguishable from their predicate FDA-cleared assay. IHC assay calibration represents an important transition for companion diagnostic testing. The new tools will improve patient treatment stratification, test harmonization, and foster accuracy as tests transition from clinical trials to broad clinical use.

Analytic Sensitivity of 3 Nucleic Acid Detection Assays in Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription PCR is the primary method to diagnose coronavirus disease 2019 (COVID-19). However, the analytic sensitivity required is not well defined and it is unclear how available assays compare. METHODS: For the Abbott RealTime SARS-CoV-2 assay (m2000; Abbott Molecular), we determined that it could detect viral concentrations as low as 26 copies/mL, we defined the relationship between cycle number and viral concentrations, and we tested naso- and oropharyngeal swab specimens from 8538 consecutive individuals. Using the m2000 as a reference assay method, we described the distribution of viral concentrations in these patients. We then used selected clinical specimens to determine the positive percent agreement of 2 other assays with more rapid turnaround times [Cepheid Xpert Xpress (GeneXpert; Cepheid); n = 27] and a laboratory developed test on the Luminex ARIES system [ARIES LDT (Luminex); n = 50] as a function of virus concentrations, from which we projected their false-negative rates in our patient population. RESULTS: SARS-CoV-2 was detected in 27% (95% CI: 26%-28%) of all specimens. Estimated viral concentrations were widely distributed, and 17% (95% CI: 16%-19%) of positive individuals had viral concentrations <845 copies/mL. Positive percent agreement was strongly related to viral concentration, and reliable detection (i.e., ≥95%) was observed at concentrations >100 copies/mL for the GeneXpert but not the ARIES LDT, corresponding to projected false-negative rates of 4% (95% CI: 0%-21%) and 27% (95% CI: 11%-46%), respectively. CONCLUSIONS: Substantial proportions of clinical specimens have low to moderate viral concentrations and may be missed by methods with less analytic sensitivity.

Development and Validation of Measurement Traceability for In Situ Immunoassays.

BACKGROUND: Immunoassays for protein analytes measured in situ support a $2 billion laboratory testing industry that suffers from significant interlaboratory disparities, affecting patient treatment. The root cause is that immunohistochemical testing lacks the generally accepted tools for analytic standardization, including reference standards and traceable units of measure. Until now, the creation of these tools has represented an insoluble technical hurdle. METHODS: We address the need with a new concept in metrology-that is, linked traceability. Rather than calculating analyte concentration directly, which has proven too variable, we calculate concentration by measuring an attached fluorescein, traceable to NIST Standard Reference Material 1934, a fluorescein standard. RESULTS: For validation, newly developed estrogen receptor (ER) calibrators were deployed in tandem with an array of 80 breast cancer tissue sections in a national external quality assessment program. Laboratory performance was assessed using both the ER standards and the tissue array. Similar to previous studies, the tissue array revealed substantial discrepancies in ER test results among the participating laboratories. The new ER calibrators revealed a broad range of analytic sensitivity, with the lower limits of detection ranging from 7310 to 74 790 molecules of ER. The data demonstrate, for the first time, that the variable test results correlate with analytic sensitivity, which can now be measured quantitatively. CONCLUSIONS: The reference standard enables precise interlaboratory alignment of immunohistochemistry test sensitivity for measuring cellular proteins in situ. The introduction of a reference standard and traceable units of measure for protein expression marks an important milestone.

A Root Cause Analysis Into the High Error Rate in Clinical Immunohistochemistry.

The field of Clinical Immunohistochemistry (IHC) is beset with a high error rate, an order of magnitude higher than in other types of clinical laboratory testing. Despite the many improvements in the field, these errors have persisted over the last 2 decades. The improvements over the years include an extensive literature describing the potential causes of errors and how to avoid them. More stringent regulatory guidelines have also been implemented. These measures reflect the standard view is that fixing the broad confluence of causes of error will address the problem. This review takes a different tack. To understand the high error rates, this review compares Clinical IHC laboratory practice to practices of other clinical laboratory disciplines. What aspects of laboratory testing that minimize errors in other clinical laboratory disciplines are not found in Clinical IHC? In this review, we seek to identify causal factors and underlying root causes that are unique to the field of Clinical IHC in comparison to other laboratory testing disciplines. The most important underlying root cause is the absence of traceable units of measure, international standards, calibrators that are traceable to standards, and quantitative monitoring of controls. These tools and practices (in other clinical laboratory disciplines) provide regular accurate feedback to laboratory personnel on analytic test performance.

Selecting an Optimal Positive IHC Control for Verifying Antigen Retrieval.

Positive immunohistochemistry (IHC) controls are intended to detect problems in both immunostaining and heat-induced epitope retrieval (HIER). However, it is not known what features in a control are important for verifying HIER. Contrary to expectation, the fact that a tissue is formalin-fixed does not necessarily render it suitable in verifying proper HIER. Some tissue controls, for some immunostains, strongly stain even without HIER. Consequently, the control may verify the immunostain but provide little or no information regarding the HIER step. To sort this out, we used formalin-fixed peptide epitopes, a model that provides for precise definition of analyte concentration, epitope composition, and degree of fixation. Our data demonstrate that formalin fixation generates a variable level of protein epitope masking, depending on the epitope recognized by the primary antibody. Some epitopes are highly masked while others hardly at all. Furthermore, the ability of amino acids in the epitope to react with formaldehyde can, at least in part, account for this variability. Most important, we demonstrate the importance of selecting a positive control with a low or intermediate analyte concentration (relative to the immunostain's analytic sensitivity). High analyte concentrations can be insensitive in verifying the HIER step.

Quantitative Assessment of Immunohistochemistry Laboratory Performance by Measuring Analytic Response Curves and Limits of Detection.

CONTEXT: - Numerous studies highlight interlaboratory performance variability in diagnostic immunohistochemistry (IHC) testing. Despite substantial improvements over the years, the inability to quantitatively and objectively assess immunostain sensitivity complicates interlaboratory standardization. OBJECTIVE: - To quantitatively and objectively assess the sensitivity of the immunohistochemical stains for human epidermal growth factor receptor type 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR) across IHC laboratories in a proficiency testing format. We measure sensitivity with parameters that are new to the field of diagnostic IHC: analytic response curves and limits of detection. DESIGN: - Thirty-nine diagnostic IHC laboratories stained a set of 3 slides, one each for HER2, ER, and PR. Each slide incorporated a positive tissue section and IHControls at 5 different concentrations. The IHControls comprise cell-sized clear microbeads coated with defined concentrations of analyte (HER2, ER, and/or PR). The laboratories identified the limits of detection and then mailed the slides for quantitative assessment. RESULTS: - Each commercial immunostain demonstrated a characteristic analytic response curve, reflecting strong reproducibility among IHC laboratories using the same automation and reagents prepared per current Good Manufacturing Practices. However, when comparing different commercial vendors (using different reagents), the data reveal up to 100-fold differences in analytic sensitivity. For proficiency testing purposes, quantitative assessment using analytic response curves was superior to subjective interpretation of limits of detection. CONCLUSIONS: - Assessment of IHC laboratory performance by quantitative measurement of analytic response curves is a powerful, objective tool for identifying outlier IHC laboratories. It uniquely evaluates immunostain performance across a range of defined analyte concentrations.

The Importance of Epitope Density in Selecting a Sensitive Positive IHC Control.

Clinical Immunohistochemistry (IHC) laboratories face unique challenges in performing accurate and reproducible immunostains. Among these challenges is the use of homemade controls derived from pathological discard samples. Such positive controls have an unknown number of analyte molecules per cell (epitope density). It is unclear how the lack of defined analyte concentrations affects performance of the control. To address this question, we prepared positive IHC controls ( IHControls) for human epidermal growth factor receptor type II (HER-2), estrogen receptor (ER), or progesterone receptor (PR) with well-defined, homogeneous, and reproducible analyte concentrations. Using the IHControls, we examined the effect of analyte concentration on IHC control sensitivity. IHControls and conventional tissue controls were evaluated in a series of simulated primary antibody reagent degradation experiments. The data demonstrate that the ability of a positive IHC control to reveal reagent degradation depends on (1) the analyte concentration in the control and (2) where that concentration falls on the immunostain's analytic response curve. The most sensitive positive IHC controls have analyte concentrations within or close to the immunostain's concentration-dependent response range. Strongly staining positive controls having analyte concentrations on the analytic response curve plateau are less sensitive. These findings emphasize the importance of selecting positive IHC controls that are of intermediate (rather than strong) stain intensity.

Analytic Response Curves of Clinical Breast Cancer IHC Tests.

An important limitation in the field of immunohistochemistry (IHC) is the inability to correlate stain intensity with specific analyte concentrations. Clinical immunohistochemical tests are not described in terms of analytic response curves, namely, the analyte concentrations in a tissue sample at which an immunohistochemical stain (1) is first visible, (2) increases in proportion to the analyte concentration, and (3) ultimately approaches a maximum color intensity. Using a new immunostaining tool ( IHControls), we measured the analytic response curves of the major clinical immunohistochemical tests for human epidermal growth factor receptor type II (HER-2), estrogen receptor (ER), and progesterone receptor (PR). The IHControls comprise the analytes HER-2, ER, and PR at approximately log concentration intervals across the range of biological expression, from 100 to 1,000,000 molecules per test microbead. We stained IHControls of various concentrations using instruments, reagents, and protocols from three major IHC vendors. Stain intensity at each analyte concentration was measured, thereby generating an analytic response curve. We learned that for HER-2 and PR, there is significant variability in test results between clinical kits for samples with analyte concentrations of approximately 104 molecules/microbead. We propose that the characterization of immunostains is an important step toward standardization.

Levey-Jennings Analysis Uncovers Unsuspected Causes of Immunohistochemistry Stain Variability.

Almost all clinical laboratory tests use objective, quantitative measures of quality control (QC), incorporating Levey-Jennings analysis and Westgard rules. Clinical immunohistochemistry (IHC) testing, in contrast, relies on subjective, qualitative QC review. The consequences of using Levey-Jennings analysis for QC assessment in clinical IHC testing are not known. To investigate this question, we conducted a 1- to 2-month pilot test wherein the QC for either human epidermal growth factor receptor 2 (HER-2) or progesterone receptor (PR) in 3 clinical IHC laboratories was quantified and analyzed with Levey-Jennings graphs. Moreover, conventional tissue controls were supplemented with a new QC comprised of HER-2 or PR peptide antigens coupled onto 8 µm glass beads. At institution 1, this more stringent analysis identified a decrease in the HER-2 tissue control that had escaped notice by subjective evaluation. The decrement was due to heterogeneity in the tissue control itself. At institution 2, we identified a 1-day sudden drop in the PR tissue control, also undetected by subjective evaluation, due to counterstain variability. At institution 3, a QC shift was identified, but only with 1 of 2 controls mounted on each slide. The QC shift was due to use of the instrument's selective reagent drop zones dispense feature. None of these events affected patient diagnoses. These case examples illustrate that subjective QC evaluation of tissue controls can detect gross assay failure but not subtle changes. The fact that QC issues arose from each site, and in only a pilot study, suggests that immunohistochemical stain variability may be an underappreciated problem.

Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems.

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads' stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls ("IHControls"). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.

Role of plasmapheresis in Waldenström's macroglobulinemia.

Despite the absence of randomized trials, plasmapheresis has consistently demonstrated efficacy in treatment of Waldenström's macroglobulinemia (WM) patients with hyperviscosity syndrome (HVS). This procedure can promptly reverse most clinical manifestations of serum HVS. Early diagnosis is crucial and usually can be made from the funduscopic exam. Serial viscosity measurements can be monitored by the Ostwald tube method which is simple, reproducible, and for which there is substantial clinical correlation. The concept of a symptomatic threshold, whereby each WM patient has a distinct viscosity threshold for the development of HVS, seems valid. Maintaining serum viscosity below each patient's symptomatic threshold effectively prevents recurrent HVS. Plasmapheresis is sometimes necessary as an emergency procedure and is useful maintenance therapy in selected patients. Prophylactic plasmapheresis should be considered in patients at risk for HVS after rituximab therapy. Vigorous plasmapheresis in WM patients with syndromes because of autoreactive immunoglobulin M antibodies requires further study.

Experimental validation of peptide immunohistochemistry controls.

Peptide immunohistochemistry (IHC) controls are a new quality control format for verifying proper IHC assay performance, offering advantages in high throughput automated manufacture and standardization. We previously demonstrated that formalin-fixed peptide epitopes, covalently attached to glass microscope slides, behaved (immunochemically) in a similar fashion to the native protein in tissue sections. To convert this promising idea into a practical clinical laboratory quality control tool, we tested the hypothesis that the quality assurance information provided by peptide IHC controls accurately reflects IHC staining performance among a diverse group of clinical laboratories. To test the hypothesis, we first designed and built an instrument for reproducibly printing the controls on microscope slides and a simple software program to measure the color intensity of stained controls. Automated printing of peptide spots was reproducible, with coefficients of variation of 4% to 8%. Moreover, the peptide controls were stable at

National HER2 proficiency test results using standardized quantitative controls: characterization of laboratory failures.

CONTEXT: An important component in fostering test standardization for HER2 testing by immunohistochemistry is an appropriate positive control. We developed a new standardized, quantitative immunohistochemical HER2 control using a HER2 peptide covalently attached to glass microscope slides. The peptide controls can be formalin fixed or unfixed, providing the new capability of distinguishing errors associated with antigen retrieval from errors associated with the staining process itself. OBJECTIVE: To investigate the causes of variability in HER2 immunohistochemistry staining performance. By comparing laboratory performance with both formalin-fixed and unfixed analyte controls, we aimed to distinguish problems associated with antigen retrieval from reagent or staining protocol deficiencies. DESIGN: HER2 peptide analyte controls were printed on 2 slides that also contained unstained sections of invasive breast carcinomas and were mailed with the College of American Pathologists' 2006 HER2-B proficiency testing survey. Laboratory participants were asked to stain the 2 slides and return them for central review and quantification. This study is unique in combining central review with new quantitative HER2 controls. RESULTS: Of 109 participants who returned evaluable stained slides, staining was suboptimal in 20 (18.3%) as judged by quantification of the peptide analyte controls and review of tissue sections. Of those, 35% failed due to antigen retrieval errors, 20% failed due solely to antibody or staining protocol problems, and the remainder failed due to a combination of the two. CONCLUSIONS: In practice, errors in HER2 testing are caused by variables associated with antigen retrieval and the reagents and staining protocol, as well as interpretive error. Analyte controls help distinguish these different causes.

Bioinformatic requirements for protein database searching using predicted epitopes from disease-associated antibodies.

We describe a new approach to identify proteins involved in disease pathogenesis. The technology, Epitope-Mediated Antigen Prediction (E-MAP), leverages the specificity of patients' immune responses to disease-relevant targets and requires no prior knowledge about the protein. E-MAP links pathologic antibodies of unknown specificity, isolated from patient sera, to their cognate antigens in the protein database. The E-MAP process first involves reconstruction of a predicted epitope using a peptide combinatorial library. We then search the protein database for closely matching amino acid sequences. Previously published attempts to identify unknown antibody targets in this manner have largely been unsuccessful for two reasons: 1) short predicted epitopes yield too many irrelevant matches from a database search and 2) the epitopes may not accurately represent the native antigen with sufficient fidelity. Using an in silico model, we demonstrate the critical threshold requirements for epitope length and epitope fidelity. We find that epitopes generally need to have at least seven amino acids, with an overall accuracy of >70% to the native protein, in order to correctly identify the protein in a nonredundant protein database search. We then confirmed these findings experimentally, using the predicted epitopes for four monoclonal antibodies. Since many predicted epitopes often fail to achieve the seven amino acid threshold, we demonstrate the efficacy of paired epitope searches. This is the first systematic analysis of the computational framework to make this approach viable, coupled with experimental validation.

Accurate identification of paraprotein antigen targets by epitope reconstruction.

We describe the first successful clinical application of a new discovery technology, epitope-mediated antigen prediction (E-MAP), to the investigation of multiple myeloma. Until now, there has been no reliable, systematic method to identify the cognate antigens of paraproteins. E-MAP is a variation of previous efforts to reconstruct the epitopes of paraproteins, with the significant difference that it provides enough epitope sequence data so as to enable successful protein database searches. We first reconstruct the paraprotein's epitope by analyzing the peptides that strongly bind. Then, we compile the data and interrogate the nonredundant protein database, searching for a close match. As a clinical proof-of-concept, we apply this technology to uncovering the protein targets of para-proteins in multiple myeloma (MM). E-MAP analysis of 2 MM paraproteins identified human cytomegalovirus (HCMV) as a target in both. E-MAP sequence analysis determined that one para-protein binds to the AD-2S1 epitope of HCMV glycoprotein B. The other binds to the amino terminus of the HCMV UL-48 gene product. We confirmed these predictions using immunoassays and immunoblot analyses. E-MAP represents a new investigative tool for analyzing the role of chronic antigenic stimulation in B-lymphoproliferative disorders.