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.

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 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.

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.

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.

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.

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.

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.

A high throughput combinatorial library technique for identifying formalin-sensitive epitopes.

We present a technique for identifying the amino acids responsible for a loss of immunoreactivity in response to treating an antigen with a chemical modifier. This is of particular interest for the chemical formaldehyde, the cross-linking agent in formalin. Formalin is a commonly used fixative to preserve the cellular architecture of cells and tissues and to prevent degradation from proteases and nucleases. Formalin is also routinely used in the preparation of vaccines, to inactivate both toxins and microbes. Formalin fixation attenuates infectivity and pathogenicity by cross-linking while often preserving antigenicity. However, some epitopes are irreversibly modified by formalin while others are not. An understanding of how formalin affects epitope immunoreactivity may be useful in vaccine development or in the development of diagnostic antibody reagents for formalin-fixed tissues. In this report, we describe a method for systematically identifying formalin-sensitive and formalin-insensitive epitopes in a high throughput fashion, for any particular antibody. The data from this effort underscore the importance of certain amino acids, notably lysine, in affecting antibody immunoreactivity after formalin fixation. The method can be generally applicable in exploring the sensitivity of protein epitopes to an agent or condition of interest.

Clinical laboratory measurement of serum, plasma, and blood viscosity.

This article discusses the fundamentals for measuring the viscosity of whole blood, serum, and plasma and its application to the diagnosis of hyperviscosity syndrome. We describe some of the terminology in the field, including relevant definitions, the different units of measure, and general principles of clinical laboratory viscosity measurement. The 3 main categories of instrumentation for viscosity measurement--capillary, falling-sphere, and rotational viscometers--are discussed. We compare the various types of instrumentation for their usefulness with various types of clinical specimens. Relevant features that may be important in selecting a viscometer are described. We describe our 1.5-year experience with the viscometer that we chose. We hope the information in this review will be useful to pathologists and clinical laboratory staff in explaining the available choices for measuring serum, plasma, and whole blood viscosity.

A molecular model of antigen retrieval using a peptide array.

Even though antigen retrieval is highly denaturing, it paradoxically restores immunoreactivity after formalin fixation. It is unclear how this happens. We address this question using a peptide array to model formalin fixation and antigen retrieval. The peptides are linear stretches based on the native protein sequence, containing antibody epitopes of HER-2, estrogen receptor, progesterone receptor, and Ki-67. Of the 7 peptides, 6 retain their immunoreactivity after formalin fixation. However, if formalin fixation is performed in the presence of an irrelevant protein, immunoreactivity is abrogated, regardless of the peptides' amino acid composition. Fixation of an external protein around the antibody epitope prevents antibody binding. Antigen retrieval restores immunoreactivity. These findings demonstrate that native protein conformation is not relevant during antigen retrieval. Moreover, the loss and recovery of immunoreactivity associated with fixation and antigen retrieval, respectively, can be accounted for completely with a model of steric interference by adjacent proteins.

Antibodies immunoreactive with formalin-fixed tissue antigens recognize linear protein epitopes.

It is not clearly understood why some monoclonal antibodies bind to their antigens informalin-fixed, paraffin-embedded tissue sections but others do not. To address this question, we analyzed the protein epitopes of 9 monoclonal antibodies that are immunoreactive after formalin fixation and antigen retrieval. We identified the antibody contact sites by using phage display and synthesized corresponding peptides derived from the GenBank database sequence that contain the predicted antibody binding sites. Our data indicate that all 9 antibodies bind to linear epitopes, ie, composed of contiguous amino acids. In addition, the amino acids proline, tyrosine, glutamine, and leucine are highly represented in these antibody contact sites. The epitopes tend to be mildly to moderately hydrophilic. These findings are the first detailed studies of antibody epitopes associated with antigen retrieval and suggest that antibodies must recognize linear sequences to bind after formalin fixation and antigen retrieval.

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.

A novel quality control slide for quantitative immunohistochemistry testing.

We introduce a novel quality control technology that may improve intra- and interlaboratory immunohistochemistry (IHC) standardization. The technology involves the creation of standardized antibody targets that are attached to the same slides as the patient sample. After IHC staining, the targets turn the same color as the stained cells or tissue elements. Unlike current clinical practice, our proposed targets are neither cells nor tissue sections. To create reproducible standards that are available in unlimited supply, we use short constrained peptides as antibody targets. These peptides are attached directly to the glass slide. We show that these peptides simulate the portion of the native antigen to which the antibody binds. They are useful in detecting subtle changes in IHC staining efficacy. Moreover, the peptides do not degrade after deparaffinization or antigen retrieval treatments. This technology may be valuable in creating nationally standardized controls to quantify IHC analytical variability.