Development and Validation of Measurement

Traceability for In Situ Immunoassays

Emina E. Torlakovic,a,b,c Seshi R. Sompuram,d Kodela Vani,d Lili Wang,e Anika K. Schaedle,d

Paul C. DeRose,e and Steven A. Bogend,*

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.

Introduction

Traceability of measurement to a higher order reference

standard is a foundation of laboratory testing. In clinical

chemistry, immunology, molecular virology, hematology, and other laboratory testing disciplines, hundreds

of primary reference standards are maintained at international institutions. However, this system of traceable

measurement does not exist for immunohistochemistry

(IHC) testing, including all of its clinical applications

(1). The science of metrology, the study of measurement, has not yet extended to IHC because of technical

challenges. There is no method for creating reference

standards for in situ cellular proteins in a fashion analogous to that for soluble (e.g., serum or plasma) analytes.

The absence of reference standards and traceable units

of measure is associated with comparatively high rates of

testing discrepancies among IHC testing laboratories

(1). To address this need, we developed a system of

measurement traceability using a linked fluorescein tag

for creating reference standards for any cellular analyte

and validated it for estrogen receptor (ER) testing.

The absence of metrology tools means that we still

do not know how many molecules of ER must be present per cell before a pathologist can see the stain and,

even more important, how many are required to reliably

predict response to hormonal therapy. The lack of metrology standards for IHC makes it impossible to precisely align one laboratory’s test with the next and

impedes development of reliable IHC assays for predictive biomarkers in clinical trials (1). For this study, we

developed an ER standard to define and compare the

thresholds separating “high positive,” “low positive,”

and “negative” tests according to recently updated

American Society of Clinical Oncology and College of

American Pathologists (ASCO/CAP) guidelines (2).

The threshold between negative and positive cases is the

a

Canadian Biomarker Quality Assurance, Saskatoon, Saskatchewan, Canada; bUniversity of

Saskatchewan, Saskatoon, Saskatchewan, Canada; c

Saskatoon Health Authority,

Saskatoon, Saskatchewan, Canada; d

Boston Cell Standards LLC, Boston, MA, USA; e

Biosystems and Biomaterials Division, National Institute of Standards and Technology,

Gaithersburg, MD, USA .

*Address correspondence to this author at: Boston Cell Standards LLC c/o Tufts Medical

Center, Department of Pathology and Laboratory Medicine, Box 115, 800 Washington

Street, Boston MA 02111, USA. Fax: 617-674-3570; e-mail sbogen@bostoncellstandards.com

Received September 17, 2020; accepted December 21, 2020.

DOI: 10.1093/clinchem/hvab008

VC American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com. 763

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lower limit of detection (LOD). This study compares

each laboratory’s LOD with its test results on a series of

80 tumor samples expressing a range of ER

concentrations.

Materials and Methods

PREPARATION OF CALIBRATORS (NIST STANDARD REFERENCE

MATERIAL [SRM] 1934–TRACEABLE STANDARDS)

A 24-amino acid long peptide incorporating the linear epitope of the ER binding SP1 monoclonal antibody was covalently coupled to cell-sized (diameter: 7–8 lm) glass

microbeads (Cospheric), as described previously (3–5).

This coupling reaction was performed at 10 different peptide concentrations, resulting in a range of measurement

standards with varying ER peptide concentrations per

microbead. The peptide was conjugated with a fluorescein

molecule, schematically illustrated in Fig. 1, A, to establish

traceability of measurement. The number of peptides conjugated to each group of microbeads was measured at

NIST using methods described in the next section.

The peptide was designed to incorporate a single

SP1 epitope and a single fluorescein (at the e position of

a lysine distant from the epitope) so that the two are

equimolar. The use of peptides (that incorporate an epitope) in lieu of a native protein was described previously

(6–8). Flanking amino acids corresponding to the sequence of native ER were also incorporated into the

peptide and served as a spacer. The 1:1 relationship between the amino acids comprising the SP1 epitope and

fluorescein was confirmed by establishing 96% peptide

purity using mass spectroscopy.

ER peptide-coated microbeads were mixed with

color standard microbeads (diameter; 4.5 lm) in a proprietary clear liquid matrix, as described previously (1,

4). When 1 lL of this microbead suspension is dispensed onto a glass microscope slide, the droplet dries

within a few minutes. The matrix in the dried droplet

retains the microbeads through subsequent routine IHC

treatments. Each dried microliter droplet on the slide

incorporates approximately 5000 peptide-coated (test)

microbeads. Approximately half of the test microbeads

bear the relevant analyte, whereas the other half has an

antigenically irrelevant peptide, serving as a negative internal control.

ESTABLISHING TRACEABILITY TO NIST SRM 1934

Fluorescence intensity values were used to assign concentration values to the microbeads in terms of equivalent

reference fluorophore (ERF) values. The ERF value assignment was performed according to a standard operating procedure (9) that includes 4 steps. First,

fluorescence measurements are carried out on serial dilutions of the fluorescein reference solution using a

spectrofluorometer equipped with a 488-nm laser excitation. The relative radiometric accuracy as a function of

wavelength of the fluorescence detection system was corrected using a calibrated light source, traceable to the

NIST realization of the International System of Units

(SI) (10–14). All fluorescence measurements were taken

at 21C (SD: 1.0 C) using a 90transmitting geometry

with the excitation beam incident on and normal to one

of the polished surfaces of the sample cuvette. All emission spectra were corrected for the responsivity of the detection system and normalized to the mean laser

intensity measured over the same time period as each

spectrum was taken. Fluorescence intensity was measured

by integrating a fluorescence emission spectrum from

515 nm to 535 nm. The resulting fluorescence intensities

and the corresponding concentrations of fluorescein solutions are used to calibrate the response of the fluorimeter.

In the second step, the fluorescence intensities of

calibrator microbeads were measured using the spectrofluorometer calibrated in the first step. The objective of

the second step is to determine the equivalent concentration of fluorescein reference fluorophore that gives

the same fluorescence intensity as the suspension of

microbeads. Next, as a third step, a light obscuration–

based, liquid particle counter and an Attune NxT flow

cytometer from Thermo Fisher Scientific were used to

measure the concentration of calibrator microbeads in

the suspensions used for the fluorescence measurements.

The light obscuration counter was a PAMAS model

SVSS-C with an HCB-LD-25/25 sensor head (S/N

U32757). Particle concentration was obtained by dividing particle count by the sample volume. Traceability to

the SI was assured by determining the confidence that

all particles within the sample volume were counted.

Flow cytometry was used to confirm the light obscuration–based microbead concentration. This was done using TruCount microbeads from BD Biosciences as an

internal standard in the calibration microbead suspension. The light obscuration measurement was used as

the primary method for microbead concentration measurements because the uncertainties in the light obscuration measurement are more thoroughly understood and

traceable to the SI (15, 16). Flow cytometry was used to

ensure that calibrator microbeads were monodispersed.

Last, the ERF values of the microbeads at different intensity levels were calculated by dividing the respective

ERF values for the microbead suspensions determined

in the second step by the concentrations of the

microbead suspensions (9).

BIOSPECIMEN MATERIALS

Ethics approval for the use of human tissues was

obtained from the Institutional Review Board of the

University of British Columbia and the British

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Columbia Cancer Agency. Human biospecimen tier 1

items are summarized in online Supplemental Table 1

according to Biospecimen Reporting for Improved

Study Quality (BRISQ) recommendations (17). A tissue

microarray (TMA) was constructed by using 80 cores,

each 0.6 mm, of 80 routinely formalin-fixed, paraffinembedded breast carcinomas and 2 benign-tissue negative control samples including benign liver and kidney

cortex. Tumors were classified according to the ASCO/

CAP guidelines based on ER expression by IHC (2), as

measured by the Canadian Immunohistochemistry

Quality Control (CIQC) reference laboratory

(Vancouver, BC, Canada). The cohort of tumors comprised approximately 35% ER negative, 25% ER-low

positive, and 40% ER-high positive tumors, representing the spectrum of ER expression.

CIQC ER SURVEY

IHC laboratories participating in the CIQC survey received a slide with a 4-lm–thick section of the breast

cancer TMA and ER calibrators. The unstained slides

were shipped at room temperature within 2 weeks after

preparation. Each participating laboratory assessed its

own staining and self-reported test results to CIQC. In

addition, the stained slides were returned to CIQC for

further expert assessment. The stained slides were also

shared with Boston Cell Standards for photomicroscopy

and image quantification of the ER calibrators. The

TMA was also evaluated by an additional expert pathologist (E.E.T) to determine the histology score (HS) for

each tumor. The percentages of negative nuclei, weakly

positive nuclei (1þ), moderately positive nuclei (2þ),

and strongly positive nuclei (3þ) were recorded for each

tumor, and the HS was calculated based on previously

published recommendations (18), as follows: HSCORE

1⁄4 P Pi(i þ 1), where i 1⁄4 1,2,3 and Pi varies from 0%

to 100%. Acceptability criteria were set as follows: minimum of 100 viable tumor cells for assessment from each

case and intraobserver reproducibility of H-score at j 

0.80. The latter was tested on a pilot sample of 30 cases.

Fig. 1. Schematic illustration of calibrator construction. The analyte is represented either by a peptide (A) or a protein (B), covalently coupled to a microbead. (A), Each sphere represents a single amino acid (single letter abbreviation) in a peptide. This particular sequence is for illustrative purposes only. Adapted from Vani et al. (3). (B), The recombinant extracellular domain of the

PD-L1 protein is depicted using a stick model. In both peptide and protein, 1 fluorescein label (illustrated as glowing yellow

spheres) provides a link for traceability of concentration. (C), Schematic illustration of the survey tool, showing the layout of calibrators and the TMA on a slide. The calibrators are organized in 2 columns with levels 1–5 on the right side and 6–10 on the

left. Level 1 has the lowest biomarker (ER) concentration; level 10 has the highest. The TMA in (C) is for illustrative purposes;

the exact number of tissue cores and the locations of positive and negative cores in the survey slide are different.

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The HS observer was unblinded for the results with ER

calibrators only after obtaining HS for the stained

TMAs. Excel 2016 (Microsoft) and SPSS version 26

(IBM Corp) were used for data entry and analyses.

Descriptive statistics were used to analyze the results.

CALIBRATOR QUANTIFICATION

Calibrators were photographed using a Zeiss Axioskop

microscope fitted with a Spot Imaging Solutions Insight

Gigabit CCD camera (Diagnostic Instruments). The

details of microbead stain intensity quantification is described elsewhere (4, 19).

Results

We describe the creation of IHC measurement traceability, how the new tools are used for measuring analytic sensitivity, and their validation in a national

laboratory proficiency testing program.

DESIGN OF IHC CALIBRATORS

According to this new system of IHC measurement

traceability, NIST SRM 1934 serves as a universal clinical IHC standard, regardless of the analyte. NIST SRM

1934 was originally developed for flow cytometry applications. It has a known fluorescein concentration, serving to standardize the fluorescence intensity

measurements of small cell-sized microbeads. If fluorescein is attached to an immunohistochemical analyte,

then NIST SRM 1934 can serve as a traceable concentration marker for the analyte. For example, because

each ER has a single fluorescein, the ER concentration

equals the fluorescein concentration. In the absence of

any existing immunohistochemical standards, we calculate ER concentration based on a linked fluorescence

measurement traceable to NIST SRM 1934.

Figure 1, A and B, schematically illustrates the construction of IHC calibrators, comprising either peptide

(Fig. 1, A) or protein (Fig. 1, B) analytes covalently coupled to cell-sized glass microbeads. We previously characterized microbead–peptide constructs for use as test

controls (3, 4, 20, 21). In this study, we describe using

this construct for the creation and use of calibrators

with traceability to an international standard, NIST

SRM 1934. The various concentrations, termed

“levels,” are pipetted and adhered onto a microscope

slide in an array where they remain during staining

(Fig. 1, C). The calibrators are processed through all of

the same antigen retrieval and staining steps as the 80

patient samples on the same slide.

For the CIQC ER national study, 3 calibrator sets

were created with different peptides for the SP1, EP1,

and 6F11 monoclonal antibody epitopes. Each laboratory received the appropriate set of calibrators and the

same set of patient samples. Fifty-eight laboratories used

the SP1 antibody, 17 used EP1, and 3 used 6F11. Of

the 58 laboratories that used the SP1 primary antibody

clone, 5 were not included because the calibrators were

inadvertently removed by the laboratory during staining.

In this study, we report the results with the SP1 clone

only because this clone was used by the overwhelming

majority of participants.

ESTABLISHING TRACEABILITY TO NIST SRM 1934

This new system of measurement traceability is unusual

because the reference standard (for fluorescein) is different than the analyte (ER). We describe this as a “linked”

system of measurement traceability. Because this article

presents the first example of IHC measurement traceability to a recognized reference standard, we briefly describe the process. The fluorescence intensity of each

calibrator microbead suspension was compared with the

fluorescence intensity of various dilutions of NIST

SRM 1934. Each calibrator’s concentration was calculated based on fluorescence intensity by first defining

the relationship between integrated fluorescence intensity as a function of reference fluorophore concentration, using serial dilutions of the SRM 1934 fluorescein

reference solution (Fig. 2, blue diamonds). A linear regression line was fitted to the plot. The integrated fluorescence intensity of each calibrator microbead

suspension was then measured using the same spectrofluorometer settings. The location of each calibrator

microbead suspension’s integrated fluorescence intensity

on the fitted straight line is shown in red triangles in

Fig. 2. When the fluorescence intensity values are interpolated onto the x axis, it provides the equivalent concentration of fluorescein, which is also the ER

concentration.

The only caveat in this linked system of fluorescein

traceability is that NIST SRM 1934 is composed of soluble fluorescein, whereas the calibrator fluorescein fluorophores are on microbeads. This difference—bound vs

soluble—can slightly change fluorescence intensity per

molecule. Therefore, calibrator fluorescein concentration is expressed as equivalent number of (soluble) reference fluorophores needed to produce a fluorescence

intensity equal to that of the (bound) microbead suspension (9). The ERF value for a single calibrator

microbead at the 95% confidence level (expansion coefficient k 1⁄4 2) is provided at the upper left of Fig. 2. It is

determined by dividing the ERF value for the

microbead suspension by the concentration of the

microbead suspension (9). A combined uncertainty

(95% confidence limits) associated with the ERF value

assigned to each calibrator microbead was calculated

from all steps of the value assignment, including weighing the fluorescein reference solution,

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spectrofluorometer calibration, charge-coupled device

(CCD) response calibration, microbead concentration

measurement by light obscuration, and the measurement of the fluorescence spectrum of the microbeads in

suspension (9). These measurement uncertainties are

also provided at the upper left of Fig. 2.

MEASUREMENT OF LOWER LOD

In IHC, the LOD represents the threshold separating a

positive result for an analyte from one that is negative.

Although the concept of a descriptive (nonquantitative)

IHC LOD has been introduced with critical assay performance controls (22), quantitative lower LODs do

not presently exist in IHC testing. With the newly developed calibrators introduced in this study, it is possible to measure the LOD of various IHC protocols.

Fig. 3, A, shows images of ER (SP1 peptide)–coated

microbeads after staining, along with their corresponding ER concentrations. The calibrators comprise negative control microbeads admixed with the positive ER

peptide-coated microbeads. This finding allows a direct

side-by-side comparison of stained and unstained

microbeads in the same images. Fig. 3, A, shows that

stain intensity gradually decreases from level 6 (592 000

molecules/microbead) to level 2 (16 500 molecules/

microbead). Therefore, the LOD is visually estimated in

this example at approximately 16 500 molecules per

microbead.

Figure 3, B, quantitatively depicts an example of

the relationship of stain intensity (y axis) as a function of

ER concentration (x axis). The LOD is the lowest analyte concentration that can be reliably distinguished

from the stain intensity of a zero-concentration sample.

For each laboratory, we identified the LOD as the mean

plus 3 SD of stain intensity. At this level, there is a 99%

probability that higher stain intensity is associated with

a nonzero analyte concentration. Using the linear regression line in this particular example (Fig. 3, B, dotted

line), the ER concentration at the LOD is 15 350 molecules per microbead. This calculated LOD closely

matches the estimated LOD based on visual inspection.

CLINICAL VALIDATION: CORRELATION OF LOD WITH TUMOR

ER EXPRESSION

A total of 5590 tissue cores from stained slides were received from all CIQC participants. Of all CIQC laboratory participants, 58 used the SP1 primary antibody

clone. This resulted in 4640 tumor samples from which

an additional 1602 were excluded because of missing tissue cores, insufficient number of tumor cells, or crush

and other tissue artifacts. A total of 3038 tissue samples

were included in the analysis. Of the 58 slides with samples, the calibrator had inadvertently been removed

from 5 during processing, leaving 53 for evaluation.

The distribution of the laboratories’ analytic sensitivity

is shown in Fig. 4. Most IHC laboratories had a LOD

between 10 000 and 25 000 molecules of ER per

microbead.

Figure 5, A, demonstrates that the percentage of

positive patient tumor samples correlates with analytic

sensitivity. Depending on the analytic sensitivity, anywhere between approximately 37% and 76% of tumor

samples would be considered ER positive. Laboratories

with highly sensitive ER assays (low LODs) detected

more positive cases, whereas those with poorly sensitive

ER tests detected fewer cases. The LOD of each laboratory correlated with the percentage of positive cases that

its IHC protocol produced (rs 1⁄4 0.767, P < 0.0001,

Spearman correlation; Fig. 5, A) and with cumulative

individual laboratory HS (rs 1⁄4 0.612, P < 0.0001,

Spearman correlation). Fig. 5, B, illustrates that the tumor samples accounting for this difference are principally “low positive,” according to the ASCO/CAP

classification (Fig. 5, B, blue dotted line). ER-high positive tumors were less affected by the differences in test

analytic sensitivity (Fig. 5, B, red dotted line). Relatively

few labs with lower LOD >25 000 were able to detect

the ER-low positive breast carcinomas, defined as 1%–

10% positive tumor cells at any intensity (2) (Fig. 5, B).

Fig. 2. Method for establishing traceability of calibrator

microbeads to NIST SRM 1934. The ER concentrations per

microbead for each calibrator (list at upper left) are in units

of ERFs. The blue diamonds are the data points from graphing fluorescence intensity (y axis) as a function of reference

fluorophore concentration in micromoles per liter (x axis)

using serial dilutions of the SRM 1934. A linear regression

line and its equation from these data points is shown. The

red triangles are the fluorescence intensity of each calibrator microbead suspension as each intercepts the linear regression line. Each is interpolated onto the x axis to find

the equivalent concentration for the calibrator at each level

(L).

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Figure 6 illustrates the actual appearance of stained

breast cancer samples from laboratories at the opposite

ends of the analytic sensitivity spectrum. These images

show just how different the results can be, depending

on the test analytic sensitivity. The upper row of images

is from the most analytically sensitive laboratory (low

LOD), whereas the lower row is from a laboratory with

the least sensitive ER test (high LOD). Representative

tumor samples considered to be high (cols. 1 and 2),

moderate (cols. 3 and 4), and low (cols. 5 and 6) ER

expressers are shown.

Discussion

The complexities of developing standards for IHC arise

mostly from the fact that IHC is a cell- and tissue-based

in situ immunoassay. The recognition of the need for

IHC reference standards dates back to 2002, when a

conference of leading organizations issued a call for a

HER2 standard (23). The conference participants envisioned the use of transformed cell lines as reference

standards, but it was technically more difficult than anticipated. We are not aware of progress since then in

Fig. 3. Derivation of lower LOD using calibrators. (A), Stained ER calibrators. The ER peptide-coated microbeads have concentrations ranging from 11 000 (level 1) to 1 810 000 (level 8) molecules per microbead. The concentrations are listed above each

photomicrograph and are the same as shown at the upper left of Fig. 2. Visually, the LOD is at level 2 (accentuated with thick

border). The red arrowhead identifies a smaller optical reference microbead, used for image quantification. These optical reference microbeads are permanently colored brown, regardless of staining. They provide an internal fixed optical reference point

for image stain intensity measurement, normalizing for variations in microscopy. (B) Graphical representation of ER calibrator

stain intensities (y axis) as a function of ER concentration (x axis). Each data point is the mean of duplicate measurements. The

empty circles represent data from higher calibrator levels that are on the analytic response plateau and thus not included in the

linear regression calculation. The horizontal dotted line represents the stain intensity of the background plus 3 SD. Background

stain intensity is measured with an antigenically irrelevant calibrator. The calculated LOD in this example is 15 350 molecules

per microbead.

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generating quantitative and traceable IHC reference

standards.

The linked system of measurement traceability

(through fluorescein) allows the rapid deployment of

calibrators to a broad range of common immunohistochemical tests, all traceable to NIST SRM 1934. In contrast, the traditional approach for creating metrology

standards would require a separate standard for each analyte. Assignment of concentration(s) would require a

gold standard assay, of which there is none for these protein analytes. In light of the fact that a typical IHC laboratory in a tertiary-care hospital has a test menu of

approximately 200 different tests, the traditional approach would involve many years of research and is a

technically challenging proposition. Even if the efforts

were focused on the most important predictive IHC

assays (e.g., PD-L1 testing for immunotherapy, HER2

in breast cancer), the traditional approach in generating

IHC standards has yet to bear fruit (23). Beyond predictive markers, there may be an important role of calibrators for many IHC tests, regardless of their ultimate

application or use. Like other qualitative testing in laboratory medicine, threshold-based laboratory assays may

benefit from a defined threshold of positivity to ensure

reproducibility and accuracy in distinguishing positive

vs negative results.

Creating standards for cell or tissue-based analytes

entails an additional complication not found with laboratory standards for serum testing. The compartmentalization of the analyte in a cell or extracellular matrix

means that it cannot be diluted to create different

concentrations. For example, a cell line expressing high

amounts of ER cannot be diluted to make ER-low cells.

In contrast, a reference standard for a soluble analyte

(e.g., for blood testing) can be serially diluted to create a

calibration scale spanning the analytic measurement

range.

The clinical validation data confirm the experimental prediction that analytic sensitivity, as measured with

NIST SRM 1934–traceable calibrators, correlates with

test results. Highly sensitive ER tests produce more ERpositive test results. Although this conclusion is hardly

Fig. 4. Analytic sensitivity of IHC laboratories’ ER tests using the SP1 antibody. The x axis reflects analytic sensitivity

as measured by lower LOD. The y axis reflects the number

of laboratories that fall within each LOD range.

Fig. 5. (A), Percentage of all positive cases as a function of

analytic sensitivity (lower LOD). (B) Same data as in (A), but

stratified by ER-high vs ER-low positive cases. In (B), the y

axis is the percentage of cases that are classified as ER-low

or ER-high, per the ASCO/CAP guidelines. The interlaboratory differences in analytic sensitivity principally affects the

ability to detect ER-low positive breast carcinomas.

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surprising, the implications of a reference standard and

traceable calibrators potentially affect the treatment of

millions of patients with breast cancer. As our data illustrate, the treatment options offered to patients with

breast and other cancers be affected by which laboratory

performs the test. ER-high positive tumors will generally

be positive regardless of the laboratory performing the

test. However, our data show that ER-low positive

tumors are highly affected by the variability in analytic

sensitivity between different clinical IHC laboratories.

These data argue for the urgent need to standardize testing to a defined analytic sensitivity range.

In this study, we used the reference standard to

characterize the LOD of each laboratory’s ER test, not

for quantifying ER concentrations in patient specimens.

As the technology matures, it will likely become possible

to quantify patient test results in terms of median number of molecules per tumor cell. Until then, several immediate clinical uses exist for calibration standards, all of

which revolve around quantitatively defining the threshold of positivity: (a) during IHC protocol development

for clinical trials and for clinical use, (b) for IHC protocol development and technical and analytical validation

in clinical laboratories, (c) as a daily quality control (e.g.,

on-slide controls to verify the LOD for each tested patient), (d) for revalidation of IHC protocols on a periodic basis, (e) for lot-to-lot validation of reagents, (f)

after major service to testing instruments, and (g) in the

context of a problem investigation (e.g., suboptimal or

poor results in proficiency testing). Furthermore, it is

only now that meaningful research can be performed to

identify clinically relevant analytic sensitivity thresholds

for classifying ER expression levels that predict patients’

responses to hormonal therapy, how they correlate to

gene expression profiling, and tumor subclassification.

Reference standards are critical not only for development of prognostic and predictive assays but also for

methodology transfer from clinical trials or published

literature to clinical laboratories. Moreover, reference

standards enable more objective design of proficiency

testing and more objective comparison of laboratory

performance with ER IHC and other IHC assays as

more reference standards are developed.

Supplemental Material

Supplemental material is available at Clinical Chemistry

online.

Nonstandard Abbreviations: IHC, immunohistochemistry; ER, estrogen receptor; LOD, limit of detection; SRM, Standard Reference

Material; ERF, equivalent reference fluorophore; TMA, tissue microarray; CIQC, Canadian Immunohistochemistry Quality Control;

HS, histology score

Author Contributions: All authors confirmed they have contributed to

the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising

Fig. 6. IHC staining of 6 breast carcinomas illustrating the range of stain intensities for the most sensitive (upper row) and least

sensitive (lower row) laboratories. Included are cases that appear to have comparatively strong staining (cases 1 and 2), moderate staining (cases 3 and 4), and weak staining (cases 5 and 6) for ER.

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the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus

ensuring that questions related to the accuracy or integrity of any part of

the article are appropriately investigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form.

Disclosures and/or potential conflicts of interest:

Employment or Leadership: S.R. Sompuram, K. Vani, and S.A.

Bogen are principals at Boston Cell Standards; A. Schaedle, Boston

Cell Standards.

Consultant or Advisory Role: None declared.

Stock Ownership: S.R. Sompuram, K. Vani, and S.A. Bogen are

shareholders in Boston Cell Standards.

Honoraria: None declared.

Research Funding: Research reported in this paper was supported in

part by the National Cancer Institute of the National Institutes of

Health under award number R44CA213476.

Expert Testimony: None declared.

Patents: Boston Cell Standards holds a patent and other patent applications on the technology used in the study. S.R. Sompuram,

EP3065704 B1; S.A. Bogen, EP3065704 B1.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of

data, preparation of manuscript, or final approval of manuscript.

Acknowledgments: We are grateful to Canadian

Immunohistochemistry Quality Control (CIQC) staff members Dr.

C. Blake Gilks, Dr. Jennifer Won, and John Garratt for their assistance in providing the tissue microarray samples and for sample distribution and collection. We are also grateful to all CIQC member

laboratories that took part in this CIQC exploratory proficiency testing

run. Since January 2020, the CIQC has evolved into Canadian

Biomarker Quality Assurance (CBQA) and Canadian Pathology

Quality Assurance. Dr. Torlakovic is currently associated with CBQA.

Disclaimer: Certain commercial equipment, instruments, and materials

are identified to specify the experimental procedure. In no case does

such identification imply a recommendation or endorsement by the

National Institute of Standards and Technology, nor does it imply that

the materials or equipment are necessarily the best available for the

purpose.

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