Diagnostic Immunohistochemistry Dabbs Pdf To Jpg

Nov 19, 2013  Diagnostic Immunohistochemistry presents the latest information and most reliable guidance on immunohistological diagnoses in surgical pathology. Dabbs, MD and other leading experts bring you state-of-the-art coverage on genomic and theranostic applications, molecular anatomic pathology, immunocytology, Non-Hodgkin s lymphoma, and more.

Standardization of controls, both positive and negative controls, is needed for diagnostic immunohistochemistry (dIHC). The use of IHC-negative controls, irrespective of type, although well established, is not standardized.

As such, the relevance and applicability of negative controls continues to challenge both pathologists and laboratory budgets. Despite the clear theoretical notion that appropriate controls serve to demonstrate the sensitivity and specificity of the dIHC test, it remains unclear which types of positive and negative controls are applicable and/or useful in day-to-day clinical practice. There is a perceived need to provide “best practice recommendations” for the use of negative controls. This perception is driven not only by logistics and cost issues, but also by increased pressure for accurate IHC testing, especially when IHC is performed for predictive markers, the number of which is rising as personalized medicine continues to develop. Herein, an international ad hoc expert panel reviews classification of negative controls relevant to clinical practice, proposes standard terminology for negative controls, considers the total evidence of IHC specificity that is available to pathologists, and develops a set of recommendations for the use of negative controls in dIHC based on “fit-for-use” principles.

BACKGROUNDGreat efforts have been made to standardize diagnostic immunohistochemistry (dIHC), especially with the establishment of external quality control assessment (EQA) services for immunohistochemistry. The UK National External Scheme for Immunocytochemistry and in situ Hybridization (UK NEQAS ICC & ISH) has been providing proficiency testing and education in dIHC for 25 years, and several other programs have subsequently developed eg, Nordic immunohistochemical Quality Control (NordiQC), College of American Pathologists-External Quality Assurance/Proficiency Testing (CAP-EQA/PT), Canadian Immunohistochemistry Quality Control (CIQC), Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP), and others.

However, it is only in the last decade that technical advances, including improved quality of antibodies (Abs), automated staining platforms, and highly sensitive detection systems have resulted in more reproducible staining methods, which have had a direct impact on interpretation of IHC results and patient care. – These standardization efforts have originated from practicing pathologists, national and international professional organizations, and industry. Although standardization of dIHC includes preanalytical, analytical, and post-analytical phases, this paper concentrates on the standardization of controls, specifically negative controls, rather than on standardization of IHC protocols overall. It is important to emphasize that the standardization of dIHC must include standardization of controls, which are essential elements of quality control (QC) (analytical phase), as well as interpretation of IHC results (post-analytical phase).

The correct interpretation of dIHC is reliant on observation of the expected staining results achieved on known positive and negative controls. Daily positive and negative controls (as defined below) are an important link between analytical and postanalytical components of IHC testing. The focus of this paper is standardization of negative controls for dIHC. DIAGNOSTIC IHC CONTROLSPositive and negative controls serve the purpose of providing evidence that each IHC test (stain) is successfully performed and is giving the expected level of sensitivity and specificity as characterized during technical optimization and validation of the IHC test for diagnostic use.

However, both positive and negative controls currently lack agreed upon criteria for standardization. In addition, the principles and approaches to the standardization of positive and negative controls are vastly different, as follows:.Positive tissue controls (PTCs) primarily monitor calibration of the system and protocol sensitivity, but also provide assurance that the right Ab is applied. Briefly, external positive tissue control (Ext-PTC) should be selected so as to have undergone fixation and processing in a manner as closely similar as possible to the test tissue.

Antigens internal or intrinsic to the test tissue (patient sample) may also be used for this purpose, if present. This type of positive control is termed here as internal positive tissue control (Int-PTC). The optimal selection of Ext-PTC and interpretation of the results of the Ext-PTC and Int-PTC requires in-depth experience and knowledge of the biological processes, in the context of the intended use of each IHC test, to establish desirable and relevant calibration of the protocol. For example, Ext-PTC for CD20 should include tissue (preferably normal tissue with predictable levels of CD20 expression), representative of both low-expression and high-expression levels to demonstrate that the overall sensitivity of the protocol is sufficient to detect CD20 expression in lesions with low CD20 expression (such as small lymphocytic lymphoma or some diffuse large B-cell lymphomas), if the intended use of the CD20 IHC test is to be able to reproducibly detect CD20 expression in such lesions. A future issue of Applied Immunohistochemistry & Molecular Morphology will focus on selection and use of positive controls.Negative controls are primarily used to evaluate the specificity of the IHC test to identify false-positive staining reactions. They include negative reagent controls (NRCs) and negative tissue controls (NTCs). Basic principles that govern the use of negative controls, including selection of the type of negative control, the frequency of use, and validity, tend to apply across all of diagnostic pathology, and are generally not subspecialty driven.

Therefore, the use of negative controls can be standardized at this time, to be more relevant to current needs of pathology practice.There is a perceived need to provide “best practice recommendations” for the use of negative controls. The perception of this need is driven not only by logistics and cost issues, but also by increased pressure for accurate IHC testing, especially in the context of predictive markers, which will continue to rise in number as personalized medicine continues to develop. The need for thorough discussion and standardization of negative controls was also highlighted by recently published recommendations regarding negative control use from the College of American Pathologists (CAP). EVIDENCE-BASED USE OF NEGATIVE CONTROLS IN DIAGNOSTIC IHCCurrently, there is no published evidence to support the diverse requirements emanating from different accreditation bodies throughout the world with respect to the selection and use of negative controls in dIHC. Hitachi dvd movie album software. False-positive reactions may occur due to characteristics of the primary Ab or “nonspecific” (unwanted) binding of the components of the detection system, which may include a secondary Ab that lacks specificity.

Although several countries have published guidelines and recommendations relating to quality assurance in IHC, there is little published evidence to support the use of particular types of negative controls in dIHC. Much of the published work relates primarily to biotin-based systems. –The recommendations in this paper are based on: (i) published literature; (ii) evidence generated in various EQA programs; and (iii) current expert opinion. COST-EFFECTIVENESS OF NEGATIVE CONTROLS IN dIHCGlobally, there is great practice variation in the use of negative controls. Laboratory accreditation requirements may on occasion be the main driver for the use of certain negative controls.

Additional slides serving as NRCs varied from 0 in some laboratories to ≥15% of the total IHC slide annual workload in other laboratories. Even in countries with a long history of laboratory accreditation and EQA, there remains a non-standardized approach to the use of NRCs; UK NEQAS reported that 48% of dIHC laboratories used NRCs, whereas specifically in neuropathology is was even less at 36% (E.E.T., unpublished data, 2012).The importance of negative controls has been emphasized for IHC protocols that use avidin-biotin detection systems, especially in combination with powerful antigen retrieval methods known to “unmask” endogenous biotin, a procedure that exacerbates the undesirable binding of avidin-based detection systems.

Although many laboratories no longer use avidin-biotin– based detection systems, the use of negative controls designed to detect such spurious binding continues unabated. This practice has resulted in the unnecessary processing of very large numbers of negative control slides, even for IHC protocols that utilize “polymer/multimer” detection systems with much less susceptibility for false-positive results.One direct consequence of this “routine unthinking” approach is that pathologists often ignore the additional NRC slides, a practice justified on the basis that evaluation of a panel of IHC Abs, in the hands of an expert interpreter, provides substantial and sufficient evidence that the achieved reactions are specific. Therefore, it may be very cost-inefficient to comply with accreditation requirements that mandate one or more NRC slides on every case.In contrast, many laboratories around the world may not run NRCs at all, either because these controls are not mandated, or their implementation would increase the cost of operations, or both.In this context, there are expert opinions that NRCs now are largely or completely unnecessary. Recently, this approach was also partly accepted by the CAP in the United States for dIHC tests utilizing polymer-based/multimer-based detection systems. The CAP anatomic pathology checklist item concerning the use of negative controls (ANP.22570) was recently revised with the use of NRCs in dIHC now being at the “discretion of the laboratory director,” so long as the avidin-biotin detection systems are not used. However, the CAP checklist still requires NTCs (see for definitions).

The revised requirements from the CAP are highly appreciated because extensive experience with the use of polymer-based/multimer-based systems has shown that NRCs are not very useful, in particular if a panel of markers are used with the same detection system. THE EXTENT OF THE PROBLEMThe use of IHC-negative controls, irrespective of type, although well established, is not standardized and the relevance and applicability of negative controls continues to challenge both pathologists and laboratory medicine budgets. Despite the clear theoretical notion that appropriate controls serve to demonstrate the sensitivity and specificity of the dIHC test, it remains unclear for most dIHC tests that which types of positive and negative controls are applicable, and/or useful in day-to-day clinical practice.This paper addresses the different types of negative controls and their recommended use in diagnostic pathology. There is a wide-spread tendency to use the general term “negative control” for all types of negative controls, including the “NRC,” which is a separate slide on which a parallel section of the patient’s test tissue sample is run omitting the specific primary Ab. This lack of clear definition is misleading, because other types of negative controls exist, having different import.It is critical to recognize that NRCs are not the only source of evidence of the specificity of the IHC test and that NRCs have limited potential to detect false-positive results due to unexpected cross-reactivity of the primary Ab. EQA experience has shown that false-positive results may unexpectedly appear with various Abs, even those that are well established and widely validated in the literature.

Recent examples from CIQC experience include Ab clones 6F11 for estrogen receptor (ER) and SP4 for cyclin D1, as well as UK NEQAS ICC & ISH and NordiQC experience with the Ab products of clones SP2 and IE2 for progesterone receptor (PR)., False-positive results were observed that were not detected by NRC slides alone. A combination of experience, knowledge of the biodistribution of the target antigen, and employment of other types of negative controls is required in these instances; in particular this includes evaluation of the NTCs. Where appropriate, absorption of the primary Ab with the target antigen (especially valuable for “polyclonal” antisera as opposed to monoclonal Abs) and the use of cell lines known not to express the antigen in question, by ELISA or mRNA comparative studies could be employed. These latter controls are beyond the scope of the “routine clinical laboratory” and usually reside within the domain of the Ab manufacturer as a part of characterization and validation; such data should be available to the user in package inserts or specification sheets.This paper examines previously published recommendations for negative controls in dIHC and introduces the concept of “IHC Specificity Evidence” (IHC-SE), to reconcile theories around the use of negative controls with current diverse practices and observed deviation from recommendations. IHC-SE includes all evidence available to pathologists, by which they can determine the likelihood of whether IHC test results are specific, with respect to cell distribution and diagnostic utility. Positive ControlsThe purpose of the positive control is to demonstrate that the IHC protocol is able to detect the antigen of interest (qualitative positive control) and is sensitive at the level of clinical relevance (calibrated positive controls; low and high expressers).

Positive controls include samples that have been previously tested for the Ab in question, or samples which are known to express the Ab at levels within the desired range for the intended use following sample preparation (including fixation and retrieval). “On-slide” external and internal negative tissue controls are illustrated. It is sound practice whenever possible to include cells (or tissue elements) that will serve as negative controls (expected to be nonreactive) when selecting tissue for the positive tissue control.

Both internal and external negative on-slide tissues are so-called “specific” negative controls because all are exposed to the specific primary antibody. Separate slide negative controls are generally used for negative reagent controls, where the primary antibody is omitted or an irrelevant primary antibody is used.

Note that reagent controls should have identical protocols to the specific immunohistochemistry test, including the same type of pretreatment, as far as is possible.Even when using an optimized and validated primary Ab (polyclonal or monoclonal), it is still possible that nonspecific labeling, other than of the target epitope, may be found. In this context, “nonspecific” is defined as staining of normal or abnormal cells or tissues that are not part of the described intended use for the particular IHC test. This outcome may be due to contamination of the commercial product, including unpurified ascites mouse monoclonal Abs causing the so-called mouse as-cites Golgi reaction in human exocrine cells from individuals with blood type A, but may also be a consequence of low affinity binding to secondary target epitopes, or to cross-reactive (molecular) species that share the primary epitope., These effects may be reduced (although not necessarily eliminated) through optimization/calibration of the entire protocol before clinical use. There are also newly developed tools to test the monospecificity of primary Abs, such as high-density protein microarray chips developed for Ab-specificity testing; for example, by using a chip spotted with 10,000 unique overexpressed proteins, it is possible to validate the specificity of an existing diagnostic Ab.

Also, it must be remembered that when elements of fixation/processing also are considered (antigen loss, masking, alteration, retrieval), then existing negative (and positive) controls may fall short, – as discussed below. Immunohistochemistry-Specificity EvidenceIHC-SE consists of all components of IHC testing that provide evidence relating to the specificity of IHC tests. Evidence may be derived from on-slide controls (both internal and external), separate slide NRCs, evidence of specificity derived from an IHC “panel” used on the same tissue block with the same detection system, informed interpretation of differential intensity of staining in the internal and external positive controls, as well as subcellular signal localization/pattern, and informed interpretation based on understanding of biology. This all-encompassing approach empowers pathologists to apply their expertise in interpretive science and allows for efficient and cost-effective practice. Internal Tissue ControlsInternal tissue controls, also referred to as intrinsic or built-in controls, utilize the tissue elements in the patient’s test sample that is being evaluated for expression (internal tissue positive control) or absence internal negative tissue control (Int-NTC) of certain epitopes. External Tissue ControlsExternal controls designate the use of tissues, or cell lines, derived from sources other than the actual patient’s test sample (therefore they are termed “external”). It is important to note that because the tissue (or cell line) is not a part of the patient’s test sample used for IHC testing, preanalytical processing, despite best efforts, will not be identical (eg, different ischemic time, different fixation time).

External controls may be tissue controls (single tissue sections or tissue microarrays), cell blocks prepared from cell lines (or body fluids such as pleural or peritoneal fluids), any combination of these, or even peptides deposited onto glass slides. – The tissues may originate from the diagnostic tissue archives or may be grown as xenografts, or faux tissues (artificial control tissue) such as “histoids” constructed from several cell lines.

– The latter may be superior when antigens are not expressed in normal tissues, such as ALK-1, an important marker in anaplastic large-cell lymphomas or certain lung cancers.External tissue controls are designated as “external positive tissue controls” (Ext-PTCs) if they express an epitope of interest and are designated as “external negative tissue controls” (Ext-NTCs) if they contain cells known not to express an epitope of interest. External controls may be run on the same slide as patient’s test sample (so-called “on-slide” controls) or on a separate slide. “On-slide” controls require the mounting of 2 cut paraffin sections on the same slide (the control section and the test section), which may be technically more demanding, but offers greater assurance of identical retrieval and staining conditions being applied for the external control and the patient’s test sample. External controls on separate slides may also be used as “batch” or “run controls,” when a large number of slides are stained with the same primary Ab/protocol.

It is worth emphasizing that several EQA programs recommend that external controls should contain both expected positive and expected negative tissue elements. Specific and Nonspecific Negative ControlsIn 2000, Taylor introduced a classification of negative controls as “specific” and “nonspecific.” “Specific” negative controls are tissue-based controls designed to test whether the reactivity with the tissue/staining is due to primary Ab-specific design for specific epitope so that the reaction will not be present if the specific primary Ab is replaced by nonimmune serum of the same immunoglobulin (Ig) subtype. “Nonspecific” negative controls are designed to test whether any part of the protocol or detection system (beyond the primary Ab) gives unexpected staining. NRC (Negative Reagent Controls)NRCs are used to confirm specific binding by the primary Ab and detection system.

NRCs are always run on a separate slide using serial sections from the same paraffin block used for IHC testing of the patient’s test sample. NTC (Negative Tissue Controls)Specific negative controls are tissues that are known to not contain the antigen of interest and are evaluated using the specific primary Ab. More broadly, specific negative controls may also be created from cell lines, in which case they are most appropriately named negative cell line control.There are 2 types of NTCs (depending on the source):.Int-NTC, if the control element is a portion of the patient’s test sample (internal, intrinsic or built-in negative control) , or.Ext-NTC, if the control element is part of the external (usually on-slide) control, which contains both positive and negative cell types or tissues “external on-slide negative control” (see above, and ).

INTERPRETATION OF NEGATIVE CONTROL RESULTSPathologists should interpret the results of all positive and negative controls before examining the test/patient’s samples. Technologists also should interpret the results of NRCs and on-slide external (positive and negative) controls. The results should be recorded as part of the routine laboratory QC.If the NRC-primAb is positive, then this is a false-positive reaction, which may be due to characteristics of the primary Ab isotype or class (eg, IgG1) that result in nonspecific attachment of “nonimmune” Ig to the section at the specific concentration and incubation time employed (see below). Whether it is the primary Ab isotype or class or some components of detection system that are causing the false-positive staining may be further explored by using various types of NRC-detSys (see below and and ).

If the NRC-detSys is positive, then the false-positive reaction is due to nonspecific binding by the secondary Ab or any other component of the detection system. Most frequently endogenous biotin is visualized as granular cytoplasmic reactivity; endogenous biotin in adrenal tissue (A) and kidney (B). On occasion, it also may be seen as nuclear reactivity shown here in endometrium (C). When strongly expressed, endogenous biotin cannot be blocked even with commercially available biotin-blocking reagents, shown here with CDX-2 IHC test (D), but when run with biotin-free detection systems false cytoplasmic positivity disappears (E). It is important to note that this is a specific reaction between avidin and biotin and therefore does not represent nonspecific background. It is also difficult to block. NRCs (NRC-primAb and NRC-detSys) (Nonspecific Negative Controls)Separate slide NRCs should be processed in the same manner as the slides for specific IHC tests, including use of identical epitope retrieval procedures.

This requirement is automatically achieved for internal and “on-slide” external negative controls, except of course that the latter will have experienced different preanalytic treatment (tissue processing).The number of slides to be used as NRCs is also determined by the number of different pretreatment procedures: 1 NRC should be prepared for each methodological variation employed in a given clinical case. For example, if 3 different epitope retrieval procedures are used in a panel of several different Abs eg, HIER (heat-induced epitope retrieval) in citrate buffer, HIER in EDTA, and protease digestion, 3 NRCs (1 processed with HIER in citrate buffer, 1 with HIER in EDTA, and 1 with protease digestion) should be prepared.However, experience has shown that this approach is not necessary when polymer-based/multimer-based detection systems are used (after initial validation), although it is recommended if an avidin-biotin–based detection system is employed. InterpretationNone of the NRCs are able to completely exclude the possibility of undesirable/unexpected cross-reactivity of the primary Ab with some epitopes. Specific negative controls (tissue controls) may detect such undesirable cross-reactivity, but detection depends on the presence of such epitopes in the internal or external control tissue, and also upon critical evaluation by the pathologist, to recognize that nonspecific staining is present. Therefore, negative results with NRCs do not ensure specificity of IHC tests in all cases, and when unexpected reactivity of the primary Ab is encountered, the potential for false-positive results still needs to be considered. Careful evaluation of NTCs is still required even if the NRCs are negative. “Fit-for-Use” PrinciplePathologists use IHC tests for different purposes in diagnostic practice, and different weighting is given to the IHC findings, commensurate with intended use.In the United States, the Food and Drug Administration (FDA) has included IHC tests under the category of diagnostic devices (Code of Federal Regulations.

The FDA classifies IHC test/devices in 3 classes (classes I to III) on the basis of risk to the patient.Class I IHC test results are interpreted in the context of morphology and other relevant laboratory or clinical information. Class I IHC tests are used after the primary diagnosis of tumor has been made by conventional histopathology using nonimmunologic stains, such as hematoxylin and eosin. These IHC tests provide the pathologist with adjunctive diagnostic information that may be incorporated into the pathologist’s report, but is not ordinarily reported to the clinician as an independent finding.Class II IHC tests are intended for the detection/measurement of target analytes to provide prognostic or predictive data that are not directly confirmed by routine histopathologic methods. Class II tests provide information that is ordinarily reported as independent diagnostic information to the ordering clinician. Claims associated with data from class II IHC tests are widely accepted and supported by valid scientific evidence.Class IHC III tests are defined (by the FDA) by exclusion, as any IHC test not falling under classes I or II.

They are of higher risk, complex, and are often used as standalone findings, not confirmed by other pathologic criteria. Class III devices are subject to Premarket Approval by the FDA, with defined guidelines for use, including controls, exemplified by the HercepTest, Dako as the prototype for the series.It is important to recognize that the FDA classification relates to IHC devices and reagents and it is up to the end user (pathologist) to determine how and for what purpose various IHC tests are used (eg, ALK-1 in lung cancer is a “predictive” IHC test vs. ALK-1 for lymphoma, which is a diagnostic IHC test). On the basis of the “fit-for-use” concept, and the level of QA required in the clinical laboratory, the Canadian Association of Pathologists National Standards Committee has put forward recommendations that IHC tests/protocols (vs.

Ext-NTCExt-NTC elements may be present in the external on-slide positive controls: as noted, ideally positive control tissues should be selected so as also to include elements that are known to be negative (nonreactive) for the evaluated marker. In principle, this goal is best achieved by using multitissue positive control blocks (a combination of strongly positive, intermediate/weak, and negative tissue samples) preferably placed on the same slide as the patient sample. Such composite on-slide controls are recommended by several external quality assurance programs. In the absence of available multitissue blocks, single tissue blocks may be used, carefully selected so as to include tissue elements known not to contain the epitope in question.Evaluation and “quality control” of the results of external positive and negative controls is normally performed by technologists/biomedical scientists before the slides are forwarded to pathologists for evaluation as part of the overall interpretation of the IHC results. Many EQA agencies require documentation of review and approval of such controls on a daily basis.

Format

Inbunden (Hardback)

Språk

Engelska

Antal sidor

960

Utgivningsdatum

2013-11-21

Upplaga

4

Illustratör/Fotograf

approx illustrations

Illustrationer

Approx. 1376 illustrations (1376 in full color)

Dimensioner

285 x 228 x 50 mm

Vikt

3084 g

Antal komponenter

1

Komponenter

Hardback and Internet Resource

ISBN

9781455744619

Theranostic and Genomic Applications, Expert Consult: Online and Print

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'This is a great contribution to the field of pathology. The book will be very useful for anyone who wants to know immunohistochemistry and theranostics in detail.'-Tejashree Karnick, MBBS(University of Kansas Medical Center) Doody Review: 5 stars

1. Techniques of Immunohistochemistry: Principles, Pitfalls and Standardization

2. Molecular Anatomic Pathology: Principles, Technique and Application to Immunohistologic Diagnosis

3. Immunohistology of Infectious Diseases

4. Immunohistology of Neoplasms of Soft Tissue and Bone

5. Immunohistology of Hodgkin Lymphoma

6. Immunohistology of Non-Hodgkin Lymphoma

7. Immunohistology of Melanocytic Neoplasms

8. Immunohistology of Metastatic Carcinoma of Unknown Primary Site

9. Immunohistology of Head and Neck Lesions

10. Immunohistology of Endocrine Tumors

11. Immunohistology of the Mediastinum

12. Immunohistology of Lung and Pleural Neoplasms

13. Immunohistology of Skin Tumors

14. Immunohistology of the Gastrointestinal Tract

15. Immunohistology of the Pancreas and Hepatobiliary Tract

16. Immunohistology of the Prostate

17. Immunohistology of the Urinary Bladder, Kidney, and Testis

18. Immunohistology of the Female Genital Tract

19. Immunohistology of the Breast

20. Immunohistology of the Nervous System

21. Immunocytology

22. Immunohistology of Pediatric Neoplasms

23. Imaging and Quantitative Immunohistochemistry