Saturday 18 January 2014
Table of Contents
2014Month :JanuaryVolume :Vol3Issue :Issue 2Page :322-325METASTATIC BREAST CARCINOMA WITH MULTIPLE OSTEOLYTIC LESIONS - A DIAGNOSTIC DILEMMA
Wednesday 31 July 2013
Wednesday 12 June 2013
Fite-Faraco Staining Protocol
Principle
Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are ß-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species (Lyon H 1991).
The leprosy bacillus is much less acid and alcohol fast than the tubercle bacillus, therefore alcohol is removed from the hydrating and dehydrating steps and 10% sulphuric acid is used as a decolouriser in place of acid / alcohol solution. The sections are also deparaffinised using peanut oil/xylene mixture, this helps to protect the more delicate waxy coat of the organisms.
Technical Points
(step 7) It is important not to over-stain with methylene blue, as it will not be possible to remove the excess dye in alcohol.
Method
1. Warm sections and de-paraffinize in a mixture of two parts xylene/one part vegetable oil for 15 mins.
2. Blot dry and wash in water. Repeat if any xylene-oil remain on the section.
3. Filter on carbol fuchsin solution, DO NOT HEAT, for 20 mins.
4. Wash in running tap water.
5. Differentiate in 10.0% sulphuric acid for 2 mins.
6. Wash well in running tap water, rinse distilled water.
7. Counterstain in 0.25% methylene blue for 20 seconds.
8. Wash and blot dry. DO NOT DEHYDRATE IN ALCOHOL.
9. Clear in xylene. Repeat the blotting-xylene treatment until section is clear.
10. Mount in a DPX type mountant.
Results
- Leprosy bacilli , hair shafts ……………magenta
- Nuclei, background…………………………blue
- Red blood cells……………………………… pale pink
Reagent Formulae
1. xylene/peanut oil
xylene ----------------------- 2 parts
peanut oil ------------------- 1 part
2. carbol fuchsin obtain from MedVet
3. sulphuric acid 10% *add acid to water*
distilled water --------------- 90.0 ml
conc. sulphuric acid -------- 10.0 ml
4. acetified methylene blue methylene blue (CI 52015) ----- 0.25 gm
distilled water ------------------- 99.0 ml
glacial acetic acid --------------- 1.0 ml
Dissolve the dye in the water. Add the acid and mix. Filter into the reagent bottle. The stain keeps well.
Wednesday 24 April 2013
Role of Evidence Based Medicine in Diagnostic Surgical Pathology
Evidence-based medicine (EBM) has been defined as “the conscientious, explicit, and judicious use of current best evidence in making decisions about the care of individual patients” or as “the integration of best research evidence with clinical expertise and patient values”. It is an evolving discipline that applies analytical and quantitative methods to evaluate the validity of available medical information, with the overall goal of identifying scientifically-sound data or “best evidence”. This evidence is integrated to improve medical practice through clinical guidelines and other tools that are used for education, standardization of care, quality initiatives and coverage decisions. The ideas of EBM have spread rapidly through medicine during the past decade and are recently eliciting a growing interest in Anatomic Pathology and Laboratory Medicine.
Basic Concepts of Evidence-Based Medicine
EBM investigators attempt to identify the best current and relevant research information available for a particular problem and to integrate the “evidence” into guidelines, rules or other tools that will assist medical practitioners in their daily practice.
Basic Process for the identification of best evidence and its integration into guidelines, rules or other protocols.
Basic Process for the identification of best evidence and its integration into guidelines, rules or other protocols.
1. Formulate specific questions regarding the diagnosis, prognosis, causation and/or treatment of individual patients with a particular clinical problem
2. Search for specific information in the scientific literature
3. Appraise the internal and external validity of the available evidence, and its impact, applicability and usefulness in daily practice
4. Incorporate “best evidence” from several reliable sources along with personal clinical exexperience into” guidelines, rules or other protocols
5. Evaluate the effectiveness and efficiency of those “Evidence-based” recommendations
2. Search for specific information in the scientific literature
3. Appraise the internal and external validity of the available evidence, and its impact, applicability and usefulness in daily practice
4. Incorporate “best evidence” from several reliable sources along with personal clinical exexperience into” guidelines, rules or other protocols
5. Evaluate the effectiveness and efficiency of those “Evidence-based” recommendations
Bayesian approach to the analysis of data : influence of the prior probability of findings of interest.
Descriptive statistical tests offer limited information about other features that can influence the outcome of observational studies, such as the prevalence of a disease within the population study and in the control group and the prior probability of a finding. For example, it is well known that lymph node status has a statistically significant prognostic significance in most patients with cancer. However, in patients with Stage IV neoplasms who have a high “prior probability” of dying from their disease, the prognostic value of the feature lymph node status is probably rather limited. These considerations are intuitively used in daily practice by most pathologists, but there are few, if any, available evidence-based guidelines or other protocols that take into consideration the prevalence and prior probability of various findings into the selection and/or interpretation of diagnostic features, immunostains or other ancillary tests in Surgical Pathology. Another consideration that has not been addressed in most observational studies in Pathology is the need to divide the data into “training” or “testing” sets (“study” and “holdout” cases) in research projects attempting to derive classification or prognostic models. Most clinico-pathological categorization has been based on data derived from analyzing 100% of the data from study groups and control groups with descriptive univariate, and less often multivariate statistical methods. However, multiple studies using Bayesian methods have shown that models derived by the use of 100% of a dataset usually have limited external validity as there is a certain element of “circular reasoning” in the modeling methodology.
Evaluating the Quality of Published Studies in the Medical Literature.
Ebell has proposed a system for classifying published medical evidence into 4 levels, with “grade I” being the best (most reliable). Grade I studies are those that include data validated with a “test” group that is from a different and distinct population from the “training” cohort. Grade II studies report data that are obtained from the same population, the members of which are divided into independent “training” and “validation” subsets and evaluated prospectively. Grade III analysis also include “training” and “validation” subsets from the same population, but data are collected contemporaneously rather than prospectively. Grade IV studies are those in which the “training” group is also used as the “validation group”. According to this scheme, most studies in the pathology literature would probably be classified as Grade IV, the most vulnerable to external validity problems.
Ebell has proposed a system for classifying published medical evidence into 4 levels, with “grade I” being the best (most reliable). Grade I studies are those that include data validated with a “test” group that is from a different and distinct population from the “training” cohort. Grade II studies report data that are obtained from the same population, the members of which are divided into independent “training” and “validation” subsets and evaluated prospectively. Grade III analysis also include “training” and “validation” subsets from the same population, but data are collected contemporaneously rather than prospectively. Grade IV studies are those in which the “training” group is also used as the “validation group”. According to this scheme, most studies in the pathology literature would probably be classified as Grade IV, the most vulnerable to external validity problems.
Integration of “best evidence” from the literature with personal clinical experience into “evidence-based” guidelines, rules or other protocols.
Advocates of EBM have attempted to organize “best evidence” from the scientific literature and their own experience into algorithms, protocols, guidelines or “rules” that guide individual patient care by practitioners. Pathologists may benefit from emulating this approach, in future efforts at constructing “patient-based” prognostic and predictive models. For example, immunostains are most often used to distinguish between various neoplasms in a descriptive manner. Studies using immunostains in the pathology literature usually list the percentage of lesions that label for particular epitopes, as well as the sensitivity, specificity and predictive values of such markers in narrow morphological contexts. However, few studies have assessed these data with meta-analysis or calculated likelihood ratios (LR) or other probabilistic measures as applied to panels of markers in selected differential diagnoses. At an even more basic level, the relative statistical values attending particular morphological findings has seldom been analyzed in the same fashion. In contrast, several prognostic scoring models or “rules” that integrate multivariate pathological, clinical, imaging and other information are being developed by other specialists. For example, Kattan and associates have developed pretreatment nomograms that combine clinical and pathological data from prostate cancer patients and predict 5-year probability of metastasis.
Data collected from surgical pathology specimens can also be integrated with the use of “tools for reasoning with uncertainty” such as rule-based expert systems, multivariate statistics, Bayesian belief networks and neural networks.
Evolving Role of Immunohistochemistry in Surgical Pathology
Immunohistochemistry has far surpassed it’s initial expectation as an invaluable tool in the correct recognition of tumours. It is now being increasingly sought after for prognostication of tumours and as a justification for initiaton of expensive targeted therapy in oncology practice. The wider application of IHC has increased the demands from a surgical pathologist who no longer restricts to giving a correct label but also actively participates in the subsequent clinical decision making process.
However many physicians have this notion that IHC is akin to a biochemical test. The IHC result is considered as representing enough “evidence” to neatly categorise disease entities and resolve messy diagnostic dilemmas. In fact clinicians often demand “IHC confirmation” of surgical pathology reports (even where IHC may not be needed and is unlikely to add any value) and ever so often pathologists who lack access to IHC facilities often end their reports stating “IHC confirmation necessary” (almost as a disclaimer) for every possible lesion.
This situation has risen partly due to the belief (more aptly misbelief ) that the “IHC test” can be translated into “a” particular diagnosis. The “IHC test” is looked upon as a tool to usher in the much needed objectivity in routine surgical pathology practice and this is accompanied with an underlying perception that IHC can make-up for lack of diagnostic skill and experience in the complex task of giving a correct histological diagnosis.
The moot point , therefore, is : “ How good is IHC evidence ?” This issue will be addressed under the following headings :
A. Generation of the IHC result
B. Search engines for information
C. Application of IHC result to resolve frequent diagnostic dilemmas
D. Clinical Decision support from IHC result
E. Data warehousing
F. Quality control in IHC
B. Search engines for information
C. Application of IHC result to resolve frequent diagnostic dilemmas
D. Clinical Decision support from IHC result
E. Data warehousing
F. Quality control in IHC
A. Generation of IHC result : Several factors influence the final IHC result and no two tissue specimens will react in the same way even though they could be representing the same anatomic site and could also be matched for stage and grade. The factors responsible for this variation can be Pre-analytical, Analytical, and Post-analytical.
The pre-analytical factors : Optimal preservation of the antigenic epitope is vital. IHC can be affected by the duration of anoxia at surgery, time gap between resection to fixation, the type of fixative, the duration in the fixative, the size of the tissue, the thickness, and whether freezing was done. Finally, the quality of reagents such as the company, the batch, and the shelf-life of antibodies can affect the kind of result obtained. Of these optimal fixation is of special interest because it is a critical yet manageable step.
The analytical factors : include the various techniques used for antigen retrieval namely heat, Microwaving, Pressure cooking, trypsin digestion, autoclaving with different buffers etc. Proper endogenous peroxidase blocking is vital to prevent background staining. Further, whether an autostainer is being used or the staining is done by hand will influence the end result. The biochemical process involved is important, for example the relatively recent “catalyzed signal amplification” method is considered more sensitive than avidin-biotin or extraavidin methods.
The postanalytical (interpretative) factors : Finally, a very much underrated factor is the actual interpretation of the IHC result by the surgical pathologist. Several large studies have addressed the issue of inter and intra-observer errors .The positive or the negative interpretation has several subtle nuances which only a busy practitioner of IHC will realise. Suffice it to say a combination of several observations such as intensity, quantity and localisation of the IHC reaction and visualization of the immunostain in the lesional cells - as opposed to the immuno reaction seen in normal tissues, reactive tissues and other ‘bystanders’ (often referred to as “background” staining) – are features vital to the the final interpretation. Nevertheless the lack of a prescribed threshold level for interpreting a reaction as positive leaves immense scope for inter and intra observer errors. Finally, whatever the interpretation – it is the integration of the information obtained from the IHC test in the histopathologic picture is what matters most in the final interpretion.
B. Search engines : There is burgeoning literature on new antibodies, and newer application of old antibodies. Sharing of epitopes and significant immunoreactivity as an epiphomena rather than a common trait (eg. bcl2, CD34, c-kit), is well-recognized.
Hence, it is necessary to refer to sites which give information of the possible range of reactivity and some of these are as follows :
Hence, it is necessary to refer to sites which give information of the possible range of reactivity and some of these are as follows :
http://immunoquery.com, http://immunohypermart.net,
http://www.ncbi.nlm.nih.gov/prow
http://www.ncbi.nlm.nih.gov/prow
as also the various journals on Immunohistochemistry.
C. Application of IHC to resolve some diagnostic dilemmas in surgical pathology :
Several vexatious diagnostic dilemmas are well-known in surgical pathology practice.
To mention some of the frequent queries include distinguishing mesothelioma vs adenocarcinoma, the subtype of the malignant round cell tumour and poorly differentiated malignancies, the possible primary in case of metastasis from unknown primary (MCUO), neuroendocrine vs neuroectodermal lesions, and many others. Lineage identification by IHC is particularly helpful in lymphomas, melanomas, astrocytomas and pecomas. Algorithms constructed on the basis of previously demonstrated immunoreactivity on a large number of test cases have proved to be accurate in identifying the primary in 67% of MCUO. The IHC approach to find the possible primary is much more cost-effective as opposed to extensive work-up with radiological studies and various endoscopy procedures.
Several vexatious diagnostic dilemmas are well-known in surgical pathology practice.
To mention some of the frequent queries include distinguishing mesothelioma vs adenocarcinoma, the subtype of the malignant round cell tumour and poorly differentiated malignancies, the possible primary in case of metastasis from unknown primary (MCUO), neuroendocrine vs neuroectodermal lesions, and many others. Lineage identification by IHC is particularly helpful in lymphomas, melanomas, astrocytomas and pecomas. Algorithms constructed on the basis of previously demonstrated immunoreactivity on a large number of test cases have proved to be accurate in identifying the primary in 67% of MCUO. The IHC approach to find the possible primary is much more cost-effective as opposed to extensive work-up with radiological studies and various endoscopy procedures.
D. Clinical Decision support :
Several immunohistological markers which influence tumour behaviour and help tumour prognostication for microstaging, predicting response to therapy, monitoring drug resistance, detecting growth factors and receptors, evaluating tumour angiogenesis etc. have been described in the literature. Most of these have not as yet been incorporated in a routine diagnostic setting. There are other markers such as CD20, C-kit and HER-2/NEU which are increasingly requested to clinically justify targeted therapy. The application of this information for the Indian patients could be guided by the general guidelines for evidence based medicine with the following considerations: a) will the IHC result make a difference in the management or will the patient benefit from the information given by the IHC result; b) what about the cost to the patient and the delay in the report? c) is the treatment feasible in our socioeconomic setting? Generating IHC results which are not going to affect patient management or are not applicable to an individual patient only burden the IHC laboratory with no value added to an individual patient although they make the typed report look more impressive.
Several immunohistological markers which influence tumour behaviour and help tumour prognostication for microstaging, predicting response to therapy, monitoring drug resistance, detecting growth factors and receptors, evaluating tumour angiogenesis etc. have been described in the literature. Most of these have not as yet been incorporated in a routine diagnostic setting. There are other markers such as CD20, C-kit and HER-2/NEU which are increasingly requested to clinically justify targeted therapy. The application of this information for the Indian patients could be guided by the general guidelines for evidence based medicine with the following considerations: a) will the IHC result make a difference in the management or will the patient benefit from the information given by the IHC result; b) what about the cost to the patient and the delay in the report? c) is the treatment feasible in our socioeconomic setting? Generating IHC results which are not going to affect patient management or are not applicable to an individual patient only burden the IHC laboratory with no value added to an individual patient although they make the typed report look more impressive.
E. Importance of data warehousing :
Unlike blood chemistry results there are no readily available reference ranges for the several IHC antibodies with respect to age, normal tissue, physiological alterations, benign tumours and malignant tumours. In the Indian context IHC is in its infancy and there is a lack of information on Indian co-horts. Geographic variation could influence IHC results such as percentage of ER/PR positivity in breast tumours, ALK1 & CD30 in ALCL, and ALK-1 in B- cell NHL. This highlights the importance of data warehousing to obtain information on Indian patients
Unlike blood chemistry results there are no readily available reference ranges for the several IHC antibodies with respect to age, normal tissue, physiological alterations, benign tumours and malignant tumours. In the Indian context IHC is in its infancy and there is a lack of information on Indian co-horts. Geographic variation could influence IHC results such as percentage of ER/PR positivity in breast tumours, ALK1 & CD30 in ALCL, and ALK-1 in B- cell NHL. This highlights the importance of data warehousing to obtain information on Indian patients
F. Quality control / assurance in Immunohistochemistry :
Qualitive assurance is easily applicable to results which are quantifiable and objective. Attempts at quality assurance in IHC comprise establishment of standardized procedures to ensure technical reproducibility, uniformity in interpretation and evaluation and quantification of extent of immunoreaction by the use of a scoring system to bring in objectivity. An interlaboratory trial involving 172 pathologists and 3526 immunostains brought out some unexpected findings. There is a general belief that the staining quality is a major problem in the application of IHC; however a multivariate model in which each step of the diagnostic pathway, beginning with a pre IHC tentative diagnosis, was introduced, revealed that only (i) the correct tentative diagnosis, (ii) the interpretation of the IHC stain and (iii) the conclusions drawn from the IHC stain were independently predictive of the correct final diagnosis. Neither antibody selection nor quality of immunostain correlated independently to the correct final diagnosis. A definitive diagnosis could not be rendered if the morphological tentative diagnosis was incorrect or not included in the differential diagnosis. The results of another large study, evaluating interlaboratory and interobserver agreement for semiquantitative assessment of estrogen receptor (ER) using Tissue array technology wherein 172 laboratories participated, suggested that neither the pH of the formalin buffer or the duration in the fixative greatly influenced the detection of ER. Variability in subsequent IHC practices (such as antigen retrieval) and interpretation of results was a greater source of diagnostic error. The antibody which has been subjected to a lot of scrutiny with respect to overall evaluation of accuracy, specificity, sensitivity and reproducibility is HER 2/neu overexpression to identify the 20%-30% of breast cancer women who could benefit from Herceptin therapy (which is a humanized monoclonal antibody). A comparative study involving results from 94 Laboratories in 21 countries (predominantly European) participating in the National External Quality Assessment scheme for Immunocytochemistry (UK NEQAS-ICC) concluded that the reliability of the Her-2 assay could be greatly improved by stringent quality control and an ongoing quality assurance program using a standard reference obtained from cell lines.
Qualitive assurance is easily applicable to results which are quantifiable and objective. Attempts at quality assurance in IHC comprise establishment of standardized procedures to ensure technical reproducibility, uniformity in interpretation and evaluation and quantification of extent of immunoreaction by the use of a scoring system to bring in objectivity. An interlaboratory trial involving 172 pathologists and 3526 immunostains brought out some unexpected findings. There is a general belief that the staining quality is a major problem in the application of IHC; however a multivariate model in which each step of the diagnostic pathway, beginning with a pre IHC tentative diagnosis, was introduced, revealed that only (i) the correct tentative diagnosis, (ii) the interpretation of the IHC stain and (iii) the conclusions drawn from the IHC stain were independently predictive of the correct final diagnosis. Neither antibody selection nor quality of immunostain correlated independently to the correct final diagnosis. A definitive diagnosis could not be rendered if the morphological tentative diagnosis was incorrect or not included in the differential diagnosis. The results of another large study, evaluating interlaboratory and interobserver agreement for semiquantitative assessment of estrogen receptor (ER) using Tissue array technology wherein 172 laboratories participated, suggested that neither the pH of the formalin buffer or the duration in the fixative greatly influenced the detection of ER. Variability in subsequent IHC practices (such as antigen retrieval) and interpretation of results was a greater source of diagnostic error. The antibody which has been subjected to a lot of scrutiny with respect to overall evaluation of accuracy, specificity, sensitivity and reproducibility is HER 2/neu overexpression to identify the 20%-30% of breast cancer women who could benefit from Herceptin therapy (which is a humanized monoclonal antibody). A comparative study involving results from 94 Laboratories in 21 countries (predominantly European) participating in the National External Quality Assessment scheme for Immunocytochemistry (UK NEQAS-ICC) concluded that the reliability of the Her-2 assay could be greatly improved by stringent quality control and an ongoing quality assurance program using a standard reference obtained from cell lines.
To conclude, standardization in IHC is a daunting task. The multiple variables which affect the final result are not easy to control but standardization of technical steps, and hopefully availability of a reference for most lesions in future (such as in HER-2/neu assays) will bring in uniformity in IHC results regardless of where they are generated.
As of now it will be difficult to find a pathologist who has not, at some point or other, decided to neglect the IHC result.
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