By Bahram Robert Oliai, M.D.
In this second series of three newsletters, I would like to walk the reader through the pathologic examination of prostate needle biopsies, specifically pertaining to cancer diagnosis. My goal is to explain the entire process from biopsy to diagnosis, along the way sharing some insights that I have gleaned in my years of training at The Johns Hopkins Hospital as both a resident and a fellow and in my time spent in private pathology practice. In the world of the clinical laboratory we describe tests in terms of pre-analytic, analytic, and post-analytic factors, and I feel that this division is particularly well suited to describe the process of prostate biopsy interpretation.
Pre-analytic Factors
Fixatives
Immediately after the core biopsy specimens are taken by the urologist, they need to be placed in a fixative solution. This fixative prevents tissue degradation, allowing us the clearest snapshot of what is happening in the prostate when we examine it microscopically. Without proper fixation tissue begins to degrade, obscuring the vital, and yet sometimes subtle, morphologic findings so necessary in making a durable diagnosis. There is quite an array of fixatives available from vendors and I have reviewed biopsies submitted in various types of fixative solutions; however, for best results I strongly recommend using only 10% buffered formalin. I stronglydiscourage the use of alternative fixatives such as Bouin’s, B5, or alcohol based solutions. Another practice I have observed which I stronglydiscourage is the heavy soaking of cores in eosin to stain them so as to observe them better in the block(s); if necessary I recommend painting cores with ink for this purpose. There are several reasons to avoid alternative fixatives, perhaps the most compelling of which is the artifacts these solutions introduce. Bouin’s and eosin tend to make nucleoli very prominent in benign prostatic ducts/acini, and nucleolar prominence is a key feature we use in diagnosing both high grade prostatic intraepithelial neoplasia and cancer; thus, these solutions introduce a potential for overdiagnosis. Additionally, Bouin’s fixatives have a reputation for ruining the immunoreactivity of tissue, potentially making it impossible to perform valuable ancillary studies (i.e. immunoperoxidase stains for high molecular weight cytokeratin, p63, and Racemase/P504S, please refer to April 2004 Immunohistochemistry newsletter for more on this procedure). Prostate biopsy kits containing multiple formalin filled containers are readily available at no charge to our clients and can be easily ordered via our website (see Order Supplies link of the ProPath website under the client services menu to order online).
Core Submission
Once the cores have been obtained and the jars of 10% formalin are at hand, the next issue becomes how best to submit the cores in formalin. I stronglyrecommend that each core be submitted separately in its own container, labeled with the location of that core and of course the patient’s name/identification. While it is technically possible to submit more than one core per container, I recommend that if possible no more than two cores be submitted in each container. Why? First of all, when multiple biopsies are submitted together (as for example, when multiple biopsies are submitted from the left side in one container, and the rest from the right in the other), the cores tend to fragment during processing resulting in a loss of valuable tissue (FIG. 1). In addition, embedding multiple or fragmented cores in a paraffin block is extremely difficult for even the best, most experienced histotechnologists. This often results in a reduction of tissue available for review on the slides, even when examining multiple levels. Finally, the combination of poor histology and fragmented cores may make establishing a definitive diagnosis of carcinoma (especially when few atypical glands are noted) virtually impossible, preventing a definitive diagnosis, sometimes delaying diagnosis, and often subjecting the patient to additional round(s) of biopsy. Gupta et al. noted a significant reduction in atypical diagnoses with atypical rates dropping from 3.5% of cases to 1.8% using 6-12 container kits to optimize biopsy submission (fewer cores in each container, labeled with location, 3).
Additionally, in cases in which a previous atypical diagnosis has been made, separate submission and labeling of cores facilitates re-biopsy of the atypical focus (1). While I certainly understand that in some cases increasing the number of parts/containers submitted may increase the cost of the case, in the long run this may well be a more cost effective approach saving some additional biopsies and perhaps even some delayed diagnoses.
Specimen Grossing
At ProPath all prostate biopsies are grossed by experienced Pathology Assistants and/or senior Pathology Residents. Cores are carefully separated from each other and flattened/stretched. This serves to overcome the curling of core tissue that occurs after fixation, thereby increasing the tissue surface area for review. Finally, the number of cores per part and their individual lengths are precisely recorded in the gross report and the tissue from each part is submitted in a cassette for embedding and processing.
Specimen Processing
After additional chemical processing in specialized machines, the core tissue is embedded in paraffin wax cubes referred to as blocks. These blocks are then cut with extremely sharp blades, placed on glass slides, and stained with Hematoxylin and Eosin in staining machines. Levels is an another pathology term which refers to the number of cuts through the tissue in different planes that are examined. Several reports have shown that cutting core sections at different levels can improve the detection of small foci of carcinoma. Although there is no definite rule as to how many levels are necessary to appropriately evaluate cores, van der Kwast et al. suggest that in adequately grossed specimens two true levels per core may be optimal (5). Intervening levels may be cut up front and saved unstained or tissue protection Immunohistochemistry can be performed on an original atypical focus allowing for the opportunity to evaluate these often small yet challenging foci to the fullest extent by utilizing immunoperoxidase stains for basal cells. Green and Epstein described a procedure for cutting unstained levels up front and demonstrated that although there is some additional cost initially, in the long run it is very cost effective in saving patients from additional biopsies (2). At ProPath our cutting edge Immunohistochemistry laboratory under the direction of internationally known expert Dr. Rodney Miller affords us the luxury to rapidly perform tissue protection immunohistochemistry in house with excellent results (4).
True levels vs. Ribbons
One of my biggest pet peeves in pathology is when I am given a slide which is completely crowded and overwhelmed by several rows of tissue and told that they are levels. These most often are not true levels but simply ribbon cuts akin to a belt of ammunition feeding into a machine gun. Ribbons (FIG. 2) offer little in the way of improved diagnostic sensitivity which a true level does, and often do nothing more than clutter a slide making it more difficult to read! I have compared ribbons to true levels in (Table 1).
It is my opinion that optimally, tissue sections should be placed on the slide in a single row and two-three true levels be examined per core (FIG. 3).
As you can see, at ProPath we are committed to supporting our clients to achieve the ultimate in patient care and constantly strive to provide their patients the most timely, accurate, and up to date diagnoses. This is accomplished through superior laboratory technique and diagnostic practice.
References:
1. Allen EA, Kahane H, Epstein JI. Repeat biopsy strategies for men with atypical diagnoses on initial prostate needle biopsy. Urology 1998; 52: 803-807.
2. Green R and Epstein JI. Use of Intervening Unstained Slides for Immunohistochemical Stains for High Molecular Weight Cytokeratin on Prostate Needle Biopsies. The American Journal of Surgical Pathology 1999: 23(5): 567-571.
3. Gupta C, Ren JZ, Wojno KJ. Individual Submission and Embedding of Prostate Biopsies Decreases Rates of Equivocal Pathology Reports. Urology 2004: 63(1): 83-86.
4. Kubier P and Miller RT. Tissue Protection Immunohistochemistry (TPI): A Useful Adjunct in the Interpretation of Prostate Biopsies and Other Selected Cases Where Immunostains are Needed on Minute Lesions. American Journal of Clinical Pathology 2002: 117: 194-198.
5. van der Kwast TH, Lopes C, Santonja C, et al. Guidelines for Processing and Reporting of Prostate Needle Biopsies. Journal of Clinical Pathology 2003: 56: 336-340.
Date of last revision: January 2007.