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A Sound Foundation Is Key

In 1990, when I was a young academician, I presented a talk at the International Conference on the Computerized Cytology and Histopathology Laboratory entitled, “Fixation of Specimens for Quantitative DNA Analysis by Slide-Based Fuelgen Microspectrometry.”1 I had done some simple, basic work that pointed out the problems of not taking fixation and specimen preparation into account. I did not consider it to be a particularly exciting topic. However, a lasting impression was made upon me by the kind and positive comments by Dr. Leo Koss about the importance of my paper and others like it that focused upon validating one of the “first steps” in marker studies: choosing the optimal specimen preparation and fixation, and defining the variables related to those processes. Dr. Koss talked about the many published papers touting a correlation with some clinical condition that were useless because the variables of specimen preparation and fixation had not been taken into account. The next year, at the 10th International Congress of Cytology, Buenos Aires, I gave another presentation,2 which Dr. William Johnston, the moderator, called “elegant.” My co-authors and I had discovered a pitfall in methenamine silver stain that could result in a false negative when looking for Pneumocystis carinii. The problem was resolved by using a Pneumocystis control instead of a fungal control; the former previously was not thought to be necessary. We discovered that Pneumocystis stains inconsistently, and when the fungal control stains lightly, one cannot be sure whether any Pneumocystis in the test specimen will stain. We wondered whether the few previous reports of negative Grocottmethenamine-silver in the face of positive, foamy alveolar casts (FACs) had in fact been due to staining failures rather than the purported nonspecificity of FACs. This was a seemingly picky little point that could make a great difference in how a case was interpreted or mistakenly belittle the importance of the finding of FAC in a clinical specimen. The paper by Shidham et al in this issue, “Methods of Cytologic Smear Preparation and Fixation: Effect on the Immunoreactivity of Commonly Used Anticytokeratin Antibody AE1/AE3,” is another one of those simple-yet-elegant basic studies that are so important to building the foundation upon which other more exciting and clinically useful findings can be made. The authors found that the stain intensity and proportion of cells staining were affected to variable degrees by the method of smear preparation and fixation used. In their study, saline-rehydrated air-dried smears postfixed in alcoholic formalin yielded optimal results similar to those with formalin-fixed, paraffin-embedded tissue and smears wet fixed in 95% ethanol. The lowest intensity and proportion scores were obtained with air-dried smears that were postfixed with ethanol/acetic acid without saline rehydration. It follows that if clinical or diagnostic decisions are based upon negative or positive immunochemical results, inadvertently choosing a suboptimal method of smear preparation or fixation could have important consequences. The authors point out in their discussion that immunostaining can be affected by other smear preparation and fixation variables, such as the length of time an air-dried smear sits before immunostaining, and that saline rehydration itself, while producing optimum results for AE1/AE3 antigen, may yield false negative results with other immunomarkers of different molecular size or solubility. The bottom line is that before a valid determination can be made that the presence or absence of a particular antigen correlates with some clinical con-