Dear Editor,

We fully support the integrated approach to hematopathological diagnostics, which has been advanced by the WHO 2008 classification and we acknowledge the prominent role of immunophenotyping in this classification (1). The integrated approach is still not fully applied even in the most advanced countries. In some other countries, (hemato)pathologists still struggle, having only bone marrow (BM) tissue core biopsies to work with. In some centers and countries, there may be a partial redundancy in immunophenotyping with both flow cytometry and immunohistochemistry (IHC) methods available.

This is not universally the case and one must rely much more on one or the other source of information. If the histopathologists are restricted to histology, they need to be assured that the applied IHC methodology is accurate. Quality assurance (QA), quality control (QC), proficiency testing, reagent documentation and validation are standard parts of everyday practice in clinical laboratories (2). IHC is one area of laboratory practice where such standardization and quality assurance procedures have been addressed relatively late (2,3). The complexity of IHC presents challenging issues within pre-analytical, analytical, and post-analytical components, which still make true standardization difficult. However, optimization in this area has become possible due to rapidly advancing technologies. Also, we have increased our understanding of each of these components, which makes it possible to start the standardization process (3,4).

Tissue processing has been recognized as an essential pre-analytical component that greatly determines the outcome of IHC stains (5,6). When very few IHC markers were employed in the evaluation of BM biopsies, there was no emphasis on QA/QC issues. Currently we face a challenge in the light of increasing number of applied markers and the variation in tissue processing of the BM biopsies. The results of IHC testing in BM biopsies are not exempt from the principles that apply for any other tissue biopsy. This means that none of the components of the IHC testing can be neglected. Even in the situation where one type of fixative is used and no decalcification is applied, pre-analytical components must be subjected to the standardization process. Analytic variables such as the antigen retrieval protocol, the detection system, and the immunostaining platform play an important role in the quality of IHC stains.

Our pilot study included only a fraction of European Bone Marrow Working Group laboratories. However, it disclosed that even in this limited number of laboratories, a large variety of tissue processing and staining methods were in use. It clearly shows that a currently used approach to external QA/proficiency testing is not possible for BM biopsies. At the same time our study shows that there is a need of such external QA/QC to assure reproducibility of the results. BM biopsy remains excluded from the good practices of QC/QA, which are already established in surgical pathology. This is important in the light of the increase in number of tests applied to BM biopsy, which is happening despite advances in other, possibly more sophisticated methods of tissue analysis. Moreover, introducing class II tests to this type of tissue, a new landmark in BM tissue biopsy IHC is approaching.

The priority must be given to stand-alone IHC tests. However, we disagree with Dr.Orazi that standardization and external QA/QC should be limited to class II tests. Many of the class I tests are equally complex to perform and may have critical impact for the final diagnosis (5). If any clinical test deserves to be done, it also deserves to be performed optimally and interpreted correctly. The integrated approach of WHO 2008, at least in our minds, was never constructed to support integration of either false-negative or false-positive IHC results or to obscure deficiencies of technically suboptimal results.

Sincerely yours,

Emina Emilia Torlakovic, MD, PhD, Kikkeri Naresh, M.B.B.S.; D.C.P.; M.D.;F.R.C.Path., Marcus Kremer, MD, Jon van der Walt, M.D., F.R.C.Path., Elizabeth Hyjek, MD, PhD Anna Porwit, MD, PhD,

References:

1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. WHO Press, Geneva, 2008.

2. Taylor CR. Editorial--a personal perspective. Appl Immunohistochem Mol Morphol. 2007;15:121-3.

3. Taylor CR. Quality assurance and standardization in immunohistochemistry. A proposal for the annual meeting of the Biological Stain Commission, June, 1991. Biotech Histochem. 1992;67:110-7.

4. Bogen SA, Vani K, McGraw B, Federico V, Habib I, Zeheb R, Luther E, Tristram C, Sompuram SR. Experimental validation of peptide immunohistochemistry controls. Appl Immunohistochem Mol Morphol. 2009;17:239-46.

5. Goldstein NS, Hewitt SM, Taylor CR, Yaziji H, Hicks DG; Members of Ad- Hoc Committee On Immunohistochemistry Standardization. Recommendations for improved standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol. 2007;15:124-33.

6. Taylor CR, Shi S-R, Barr N, Wu N. Techniques of Immunohistochenmistry: Principles, pitfalls, and standardization. In: Dabbs D. Diagnostic Immunohistochemistry. Churchill Livingstone, Philadelphia, PA, USA, 2nd Ed. 2006:17-19.

Dear Editor

The authors use sweeping generalizations to make their case. This is just one example: "while the role of external quality assurance programs is well established as a valuable tool for QA/QC in diagnostic immunohistochemistry in surgical pathology, such approaches are notably lacking in bone marrow biopsy pathology". However, in reality, no published data support a need for an external QA/QC program for bone marrow biopsies, as proposed by the authors. A long list of unpublished results from the NordiQC cannot be considered as equivalent to published peer-reviewed evidence and should not have been included among the references. Moreover, the authors themselves do not provide any data which may suggest a need for external quality assurance for class I tests, the only tests that they have evaluated in this study. The markers that the authors have tested are and they should most definitely remain class I tests. If the authors’ intent was to address the issue of analyzing potential class II markers which can be used on bone marrow biopsy, those are the ones they should have studied. In regard to a need for class II markers in bone marrow biopsy evaluation, it is unclear, at least to this reader, why immunohistology should be entitled to such a prominent place within a complex diagnostic algorithm. Most of the neoplastic conditions in which immunophenotyping can be useful, greatly benefit from a balanced integrated diagnostic approach. Such an approach is the one mandated by WHO 2008 classification. As they stand now, the conclusions that are provide by this paper are subjective ones and most definitely not based on evidence.

Attilo Orazi