College of American Pathologists

  Q and A





cap today



October 2006

Richard A. Savage, MD

Q: We recently had a question concerning nonvalidated sample types for clinical chemistry testing—specifically, assaying body fluids on tests where the manufacturer never specified that body fluids were acceptable sample types. What, if anything, do we need to satisfy CAP requirements for test validation?

This is a difficult question to answer. In an ideal world, tests in matrices other than blood, serum, or plasma would be validated, but in most cases, this is not practical. Effusions from different patients vary in composition, so the fluid from one patient can be considered to have a different kind of matrix than that from every other patient. It may be impossible for laboratories to obtain sufficient material to perform validation studies for a particular fluid and, even if such studies were performed, their applicability to fluids in the general patient population would be questionable. It should be left to the medical director to decide whether to validate these tests in these fluids and how to do so. If the laboratory is unable to verify or establish reference ranges, or normal ranges, for different types of body fluid specimens, it must have a system in place to inform the clinician, such as an educational mechanism or a disclaimer on the patient’s report. This shifts the responsibility from the laboratory to the clinician. I recommend that you discuss with your risk management department reporting methods that are not validated and the use of disclaimers.

Linda D’Agostino, MT(ASCP)
Senior Technical Specialist
Laboratory Accreditation Program
College of American Pathologists
Northfield, Ill

Q: How do you use CD20, CD79a, and PAX-5 for B-cell determination? Are there situations where one is especially useful, as in Hodgkin versus T-cell-rich B-cell lymphoma?

CD20 is a marker of mature B cells and is expressed by the neoplastic cells in B-cell lymphoproliferative disorders and by the neoplastic lymphocytic and histiocytic, or L&H, cells in nodular lymphocyte predominant Hodgkin lymphoma, or NLPHL. In classical Hodgkin lymphoma, or cHL, CD20 is expressed by neoplastic Reed-Sternberg cells and mononuclear variants in a significant minority of cases, although staining for CD20 may be weak and variable. Recent tissue microarray studies found that 18 to 33 percent of cHL cases exhibited weak to moderate immunostaining for CD20 by Hodgkin and Reed-Sternberg cells.1,2

CD79a is a pan-B-cell marker expressed throughout B-cell development and is widely expressed in B-cell neoplasms, including precursor B lymphoblastic lymphoma/ lymphoblastic leukemia. It is expressed by neoplastic L&H cells in NLPHL but is less often present in cHL. Recent tissue microarray studies found that 10 to 18 percent of cHL cases exhibited immunostaining for CD79a by Hodgkin and Reed-Sternberg cells. 1,2

PAX-5, or B-cell-specific activator protein, called BSAP, is expressed throughout B-cell development until the plasma cell stage. It is widely expressed by B-cell lymphoproliferative disorders as well as by the neoplastic cells in NLPHL and cHL. In a study of 592 routinely fixed and embedded biopsies, PAX-5 immunostaining was observed in all cases of precursor and mature B-cell leukemias and lymphomas. All 58 cases of NLPHL exhibited weak to strong PAX-5 immunostaining (as well as CD20 immunostaining), and all but three of 117 cases of cHL (97 percent) exhibited weak to strong PAX-5 immunostaining by Reed-Sternberg cells.3 A recent microarray study found that 62 of 273 cases of cHL were strongly immunoreactive and 211 of 273 cases were weakly immunoreactive for PAX-5.2

In summary, B-cell lymphoproliferative disorders are immunoreactive for CD20, CD79a, and PAX-5, as is NLPHL. Classical Hodgkin lymphoma is immunoreactive for PAX-5 as well, but is less often immunoreactive for CD20 or CD79a. Other immunophenotypic markers may be helpful in characterizing these lymphoid neoplasms. Recently, Browne and co-workers studied expression of the B-cell transcription factors Oct-2, BOB.1, as well as PAX-5, and found that strong immunostaining for all three was typical in diffuse large B-cell lymphoma, including T-cell-rich B-cell lymphoma, and NLPHL. They also found that cases of cHL were typically not immunoreactive for all three B-cell transcription factors, and immunostaining for Oct-2 and BOB.1 was weak to moderate, when present.4 CD45 (leukocyte common antigen) is typically expressed by neoplastic cells in diffuse large B-cell lymphoma, including T-cell-rich B-cell lymphoma, as well as in NLPHL, but not in cHL. Conversely, the majority of cases of cHL are immunoreactive for CD15 (Leu M1), which is typically not expressed in NLPHL and diffuse large B-cell lymphoma.5


  1. Tzankov A, Zimpfer A, Pehrs AC, et al. Expression of B-cell markers in classical Hodgkin lymphoma: a tissue microarray analysis of 330 cases. Mod Pathol. 2003;16:1141–1147.
  2. Garcia-Cosio M, Santon A, Martin P, et al. Analysis of transcription factor OCT.1, OCT.2 and BOB.1 expression using tissue arrays in classical Hodgkin’s lymphoma. Mod Pathol. 2004;17:1531–1538.
  3. Torlakovic E, Torlakovic G, Nguyen PL, et al. The value of anti-pax-5 immunostaining in routinely fixed and paraffin-embedded sections: a novel pan pre-B and B-cell marker. Mod Pathol. 2002;26:1343–1350.
  4. Browne P, Petrosyan K, Hernandez A, et al. The B-cell transcription factors BSAP, Oct-2, and BOB.1 and the pan-B-cell markers CD20, CD22, and CD79a are useful in the differential diagnosis of classic Hodgkin lymphoma. Am J Clin Pathol. 2003;120:767–777.
  5. Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Hematopoetic and Lymphoid Tissues. Lyon, France:IARC Press;2001.