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Applications of Flow Cytometry and Immunohistochemistry to Diagnostic Hematopathology

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Applications of Flow Cytometry and Immunohistochemistry to Diagnostic Hematopathology Review Article Applications of Flow Cytometry and Immunohistochemistry to Diagnostic Hematopathology Cherie H. Dunphy, MD c Objective.ÐDiagnostic hematopathology depends on selected studies to ensure reliable comparison of reported the applications of ¯ow cytometric immunophenotyping data. and immunohistochemical immunophenotyping combined Data Synthesis.ÐFlow cytometric immunophenotyping with the cytomorphology and histologic features of each offers the sensitive detection of antigens for which anti- case. Select cases may require additional ancillary cyto- bodies may not be available for paraf®n immunohisto- genetic and molecular studies for diagnosis. The purpose chemical immunophenotyping. However, paraf®n immu- of this review is to focus on the applications of ¯ow cy- nohistochemical immunophenotyping offers preservation tometric and immunohistochemical immunophenotyping of architecture and evaluation of expression of some pro- of paraf®n-embedded tissue to diagnostic hematopatholo- teins, which may not be available by ¯ow cytometric im- gy. Advantages and disadvantages of these techniques are munophenotyping. These techniques should be used as examined. complimentary tools in diagnostic hematopathology. Data Sources.ÐThe literature is extensively reviewed Conclusions.ÐThere are extensive applications of ¯ow (PubMed 1985±2003) with an emphasis on the most recent cytometric and immunohistochemical immunophenotyp- applications and those that are most useful clinically, both ing to diagnostic hematopathology. As cytogenetic and mo- diagnostically and prognostically. lecular ®ndings evolve in diagnostic hematopathology, Study Selection.ÐStudies were selected based on statis- there may be additional applications of ¯ow cytometric tically signi®cant results in large studies with reported ad- and immunohistochemical immunophenotyping to this equate clinical follow-up. ®eld of pathology. Data Extraction.ÐThe methodology was reviewed in the (Arch Pathol Lab Med. 2004;128:1004±1022) iagnostic hematopathology relies heavily on combin- 2. Dead cells may be gated out of the analysis. D ing cytomorphology and histology with ancillary 3. Weakly expressed surface antigens may be detected. techniques that apply immunophenotyping and molecu- 4. Multicolor (2-, 3-, 4-) analysis may be performed, al- lar/cytogenetic analysis. This review will focus on the ap- lowing for an accurate de®nition of the surface antigen plications of ¯ow cytometric and paraf®n immunohisto- pro®le of speci®c cells. chemical immunophenotyping to diagnostic hematopa- 5. Two simultaneous hematologic malignancies may be thology. detected within the same tissue site. 6. Tissue biopsy may be obviated by the relatively non- FLOW CYTOMETRY invasive diagnostic evaluation of body ¯uids and FNA General Overview specimens. Flow cytometric immunophenotyping (FCI) is a useful Disadvantages of FCI include the following: tool in diagnostic hematopathology. Types of specimens suitable for FCI include peripheral blood, bone marrow 1. Sclerotic BM may yield too few cells for adequate (BM) aspirates, and core biopsies,1 ®ne-needle aspirates analysis. (FNAs),2 fresh tissue biopsies, and all types of body ¯u- 2. A markedly hypercellular or ``packed'' BM may yield ids.3 too few cells for analysis. Advantages of FCI include the following: 3. Sclerotic tissue may be dif®cult to suspend for indi- vidual cellular analysis. 1. Distinct cell populations are de®ned by their size 4. There is loss of architectural relationships. (forward light scatter) and granularity (side light scatter). 5. A small population of monoclonal B cells may not be detected in a T-cell±rich or lymphohistiocytic-rich B-cell Accepted for publication May 11, 2004. lymphoma. From the Department of Pathology and Laboratory Medicine, Uni- 6. T-cell lymphomas that do not have an aberrant im- versity of North Carolina, Chapel Hill. munophenotype may not be detected. The author has no relevant ®nancial interest in the products or com- 7. An aberrant T-cell immunophenotype (ie, absence or panies described in this article. Reprints: Cherie H. Dunphy, MD, Department of Pathology and Lab- down-regulation of pan±T-cell antigens, particularly CD7) oratory Medicine, CB#7525, University of North Carolina, Brinkhous- does not necessarily indicate malignancy and may be ob- Bullitt Building, Chapel Hill, NC 27599-7525 (e-mail: cdunphy@ served in infectious mononucleosis,4 reactive dermatoses, unch.unc.edu). and in¯ammatory disorders.5±7 1004 Arch Pathol Lab MedÐVol 128, September 2004 FCI and IHC Immunophenotyping in HematopathologyÐDunphy 8. Partial tissue involvement by lymphoma with sam- FCI has been reported to be attainable in 71% to 77% of pling differences or poor tumor preservation may result positive cases. Furthermore, evaluation of cell size by com- in falsely ``negative'' FCI results.8 bining FCI and cytomorphology has recently been stud- 9. Inability to detect/diagnose Hodgkin lymphoma ied; large cell lymphoma/transformation may be diag- (HL) due primarily to the low number of neoplastic cells nosed reliably if greater than 40% large cells are present.9 normally present in this disease. The following situations require biopsy, based on FNA and FCI results: NHL of follicle center cell origin with a Due to these disadvantages, FCI data should always be mixed cellular composition, indeterminate results, the correlated with light microscopy if no FCI abnormalities presence of necrosis and polymorphonuclear cells in eval- are detected. Immunohistochemistry (IHC) may need to uation of recurrent NHL, fewer than 10% neoplastic cells be performed in selected cases. In addition, as mentioned, detected by FCI, a predominance of small cells detected an aberrant T-cell immunophenotype does not necessarily by cytomorphology or by FCI with clinical signs of trans- indicate malignancy and requires correlation with light formation, and evaluating for an initial diagnosis of or re- microscopy as well as clinical data and additional ancil- currence of HL, due to the possibility of a composite lym- lary studies (ie, molecular/cytogenetic analysis), in some phoma or a subsequent NHL. situations. In FNA specimens as well as in excised tissue speci- In addition, when a monoclonal or aberrant B cell pop- mens, FCI may be useful in differentiating ¯orid FH from ulation or an aberrant T-cell population, characterized by FL. This differentiation is most often accomplished by the a loss of a T-cell antigen, is identi®ed, HL may be excluded detection of a monoclonal B-cell population by FCI. How- only after correlation with the histology, in order to ex- ever, there may be occasional cases of ¯orid FH in which clude the possibility of a composite lymphoma. In cases the clonality of the germinal center B cells is indeterminate in which FCI data is diagnostic, microscopic observations by FCI. These cases may be distinguished by HLA-DO, a may provide additional information, not only due to sam- ¯ow cytometric marker that is markedly down-regulated pling, but also due to patterns of involvement and the in CD101 germinal center B cells of ¯orid FH, in compar- cytological features of the malignant cells. ison to CD102 polytypic B cells and to CD101 neoplastic The following list represents applications of FCI to di- cells of FL.10 In addition, multicolor ¯ow cytometric anal- agnostic hematopathology: diagnosis and subclassi®cation ysis of expression of Bcl-2, CD10, and CD20 may be useful of non-Hodgkin lymphoma (NHL) in FNA specimens; in distinguishing FH from FL. In a recent study by Cook distinguishing between follicular hyperplasia (FH) and et al,11 the presence of CD101 cells with high Bcl-2 ex- follicular lymphoma (FL); detection of the lack of surface pression predicted the presence of FL rather than FH with immunoglobulin (sIg) and light-chain expression by a sig- a positive predictive value of 100%. The analysis was per- ni®cant number of B cells indicating malignancy; subtyp- formed on lymph node and BM specimens. ing B-cell lymphomas/leukemias composed predomi- As detection of monoclonal B cells by FCI may be useful nantly of small cells; identifying prognostic markers in in establishing a diagnosis of B-cell NHL, it should also chronic lymphocytic leukemia (CLL); differentiating lym- be noted that the lack of sIg light-chain expression by FCI phoplasmacytic lymphoma or other types of B-cell lym- helps identify peripheral B-cell lymphoma. In a recent phomas from plasma cell dyscrasias (PCDs); identifying study by Li et al,12 cases with greater than 25% B cells prognostic markers in PCDs; differentiating various types lacking sIg light-chain expression all represented lympho- of large B-cell lymphomas (LBCLs) from anaplastic PCDs 1 ma. By FCI, the identi®ed sIg light-chain±negative popu- and from anaplastic CD30 large cell lymphoma (LCL); lation was distinctly separate from the normal polytypic immunophenotyping B-cell lymphomas/leukemias; dis- B cells; in 90% of cases, the identi®ed population was larg- tinguishing between hematogones and neoplastic lympho- er by forward angle light scatter than the reactive T cells blasts; differentiating NHL from HL and T-cell from B-cell and polytypic B cells. In their review of reactive cases, no NHL; identifying composite lymphomas; distinguishing reactive case revealed greater than 17% sIg-negative B between T-cell lymphoblastic lymphoma (T-LL) and thy- cells. moma; and immunophenotyping T-cell lymphomas/leu- Flow cytometric immunophenotyping is particularly kemias, natural killer (NK) cell lymphoproliferative
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