Best practices in contemporary diagnostic immunohistochemistry: panel approach to hematolymphoid proliferations.
REASONS TO USE DIRECTED IHC PANELS IN THE EVALUATION OF LYMPHOID PROLIFERATIONS
One of the major purposes of paraffin section IHC panels is to serve as an aid in making a series of diagnostic decisions when lymph node biopsies or hematopoietic/ lymphoid infiltrates at extranodal sites are evaluated. Those decisions include determining whether a proliferation is benign or malignant; if malignant, whether it is a lymphoma; if a lymphoma, whether it is of Hodgkin or non-Hodgkin type; and then what precise type of Hodgkin or non-Hodgkin lymphoma. The stains are used to provide information that is not morphologically apparent, such as whether plasma cell populations are light chain class restricted; they are used to draw our attention to subtle morphologic features that otherwise might be missed, such as identifying small foci of lymphoma in otherwise hyperplastic lymph nodes; and in helping to identify the increasingly recognized "early" or "in situ" involvement of lymph nodes by mantle cell lymphoma (MCL) or follicular lymphoma. (2,3) Finally--and from our perspective, equally important--they function as an internal "private" quality control by either helping confirm our histologic impressions or letting us know (in the privacy of our own offices) that we had better rethink our original appraisal of a case. Even if one thinks the routine stained sections are obvious, there will be cases where even the most sophisticated experts may be fooled.
To accomplish the above goals, it is important to use relatively comprehensive panels of IHC stains, because individual stains or an extremely focused panel may be misleading. For example, doing a CD20, CD3, and BCL2 stain just to confirm a diagnosis of follicular lymphoma could be misleading if, in fact, one had a very follicular-appearing MCL, because one would have BCL2-positive follicles in both situations. On the other hand, indiscriminate use of IHC stains where one uses every stain in one's armamentarium is also not advised. Aside from the added expense and an open invitation for greater outside interference in the practice of pathology, use of large nondirected antibody panels can be misleading because they increase the chance of misinterpreting a finding that otherwise would not have been detected. This may occur because of an unusual immunoreactivity, an artifactual positive or negative staining pattern, or unfamiliarity with the expected staining pattern.
IHC PANELS IN DIAGNOSTIC HEMATOPATHOLOGY
Two IHC panels will be discussed, together with additional IHC stains that may be added in selected circumstances. The bulk of our discussion will cover a panel whose major purpose is to evaluate potential B-cell neoplasms (Table 1). However, although not covered here because of space constraints, the panel can also provide important clues as to the presence of a T-cell ([CD3.sup.+], [CD5.sup.+]), natural killer cell ([CD3.sup.+], [CD5.sup.-]), or even Hodgkin lymphoma. A basic and expanded panel to use in cases of suspected Hodgkin lymphoma will be discussed briefly (Table 2). These 2 panels can be used as part of the workup for most malignant lymphomas in the United States.4,5 Even more limited screening panels may be used in some circumstances, or sometimes none at all, depending on the histopathologic differential diagnosis, the degree of clinical suspicion, and the comfort level of the pathologist.
MULTIPURPOSE PANEL FOR THE EVALUATION OF SUSPECTED NON-HODGKIN LYMPHOMAS
The most widely used panel in our practice and the way in which we use it is described in Table 1.6,7 It will help resolve many cases of suspected B-cell lymphomas and provide clues to the diagnosis of T/natural killer cell or Hodgkin lymphomas. It is important to know not only the basic reactivity of each antibody but also what to look for that suggests the presence of a neoplasm and, if a lymphoma is recognized, how to use the findings to help with the precise classification. Likewise, the stains should be interpreted with attention not only to simple extent of positivity, but also to the architectural distribution and cytology of the positive (and negative) cells.
It should be noted that we do not routinely perform k and X stains on our paraffin-embedded sections because in our laboratory and many (but not all) other laboratories, these stains fail to reveal specific surface immunoglobulin staining in most cases. Most B-cell lymphomas have surface rather than significant amounts of cytoplasmic immunoglobulin expression, and so often the stains only show bothersome nonspecific staining or highlight a re active plasma cell population. In situ hybridization stains for [kappa] and [lambda] may deal with the nonspecific staining problems but do not provide significantly greater sensitivity. Detection of surface k and X expression is one of the major strengths of flow cytometric immunophenotypic studies. Nevertheless, paraffin section IHC stains for [kappa], [lambda],and sometimes the immunoglobulin heavy chains are useful in evaluating potential lymphoid or plasmacytic neoplasms, especially if there is plasmacytoid differentiation or overt plasma cells. We tend to add [kappa] and [lambda] stains when evaluating potential marginal zone lymphomas, because they often fail to have an "aberrant" B-cell phenotype, and a moderate number (based on the site and type) may have plasmacytic differentiation. Remember, however, that light-chain class restriction is not an absolute indicator of neoplasia!
HOW DO WE USE THE PANEL TO SUPPORT THE PRESENCE OF A LYMPHOID NEOPLASM?
Some of the more common ways in which the basic IHC panel can be used to support the presence of a lymphoid neoplasm are described below.
1. Identify BCL2-positive follicular center B cells, because most normal follicular center cells are BCL2 negative (Figure 1). One does need to beware of "positive" nodules of normal BCL2-positive, non-follicular center B cells (eg, primary follicles or BCL2-positive mantle zones cut tangentially; Figure 2) or follicles with numerous T cells. One must also remember that follicles colonized by non-follicular type lymphomas can also be BCL2 positive.
2. Identify areas with CD5 coexpression on B cells based on a comparison of the CD3, CD5, and CD20 stains. Whereas small populations of [CD5.sup.+] B cells are routinely seen by flow cytometry in normal individuals, recognizable areas where [CD5.sup.+] B cells aggregate are usually not identified in paraffin sections (Figure 3). We do occasionally see some expression in normal mantle zones. Interpretation may be complicated in cases with numerous T cells.
3. Identify sheets of B cells where there should not be sheets of B cells (Figure 4). This is most useful at extranodal sites, but you must beware of B-cell-rich reactive infiltrates that have been described in the breast. (8) Lymph
nodes may also have numerous interfollicular B cells in perisinus and medullary areas. Because of the sometimes prominent perisinus B-cell populations, reactive lymph nodes cut tangential to the capsule may also demonstrate apparent sheets of B cells.
4. Identify numerous CD10+ or [BCL6.sup.+] cells outside of follicular centers (Figure 5). Normally, only scattered [CD10.sup.+] cells of B-cell or T-cell origin can be seen, in addition to [CD10.sup.+] neutrophils. Sometimes one even sees some scattered [CD10.sup.+] lymphoblasts. Likewise, one normally expects to see only scattered [BCL6.sup.+] interfollicular lymphoid cells. It should also be kept in mind that not all neoplastic CD10 or [BCL6.sup.+] cells outside of follicles are necessarily related to follicular center cells. Other lymphomas can also express these antigens, including some lymphoblastic lymphomas and some T-cell lymphomas, such as angioimmunoblastic T-cell lymphoma.
5. Identify numerous [CD43.sup.+] B cells in a fashion analogous to identifying [CD5.sup.+] B cells. CD43 will help identify a distinct but overlapping population of normal and neoplastic B cells compared with CD5, and thus provides additional information, because sheets of [CD43.sup.+] B cells are not seen in most nonneoplastic circumstances. However, nonneoplastic [CD43.sup.+] B-cell populations can be identified in paraffin sections in some circumstances, such as in subepithelial regions of the tonsils, myo/lymphoepithelial sialoadenitis, atypical marginal zone hyperplasia, as described in children, (9) and in reactive lesions of the terminal ileum (including Peyer patches). (10) CD43 expression may be difficult to evaluate because of normal reactivity of background nonneoplastic T cells and plasma cells.
6. Identify significant populations of cyclin D1-positive lymphocytes or plasma cells. Because these cell types do not normally express cyclin D1, this strongly supports the presence of a neoplasm, usually of mantle cell or plasma cell myeloma type, although rare other B-cell neoplasms (such as hairy cell leukemia) and some carcinomas may also exhibit cyclin D1 positivity. Careful examination for internal control positivity is important because lack of cyclin D1 expression may sometimes be partly due to technical factors. (8) It is also important not to interpret nonlymphoid cell staining as abnormal (Figure 6).
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HOW DO WE USE THE PANEL TO HELP CLASSIFY THE MORE COMMON B-CELL LYMPHOMAS?
Use of a phenotypic algorithm based on many of the antibodies included in this panel is a critical aid in the recognition and confident classification of many of the B-cell lymphomas (Figure 7). However, exceptions to the standard phenotypes occur not infrequently, and this schema should only be used as a general guideline.
Once one is dealing with a B-cell neoplasm, one can think of those that are usually [CD5.sup.+] and those usually [CD5.sup.-]. Most [CD5.sup.+] B-cell lymphomas are of chronic lymphocytic leukemia/small lymphocytic lymphoma or MCL type, with a minority of diffuse large B-cell lymphomas (DLBCLs) falling into this category, as well as occasional other B-cell neoplasms. Rare cyclin D1-negative MCLs are reported11; however, most are positive, (12) distinguishing them from chronic lymphocytic leukemia/small lymphocytic lymphoma in which, with possible rare exception, cyclin D1 is negative or only positive in proliferation centers. (13) CD23 expression, which can be detected in paraffin sections, is not a specific finding but also would favor the diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma over MCL. The CD5-negative B-cell lymphomas are next thought of in terms of those that are CD10 positive or negative. The former group, which also is usually BCL6 positive, includes most follicular lymphomas, some DLBCLs, and Burkitt lymphomas (BL; see below). The CD10-negative cases, which also are BCL6 negative, include the different types of marginal zone lymphoma, lymphoplasmacytic lymphoma, some DLBCLs, a minority of MCLs, and even some chronic lymphocytic leukemia/small lymphocytic lymphoma. Finally, there are [CD10.sup.+] [BCL6.sup.+] lymphomas, which mostly include DLBCLs, some follicular lymphomas, and particularly the diffuse cases of primary cutaneous follicle center lymphomas. (14) Bcell lymphomas of lymphoblastic origin may lack CD20 expression and can be found in the CD5-negative, CD10positive or CD10-negative groups. They usually also would express TdT.
Addition of a Ki-67 stain can also be useful in a number of circumstances, although precise agreed-upon rules for its interpretation in varied situations remain elusive, and it is one of the antibodies where reproducibility is an issue. The stain is probably most useful when there is suboptimal cytologic preservation, such as in many needlecore biopsies, in part because the nuclear morphologic fea tures are often better appreciated. What appears to be a diffuse proliferation of small lymphoid cells can turn into a large cell proliferation when one sees that the nuclei actually are larger than they appeared in the hematoxylineosin-stained section and that they have more vesicular chromatin and nucleoli. Remember, however, that Ki-67 will also be positive in some nontransformed small lymphoid cells that are cycling. Nevertheless, it can serve as another form of internal quality assurance, because if one was contemplating diagnosing chronic lymphocytic leukemia/small lymphocytic lymphoma and the Ki-67 stain shows sheets of positive nuclei throughout, one might want to carefully reexamine the histopathologic and other ancillary findings before rendering a diagnosis. In addition to improving cytologic detail in some cases, Ki-67 stains may be of value in assessing follicular lymphomas, as cases with an average proportion of Ki-67-positive intrafollicular cells of greater than 30% have been reported to be associated with a course more like that seen with grade 3 cases, even if histologically the lymphoma is of grades 1 to 2. (15) Prospective studies further evaluating this finding, however, are needed, and it must be remembered that there are also many grade 3 follicular lymphomas reported in some studies with less than 30% Ki-67-positive cells. (16) Ki-67 staining also has been used in the prognostication of MCLs, with varied cutoffs for what should be considered high (>60%, >40%, or even lower cutoffs).1 Its use in the differential diagnosis between BL and DLBCL is discussed below.
[FIGURE 3 OMITTED]
Much of the above discussion focuses on the small B-cell lymphomas. In contrast to many of the small B-cell neoplasms, where identification of a definite neoplasm can be a major challenge, many of the large cell, blastoid, and other transformed-appearing neoplasms are obviously neoplastic, and the goal of the immunostains is limited more to assessing the precise cell type present. As discussed above, the panel will identify the blastoid MCL. It is important to add a TdT and sometimes other markers of immaturity (eg, CD34 or, in T-cell proliferations, CD1a) when a truly blastic neoplasm is in the differential. It may also be necessary to add additional pan-B-cell markers, such as PAX-5 or CD22, and other pan-T-cell markers to establish the lineage of some lymphoblastic neoplasms. The sometimes very problematic distinction of DLBCL from BL is beyond the scope of our discussion here, but the presence of BCL2 expression that would be identified by our panel is a major strike against the latter diagnosis. (17-20) One could then add a Ki-67 stain looking for nearly 100% positivity to support the diagnosis of a BL, although the finding is not specific and may be found in some DLBLCs. Cytogenetic fluorescence in situ hybridization studies looking for MYC, BCL2, and BCL6 translocations can also be very helpful in these circumstances, but it must be remembered that MYC translocations are not specific for BL. (21-23) With the addition of a MUM-1 antibody, this panel will also provide the information needed to use the Hans algorithm for the distinction of DLBCLs of germinal center from those of non-germinal center type. (24) Whether this algorithm is of prognostic utility in the immunochemotherapy era or is the best of the available algorithms to accomplish this purpose is also beyond the scope of this manuscript. Furthermore, reproducibility is another major problem with immunophenotypic prognos tication of DLBCL.25 Our panel will not be sufficient for plasma cell or plasmablastic neoplasms that will require markers, such as CD138 (which identifies plasma cells but also some epithelial cells) and kappa and lambda stains. We also have not discussed the CD20-negative potential B-cell neoplasms that are best evaluated using one or more of the other pan-B-cell markers, such as PAX-5, CD79a, or CD22. (26)
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[FIGURE 6 OMITTED]
Although we have dropped the use of an antibody to CD43 from our large cell panel because it does not generally help in the differential diagnosis of these usually overt neoplasms, it should be remembered that CD43 expression is found not only in many B- and T-cell lymphomas but also in many myeloid sarcomas. Other antibodies useful to help evaluate potential myeloid sarcomas include CD34 (blasts) and CD117 (immature myeloids, including myeloblasts plus some lymphoblasts), and the myeloid/ monocytic-associated markers, such as myeloperoxidase, CD33, lysozyme, neutrophil elastase, CD68 (PGM1 clone more specific for monocytic differentiation than KP-1), CD14, and CD163 (relatively specific monocytic marker).
PANEL FOR SUSPECTED HODGKIN LYMPHOMA
A basic panel for evaluating cases of potential Hodgkin lymphoma will be sufficient in most cases (Table 2). One should be aware that some Hodgkin lymphoma cases will have an "atypical" phenotypes that one may find, for example, absence of CD15 expression. Not all hematopathologists find the CD45 stain to be as useful as others. In problematic cases, depending on which features are causing confusion, one or more additional markers may be used. It is important to realize, however, that some cases will ultimately best be classified as a gray-zone lymphoma with features intermediate between DLBCL and Hodgkin lymphoma. (1,27) PAX-5, the pan-B-cell marker, can be used if the problem is a differential diagnosis with a T-cell neoplasm because most Reed-Sternberg cells are weakly positive. (28-30) If an EMA+, CD15", PAX5- population is identified, ALK staining is recommended to help exclude an anaplastic large cell lymphoma, which may morphologically mimic classical Hodgkin lymphoma.31 Remember that some T-cell neoplasms may include a population of large B cells. IRF4 MUM-1 is an excellent marker expected to be strongly positive in Reed-Sternberg cells and can be used to highlight their presence, even though it is not at all specific and is also present in anaplastic large cell lymphoma. (30) In the differential diagnosis with DLBCL, Oct2 and Bob.1 stains can be used because the Reed-Sternberg cells in most cases of classical Hodgkin lymphoma should be negative for one or both of these antigens, and DLBCL should be positive with both. (28,32,33) Difficulties with a differential diagnosis of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and a T-cell/histiocyte-rich large B-cell lymphoma can sometimes be dealt with by adding a CD21 stain to identify the follicular nodules that are characteristic of NLPHL, a J-chain stain that helps in some cases to identify the neoplastic cells in NLPHL, and a CD57 stain that in most cases of NLPHL identifies numerous positive cells, sometimes forming rosettes around the neoplastic cells. Recently, the utility of a PD-1 stain to identify T-cell rosettes in NLPHL has been reported. (49) Sometimes a BCL6 stain that identifies the neoplastic cells in NLPHL may also be helpful, although it is very nonspecific.
[FIGURE 7 OMITTED]
We have seen how the use of a relatively limited panel of IHC stains can help deal with many of the more common neoplastic hematopathologic diagnoses (Table 3). Nevertheless, some cases will require additional stains (Table 4), and others fewer. Cytogenetic and/or molecular studies also may be critical in some cases. The complexity of the cases you are dealing with will affect the number of immunostains you need to perform and the need for additional ancillary studies. Finally, remember that IHC stains always must be put into the context of the histopathologic studies and the other ancillary studies that have been performed.
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Christine F. Garcia, Steven H. Swerdlow
Accepted for publication October 16, 2008.
From the Division of Hematopathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
The authors have no relevant financial interest in the products or companies described in this article.
This article is provided for educational purposes only and is not intended to suggest either a practice standard or the only acceptable pathway for diagnostic evaluation. The views presented reflect the authors' opinions. The application of these opinions to a particular medical situation must be guided by the informed medical judgment of the responsible pathologist(s) based on the individual circumstances presented by the patient. The College of American Pathologists has no responsibility for the content or application of the views expressed herein.
Reprints: Steven H. Swerdlow, MD, Division of Hematopathology, University of Pittsburgh Medical Center Presbyterian, Room G335, 200 Lothrop St, Pittsburgh, PA 15213 (e-mail: firstname.lastname@example.org).
Table 1. Multipurpose Panel for the Evaluation of Suspected Non-Hodgkin Lymphomas Antigen Major Normal Reactivity Purpose CD20 Most B cells, small To identify B lineage in the subset of T cells absence of any features to the contrary. Beware, some T-cell lymphomas are positive, (34, 35) and some B-cell lymphomas are very weakly positive (eg, CLL/SLL) or negative. CD3 T cells and natural To identify T or natural killer cells killer cell lineage in the absence of any features to the contrary; may be lost in some T-cell neoplasms. CD5 T cells, small B-cell To identify major [CD5.sup.+] subset (36) B-cell populations. (7, 37, 38) Also should be positive in almost all normal T cells but negative in natural killer cells. [CD5.sup.-] [CD3.sup.+] populations need to be further pursued. CD43 T cells, small B-cell To identify major [CD43.sup.+] subset, some plasma cells B-cell populations. and myeloid cells. (39) Some would be less enthusiastic about the inclusion of CD43 in a panel such as this. CD10 Most follicular/GC B To identify [CD10.sup.+] B cells, subset of lym- cells which, if mature, phoblasts, small subset supports their GC origin, and of mature T cells, assess their distribution (are neutrophils, and some they restricted to GC?). Also epithelial cells. (40-42) will identify [CD10.sup.+] T cells with generally crisper, more intense staining, the characteristic phenotype of the neoplastic cells in AILT. (43) Will also highlight subset of lym- phoblasts that can even be present in reactive hyperplasias. (44) BCL6 Most GC B cells, small To help identify GC B cells, T-cell subset, and some particularly when CD10 is very other cells. (30) Weak weak or negative and in staining may not have algorithms used to further the same implications as classify DLBCL. [CD10.sup.-], stronger staining. This [BCL6.sup.+] GCs are not a is a less specific but specific finding but are often more sensitive marker found in MZL with follicular for GC cells. colonization. ALCL and T-LBL are often positive. (30, 45) BCL2 Many lymphoid cells but Primary uses are to identify not many normal GC B clearly abnormal positive GC, cells. (30, 46) highlight potentially normal GC and aid in the differential diagnosis between DLBCL (variable) and BL (negative). Cyclin D1 Normal lymphoid cells To identify abnormal cyclin negative, some endothelial D1-positive lymphoid or and histiocyte nuclei plasma cells. positive that function as internal positive controls. (12, 30) Abbreviations: AILT, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; BL, Burkitt lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; DLBCL, diffuse large B cell lymphoma; GC, germinal center; MZL, marginal zone lymphoma; T-LBL, T-lymphoblastic leukemia/lymphoma. Table 2. Basic Hodgkin Lymphoma Panel Neoplastic Cell Phenotype Normal Hematopoietic/ Antigen Lymphoid Reactivity cHL CD20 B cells, few T cells Most cases negative; some cases positive, usually with staining of only a subset of R-S CD3 T cells, NK cells Only rarely positive CD15 Granulocytes, some Most (~85%) but not all cases histiocytes, some positive (48) epithelial cells and carcinomas, CMV-infected cells (47) CD30 Activated lymphoid cells Positive LCA Leukocytes Negative EMA Some plasma cells Only rarely positive Neoplastic Cell Phenotype Antigen NLPHL B-NHL CD20 Positive Most cases positive CD3 Negative Negative CD15 Negative Most cases negative CD30 Usually negative; Often neg- occasionally ative positive; nonneoplastic positive cells may be seen LCA Positive Usually positive EMA Some positive Usually negative Abbreviations: B-NHL, B-cell non-Hodgkin lymphoma; cHL, classical Hodgkin lymphoma; CMV, cytomegalovirus; EMA, epithelial membrane antigen; LCA, leukocyte commmon antigen; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; R-S, Reed-Sternberg cells. Table 3. Take-Home Messages Establish a differential diagnosis based on clinical and morphologic evaluation prior to ordering additional ancillary studies on paraffin-embedded tissues. Interpret the stains with your differential diagnosis in mind--do not interpret the stains blindly (simply with a long list of antibodies and 1 +,2+,3+ designations), and do not interpret them wildly (know the implications of your observations). Use panels of immunohistochemical stains, and be sure that everything adds up--discrepant staining patterns may be an important clue to a specific diagnosis, to problems with a given stain, or to problems with your proposed diagnosis Table 4. Diagnostically Useful Antigens Detectable Using Paraffin-Reactive Antibodies Cell Type Antigens Detectable B cells CD20, CD79a, CD22, PAX5(BSAP) Markers useful for defining CD10, BCL6, CD23, BCL2, CD5, B-cell subsets and/or CD43, cyclin D1, IRF4/MUM1 classifying B-cell lymphomas T cells (all but CD5 also on CD2, CD3, CD5, CD7 NK cells) T-cell subsets CD4, CD8, CD56, CD57, TIA-1, granzyme-B, [beta]-F1, CXCL13, PD-1 NK-associated CD56, CD57 Hodgkin lymphoma-associated CD30 and CD15 (classical), J chain (NLPHL), Oct2 and/or Bob-1 absent or minimal (classical) Other markers used in CD45 (LCA), CD30, EMA, ALK-1, lymphoma diagnosis clusterin, Ki-67 Plasma cells CD138, VS38c, [kappa], [lambda], IgG, IgM, IgA, IgD Follicular dendritic cells CD21, CD23 (subset), CD35, clusterin Other dendritic cells CD1a (LC), Langerin (LC), S100 (LC and IDC), CD123 (plasmacytoid dendritic cells as well as some AML, HCL) Immature/precursor cells CD34, CD99 (not specific), TdT, CD1a (thymocytes), CD117 (mostly myeloid) Myeloid/monocytic Lysozyme, myeloperoxidase, CD33, neutrophil elastase, CD117, CD68, CD163, CD14, glycophorin (erythroid), VWF/FVIIIRA (megakaryocytes), CD42b (megakaryocytes) Mast cells Tryptase, CD117 (strongly positive) Viral markers EBV- LMP-1 (plus EBER ISH), HHV-8 (KSHV), CMV, other Abbreviations: AML, acute myeloid leukemia; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HCL, hairy cell leukemia; HHV-8, human herpesvirus 8; IDC, interdigitating dendritic cells; ISH, in situ hybridization; KSHV, Kaposi sarcoma herpesvirus; LC, Langerhans cells; mega, megakaryocytes; NK, natural killer; NLPHL, nodular lymphocyte-predominant Hodgkin lymphoma; VWF/FVIIIRA, vonWillebrand factor/factor VIII--related antigen.
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|Author:||Garcia, Christine F.; Swerdlow, Steven H.|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||May 1, 2009|
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