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Practical Immunohistochemistry in Hematopathology:A Review of Useful Antibodies for Diagnosis

Ji Lu, MD and Karen L. Chang, MD

Abstract: This review article offers some useful panels ofimmunohistochemical stains and discusses their use in determininga hematopathology diagnosis. As a comprehensive review of thevast array of hematolymphoid malignancies is beyond the scope ofthis study, the suggestions are based on broad morphologiccategories such as follicular proliferations, paracortical expansions,diffuse small-cell infiltrates, diffuse large-cell infiltrates, andHodgkin-like infiltrates. The review article also discusses the mostcommon hematolymphoid malignancies and their immunohisto-chemical profiles, and how to use immunophenotyping todifferentiate them from other entities. Common diagnostic pitfallsand misconceptions about certain antibodies will also be discussed.New antibodies, such as SOX11, will also be explored in thecontext of specific disease entities for which they may be of use.

Key Words: hematopathology, immunohistochemistry, B-cell

lymphoma, T-cell lymphoma, Hodgkin lymphoma, leukemia,

histiocytic neoplasm, immunophenotype

(Adv Anat Pathol 2011;18:133–151)

Many of the hematolymphoid disorders are derivedfrom the same cell types in different stages of

maturation and their morphologic differences may besubtle. With experience, one may arrive at the correctdiagnosis based on the hematoxylin and eosin (H&E)-stained sections alone. This may be quite challenging,particularly if one does not see these specimens frequently.A panel of immunohistochemical stains may help todetermine lineage, subclassify tumors, and screen forcommon morphologic mimics. The purpose of this reviewarticle is to suggest some useful panels of immunostains andto discuss their use in arriving at the final diagnosis. As acomprehensive review of the vast array of hematolymphoidmalignancies is beyond the scope of this study, thesuggestions are based on broad morphologic categoriessuch as follicular proliferations, paracortical expansions,diffuse small-cell infiltrates, diffuse large-cell infiltrates, andHodgkin-like infiltrates. These panels, which serve as astarting point to think about a vast differential diagnosis,require alteration for each individual case, based on themorphology. The authors stress that morphology is morediagnostic than immunophenotype, but where discrepant,the pathologist may seek assistance from a specially trainedhematopathologist. The first part of this review articleoffers suggested antibody panels and how to use them to

narrow the differential diagnosis. The second section is adiscussion of the most common hematolymphoid malig-nancies, their immunohistochemical profiles, and how touse immunophenotyping to differentiate them from otherentities. Common diagnostic pitfalls and misconceptionsabout certain antibodies will also be discussed. New anti-bodies, such as SOX11, will also be explored in the contextof specific disease entities for which they may be of use.

HELPFUL IMMUNOHISTOCHEMICAL PANELSImmunohistochemical panels are selected based on the

morphologic examination. Recognition of more specificfeatures on the H&E stained sections will result in moretailored and targeted immunohistochemical panels. For thissection, we will assume that only very general architecturalpatterns are present, so the panels will be very broad andencompass many entities in the differential. Then, depend-ing on the staining profile of these initial panels, one cangenerate a list of differential diagnoses and refer to section IIfor more detailed discussion of each disease. Alternatively,if the morphology is pathognomonic, one may choose tobypass the initial panel and skip straight to confirmatorystains for that tumor.

General StainsMost lymph nodes and even extranodal lymphoid

tissue consistently show some degree of reactive changes.Certain patterns of reactive hyperplasia may suggest anunderlying etiology. The morphologic differentiation ofthese reactive conditions is beyond the scope of this review.Perhaps, more important than determining the etiology of areactive lymphoid hyperplasia is to exclude a lymphoma. Inlymph nodes, it is helpful to note that most cases of reactivehyperplasia show a combination of follicular, paracortical,and/or sinus hyperplasia, although one pattern maypredominate. When hyperplasia of a single component isseen to the exclusion of the others, one should be suspiciousof a neoplastic process.

Several stains compose the backbone of most of thepanels listed below. Most of the stains are aimed at B-celllesions, which comprise the majority of lymphomas. Theseinclude CD20, CD3, bcl-2, and CD43. CD30 may also beadded to screen for Hodgkin lymphoma (HL). One shouldunderstand the normal staining pattern of these markers inbenign/reactive lymphoid tissues. CD20 should highlightlocalization of B-cells mainly within follicles (Fig. 1A).Primary follicles express bcl-2, but lack CD10 and bcl-6.Secondary follicles contain a germinal center and a mantlezone. The germinal center cells express CD10 and bcl-6 butlack bcl-2 (Figs. 1B, C). The mantle zone is composed ofsimilar cells as the primary follicle. Scattered B-cells,including large immunoblasts, may be present in theparacortical region. Immunoblasts show weak, variableCopyright r 2011 by Lippincott Williams & Wilkins

From the Department of Pathology, City of Hope National MedicalCenter, Duarte, CA.

Reprints: Karen L. Chang, MD, Department of Pathology, City ofHope National Medical Center, NW2241C, 1500 East Duarte Road,Duarte, CA 91010 (e-mail: [email protected]).

REVIEW ARTICLE

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CD30 positivity, which characteristically varies in intensityfrom cell to cell (Fig. 1D). Both paracortical B-cells andimmunoblasts may be increased in reactive conditions (Fig.1A) but are always outnumbered by the paracortical T-cells. Diffuse sheets of CD20-positive cells outside follicles

should raise suspicion for B-cell lymphoma (Fig. 4). TheCD3 immunostain highlights T-cell localization predomi-nantly to the paracortical region, with only scattered T-cellsinside germinal centers. CD43 should always be performedin parallel with CD3 and CD20 to assess the distribution of

FIGURE 1. Reactive lymph node. A, CD20 highlights B-cells primarily within follicles. In cases with paracortical hyperplasia,interfollicular B-cells may be increased but do not form sheets. B, Bcl-6 is positive in the germinal center (right) and negative in theprimary follicle (left). C, Bcl-2 is negative in the germinal center (right) and positive in the primary follicle (left). Note the bcl-2 positivemantle zone B-cells, interfollicular T-cells, and scattered germinal center T-cells. D, CD30 highlights immunoblasts. Note the variableintensity staining from cell to cell. E, Ki-67 is very high in reactive germinal centers and variably lower in the interfollicular regions.Figures B and C: courtesy of Dr Lawrence Weiss.

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staining. In most reactive conditions, the CD43 stain shouldparallel that of CD3, with no co-localization on CD20-positive cells. The bcl-2 staining pattern also parallels that ofCD3 with additional staining in primary follicles and mantlezones (Fig. 1C). CD5 is another helpful marker for B-cell andT-cell malignancies. However, most B-lymphomas thatexpress CD5, such as chronic lymphocytic leukemia/smalllymphocytic lymphoma (CLL/SLL) and mantle cell lympho-ma (MCL), are also positive for CD43. Therefore, CD5 is notincluded in the “general” panel but still helpful under certainconditions detailed below.

Follicular PatternThis pattern refers to a proliferation of many nodules

of lymphocytes, which resemble follicles. The main differen-tial is between reactive follicular hyperplasia and follicularlymphoma (FL). Multiple well-described morphologic fea-tures are helpful in making this distinction, but one findingalone is not sufficient. When faced with ambiguous cases, apanel of immunohistochemical stains may be helpful. Mostcases can be clarified using the basic panel of CD20, CD3,bcl-2, CD43, and/or CD5, with the addition of CD10 and/or bcl-6 (Table 1). Kappa and l immunostains are helpfulin a minority of cases; flow cytometry has greater sensitivityfor light chain evaluation. Reactive germinal center B-cellsstain for CD10 and bcl-6, but not bcl-2 (Fig. 1). Germinalcenters that are bcl-2 positive are almost always lymphoma(Fig. 2). However, 3 caveats exist. First, only germinalcenters should be assessed, because normal primary folliclesand mantle zones are bcl-2 positive (Fig. 1C). Co-localization of CD10, bcl-6, and bcl-2 positivity is helpfulas CD10 and bcl-6 are not expressed in primary follicles ormantle zone B-cells (Figs. 1, 2). Second, scattered bcl-2-positive cells within germinal centers likely representnormal follicular helper T-cells and should not beinterpreted as neoplastic (Fig. 1C). The exception is whenincreased, scattered bcl-2-positive neoplastic B-cells insidethe germinal center show stronger staining than theadjacent bcl-2-positive T-cells or the mantle cells(Fig. 2D). This finding is suggestive of FL-in-situ. Lastly,lack of bcl-2 staining does not exclude a diagnosis of FL,especially in the pediatric population, in which up to two-

thirds of cases are bcl-2 negative.1 Bcl-2 is not specific forFL and does not distinguish it from other small B-celllymphomas that may colonize follicles, such as MCL or lesscommonly marginal zone lymphoma (MZL). CD43 posi-tivity with lack of CD10 expression is uncharacteristic forFL and raises the possibility of MCL or MZL. Coexpres-sion of CD5 on the B-cells is suggestive of MCL or CLL/SLL. Ki-67 may be helpful, showing a higher proliferationrate in reactive germinal centers and often lower numbersof positive nuclei in FL (Figs. 1, 2). CD20 is useful in establish-ing B-lineage and assessment of diffuse versus folliculararchitecture. One may also see a vaguely nodular patterncomposed of larger and more spaced-out nodules thannormally seen in FL, composed of a mixture of B andT-cells (Fig. 3). Consideration should be given to nodularlymphocyte-predominant HL (NLPHL) and careful histo-logic examination for lymphocyte-predominant cells (LP cells,formerly L&H cells) and additional stains are warranted.

Paracortical Hyperplasia PatternThe paracortical region is the space between the

follicles and is usually composed of T-cells with a lesserdegree of admixed B-cells, including large immunoblasts,dendritic cells, macrophages, plasma cells, and/or eosino-phils and plasmacytoid dendritic cells. Predominant reac-tive hyperplasia of the paracortical region is also relativelycommon but more difficult to distinguish from the multi-tude of lymphomas, both B- and T-cells, which mimic it.Importantly, reactive paracortical hyperplasia is almostalways accompanied by follicular and/or sinus hyperplasia.In some instances, such as with acute infectious mono-nucleosis, the paracortical hyperplasia may overshadow thefollicular hyperplasia, although reactive follicles should stillbe evident. Lymphomas may also preferentially involve theparacortical region with some residual reactive follicles stillpresent. The tumors that mimic reactive paracorticalhyperplasia are usually those with a mixed infiltrate. Themain neoplastic entities to consider include T-cell/histio-cyte-rich large B-cell lymphoma (THRLBCL), classical HL(cHL), and NLPHL, and T-cell lymphomas, includingperipheral T-cell lymphoma, not otherwise specified(PTCL, NOS), and angioimmunoblastic T-cell lymphoma.

TABLE 1. Useful Stains for Follicular Pattern

Stain Normal Pattern Use

CD20 B-cells, mostly in follicles, scattered paracortical Highlights nodal architecture. Confirm B-lineage.Assess for diffuse B-cell areas

CD3 T-cells, mostly paracortical, scattered ingerminal centers

Highlights nodal architecture. Use in conjunction with bcl-2,CD5, and CD43 to evaluate for aberrant B-cell expression

Bcl-2 T-cells, primary follicle B-cells, mantle zoneB-cells

Bcl-2-positive germinal center indicates malignancyif primary follicle is excluded

CD43 T-cells, granulocytes, some histiocytes Assess for aberrant expression on B-cells. CD43+ neoplasmmore likely follicular colonization by MZL, CLL/SLL, or MCL

CD5 T-cells Assess for aberrant expression on B-cells. CD5+ neoplasmmore likely follicular colonization by CLL/SLL or MCL

CD10 Germinal centers Confirms follicular center cell origin. Excludes primary follicleBcl-6 Germinal centers Confirms follicular center cell origin. Excludes primary follicleKi-67 High in germinal centers. Low in paracortical

regionLow Ki-67 in germinal center suggests FL

K/L K:L ratio is 2-3:1 Restriction indicates malignancy

CLL/SLL indicates chronic lymphocytic leukemia/small lymphocytic lymphoma; FL, follicular lymphoma; K, k light chain; L, l light chain; MCL, mantlecell lymphoma; MZL, marginal zone lymphoma.

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For all of the entities, the initial panel of immunostainsis similar and should include CD20, CD3, bcl-2, CD43,CD30, and Ki-67 (Table 2). In a child, Epstein-Barr virus(EBV) stains should also be added to exclude acuteinfectious mononucleosis. Some small B-cell lymphomasmay preferentially involve the paracortical region and sparethe follicles; these are easily recognized as sheets of CD20-positive B-cells (Fig. 4). As the paracortical regioncomprises predominantly T-cells admixed with other celltypes, involvement by T-cell lymphoma may be harder todiagnosis. Expansion of the CD3-positive areas to push theCD20-positive B-cells to the periphery of the lymph nodeindicates effacement of the architecture and is highlysuggestive of lymphoma (Fig. 5). Loss of bcl-2 expressionis also suggestive of T-cell lymphoma. Assessment of bcl-2expression on B-cells is not as helpful in this setting as theyare difficult to separate from the paracortical T-cells. CD43expression is expected to follow CD3 and is not generallylost in T-cell lymphoma. Ki-67 is normally low in theparacortical region whereas PTCL generally has anelevated proliferative rate. The CD20 stain may emphasizea vaguely nodular architecture not evident on the H&E,raising the possibility of NLPHL (Fig. 3). Rare, scattered,large, atypical cells with uniformly strong CD20 expressionis concerning for THRLBCL.

The CD30 staining pattern deserves special attention.In lesions resembling reactive paracortical hyperplasia,CD30 is used to screen for cHL. In cHL, the Hodgkin cellsshow a membrane and/or paranuclear/Golgi stainingpattern that is of fairly uniform intensity from cell to cell(Fig. 6A). Diffuse cytoplasmic staining alone should not beconsidered positive. The pitfall is that reactive paracorticalimmunoblasts, seen in reactive paracortical hyperplasia andeven some T-cell lymphomas, may also be CD30 positive.In contrast to the Hodgkin cells, CD30 staining inimmunoblasts is usually weaker and show variability inintensity from cell to cell (Fig. 1D). Cases with concurrentfollicular hyperplasia, no good Hodgkin or Reed Sternbergcells, and variable CD30 staining do not require furtherworkup for cHL. Others may require additional stains.

Classical Hodgkin Lymphoma-like PatternClassical Hodgkin lymphoma characteristically shows

lymph node effacement by rare Hodgkin cells in a cellularmilieu composed of small T-cells, histiocytes, eosinophils,plasma cells, and neutrophils. The most common nodularsclerosis subtype also shows broad fibrous bands thatradiate from a thickened capsule and entrap nodules ofinfiltrate. Occasionally, cHL may only involve the para-cortical regions, the so-called interfollicular pattern, and is

FIGURE 2. Follicular lymphoma. A, CD10 is positive in neoplastic follicles. This confirms follicular center origin and excludes a primaryfollicle, which is also bcl-2-positive. B, Bcl-2 positivity in germinal centers is virtually diagnostic of malignancy. C, Ki-67 is low inneoplastic follicles. Contrast this with the high Ki-67 in reactive germinal centers. D, Bcl-2 stain in follicular lymphoma in situ. Note theincreased numbers and intensity of bcl-2-positive cells inside this germinal center.



most often seen with mixed cellularity subtype. Conversely,in some reactive conditions, especially acute infectious mono-nucleosis, immunoblast numbers may be greatly increased,clustered, and even show bilobated and multinucleatedforms resembling Hodgkin cells. When cHL is suspected,the initial panel of immunohistochemical stains shouldinclude CD20, CD3, CD15, CD30, and CD45 (Table 3). If

available, PAX-5 is also a helpful addition to this panel. Inaddition to consistent and uniform CD30 positivity, Hodgkincells are also usually positive for CD15 (85%) and PAX-5(90%), with a subset positive for CD20 (20% to 50%), andare consistently negative for CD45.2–6 In contrast, reactiveimmunoblasts are consistently negative for CD15 andvariably positive for CD20, CD45, and PAX-5. Keep in

TABLE 2. Useful Stains for Paracortical Hyperplasia Pattern


CD20 B-cells, mostly in follicles, scatteredinterfollicular

Highlights nodal architecture and concurrent follicular hyperplasia.Diffuse sheets indicate B-cell lymphoma. B-cells pushed to the capsulesuggest T-cell lymphoma. Vaguely nodular architecture may be NLPHL

CD3 T-cells, mostly interfollicular, scatteredin germinal centers

Highlights nodal architecture. Use in conjunction with bcl-2 and CD43to evaluate for aberrant B-cell expression

Bcl-2 T-cells, primary follicle B-cells, mantlezone B-cells

Bcl-2 loss in T-cells suggests lymphoma. Aberrant B-cell expressionextremely difficult to assess

CD43 T-cells, granulocytes, some histiocytes Usually not lost in T-cell lymphoma. Aberrant B-cell expression extremelydifficult to assess

CD30 Immunoblasts Variable intensity staining suggests reactive immunoblasts. Uniformlystrong membranous and/or paranuclear/Golgi staining in cHL

Ki-67 High in germinal centers. Low inparacortical region

High Ki-67 in paracortical region suggestive of lymphoma

cHL indicates classical Hodgkin lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma.

FIGURE 3. Nodular LP Hodgkin lymphoma. A, Hematoxylin and eosin-stained section showing no distinct nodular architecture. B,CD20 stain highlighting large B-cell nodules. These nodules are more vague and larger than those of follicular lymphoma. C, PD1showing expanded follicular helper T-cells with characteristic ringing around LP cells. LP indicates lymphocyte predominant.



mind that CD15 is positive in granulocytes, which are smalland multilobated. These should not be confused withHodgkin or Reed Sternberg cells. CD15 may show variablepositivity between different Hodgkin cells of the same case.PAX-5 is a nuclear stain and evaluation should be performedat high power as it shows characteristic weak positivity incHL (Fig. 6B). Evaluation of CD45 is often difficult becauseof the abundant admixed T-cells that often surround Hodgkincells. To be considered a positive stain, the large cells shouldshow circumferential membranous staining that is equal orstronger when compared with the surrounding lymphocytes(Fig. 6C). Cases where CD30 is positive and CD15 is nega-tive pose the most difficulties in diagnosis and may requirefurther workup to exclude reactive paracortical hyperpla-sia, anaplastic large cell lymphoma (ALCL), other T-celllymphomas, and certain types of diffuse large B-celllymphoma (DLBCL).

Diffuse Small Cell InfiltrateB-cell lymphomas may be separated by the predomi-

nant size of the neoplastic cells. Using a histiocyte or endo-

thelial cell nucleus as reference, small cells are smaller thanan endothelial cell nucleus, intermediate cells are about thesame size, and large cell are larger. Most of these entities areeasily identified as malignant based on the morphologyalone. A monomorphic proliferation is usually evidenton low power, with at least focal effacement of the lymphnode architecture. One should keep in mind that most ofthese malignancies are actually composed of a range of cellsizes, which is more obvious on high power. However,patternless sheets of large cells are usually indicative oflarge cell transformation. The following is a proposeddiagnostic approach for classifying so-called small-cellor low-grade B-cell lymphomas based on immunohisto-chemistry.

For lymphomas composed primarily of small cells, abasic panel should include CD20, CD3, CD5, CD10, CD43,and bcl-2. Cyclin-D1 (bcl-1) and CD23 may also be addedto the initial panel, along with k and l, especially ifplasmacytoid features are present (Table 4). The pattern ofCD20 staining is important to confirm the initial morpho-logic impression of a B-cell malignancy by showing anexpanded B-cell population. The CD20 stain may also beused to highlight architectural findings that may not beobvious on the H&E-stained sections, such as nodularity inFL. CD10 positivity, along with lack of CD5 and CD43expression, is typical of FL. CLL/SLL is usually positivefor CD5, CD23, and CD43. MCL is also positive for CD5and CD43, with cyclin-D1 expression. Bcl-2 positivity isseen in most small B-cell lymphomas and is helpful fordistinguishing MZL from reactive monocytoid B-cellproliferations in the lymph node. Although much lesscommon, T-cell lymphomas such as PTCL, NOS can alsocomprise a diffuse small cell infiltrate. T-cell lymphomaswill generally show expression of CD3 and CD43 withvariable loss of bcl-2 and CD5.

Diffuse Large Cell InfiltrateFor an infiltrate composed of patternless sheets of

large hematolymphoid cells, DLBCL is the most commonentity and usually the diagnosis is straightforward. How-ever, there is a broad differential diagnosis that needs to beconsidered, including carcinoma, melanoma, reactive para-cortical hyperplasia, HL, and other non-Hodgkin hemato-lymphoid neoplasms. The latter includes lymphoblasticlymphoma (LBL), Burkitt lymphoma, blastic MCL, FL,acute myeloid leukemia, T-cell lymphomas, and precursorplasmacytoid dendritic cell neoplasm. Although this listmay seem daunting, an initial panel of paraffin immuno-histochemistry should help to sort out most of this list withrelative ease. The distinction from carcinoma and melanomais readily made using pancytokeratin and S100. Assuming ahematolymphoid disorder has already been established, ahelpful panel may include CD20, CD3, CD30, cyclin D1,terminal deoxynucleotidyl transferase (TdT), CD43, and Ki-67 (Table 5). If morphologic features are highly suggestiveof DLBCL, then bcl-2, bcl-6, CD10, and MUM1 may beperformed initially to save time. The straightforward case ofDLBCL will be positive for CD20, with a Ki-67 that iselevated but less than 80%, and negative for CD3, CD30,cyclin D1, TdT, and CD43. Positivity for any of the lattermarkers does not exclude DLBCL but rather acts as a screen-ing test for the other entities in the differential diagnosisand should warrant additional workup. The following isa brief list of positive stains and the entities for whichthey should raise suspicion: CD3—T-cell lymphomas

FIGURE 5. Peripheral T-cell lymphoma, not otherwise specified.CD20 stain shows B-cells pushed to the periphery of the lymphnode. Figure courtesy of Dr Lawrence Weiss.

FIGURE 4. Chronic lymphocytic leukemia/small lymphocyticlymphoma. CD20 stain highlights diffuse sheets of B-cells, anindication of malignancy.



including ALCL and T-LBL; CD30—ALCL, cHL, andreactive paracortical hyperplasia; cyclin D1—blastic MCL;TdT—LBL, acute myeloid leukemia, and precursor plas-macytoid dendritic cell neoplasm; CD43—Burkitt lympho-

ma, T-cell lymphomas, acute myeloid leukemia, and LBL;and Ki-67 approaching 100%—Burkitt lymphoma. A fewspecial considerations should be noted. First, in a child withtonsillar or cervical lymphadenopathy, be extremely wary

FIGURE 6. Classical Hodgkin lymphoma. A, CD30 stain is fairly uniform in a membranous and perinuclear pattern in these Hodgkincells. B, PAX-5 is characteristically weakly positive compared with the strong staining in the nearby B-lymphocytes. C, CD45 is difficult tointerpret because of the abundant T-cells. Note that in areas where the Hodgkin cell is not in contact with a lymphocyte, there is a gap inmembranous staining of CD45. CD45 negativity is easiest to prove when 2 Hodgkin cells abut each other.

TABLE 3. Useful Stains for Classical Hodgkin Lymphoma-like Pattern



Highlights nodal architecture and concurrent follicular hyperplasia in reactiveproliferations and effacement in lymphomas. Positive in LP cells of NLPHLand large cells of THRLBCL. Variably positive in Hodgkin cells. Increasedbackground B-cells may be NLPHL versus lymphocyte-rich cHL

CD3 T-cells, mostly interfollicular,scattered in germinal centers

Highlights nodal architecture. Positive in large cells of ALCL unless thereis aberrant loss

CD15 Granulocytes Positive in Hodgkin cells of cHL. Negative in large cells of most mimicsCD30 Immunoblasts Variable intensity staining suggests reactive immunoblasts. Uniformly strong

membranous and/or paranuclear/Golgi staining in cHL and ALCL. Negativein LP cells of NLPHL and large cells of THRLBCL

CD45 Almost all hematolymphoid cells Negative in Hodgkin cells of cHL. Positive in large cells of most mimics. Maybe negative in ALCL

PAX-5 B-cells Weakly positive in Hodgkin cells of cHL. Stronger staining in LP cellsof NLPHL and large cells of THRLBCL. Negative in ALCL

ALCL indicates anaplastic large cell lymphoma; cHL, classical Hodgkin lymphoma; LP, lymphocyte predominant; NLPHL, nodular lymphocytepredominant Hodgkin lymphoma; THRLBCL, T-cell/histiocyte-rich large B-cell lymphoma.



of diagnosing DLBCL and always perform EBV studiesto exclude acute infectious mononucleosis. Second, remem-ber that low-grade B-cell lymphomas may have scatteredlarge cells and groups of large cells in proliferation centers.Only use this panel if large cells are present in pattern-less sheets to avoid misdiagnosing a case of low-gradeB-cell lymphoma as DLBCL. Third, rare cases may showimmunoblastic morphology but lack all of the abovemarkers, such as in plasmablastic lymphomas or plasma-blastic plasmacytoma. Primary effusion lymphomas alsotypically lack pan-B-cell markers, but the clinical set-ting should help with this diagnosis. A very rare type oflarge-cell lymphoma called anaplastic lymphoma kinase(ALK)-positive large B-cell lymphoma is negative for pan-B-cell markers and CD30. CD138, k, l, and ALK1

immunostains along with EBV-encoded RNA (EBER) in-situ hybridization may be helpful in these null phenotypecases.

COMMON HEMATOLYMPHOID NEOPLASMS

Small B-cell Lymphomas

Follicular LymphomaFL is a lymphoma composed of centrocytes and

centroblasts with at least a focal follicular pattern. Thefollicles are usually equally sized and crowded; they may ormay not have a mantle zone and they lack polarization.Centrocytes are usually small cells with irregular/cleavednuclei, condensed chromatin, and inconspicuous nucleoli.

TABLE 4. Useful Stains for Diffuse Small Cell Pattern



Highlights effacement of nodal architecture by diffuse sheets of B-cells.B-cells pushed to the capsule suggest T-cell lymphoma. Vaguely nodulararchitecture may be NLPHL


Scattered T-cells may still be present in B-cell lymphomas. Use inconjunction with bcl-2, CD5, and CD43 to evaluate for aberrantB-cell expression

Bcl-2 T-cells, primary follicle B-cells,mantle zone B-cells

Aberrant B-cell expression suggests B-cell lymphoma. Bcl-2 loss in T-cellssuggests T-cell lymphoma

CD43 T-cells, granulocytes, somehistiocytes

Usually not lost in T-cell lymphoma. Aberrant B-cell expression suggestsCLL/SLL, MCL, or MZL. CD43-negativity suggest FL or MZL

CD5 T-cells Assess for aberrant expression on B-cells. Positive in CLL/SLL and MCL.Aberrant loss suggests T-cell lymphoma

CD10 Germinal centers Positive in FLCyclin D1 (bcl-1) Endothelial cells Positive in MCL (diffusely). May be positive in plasma cell neoplasms,

HCL (subset), and CLL/SLL (weak)CD23 Follicular dendritic network Positive in CLL/SLLK/L K:L ratio is 2-3:1 Restriction indicates malignancy

CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; FL, follicular lymphoma; HCL, hairy cell leukemia; K, k light chain; L, l lightchain; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma.

TABLE 5. Useful Stains for Diffuse Large Cell Pattern



Highlights effacement of nodal architecture by patternless sheets of B-cellsin DLBCL, BL, and blastic MCL. Variably positive in B-ALL


Positive in ALCL and T-ALL

CD30 Immunoblasts Uniformly strongly positive in cHL and ALCL. May be variably positivein DLBCL

Cyclin D1 (bcl-1) Endothelial cells Positive in blastic MCLTdT Thymic lymphocytes Positive in ALL. May be positive in AML and BPDCCD43 T-cells, granulocytes, some

histiocytesPositive in BL, AML, ALL, and T-cell lymphomas including ALCL.Often negative in DLBCL

Ki-67 High in germinal centers. Low inparacortical region

100% positive cells suggestive of BL but not specific

CD138 Plasma cells Positive in PCN and B-cell neoplasms with plasmacytoid differentiationsuch as plasmablastic lymphoma. Positive in some carcinomas, sarcomas,and melanomas

K/L K:L ratio is 2-3:1 Establishes hematolymphoid differentiation if CD138 is the only positivemarker

ALK1 Negative Positive in ALCL and ALK+ DLBCL

ALCL, anaplastic large cell lymphoma; ALK, anaplastic lymphoma kinase; AML, acute myeloid leukemia; B-ALL, B-cell lymphoblastic lymphoma; BL,Burkitt lymphoma; BPDC, blastic plasmacytoid dentritic cell neoplasm; cHL, classical Hodgkin lymphoma; DLBCL, diffuse large B-cell lymphoma; K, k lightchain; L, l light chain; MCL, mantle cell lymphoma; PCN, plasma cell neoplasm; T-ALL, T-cell lymphoblastic lymphoma; TdT, terminal deoxynucleotidyltransferase.



Centroblasts are large cells with round/noncleaved nuclei,vesicular chromatin, and 1 or more peripheral nucleoli. FLis positive for all pan-B-cell markers including CD20,CD79a, PAX-5, and the B-cell transcription factors BOB.1and OCT-2. The germinal center markers CD10 and bcl-6are positive in most cases of FL (Fig. 2A), and aberrantcoexpression of CD5, CD23, or CD43 is rare. Bcl-2 ispositive in most cases (Fig. 2B), with the exception of grade3, which can be bcl-2-negative in up to 25% of cases.7

Although grading is based on morphology, assessment ofdiffuse architecture is best performed on immunohisto-chemical stained sections, such as CD20. Note that CD10may be negative in the interfollicular portion of FL.8 Asdiscussed earlier, one may find FL-in-situ, characterized bybcl-2-positive neoplastic B-cells inside the germinal centerthat show stronger staining than the adjacent bcl-2-positiveT-cells or the mantle cells (Fig. 2D, Table 6).9

Chronic Lymphocytic Leukemia/SmallLymphocytic Lymphoma

CLL/SLL involving lymph nodes shows a vaguelynodular appearance because of the presence of proliferationcenters containing prolymphocytes and paraimmunoblastsor immunoblasts. In extranodal sites or small biopsies, thesubtle nodular architecture may not be appreciated.CLL/SLL also expresses pan-B-cell markers although thereactivity for CD20 is usually weak. Most cases are positivefor CD5, CD23, CD43, and bcl-2, and are negative forCD10 and bcl-1 (cyclin D1). The CD23 positivity and bcl-1negativity is useful in distinguishing CLL/SLL from MCL.Rare cases of CLL/SLL may be CD23 negative and evenbcl-1 positive. In difficult cases, the distinction rests withmorphologic identification of proliferation centers, whichexcludes MCL. Identification of the t(11;14) translocation,although earlier reported in CLL/SLL, is now thought toexclude this diagnosis.10 Positivity for CD5 and CD23 helpsin making the distinction from MZL. However, rare casesof MZL may be positive for CD5 and even CD23,therefore, morphologic examination is still most important,with rare cases necessitating flow cytometry or cytogenetics.ZAP-70 is positive in about half of the cases and correlateswith poorer prognosis, although evaluation is difficult inparaffin sections.11 Immunoglobulin studies in paraffinsections are usually negative unless the cells exhibit someplasmacytoid features. Ki-67 usually shows low prolifera-tion in diffuse areas and high rates in proliferation centers.

MUM1 is also positive in proliferation centers and may beuseful.

Mantle Cell LymphomaMCL usually shows expression of CD5 and CD43, but

with slightly less frequency than CLL/SLL.12,13 CD10 andCD23 are usually negative, which is helpful in thedistinction from FL and CLL/SLL, respectively. CyclinD1 (bcl-1) expression is seen in almost all cases and isassociated with the t(11;14)(q13;32) translocation betweenthe genes CCND1 (BCL-1) and Ig heavy chain.14 Cyclin D1staining is nuclear and characteristically varies in intensityamong the tumor cells in a given lymphoma. One should becareful to recognize positive nuclear staining in endothelialcells, which serves as an internal control. Cyclin D1-negative cases may show overexpression of cyclin D2 orD3.15 Rare cases may lack expression of the cyclin proteinsand may also lack the t(11;14)(q13;q32) translocation, andthe diagnosis of MCL should be made only if all otherfeatures are classic. A recently described nuclear stainingantibody, SOX11, shows similar sensitivity as cyclin D1 insmall studies of MCL. In fact, 1 cyclin D1-negative MCLhas been reported to be SOX11 positive.16 Other lymphoidneoplasms that may also show cyclin-D1 positivity includeplasma cell neoplasms (up to 25% of cases),17 hairy cellleingukemia (HCL; up to 50% of cases but usually only asubset of cells),18,19 and CLL/SLL (rare cases showing weakstaining in proliferation centers).20 Of these, only the lattershows morphologic similarities. MCL does not containadmixed large cells or proliferation centers, a helpfulfeature in distinguishing this from the other common smallcell lymphomas such as FL, MZL, and CLL/SLL. Theblastoid and pleomorphic variants of MCL contain largecells, but all the neoplastic cells are large, and in theblastoid variant, they are uniform in appearance, resem-bling a LBL. Both of these variants are also positive forcyclin D1, helping to make the distinction from otherentities such as LBL, DLBCL, and Burkitt lymphoma.

Marginal Zone B-cell LymphomaMZL is separated into extranodal, nodal, and splenic

types, all of which show similar immunophenotyping. Allexpress CD20 and are usually negative for CD5, CD10,CD23, and cyclin D1. Expression of CD43 is present inone-third of cases.13 Plasmacytic differentiation is oftenpresent and quite striking, usually with demonstrable

TABLE 6. Differentiation of Small B-cell Lymphomas

+ +/� �

FL* CD10 Bcl-1, CD5, CD23, CD43CLL/SLL* CD5, CD23, CD43 Bcl-1w, CD10wMCL* Bcl-1, CD5, CD43 CD10w, CD23wMZL* CD43, CD138, K/L restriction Bcl-1, CD5w, CD10w, CD23w, DBA.44w,

TRAPwLPL* CD138, K/L restriction CD43 Bcl-1, CD5w, CD10w, CD23wPCN CD138, K/L restriction Bcl-1, CD43, CD79a, MUM-1,

CD56CD10w, CD20w, CD45w, PAX-5w

HCL CD20, annexin A1, DBA.44, TRAP Bcl-1 CD5w, CD10w, CD23w, CD43w

*Positive for pan-B-cell markers CD20, CD79a, PAX-5, BOB.1, and OCT-2.wRare cases may be positive.CLL/SLL indicates chronic lymphocytic leukemia/small lymphocytic lymphoma; FL, follicular lymphoma; HCL, hairy cell leukemia; K, k light chain;

L, l light chain; LPL, lymphoplasmacytic lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; PCN, plasma cell neoplasm.



CD138 expression and light chain restriction. The lack ofCD5 and CD10 help to differentiate MZL from CLL/SLL,MCL and FL with relative ease in most cases. A moredifficult distinction is between MZL and reactive prolifera-tions such as reactive monocytoid B-cell hyperplasia inlymph nodes or severe chronic gastritis. The finding ofmonotypic immunoglobulin light-chain restriction is diag-nostic of malignancy, but this is usually only found inparaffin sections when plasmacytoid features are present. Inlymph nodes, bcl-2 positivity in a monocytoid B-cellpopulation is diagnostic of malignancy. Bcl-2 positivity insmall biopsies of extranodal sites may be very difficult tointerpret and should not be used as the sole indicator ofmalignancy. Aggregates of bcl-2-positive B-cells in astomach biopsy may represent primary follicles or reactivemantle or marginal zone B-cells. Reactive marginal zonehyperplasia of the spleen, abdominal lymph nodes, or ilealmucosa may express bcl-2 in or near the follicles.21

In extranodal sites such as the stomach, CD43 positivityin the B cells may be the most easily identified featureof malignancy. Another indicator of malignancy is thepresence of sheets of CD20-positive cells without an asso-ciated, compartmentalized T-cell population. Many casesof extranodal MZL will not show any of the above-mentioned immunohistochemical criteria for malignancy.One must rely on morphologic features and if necessary,gene rearrangement studies.

Lymphoplasmacytic LymphomaLymphoplasmacytic lymphoma (LPL) is often associ-

ated with Waldenstrom macroglobulinemia (WM), definedin the 2008 World Health Organization (WHO) Classifica-tion scheme as any concentration of IgM monoclonalgammopathy combined with bone marrow involvement byLPL.22,23 LPL most commonly but not always produces anIgM paraprotein. LPL is characterized morphologically byan infiltrate composed of small lymphocytes, plasmacytoidlymphocytes, and plasma cells. One sees immunohistochem-ical expression of pan-B-cell markers and frequentlyCD43,13 usually with no expression of CD5, CD10, CD23,or cyclin D1. As with most lymphomas with plasmacyticdifferentiation, monotypic light-chain expression is usuallydetectable in paraffin sections. The mature plasma cellsare CD138 positive. IgM paraprotein is not unique toLPL; WM-like clinical presentations may be seen in otherlymphomas including CLL/SLL and splenic MZL. LPLusually does not stain for CD5 or CD10, a feature thatoffers distinction from most other small B-cell lymphomas.However, differentiation from MZL may be difficultbecause of similar immunophenotypes and only subtlemorphologic differences, such as the monocytoid appear-ance and plasma cell compartmentalization of MZL.Sometimes, the diagnosis of “small B-cell lymphoma withplasmacytic differentiation,” as recommended by the WHO,may be necessary.23 The presence of a spectrum of cells withlymphoid and plasmacytoid features, along with CD20expression, helps to distinguish LPL/WM from pure plasmacell neoplasms.

Plasma Cell NeoplasmsThis contains a pure population of plasma cells that

usually appear mature but occasionally show atypical orplasmablastic/immunoblastic cytology. Plasma cell neo-plasms consistently express CD138, and frequently expressCD79a, MUM-1, and CD43. Aberrant expression of CD56

and CD117 is common in neoplastic but not reactiveplasma cells.24,25 There is usually no expression of CD45,CD20, or PAX-5. Monotypic light-chain restriction isdemonstrable in paraffin sections, usually with expressionof IgG and IgA. Cyclin D1 expression is seen inapproximately one-quarter to one-third of cases andcorrelates with the presence of the t(11;14) translocation.17

SOX11 protein expression is not seen, even in plasma cellneoplasms with this translocation.16 The differential diag-nosis includes reactive plasmacytosis, Castleman disease,and small B-cell lymphomas that contain a prominentplasma cell infiltrate such as LPL, MZL, and CLL/SLL.The initial panel should include CD138, k, l, CD43, CD45,CD20, and CD3. Monotypic light-chain restriction differ-entiates a plasma cell neoplasm from reactive plasmacy-tosis. If the light chain stains are noncontributory, CD56may be helpful. The plasma cells in Castleman disease mayshow l restriction (but not k); human herpesvirus 8 (HHV-8) positivity may be helpful if there is clinical suspicion. Thelack of a CD20-positive/CD45-positive infiltrate helps todistinguish a plasma cell neoplasm from small B-celllymphomas that may contain plasmacytoid differentiationor plasma cell infiltrates. Plasmablastic plasma cell neo-plasms cannot be distinguished from plasmablastic lym-phoma by morphology or immunohistochemistry andrequires clinical correlation. CD138 expression is notspecific for plasma cell neoplasms and may be seen inseveral nonhematolymphoid malignancies such as carcino-ma, sarcoma, and melanoma.26 In cases with plasmablasticmorphology and CD138 positivity, one must also establisha hematolymphoid origin with a panel that may includeCD45, CD79a, CD43, k, l, CD20, CD3, CD30, pancyto-keratin, and S100. CD43 is fairly specific for hematolym-phoid malignancies (both lymphoid and myeloid), and hasonly rarely been reported in other tumors.27 In contrast,MUM-1, although present in plasma cell neoplasms andother lymphomas, is also expressed in melanoma.28

Hairy Cell LeukemiaHairy cell leukemia (HCL) is an indolent B-cell

neoplasm composed of cells with oval nuclei, abundantcytoplasm, and “hairy” cytoplasmic projections, whichimpart a “fried-eggs” appearance in histologic sections. Thedisease primarily involves the bone marrow and spleen;occasional liver, lymph node, and skin involvement areseen.23 Bone marrow involvement shows a characteristicinterstitial pattern that is distinctive from other small B-cellneoplasms. However, its histologic appearance in the lymphnode and spleen overlaps with MZL. A helpful panel ofimmunostains may include CD20, CD3, CD43, annexinA1, DBA.44, tartrate-resistant acid phosphatase (TRAP),bcl-1, CD5, and/or CD23. HCL is generally positive forCD20, CD45, annexin A1, DBA.44, TRAP, and bcl-2.29,30

A subset of cells show weak expression of bcl-1 in somecases.14,31 Although DBA.44 and TRAP are sensitivemarkers of HCL, they both may be positive in some casesof MZL.30 SOX11 staining correlates with bcl-1-positivecases.16 The cells are negative for CD5, CD23, and CD43.Bcl-1 is not a very sensitive marker for HCL but isconsistently negative in MZL. Perhaps, the most sensitiveand specific marker is annexin A1, which shows cytoplas-mic staining in almost all HCL cases and is negative in allother B-cell lymphomas, including MZL.29



Large B-cell Lymphomas

Diffuse Large B-cell LymphomaThis is a heterogeneous group of lymphomas with

multiple variants and subtypes that have been dividedbased on morphologic, immunohistochemical, genetic, andclinical features.23 The morphologic variants includecentroblastic, immunoblastic, and anaplastic. The molecu-lar and immunohistochemical subgroups include germinalcenter B-cell-like (GCB) and activated or nongerminalcenter B-cell-like (ABC). With the use of rituximab therapy,the clinical significance of these variants is no longer clear.32

These variants are currently all combined into the categoryof DLBCL, NOS (DLBCL, NOS) under the current WHOclassification. These lymphomas are positive for CD20,CD79a, and PAX-5 as well as the B-cell transcriptionmarkers BOB.1 and OCT-2, but stains other than CD20 areusually only needed in morphologically difficult cases. If adiagnosis of DLBCL, NOS is suspected by the initial panel,the addition of bcl-2, bcl-6, CD10, and MUM1 may beconsidered. For patients above 50 years of age, EBV-latentmembrane protein 1 (LMP1) may be added under certaincircumstances to exclude EBV-positive DLBCL of theelderly. DLBCL, NOS is positive for CD10 in approxi-mately half of cases; bcl-6 is positive in approximatelythree-quarters; MUM-1 is positive in approximately one-half; bcl-2 is positive in approximately one-half; and somecases may also be positive for CD5, CD23, and CD43.33–39

Ki-67 expression varies from 30% to 100%. Some cases,especially the anaplastic variant of DLBCL, are alsopositive for CD30. These cases are negative for ALKprotein and T-cell markers. Using immunohistochemistryto divide DLBCL into GCB and ABC groups is optional.The GCB type is positive for CD10 (>30% of cells) and/orbcl-6, but negative for MUM-1. The ABC type is typicallynegative for CD10 and positive for bcl-6 and MUM-1.34,36

The distinction between DLBCL and Burkitt lympho-ma may be very difficult. Burkitt lymphoma is suspected ifone sees a monomorphic appearance with a starry-skypattern, or if Ki-67 approaches 100% and the cells arepositive for CD10 and bcl-6. Bcl-2 negativity, seen inapproximately two-thirds of Burkitt lymphomas, andCD43 positivity, seen in approximately 50% of Burkittlymphomas and only 10% of DLBCL, also raises suspicion

for Burkitt lymphoma.7,13 Fluorescence in situ hybridiza-tion studies are recommended when Burkitt lymphoma ishighly suspected, with the caveat that 10% of DLBCL alsocontain the c-myc translocation, although usually in abackground of complex karyotype.35,40

In the presence of CD30 positivity in DLBCL, theother considerations are ALCL and cHL. In most cases ofDLBCL, strong CD20 expression in sheets of large cells isseen, with only focal variable positivity for CD30 (Fig. 7).These cases are not uncommon and can be comfortablydiagnosed as DLBCL without further workup. Difficultcases include those with less large cells and moreinflammatory background and fibrosis, such as the casewith primary mediastinal large cell lymphoma. A helpfulpanel to distinguish these cases from cHL may includeCD15, CD45, CD79a, BOB.1, OCT-2, and PAX-5(Table 7). Usually, not all of these markers are necessaryto make the distinction. Consistently strong CD20 mem-brane positivity of all the malignant cells favors DLBCL,whereas variable intensity staining in a subset of cells favorscHL. CD15 is a very helpful marker, because it is positive incHL and negative in DLBCL. CD45, CD79a, BOB.1, andOCT-2 are usually negative in cHL and positive in DLBCL.The intensity of PAX-5 (weak in cHL and strong inDLBCL) may also be helpful.

The 2008 WHO classification divides DLBCL into 4subtypes and 8 distinct disease entities (Table 8), a few of

FIGURE 7. Diffuse large B-cell lymphoma. A, CD20 shows strong, diffuse positivity. B, CD30 may be focally and variably positive but theCD20 staining pattern excludes a Hodgkin lymphoma or anaplastic large cell lymphoma.

TABLE 7. DLBCL Versus cHL Versus NLPHL

DLBCL cHL NLPHL

CD30 +/� (Variable) + +/� (Weak)CD15 � + �

CD45 + � +CD20 + +/� +CD79a + � +MUM-1 + + �

BOB.1 + � +OCT-2 + � +PAX-5 Strong Weak Moderate

cHL indicates classical Hodgkin lymphoma; DLBCL, diffuse largeB-cell lymphoma; NLPHL, nodular lymphocyte predominant Hodgkinlymphoma.



which involve the lymph nodes and may show a clinicalpresentation similar to DLBCL, NOS.23 For some, thedistinction from DLBCL, NOS requires morphology andimmunohistochemistry. These include THRLBCL, EBV-positive DLBCL of the elderly, and ALK-positive largeB-cell lymphoma.

T-cell/Histiocyte-rich Large B-cell LymphomaThis is a subtype of DLBCL in which the neoplastic B-

cells are scarce compared with the inflammatory infiltrate.The requirement of less than 10% neoplastic cells has beenremoved in the 2008 WHO and replaced by a moredescriptive definition with scattered, singly dispersed largeB-cells that do not aggregate or form sheets.23,41 As thename suggests, the background is composed of mostly of T-cells and histiocytes. Scattered plasma cells and eosinophilsare also present. The immunohistochemical profile of thelarge cells is similar to DLBCL, NOS. The cells showexpression of pan-B-cell markers, are usually negative forCD30 and CD15, and invariably negative for EBV. Thesehelp to distinguish this entity from cHL, acute infectiousmononucleosis, and other EBV-associated neoplasms suchas lymphomatoid granulomatosis and EBV-positiveDLBCL of the elderly. The distinction with PTCL requiresrecognition of morphologic atypia in the small and thelarge cells, with a confirmatory T-cell panel if suspicionarises. The most difficult entity in the differential diagnosisis NLPHL, the discussion of which is presented in theNLPHL section (Table 7).

EBV-positive DLBCL of the ElderlyThis occurs in patients who are above 50 years of age

and have no other causes for immunosuppression otherthan aging. The disease initially involves lymph nodes andcommonly involves extranodal sites such as skin, lung,tonsil, and stomach.42 Morphologically, 2 forms have beendescribed. One is histologically indistinguishable fromDLBCL, NOS and the differential diagnosis is similar.The other resembles cHL, with a polymorphous infiltrateand scattered Hodgkin-like cells. The immunohistochem-ical profile of both forms is similar to DLBCL, NOS,including variable CD30 expression, with the exception ofEBV positivity, which is definitional. However, EBV

positivity is not completely specific: lymphomatoid granu-lomatosis, plasmablastic lymphoma, primary effusionlymphoma, and DLBCL associated with chronic inflam-mation are all EBV positive and show CD20 staining in thelarge neoplastic cells. Clinical information and otherimmunostains will help to distinguish among these entities.EBV stains are advised for DLBCL if the patient is above50 years of age, necrosis is present, the cells showpleomorphism and/or Reed Sternberg-like cells, or extra-nodal involvement is present. The differential with HL isdiscussed above and in the cHL section later in this review.

ALK-positive Large B-cell LymphomaThis represents a small subset of B-cell lymphomas

that morphologically resemble immunoblastic DLBCL,with a sinusoidal growth pattern. Immunohistochemistryshows a granular and dot-like cytoplasmic ALK proteinreactivity, which indicates a variant translocation. Thesetumors are negative for pan-B-cell markers such as CD20,negative for CD30, only weakly positive for CD45, andoften show aberrant expression of CD4 and CD57.43,44 Thetumors are also positive for plasma cell-associated markersCD138, epithelial membrane antigen (EMA), and MUM1,and often show light-chain restriction.43,44 Proof ofB-lineage may require gene rearrangement studies. Themorphologic differential diagnosis includes ALK-positiveALCL and plasmablastic lymphoma. ALCL can be easilyexcluded by its positive CD30 stain, which is negative inALK-positive large B-cell lymphoma. ALK positivityexcludes plasmablastic lymphoma.

DLBCL-related, Clinically DistinctLarge B-cell Lymphomas

These have characteristic presentation and sites ofinvolvement, which usually hint at the diagnosis. Theseinclude primary DLBCL of the central nervous system,primary cutaneous DLBCL, leg type, primary mediastinallarge B-cell lymphoma, intravascular large B-cell lympho-ma, DLBCL associated with chronic inflammation, lym-phomatoid granulomatosis, plasmablastic lymphoma, largeB-cell lymphoma arising in HHV-8-associated multicentricCastleman disease, and primary effusion lymphoma. Mostof these show similar immunophenotype as DLBCL, NOSand are primarily defined by the clinical presentation. A fewwith distinct immunophenotypes are discussed below.

Primary Cutaneous DLBCL, Leg TypeThis is a primary cutaneous B-cell lymphoma that

preferentially affects the lower legs of elderly women andshows monomorphic sheets of centroblasts and immuno-blasts. Any primary cutaneous B-cell lymphoma with thismorphology should fall under this category, even if notpresent on the leg. These tumors express pan-B-cellantigens, including CD20, as well as bcl-2, bcl-6, MUM-1, and FOX-P1, and are usually negative for CD10.45,46 Themain differential is with primary cutaneous follicle centerlymphoma and distinction can be made with recognition ofa follicular architecture and using a typical DLBCL panelcontaining bcl-2, bcl-6, CD10, and MUM-1. Both are bcl-6positive, but primary cutaneous DLBCL, leg type usuallyshows expression of bcl-2, MUM-1, and FOX-P1 whereasprimary cutaneous follicle center lymphoma is usuallynegative for these markers.

TABLE 8. Classification of Diffuse Large B-cell Lymphoma23

Diffuse large B-cell lymphoma subtypesT-cell/histiocyte-rich large B-cell lymphomaPrimary DLBCL of the CNSPrimary cutaneous DLBCL, leg typeEBV-positive DLBCL of the elderly

Distinct clinical entities related to DLBCLPrimary mediastinal (thymic) large B-cell lymphomaIntravascular large B-cell lymphomaDLBCL associated with chronic inflammationLymphomatoid granulomatosisALK-positive large B-cell lymphomaPlasmablastic lymphomaLarge B-cell lymphoma arising in HHV8-associated multicentricCastleman diseasePrimary effusion lymphoma

ALK indicates anaplastic lymphoma kinase; CNS, central nervoussystem; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus;HHV-8, human herpesvirus 8.



DLBCL Associated With Chronic InflammationThis encompasses pyothorax-associated lymphoma

and DLBCL arising after other long-standing chronicinflammation such as chronic osteomyelitis, metallicimplants, or chronic skin ulcers. Pyothorax-associatedlymphoma, unlike primary effusion lymphoma, presentswith a tumor mass. Its phenotype is similar to DLBCL,with some cases showing plasmacytic differentiation andaberrant T-cell antigen expression. EBV-LMP and EBV in-situ hybridization are usually positive.47

Lymphomatoid GranulomatosisThis is an EBV-driven, extranodal angiocentric and

angiodestructive large cell lymphoma with admixed reactiveT-cells. The large cells are EBV-positive and express CD20,variably express CD30, and do not express CD15.48

Although EBV-LMP may be positive, EBER in situhybridization is the standard and used for grading, whichis based on the amount of EBER-positive large cells inrelation to the reactive mixed inflammatory background.Diffuse sheets of large cells without the admixed reactiveinfiltrate are not considered lymphomatoid granulomatosisand should be classified under other clinicopathologicentities such as DLBCL, NOS or EBV-positive DLBCLof the elderly. Distinction from the other main angiodes-tructive lymphoma (extranodal natural killer/T-cell lym-phoma, nasal type) can be made from the CD20 positivity.Lower-grade lesions with fewer large cells and CD30positivity may need to be distinguished from cHL with asimilar strategy outlined above (Table 7).

Plasmablastic LymphomaThis is associated with immunodeficiency states such

as human immunodeficiency virus and advanced age. Thecells may resemble immunoblasts or show more immatureplasmacytic differentiation. The immunophenotype issimilar to plasma cell neoplasms, but CD56 expressionis less common. EBV-LMP is usually negative but EBVin-situ hybridization is usually positive. HHV-8 isnegative.49,50

Large B-cell Lymphoma Arising in HHV-8-associatedMulticentric Castleman Disease

This is recognized by sheets of plasmablasts, whichefface the normal nodal architecture, in a background ofmulticentric Castleman disease. Most of these patients arehuman immunodeficiency virus-positive. These cells arepositive for HHV-8 latent nuclear antigen 1, a nuclear stain,and show IgM heavy chain and l light chain restriction.EBV is usually negative.51

Primary Effusion LymphomaThis usually presents as a serous effusion without an

associated tumor mass. The effusion contains cells thatcytologically resemble plasmablastic lymphoma. Immuno-phenotypically, CD45, CD138, and EMA are usuallypositive, but CD20, CD79a, and PAX-5 are negative.52

EBV-LMP is usually negative, but EBV may be shown byin situ hybridization.53 Similar to plasmablastic lymphomaand normal plasma cells, BOB.1 and OCT-2 are positive.54

HHV-8 is invariably positive.

Lymphoblastic Lymphoma/LeukemiaThis is a monomorphous proliferation of small to

intermediate-sized cells with scant cytoplasm, fine chromatin,and inconspicuous nucleoli. B- and T-acute lymphoblasticleukemias (ALL)/LBL have different clinical presentationsbut similar morphology. Both are consistently positive forTdT, CD43, and CD99, and frequently positive forCD34.13,55,56 CD45 is present on most but not all cases.57

Both may also express myeloid markers such as CD33, butthey never express myeloperoxidase. Flow cytometry andcytochemistry may help to establish the lineage of a blasticneoplasm. With paraffin immunohistochemistry, PAX-5 isthe most sensitive and specific marker to assign B-lineage.58

Most B-ALL/LBL will also be positive for CD10, CD79a,and BOB.1, whereas CD20 and OCT-2 are often negative.54

No expression of T-cell antigens is seen. T-ALL/LBLusually shows expression of CD3 (cytoplasmic) and CD7,although only CD3 is considered lineage specific. Theremaining T-cell antigens CD1a, CD2, CD4, CD5, andCD8 are variably expressed. Coexpression of CD4 andCD8 or lack of both may be seen.59–61 A panel containingTdT, CD43, CD3, PAX-5, CD79a, CD10, myeloperox-idase, CD20, bcl-1, and Ki-67 is helpful to assign lineageand to exclude morphologic mimics such as acute myeloidleukemia (myeloperoxidase positive), blastic MCL (bcl-1positive and TdT negative), and Burkitt lymphoma (CD20positive, TdT negative, and Ki-67 approaching 100%).TdT-positive neoplasms should be stained for myeloperox-idase because some myeloid lineage leukemias, particularlythose with t(8;21), may express TdT and/or PAX-5.62

CD79a and CD10 expression may sometimes be seen inT-ALL/LBL.63

Burkitt LymphomaThis shows a monomorphous proliferation of inter-

mediate-sized cells with moderate amounts of basophiliccytoplasm, vesicular chromatin, and 1 or more mediumnucleoli. Admixed tingible body macrophages impart astarry-sky appearance, whereas admixed reactive inflam-matory cells are sparse. Burkitt lymphoma shows consistentexpression of pan-B-cell markers CD20, CD79a, PAX-5,BOB.1, and OCT-2, as well as CD10 and bcl-6.64,65 Bcl-2 isusually negative but may be weakly positive in up to one-third of cases.7 CD43 coexpression is common.13 The Ki-67stain shows nuclear positivity in nearly 100% of cells.Burkitt lymphoma does not stain for TdT, myeloperox-idase, CD117, CD34, CD5, or cyclin D1.10,64 These help todistinguish it from most other monomorphous medium-sized cell infiltrates. Differentiation from DLBCL isdiscussed in the DLBCL, NOS section.

Hodgkin Lymphoma

Classical Hodgkin LymphomaThis is a B-lineage lymphoma derived from germinal

center B-cells. The diagnosis relies on identification ofclassic Reed-Sternberg cells and/or so-called Hodgkin cells,which are basically uninucleated Reed-Sternberg cells. Theneoplastic cells often represent less than 10% of theinfiltrate, which comprises a mixture of reactive T-cells,histiocytes, eosinophils, neutrophils, and plasma cells. Theneoplastic cells are consistently positive for CD30, usuallypositive for CD15, weakly positive for PAX-5, variablypositive for CD20, and negative for CD45 and CD3(Fig. 6).2–6 Characteristics and pitfalls of evaluating these



first-line stains are discussed in Section I. In addition,Hodgkin cells are positive for MUM-1 and bcl-2, andnegative for CD79a, BOB.1, OCT-2, CD43, CD138, and T-lineage markers (Table 7).66–70 EBV-LMP is positive inone-third of cases and is strongly associated with childrenand the elderly, and with mixed cellularity and lymphocytedepletion subtypes.71 Bcl-6 is also positive in one-third ofcases.72,73 The background reactive inflammatory infiltrateusually shows abundant CD3-positive and CD45-positiveT-cells, with the exception of the lymphocyte-rich subtype,which shows abundant CD20-positive B-cells, and thelymphocyte depletion subtype, in which lymphocytesare replaced by histiocytes and other inflammatory cells.

When the morphology is classic and CD15 is positive,the diagnosis is usually straightforward. Difficulties arisebecause CD15 is negative in 15% of cHLs and almost allentities within the differential diagnosis are also CD15negative.2–6 In cases that are also CD20 negative, especiallywhen abundant Hodgkin cells are present and form sheets,one must exclude ALCL, which lacks CD45 and CD3expression in 33% and 60% of cases, respectively.57,74 Theaddition of PAX-5, EBV-LMP, CD43, ALK, and cytotoxicmarkers, such as TIA-1, granzyme B, and/or perforin,should clarify most cases. ALCLs are consistently negativefor PAX-5 and EBV, and most ALCL cases are positive forCD43 and cytotoxic markers.75,76 In CD30-positive/CD15-negative tumors that are also CD20-positive, one shouldconsider the diagnosis of THRLBCL, which is CD30positive in 5% of cases and universally CD20 positive. Thisentity is also consistently positive for CD45, but assessmentof CD45 expression is not always straightforward, given thediffuse T-cell infiltrate. The addition of BOB-1 and OCT-2are helpful in these cases. These transcription factorsare expressed in almost all DLBCLs and only 10% ofcHLs.54,69 Reactive paracortical hyperplasia is usuallyeasily distinguished from cHL because of its accompanyingfollicular hyperplasia. A common mistake is to focus onlyon the high-power appearance of the reactive immuno-blasts, which may resemble Hodgkin cells because ofprominent nucleoli and variable CD30 positivity. Occa-sionally, the mixed cellularity subtype of cHL may show aninterfollicular pattern. The combined features of CD15negativity, variable CD45 positivity, and variable CD30positivity can usually lead one to make a diagnosis ofreactive paracortical hyperplasia and not interfollicular HL(Fig. 1). The lymphocyte-rich subtype of cHL may show anodular pattern of B-cells surrounding Hodgkin cells,which may closely resemble NLPHL (see below).

Nodular Lymphocyte PredominantHodgkin Lymphoma

As the name implies, NLPHL shows a nodular ornodular and diffuse infiltrate of reactive B-cells, sometimeswith increased epithelioid histiocytes. The diagnosis relieson identification of LP cells (formerly known as L&H cells),which are characterized by large multilobated cells withvesicular chromatin and one or more peripherally locatednucleoli. Nodularity is not always evident on the H&E-stained section, but the CD20 immunostain should high-light the characteristic nodular pattern, because themajority of the internodular areas are composed of T-cells(Fig. 3). The nodules are larger and more widely spacedthan cases of FL but still show a meshwork of CD21-positive/CD23-positive/CD35-positive follicular dendritic

cells. The LP cells are CD30 negative, CD15 negative,and consistently CD20 and CD45 positive (Table 7).77 Theyare also usually positive for CD79a, PAX-5 (moderatestaining, in contrast to weak or faint staining of cHL ReedSternberg cells), BOB.1, OCT-2, and bcl-6.69,78 Bcl-2 ispositive in one-third of cases.79 The LP cells are negativefor MUM1, CD43, and T-cell markers.70 In addition to alarge number of B-cells, the nodules also contain increasednumbers of T-cells, which have a follicular helper pheno-type (positive for CD57, PD1, and bcl-6).80 The authorsprefer using the CD57 and PD1 stains because they arenegative in LP cells and will highlight the T-cells bycharacteristically encircling LP cells (Fig. 3). Therefore, ifNLPHL is suspected, the immunohistochemical panelshould include CD20, CD3, CD45, CD30, CD15, CD57,and PD1. If the nodules are still not evident on the CD20stain, addition of CD21, CD23, or CD35 is helpful to bringout the nodularity.

In practice, CD30 may be weakly positive in LP cells inup to one-third of cases.81 CD15 may also be positive in asmaller percentage of cases.82 In ambiguous cases of cHLversus NLPHL, the addition of MUM-1, BOB.1, and OCT-2can usually help. cHL is positive for MUM-1 and negativefor BOB.1 and OCT-2. NLPHL shows the opposite pattern,negative for MUM-1 and positive for BOB.1 and OCT-2.69,70

If these stains are unavailable, bcl-6 and EBV-LMP may behelpful. Approximate 40% of cHLs are EBV-LMP positiveand most are bcl-6 negative, whereas LP cells are consistentlyEBV-LMP negative and bcl-6 positive.79,83 Once the nodulararchitecture is appreciated, reactive paracortical hyperplasiais generally not considered in the differential, but anotherreactive pattern, progressive transformation of germinalcenters, may be considered. This is also a fairly easydistinction, because progressive transformation occurs in abackground of follicular hyperplasia, with large nodulesintermixed between reactive follicles, whereas NLPHL showseffacement of the nodal architecture and is usually separatefrom areas containing residual reactive follicles. The mostdifficult differential diagnosis is often with THRLBCL. TheLP cells and neoplastic cells in THRLBCL may showidentical immunophenotypes, therefore, the distinction restsin the identification of B-cell nodules and CD57-positive/PD-1-positive cells encircling LP cells. This may be extremelydifficult to identify, especially because longstanding NLPHLcases tend to contain more diffuse areas, more T-cells, andless CD57-positive cells than early NLPHL lesions.84

T-cell Lymphomas

Peripheral T-cell Lymphoma, Not Otherwise SpecifiedThis may show a variety of morphologies, which range

from lesions that resemble reactive paracortical hyperplasiato those composed of abundant large atypical cells.Usually, but not always, the nodal architecture is effacedand there is no accompanying reactive follicular hyperpla-sia (Fig. 5). T-cell lymphomas, in general, are rare and theinitial immunohistochemical panel usually does not differfrom that of B-cell panels, with inclusion of CD20, CD3,bcl-2, CD30, and CD43. Lack of morphologic andimmunophenotypic features of B-cell lymphoma or HLwould raise suspicion for a T-cell lymphoma. Unfortu-nately, there is no equivalent of immunoglobulin light-chain restriction that provides definitive and direct evidenceof a neoplastic clone. Rather, evaluation depends onassessment of loss of pan-T-cell markers and/or bcl-2.



A general panel for T-cell lymphoma includes the pan-T-cell markers CD2, CD3, CD5, CD7, along with bcl-2,Ki-67, CD30, and CD20. CD4 and CD8 may be added, butthe CD4:CD8 ratio is not similar to the k:l ratio fordetermining malignancy. PTCL, NOS shows a matureT-cell phenotype and should express at least one of the pan-T-cell markers and should not express CD1a, TdT, orB-lineage markers such as CD20, CD79a, PAX-5, BOB.1,or OCT-2. There is often loss of expression of one or moreof the pan-T-cell markers, sometimes with aberrant loss ofbcl-2; either one provides strong evidence for a T-cellmalignancy. Most cases are composed of CD4+/CD8� T-helper cells.85 The Ki-67 is usually elevated, an unusualfinding in the paracortical region of benign lymph nodes.Scattered CD30-positive reactive immunoblasts may beseen, but these should be scattered, with variable strengthstaining (Fig. 1D). EBV infection is common but likelyrepresents infected reactive B-cells. In some cases, antigenexpression is not lost and one must rely on T-cell receptorgene rearrangement studies to confirm the morphologicimpression.

Angioimmunoblastic T-cell LymphomaThis is probably derived from follicular helper T-cells.

This entity is characterized by a polymorphous proliferation ofintrafollicular T-helper cells that characteristically show clearcytoplasm, especially surrounding prominent high endothelialvenules, and an irregular proliferation of follicular dendriticnetworks, some encasing high endothelial venules. Varyingamounts of admixed histiocytes, eosinophils, and plasma cellsmay be present. Unlike PTCL, NOS, the neoplastic cells donot frequently lose pan-T-cell antigens.86 However, there is acharacteristic immunohistochemical profile of these intrafol-licular T-helper cells that is very helpful in the diagnosis. Thediagnostic panel should include the usual T-cell markersCD2, CD3, CD5, CD7, and bcl-2, CD20, markers forintrafollicular T-helper cells bcl-6, CD10, CXCL13, and/orPD-1, and a marker for follicular dendritic cells CD21,CD23, or CD35. One should expect to see the majority ofcells positive for bcl-6, CD10, CXCL13, or PD-1 andexpanded, irregular follicular dendritic networks surroundinghigh endothelial venules, highlighted by CD21, CD23, orCD35 (Fig. 8).87,88 Ki-67 is usually not increased. Inciden-

tally, although not necessary for the diagnosis, many casesshow EBV positivity in the admixed non-neoplastic B-cells.89

Anaplastic Large Cell LymphomaALCL encompasses several T-cell lymphomas that have

distinct clinical presentations but may have overlappingmorphologic features. Immunohistochemically, all ALCLhave in common the distinct feature of diffuse strong CD30expression (Fig. 9). In addition to CD30, other markers oflymphocyte activation are also consistently positive, in-cluding CD25, CD71, and human leukocyte antigen-DR.90

In addition, most ALCL cases show expression of 1 ormore of the pan-T-cell markers, CD43, and at least one ofthe cytotoxic markers TIA-1, granzyme B, or perforin.74,75

CD45 is positive in two-thirds of cases.57 Some cases showa null-phenotype, with loss of all specific T-cell markers.Demonstration of CD43 and cytotoxic markers in thesecases is helpful to confirm the diagnosis. Most cases areCD4 positive. CD15 is occasionally positive, as is CD68,although the more histiocyte-specific marker, CD163, isnegative.91,92

FIGURE 8. Angioimmunoblastic T-cell lymphoma. A, PD1-positive cells predominate. Normally, these cells are limited to the scatteredT-cells within germinal centers. B, CD21 showing expanded follicular dendritic networks entrapping high endothelial venules.

FIGURE 9. Anaplastic large cell lymphoma. CD30 shows strongmembranous staining in all the tumor cells. Compare this patternwith the variable CD30 staining in diffuse large B-cell lymphoma.



Primary cutaneous ALCL (C-ALCL), in addition tothe above immunohistochemical profile, expresses cutaneouslymphocyte antigen (CLA) in most cases, whereas primarynodal ALCL does not express CLA. However, some casesof nodal ALCL with secondary skin involvement havealso been reported to be CLA positive,93 therefore, this anti-body is not likely to be as helpful as clinical correlation formaking the distinction between primary cutaneous andnodal forms. In addition, nodal ALCL is ALK positivein approximately 60% to 85% of cases,41 with either anuclear, cytoplasmic, or nuclear and cytoplasmic distribu-tion, depending on the type of translocation. The mostcommon t(2;5)/nucleophosmin-ALK translocation is asso-ciated with nuclear and cytoplasmic staining. ALK-positiveALCL cases are also usually EMA positive.91

In contrast to other T-cell lymphomas and DLBCL,all of which may show patchy, variable intensity CD30positivity, ALCL shows sheets of strong intensity CD30-positive cells (Figs. 7, 9). As a result of this, the differentialdiagnosis is limited to acute infectious mononucleosis andcHL, especially the syncytial variant of the nodular sclerosissubtype, which may form sheets of Hodgkin cells that areCD30 positive. This differential is confounded by the factthat ALCL may lose expression of CD3, be negative forCD45, and even occasionally be positive for CD15.Therefore, in suspected cases of cHL in which sheets oflarge cells are seen, it is a good idea to exclude ALCL with apanel of stains that include PAX-5, EBV-LMP, ALK,CD43, and cytotoxic markers TIA-1, granzyme B, and/orperforin. PAX-5 and EBV positivity supports cHL, whereasexpression of the latter stains is not seen in cHL but favorsALCL. Staining for EBV is also helpful in a young patientwhere one should be wary of acute infectious mononu-cleosis, which can occasionally form sheets of large CD30-positive immunoblasts. These sheets are usually focal anddo not efface the entire nodal architecture. EBV is notassociated with ALCL unless it is immunodeficiencyassociated, such as posttransplant lymphoproliferativedisorder.94 Other T-cell lymphomas may transform intoALCL and be indistinguishable by morphologic andimmunophenotype. Correlation with clinical setting andprevious pathology is necessary to make this diagnosis.Lastly, close attention to the clinical presentation is alsoprudent to help exclude adult T-cell leukemia/lymphoma,which may express CD30, CD25, and T-lineage markers.

Histiocytic and Dendritic NeoplasmsThis group of neoplasms, including histiocytic sarco-

ma, Langerhans cell histiocytosis, interdigitating dendriticcell sarcoma, and follicular dendritic cell sarcoma, isextremely rare. Many can be classified using a panel that

includes CD163, Langerin, CD21, CD35, and S100. Inshort, CD163 is positive in histiocytic sarcoma andLangerhans cell histiocytosis; Langerin in Langerhans cellhistiocytosis only; CD21 and CD35 in follicular dendriticcell sarcoma only; and S100 without the other markers ininterdigitating dendritic cell sarcoma (Table 9).37,92,95,96

Langerin, a glycoprotein crucial to the formation ofBirbeck granules, is a highly sensitive and specific markerfor Langerhans cells, with CD1a being a comparablealternative.95 CD163 has been shown to be a more specifichistiocytic marker than CD68 or lysozyme.92,96 A combina-tion of these latter stains may be used in adjunct if CD163 isnot available.

Blastic Plasmacytoid Dendritic Cell NeoplasmThis entity, formerly blastic natural killer-cell lym-

phoma, this is a rare neoplasm that morphologicallyresembles monomorphic blastic proliferations such asLBL, acute myeloid leukemia, blastic MCL, and Burkittlymphoma. Skin involvement is almost universal, and bonemarrow and lymph node involvements are also commonlyseen.97,98 These tumors express the plasmacytoid dendriticmarker CD123, as well as CD4, CD43, CD45, CD56, andCD68. TdT is often positive, but myeloperoxidase andB- and T-cell antigens are usually not expressed. Tumors withCD43 and TdT expression include acute myeloid leukemiaand LBL. Positivity for CD123 should confirm thediagnosis of blastic plasmacytoid dendritic cell neoplasm.

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TABLE 9. Histiocytic and Dendritic Neoplasms

CD163 Langerin

CD21/

CD35 S100

Histiocytic sarcoma + � � +/�Langerhans cellhistiocytosis

+/� + � +

Interdigitatingdendritic cellsarcoma

� � � +

Follicular dendriticcell sarcoma

� � + � /+



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