10.1 Hematolymphoid Markers

10.1 Hematolymphoid Markers

Quick Answer: CD45 is the membrane leukocyte screen—weak or negative on many plasma-cell neoplasms and on classic Hodgkin Reed-Sternberg (RS) cells. B-lineage: CD20 (membrane; rituximab pitfall), PAX5 (nuclear), CD79a. T-lineage: CD3, with CD5 and CD7 as additional T antigens. Plasma cells: CD138 (also stains epithelium) and nuclear MUM1. Classic Hodgkin RS cells: CD30+/CD15+/CD45−/PAX5 dim. Nuclear cyclin D1 in CD5+ B cells is the mantle-cell pattern; nuclear TdT is lymphoblastic; ALK pattern follows the fusion. This is technologist screening, not a hematopathology textbook.

This OpenExamPrep section is independent teaching on hematolymphoid staining patterns covering published QIHC staining topic IV.B.2.a (staining patterns). It is not an ASCP publication and does not claim Board, CAP, or WHO-classification approval. Named clones below are examples, not exclusive reagents. The QIHC technologist confirms expected localization and controls; the pathologist makes the diagnosis.

Chapter 9 covered cytokeratins and organ epithelial markers. This section does not reprint CK7/CK20 maps. Hematolymphoid antibodies answer a different first question: are the atypical cells leukocyte-lineage, and which lymphoid compartment do they resemble on the slide?

Why this is a screening panel, not a sign-out

A QIHC candidate is not expected to recite a full WHO hematopoietic classification. You are expected to:

1. Name the compartment the chromogen should occupy (membrane, cytoplasm, Golgi-dot, nucleus).
2. Use on-slide internal controls (residual B cells for CD20/PAX5, paracortical T cells for CD3, small lymphocytes for CD45 when RS cells are negative).
3. Recognize pitfalls that produce a technically pretty stain with the wrong biological meaning: rituximab, CD5 on B cells, CD138 on epithelium, cyclin D1 in endothelial nuclei.

Read the H&E before you interpret brown. Residual small lymphocytes in a carcinoma-filled node are CD45-positive; that does not make the carcinoma a lymphoma. Crush, necrosis, and histiocyte cytoplasm trap chromogen. Score the atypical cells, not the inflammatory background, unless the antibody is supposed to mark that background as an internal control.

CD45 (leukocyte common antigen)

CD45 is a membrane tyrosine phosphatase present on nearly all hematopoietic cells. Expected pattern: crisp circumferential membrane on lymphocytes. Intensity is often weaker on granulocytes and some precursors. Use CD45 as a lineage screen, not as a lymphoma subtype.

Two everyday CD45-poor hematopoietic exceptions: plasma-cell neoplasms and classic Hodgkin RS cells. If mantle-zone B cells and paracortical T cells stain and the big atypical cells do not, document the pattern. Send keratin and melanoma-lineage stains when morphology does not look lymphoid (section 10.2), rather than forcing a lymphoma label onto a CD45-negative nest.

B-cell markers: CD20, PAX5, and CD79a

CD20 is a membrane marker on mature B cells from late pre-B stages through most mature B-cell lymphomas. Tonsil or appendix follicles are the usual control: circumferential membrane on mantle and follicle-center B cells. Terminally differentiated plasma cells are typically CD20-negative. Classic Hodgkin RS cells are usually CD20-negative; a weak or subset CD20 stain does not by itself exclude classic Hodgkin when the rest of the RS pattern is present.

Rituximab pitfall. Rituximab is a therapeutic anti-CD20 monoclonal antibody. Residual or relapsed B-cell lymphoma after rituximab is often CD20-negative even though the neoplasm is still B-lineage. A positive tonsil control only proves the reagent worked on untreated B cells; it does not prove the patient's tumor still displays the CD20 epitope. Screening rule: do not close the B-cell question on CD20 alone after anti-CD20 therapy.

PAX5 is a nuclear B-cell transcription factor. Score nuclei, not cytoplasmic blush. It remains useful when CD20 is lost. In classic Hodgkin disease, RS cells often show dim nuclear PAX5 next to strongly PAX5-positive small B cells—that dim nuclear stain is a teaching pattern, not follicular lymphoma. Strong nuclear PAX5 in sheets of large cells with CD20 or CD79a is a large B-cell pattern in the Hodgkin differential (including T-cell/histiocyte-rich large B-cell lymphoma); morphology belongs to the pathologist.

CD79a labels a B-cell receptor component (membrane and cytoplasmic). It marks most B-cell lymphomas and many plasma cells, which is why it can rescue a rituximab-treated CD20-negative B-cell tumor and why it is not a pure “mature B versus plasma cell” splitter. Pair it with CD138/MUM1 when the question is plasma-cell differentiation.

T-cell markers: CD3, CD5, and CD7

CD3 is the screening T-lineage marker: membrane on mature T cells, sometimes cytoplasmic in immature T cells. Tonsil paracortex is the usual control. If the atypical sheets are CD3-negative and the background small cells are CD3-positive, you have not stained the tumor as T-lineage—you have stained the reactive T cells.

CD5 and CD7 are additional T-lineage antigens. Pathologists use aberrant loss of CD7 (or CD5) as a T-cell neoplasia clue; that is interpretation, not a technologist diagnosis. Confirm that a “CD7 hole” sits in the atypical cells, not in a drying artifact or an uncovered droplet.

CD5-positive B-cell lymphomas

CD5 is not a T-cell stamp. Two common small B-cell lymphomas coexpress B-lineage markers and CD5 on the same cells:

If the same cells are CD20-positive and CD5-positive, look at CD3: it should highlight scattered background T cells, not the CD20-positive nodules. Calling that tumor “T-cell lymphoma because it is CD5-positive” is a screening error. Less common CD5-positive B-cell processes (some diffuse large B-cell lymphomas, occasional marginal-zone lymphomas) exist; they still need B-lineage markers, not a CD5-only label.

Plasma-cell markers: CD138 and MUM1

CD138 (syndecan-1) is membrane on plasma cells. It also stains many epithelia, including squamous epithelium and a subset of carcinomas. A CD138-positive sheet in marrow may be plasma cells; a CD138-positive nest next to keratin-positive epithelium may be carcinoma. Do not use CD138 as a stand-alone “myeloma stain.”

MUM1/IRF4 is nuclear in plasma cells and in some activated B and T cells. Nuclear localization is required. Cytoplasmic-only MUM1 is not a plasma-cell call.

Plasma-cell neoplasms often cluster as CD138-positive, MUM1 nuclear-positive, CD20-negative or weak, CD45-weak or negative, CD79a variable. That is a pattern cluster for the pathologist, not a WHO diagnosis from the stainer.

Classic Hodgkin RS pattern: CD30 and CD15

Classic Hodgkin lymphoma is recognized on H&E by Hodgkin and Reed-Sternberg cells in an inflammatory background. The screening immunophenotype of those large cells:

CD30 also marks anaplastic large-cell lymphoma, embryonal carcinoma, and activated lymphoid cells—so CD30-positive is not “Hodgkin.” CD15 marks myeloid cells and some adenocarcinomas. The combination plus morphology is the teaching pattern. Nodular lymphocyte-predominant B-cell lymphoma / NLPHL LP cells are a different phenotype (typically CD20-positive, CD45-positive, CD15-negative, CD30-negative or weak). Do not force classic RS rules onto LP cells.

Cyclin D1, TdT, and ALK

Cyclin D1 (BCL1) is nuclear. In a CD5-positive B-cell process, nuclear cyclin D1 in the neoplastic B cells supports mantle cell lymphoma. Endothelium and some histiocytes can show weak nuclear cyclin D1—do not score vessels as tumor. Hairy-cell leukemia and some plasma-cell neoplasms can be cyclin D1-positive; morphology and the rest of the panel still matter. Cytoplasmic-only cyclin D1 is not the mantle-cell pattern.

TdT is nuclear in lymphoblasts (B- or T-lymphoblastic leukemia/lymphoma) and in normal thymic cortex. A TdT-positive blastic tumor is a lymphoblastic pattern. Mature B- and T-cell lymphomas are TdT-negative. Cytoplasmic TdT blush without nuclear stain is not a blast call. In mediastinal resections, residual thymus is an internal TdT-positive control—and a pitfall if you mistake cortex for tumor.

ALK in ALK-positive anaplastic large-cell lymphoma: compartment follows the fusion protein. The common NPM::ALK t(2;5) produces nuclear plus cytoplasmic ALK. Other partners can be cytoplasmic only, membranous, or granular cytoplasmic. Classic Hodgkin lymphoma is ALK-negative. ALK also appears in inflammatory myofibroblastic tumor and in a subset of lung adenocarcinomas in a different clinical setting—document compartment; do not treat ALK as a Hodgkin marker.

Practical screening sequence

1. Confirm H&E: which cells are atypical?
2. CD45 on those cells? If negative with intact lymphocyte internal control, consider Hodgkin-like, plasma-cell-like, or a non-hematopoietic mimic.
3. B versus T: CD20/PAX5/CD79a versus CD3. After rituximab, do not stop at CD20-negative.
4. CD20 and CD5 on the same cells: CLL/SLL versus mantle cell; look at nuclear cyclin D1.
5. Large CD30-positive, CD15-positive, CD45-negative cells with dim PAX5: classic Hodgkin RS pattern for the pathologist.
6. Blastic chromatin: nuclear TdT. Hallmark-cell morphology: CD30 plus ALK with a recorded compartment.

Worked example

A node after rituximab for prior CD20-positive large B-cell lymphoma shows sheets of large cells. CD20 is negative on the tumor; the tonsil control is CD20-positive. Residual small T cells are CD3-positive. PAX5 shows strong nuclear staining in the large cells; CD79a is membrane-positive; CD3 is negative on the large cells; CD45 is membrane-positive on the large cells. That is a B-lineage pattern with CD20 loss after anti-CD20 therapy, not a T-cell lymphoma and not a dead CD20 vial. The technologist recognizes that CD20-negative does not close the B-cell question. The pathologist issues the diagnosis.

These are staining patterns for published QIHC topic IV.B.2.a. They do not replace flow cytometry, genetics, or hematopathology sign-out.