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CD33 Antigen Detection by PWS44

CD33 Antigen Detection by PWS44 a New Monoclonal Antibody Reactive in Paraffin Tissues Sections: Pattern of Reactivity and Potential Diagnostic Utility.


In this study we report the pattern of CD33 reactivity of a NovocastraTM clone, PWS44, in normal and neoplastic myelomonocytic lineage cells in formalin fixed paraffin-embedded (FFPE) tissues or Bouin’s fixed decalcified bone marrow specimens. CD33 (gp 67 or siglec-3) is a 67 kd glycosylated single-chain transmembrane protein, a member of the sialic acid-binding immunoglobulin-like lectin (siglec) family, that is only expressed by hematopoietic cells.1-7 CD33 is expressed by the earliest myeloid progenitors, but not hematopoieitc stem cells, is present during myelomonocytic differentiation and is downregulated to low levels on mature granulocytes. It is retained on monocytes and is expressed on resident macrophages/histiocytes, dendritic cells and mast cells.1-7 The exact function of CD33 remains unknown, but it may have a functional role in cell-to cell adhesion.

Anti-CD33 antibodies currently available have been extensively used for the immunophenotyping of acute myeloid leukemia (AML) and for the therapeutic purging of CD33 positive AMLs.8-12 Anti-CD33 antibodies have also been shown to demonstrate therapeutic activity against CD33 positive precursor acute lymphoblastic Leukemia/Lymphoma (ALL).13-14 Recently, in vitro studies have shown that the anti CD33-targeted drug gemtuzumab ozogamicin (Mylotarg) counteracts the growth of normal and neoplastic mast cells and basophils, although the exact value of anti-CD33 targeted therapy for high-grade mast cells and basophil neoplasms remains to be determined.15 Thus, the identification of CD33 expression is important both in diagnosis and as a potential therapeutic target.

Previously, CD33 could only be detected on viable cells in suspension by flow cytometry (FC) and/or in frozen tissue sections by immunohistochemistry (IHC/FS), as a suitable reagent reactive in routinely processed tissues was not available.2-8 However, there are instances where neither fresh nor frozen material is available for diagnostic work-up or evaluation of the efficacy of therapeutic agents.


No. Cases


IHC PWS44 immunoreactivity



8(+) P67.6 PE, 4(-) P67.7 PE

 8 cases (+), 4 cases (-)


15 (BM)

8(+) P67.6PE, 7(-) MY9 FITC

13 cases (+), 2 cases (-)


9 (BM)

6(+) P67.6 PE, 3(-) P67.6 PE

6 cases (+), 3 cases (-)


2 (BM)

2 (-) P67.6 PE

2 cases (-)

Granulocytic sarcoma


1(+) P67.6 PE, 1(-) P67.6PE, 3 ND

4 cases (+), 1 case (-)

CMPD(CML-2, PV-4, IMF-1, ET-1)

8 (5 BM)


Neoplastic grans. Immature (+), mature seg.neutr.(+/-), EPs (-), MKs (-)


3 (BM)


Neoplastic grans. Immature (+), mat seg. neutr.(+/-), EPs (-), MKs (-)

Systemic mastocytosis

3 (BM)


Neoplastic mast cells (+)

Langerhans cell histiocytosis



Neoplastic Langerhans cells (+)

Plasmacytoid dentritic cell tumor

2 (1 skin,



1 BM)


Neoplastic DC (+, wk)



7 (LN)


ALCL cells in 6 cases (-), in 1 case (+)

Classic Hodgkin’s lymphoma

4 (LN)


HRS (-), seg.neutr.(+/-), mast cells (+), epithelioid histiocytes (+)

Metastatic carcinoma to BM

2 (BM)


Ca cells (-), grans. Immature (+), seg.neutr. (+/-), EPs (-), MKs (-)

Total cases




Table 1. CD33 Expression by IHC Using Clone PWS44 Versus FC (MY9 FITC or P67.7 PE). Abbreviations: monocytes (monos), segmented neutrophils (seg.neutr.), granulocytic lineage cells (grans.), Erythroid precursors (EPs), megakaryocytes (MKs), mononuclear Hodgkin cells and multinucleated Reed-Sternberg cells (HRS), carcinoma cells (Ca cells), not determined (ND), lymph node (LN), bone marrow (BM).

Pattern of PWS44 Immunoreactivity in FFPE Normal Tissues

As manual IHC using Novolink polymer detection system and automated IHC on the Leica Bond-max automated immunostainer gave similar results, the majority of the staining was performed using automation. In normal bone marrow (BM) and normal peripheral blood lymphocytes (PBL), PWS44 was reactive with granulocytic lineage cells, monocytes and histiocytes/macrophages, but was non-reactive with erythroid precursors, megakaryocytes, lymphocytes, or plasma cells (Figure 1A, B). CD33 expression as detected by PWS44 was strong in immature granulocytic precursors and progressively decreased with granulocytic maturation to weak/absent in mature segmented neutrophils, but was retained on mature monocytes. PWS44 was also reactive with immature eosinophilic precursors, but was weak to absent in mature eosinophils. Immunoreactivity with basophils was difficult to assess as they were not easily recognized in paraffin tissue sections. The pattern of PWS44 immunoreactivity was membranous. In addition, some immature myelomonocytic lineage cells demonstrated a Golgi pattern of immunoreactivity. The spectrum of cellular positivity and intensity of reactivity with PWS44 was similar to that seen with other anti-CD33 antibodies used in FC.

In reactive lymph nodes, normal spleen and normal thymus PWS44 was reactive with histiocytes, including those in the sinuses, germinal centers and paracortex as well as with paracortical dendritic cells (Figure 2A, B). In Kikuchi lymph-adenitis, reactivity with plasmacytoid dendritic cells in addition to tissue macrophages/histiocytes and sinus histiocytes was observed (Figure 2C). PWS44 showed reactivity with epithelioid histiocytes in LNs with non-caseating granulomas (Figure 2D) and splenic sinus histiocytes in the spleen. Expression was relatively strong in the sinus histiocytes, tissue histiocytes, epithelioid histiocytes and variable in dendritic cells. The pattern of reactivity with these histiocytes was predominantly membranous, but some also showed Golgi positivity.

Comparison of PWS44 Immunoreactivity in FFPE Tumo

Comparison of PWS44 immunoreactivity with the results of FC using either P67.6 or MY9 conjugated antibodies in the AML and ALL cases showed excellent correlation (Figure 3A, B & Table 1).

PWS44 was reactive with bone marrow leukemic cells in 8/8 CD33 positive BM AML cases, and with the one CD33 positive extramedullary AML. All 6/6 CD33 positive pre B-ALL cases as determined by FC were also positive for CD33 expression using Mab PWS44. PWS44 was non-reactive with the leukemic blasts in 2/7 CD33 negative AML, the one CD33 negative granulocytic sarcoma (GS), 3/3 CD33 negative pre B-ALL and 2/2 CD33 negative pre T-ALL. In 5 AML, negative for CD33 by FC, immunostaining of the accompanying routinely processed BM core biopsy with PWS44 showed CD33 positive leukemic cells. These latter cases were analyzed by FC using MY9 FITC, which we have found to be less sensitive than P67.6 FITC or P67.6 PE. The difference could be explained by the difference in sensitivity. In 3 of 5 these AMLs, immunoreactivity of leukemic cells with PWS44 was also weak.

In clinical AML samples, PWS44 was reactive with leukemic blasts immature granulocytic and monocytic precursors and monocytes. Leukemic lymphoblasts in the CD33 positive pre B-ALL showed uniform membranous positivity with PWS44. The intensity of anti-CD33 staining with PWS44 correlated with brightness of CD33 expression as determined by FC in these cases. PWS44 reactivity was strong in blasts and immature granulocytic and monocytic precursors and weak/absent in mature granulocytes but retained in monocytes.

In granulocytic sarcoma (Figure 4A), chronic myeloproliferative disease (CMPD) and myelodysplastic disease (MDS) PWS44 was reactive with neoplastic granulocytic and monocytic lineage cells. As expected and consistent with reported results, strong positivity with PWS44 was demonstrated in the neoplastic mast cells in all three cases of systemic mastocytosis (Figure 4B) and the Langerhan’s cells of the two cases of Langerhan’s cell histiocytosis. In both plasmacytoid dendritic cell tumors, the neoplastic dendritic cells demonstrated weak expression of CD33, consistent with the reported level of CD33 positivity in some of these tumors.16-18 PWS44 was non-reactive with 6/7 ALCL cases; one ALCL was CD33 positive. The CD33 positivity in one ALCL, although unexpected, has been previously reported to rarely occur in ALCL19,20; however, PWS44 was non-reactive with all four cHL and both cases of carcinoma metastatic to BM that can mimic myeloid neoplasms were negative as expected. These results show that PWS44 can be used reliably for determining CD33 expression and can be helpful in identifying patients who may benefit from anti-CD33 targeted therapy when viable cells are not available for FC analysis.


  • PWS44 is reactive in both formalin fixed tissues and Bouin’s fixed decalcified with a similar level of sensitivity.
  • Normal and neoplastic myelomonocytic lineage cells displayed membrane staining, however some reactive or neoplastic immature myelomonocytic precursors may also show Golgi reactivity.
  • The specificity and sensitivity of CD33 detection by IHC using PWS44 in AML parallels that of other anti-CD33 monoclonal antibodies used in FC/FS for immunophenotypic analysis of leukemias and shows an excellent correlation with clone P67.6 PE.
  • The broad spectrum of PWS44 reactivity with neoplastic myelomonocytic lineage cells, including blasts, immature precursors and mature forms may be especially useful for the diagnosis of minimally differentiated AML (M0) and acute monocytic leukemia (M5) when other markers used for immunophenotypic analysis on paraffin tissue sections such as myeloperoxidase (MPO), lysozyme or CD68 may be negative or expressed only by a small subset of cells.
  • Some lymphomas may exhibit aberrant expression of CD33, PWS44 immunoreactivity must always be interpreted in conjunction with the expression of other markers and the clinical information for the detection of myeloid neoplasms.


Figure 1. Normal bone marrow biopsies (A,B) demonstrating spectrum of positivity with PWS44 in normal granulocytic and monocytic lineage cells, but lack of reactivity with erythroid precursors and megakaryocytes .
Normal bone marrow biopsies demonstrating spectrum of positivity with PWS44 in normal granulocytic and monocytic lineage cells, but lack of reactivity with erythroid precursors and megakaryocytes .
Figure 2. Normal lymph node demonstrating spectrum of positivity with PWS44 in normal tissues. The staining of histiocytes/dendritic cells in the paracortex (A), sinus histiocytes and macrophages in paracortex and lymphoid follicle (B), plasmacytoid dendritic cells in Kikuchi lymphadenitis (C) and epitheloid histiocytes in a non-caseating granuloma (D).
Normal lymph node demonstrating spectrum of positivity with PWS44 in normal tissues. The staining of histiocytes/dendritic cells in the paracortex, sinus histiocytes and macrophages in paracortex and lymphoid follicle, plasmacytoid dendritic cells in Kiku
Figure 3. Positive blasts and immature granulocytic precursors, in a bone marrow biopsies of an AML positive for CD33 by flow cytometry (A) and a lack of immunoreactivity in leukemic blasts in AML negative for CD33 (B).
Positive blasts and immature granulocytic precursors, in a bone marrow biopsies of an AML positive for CD33 by flow cytometry and a lack of immunoreactivity in leukemic blasts in AML negative for CD33.
Figure 4. Strong reactivity with neoplastic cells in granulocytic sarcoma involving skin (A) and in neoplastic mast cells in systemic mastocytosis in a bone marrow biopsy (B).
Strong reactivity with neoplastic cells in granulocytic sarcoma involving skin and in neoplastic mast cells in systemic mastocytosis in a bone marrow biopsy.


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