CD 45 (Leukocyte Common Antigen)


CD 45
Available from a large number of sources including Biodesign (ALB12, J.33, MEM 28, T29/33), Biogenex, Bioprobe (bra 55, ICO-46, LT46), Cymbus Bioscience (MEM 28, RVS-1, F10-89-4), Dako (T29/33, 2B11, PD7/26), Gen Trak, Immunotech (J.33, ALB12), Oncogene (MEM 28, T29/33, J.33), Pharmingen (H130, CT-1, 30F11.1), RDI (F-10-89-4, CLB-T200/1), Sanbio (BL-leuk-45), Seralab (F10-89-4), Serotec (YTH54.12, YTH24.5) and Sigma.

CD 45R
Available from Accurate/Ancell (351C5), Biodesign (DFB1, F8-11-13, MEM56), Biogenex, Bioprobe (LT45R), Cymbus Bioscience (DFB1), Gen Trak, RDI (DFB1), Seralab, Serotec and Pharmingen (HIS24, DNL-1.9, 16A, 23G2, RA3-6B2).

Available from Accurate/Ancell (UCHL1), Biodesign (UCHL1), Biotest (UCHL1), Cymbus Bioscience (UCHL1), Dako (UCHL1, OPD4), Gen Trak, Immunotech (UCHL1), Seralab (UCHL1) and Serotec (UCHL1).

Available from Accurate (YTH80.103), Biodesign (ALB11, F8-11-13), Cymbus Bioscience (F8-1-3, MEM 56), Dako (4KB5), Gen Trak, Immunotech (ALB11), RDI (F8-11-13), Sanbio (MEM-56), Seralab, Serotec (B-C15, F8-11-13) and Pharmingen (14.8).

Available from Axcel/Accurate, Cymbus Bioscience and Dako (PD7/26).

Available from Pharmingen (HIS25) and Serotec (YTH80.103).

The CD 45 antibodies that are commercially available are mostly effective in paraffin-embedded tissues as well as in frozen sections.

The CD 45 cluster of antibodies recognizes a family of proteins known as the leukocyte common antigen (LCA) exclusively expressed on the surface of almost all hematolymphoid cells and their progenitors. The CD 45 antibody is one of the most specific currently available for diagnostic use. Virtually all hematolymphoid cells, including T and B lymphocytes, granulocytes, monocytes and macrophages, with the exception of maturing erythrocytes and megakaryocytes, express CD 45. This family of proteins has been called the leukocyte common antigen and, to date, has not been conclusively shown on any non-hematolymphoid cells.
The CD 45 proteins are coded for by a single gene located on chromosome 1q31-32. The gene is composed of 33 exons that code for the cDNA sequence as well as both 5' and 3' non-translated regions.
Differential usage of three exons termed A, B and C is known to generate eight different mRNAs and at least five proteins in the CD 45 protein family. The complete CD 45 protein consists of a large cytoplasmic domain of 707 amino acids, a transmembrane region of 22 amino acids and an external domain of 391-552 amino acids depending on the pattern of exon splicing. By electron microscopy, the CD 45 proteins consist of a globular structure of 12 nm, representing the cytoplasmic domain, and a rod-like structure of 18 nm, representing the external domain. least five proteins in the CD 45 protein family. The complete CD 45 protein consists of a large cytoplasmic domain of 707 amino acids, a transmembrane region of 22 amino acids and an external domain of 391-552 amino acids depending on the pattern of exon splicing. By electron microscopy, the CD 45 proteins consist of a globular structure of 12 nm, representing the cytoplasmic domain, and a rod-like structure of 18 nm, representing the external domain.
There is high conservation of the cytoplasmic domain among mammals and it shows homology with placental tyrosine phosphatases. Consistent with this homology, the CD 45 protein has intrinsic tyrosine phosphatase activity and belongs to a family of protein tyrosine phosphatases that includes 16 other members, at least seven of which are transmembrane proteins (Trowbridge et al, 1991).
The precise function of the CD 45 proteins is not known but they appear to play an important role in early lymphocyte activation. Protein tyrosine phosphatase can counter the actions of protein tyrosine kinases, enzymes known to be induced in early T-cell activation that may represent the primary signaling event initiated by the T-cell receptor. CD 45 expression is inversely related to spontaneous tyrosine phosphorylation of multiple proteins, which has a fundamental role in regulating T-cell calcium levels. CD 45 is required for both T-cell antigen receptor and CD 2-mediated activation of T-lymphocyte protein tyrosine kinase and is physically linked to both CD 2 and the T-cell receptor on the surface of memory T lymphocytes. The difference in structure among the external domains of the different CD 45 proteins probably determines the specific target stimuli for the different cell types expressing CD 45. Similarly, CD 45 may also be important for B-cell function. Antibodies to CD 45 inhibit an early phase in the activation of resting B cells and are able to inhibit c-myc induction in B-cells.
As a result of posttranslocational change of the mRNA of the A, B and C exons, several isoforms are produced. By strict definition, CD 45 antibodies are monoclonal antibodies, which react with all isoforms of CD 45 proteins, and there are several subclusters of antibodies that detect different species of CD 45 proteins. These have molecular weights of 220 kD representing the ABC isoform, 205 kD probably representing distinct AB and BC isoforms, 190 kD representing the B isoform and 180 kD
representing the O isoform. The restricted CD 45 antibody refers to those that recognize subsets of CD 45 proteins but not the entire class and these CD 45R antibodies can be further subdivided into CD 45RA, CD 45RB and CD 45RO, depending on the isoform recognized by the antibody. To date, there are no monoclonal antibodies that specifically recognize the C isoform. CD 45RA antibodies generally precipitate the 200 and 205 kD (ABC and AB isoforms), CD 45RB the 220, 205 and 190 kD (ABC, AB, BC and B isoforms) and CD 45RO the 180 kD protein (O isoform).
Many of the CD 45 antibodies are sensitive to neuraminidase, consistent with the suggestion that these antibodies recognize epitopes that are associated with carbohydrates, and possibly, terminal sialic acids. PD 7 is a CD 45RB antibody and labels all known CD 45 proteins with the exception of the ones lacking exons A, B and C, whereas 2B11 reacts against AB protein but not others. The combination of PD 7 with 2B11 as a CD 45-CD 45RB cocktail (Dako) allows a reliable method of detecting LCA in hematolymphoid cells. CD 45 proteins are major components of the membranes of lymphocytes and form about 10% of the lymphocyte surface, accounting for much of the carbohydrate present on the membrane. The staining with CD 45 antibodies is membranous although there may be some staining of the Golgi. Histiocytes exhibit minimal cell membrane staining and phagocytic cells show immunolocalization of the antigen to secondary lysosomes (Weiss et al, 1993).

The CD 45 proteins are the most specific diagnostic antibodies currently available. A cocktail of PD7-2B11 (CD 45-CD 45RB) antibodies is a reliable marker of cells fixed in formalin as well as in cryostat sections and fresh cell preparations. It is, therefore, an essential component of the panel used to distinguish anaplastic large cell tumors, which include malignant lymphoma, melanoma and carcinoma. It is also an essential component of panels used to separate small cell tumors of lymph nodes, skin, bone and other sites, both in adults as well as in children. The reactivity of anti-LCA antibodies is between 93% and 99% for a cross-spectrum of different subtypes of B- and T-cell lymphomas.
In classic Hodgkin's disease, excluding the nodular L & H lymphocyte predominant subtype, membrane staining for leukocyte common antigen is rare, although cytoplasmic staining may be seen. Cytoplasmic staining may be spurious as similar cytoplasmic staining can be found in non-hematolymphoid neoplasms. By contrast, the majority of nodular L & H lymphocyte-predominant Hodgkin's disease shows positivity for PD 7 and/or 2B11 and this subtype is now thought to be distinctly different from classic Hodgkin's disease.
Anaplastic large cell lymphoma may show positivity for LCA in only 50-87% of cases, although this figure may be higher in frozen section material. Furthermore, anaplastic large cell lymphoma may also show staining for epithelial membrane antigen, making its immunohistochemical differentiation from anaplastic carcinoma difficult. These tumors express CD 30 and, in 60% of cases, are of activated T-cell phenotype, showing staining for CD 45RO and/or CD 43 in paraffin sections (Chott et al, 1990).
Among other hematolymphoid neoplasms, plasmacytomas show a variable degree of positivity for LCA, ranging from 0% to 20% of cases. Hairy cell leukemia has been found to be uniformly positive for PD 7-2B11 and CD 45 expression has been found in all cases of acute leukemias of T-cell lineage and in over 80% of cases of B-cell lineage. Failure of CD 45 expression in acute childhood lymphoblastic leukemia appears to be associated with other favorable prognostic features such as lower leukocyte counts and serum lactic dehydrogenase levels and is also associated with chromosomal hyperdiploidy. Mast cell disease appears to be positive for PD 7-2B11 and polycythemia vera and extramedullary hematopoiesis were reported to be negative although only a few cases were studied. In keeping with the low expression in histiocytes, true histiocytic tumors were fìnd to be negative for PD 7-2B11, whereas cases of Langerhans' histiocytosis were reported to be positive. The rare cases of interdigitating reticulum cell sarcoma which have been studied have been reported to be positive for PD 7, similar to non-neoplastic interdigitating reticulum cells.
While larger series have reported a total absence of staining for LCA in non-hematolymphoid neoplasms, there have been rare case reports of staining examples of primitive sarcoma, probably rhabdomyosarcoma.

CD 45RA (4KB5, MB1, KiB3 and MT2)
The CD 45RA group of antibodies recognize the 220 kD and 205 kD variants of CD 45 encoded by exon A. These isoforms are expressed on the surface of most B cells, as well as postthymic, naive T cells and some medullary thymocytes. MT2 is thought to recognize a carbohydrate moiety and is negative in normal germinal centers, unlike antibodies MB1 and KiB3 which appear to bind to the peptide backbone of CD 45RA, staining mantle zone and follicular center cells. In the paracortical areas of lymph nodes, there are approximately equal numbers of CD 45RO+ and CD 45 RA+cells. In paraffin-embedded sections, MB1 and 4KB5 stain over 80% of cases of B-cell lymphomas, while MT2 stains only 57% of such cases. Small lymphocytic lymphoma has the highest rate of positivity while small non-cleaved cell lymphoma has the lowest. Fifty-seven percent of cases of follicular center lymphoma are positive for MT2 and this pattern of staining has been exploited for diagnostic purposes as only weak or absent scattered positivity for MT2 is seen in reactive germinal center cells (Browne et al, 1991).
Neoplastic follicles are labeled by MT2 whereas reactive follicles are not. This difference in staining patterns with MT2 has been postulated to be due to differences in the sialation of the CD 45 protein present on these B cells. T-cell lymphoma has a much lower incidence of positivity with CD 45RA antibodies and is seen in about 10% of cases.

CD 45RO (UCHL1, A6, OPD4)
CD 45RO antibodies recognize the 180 kD (O isoform) variant of CD 45. UCHL1 antibody reacts with approximately 90% of cortical thymocytes, 50% of medullary thymocytes and approximately 50-70% of CD 2-and CD 3+ peripheral blood and lymph node T cells. It rarely, if ever, reacts with benign B cells. While most mature T cells are CD 45RO+, some normal T-cell subsets are constitutively CD 45RO- and CD 45RA+ and the CD 45RO+ cells slowly increase in number to reach the adult level of about 50% by the age of 10-20 years. CD 45RO- cells include naive CD 4+ T cells, which predominate in neonates, and some CD 8+ or CD 4- CD 8-subsets found in intestinal intraepithelial T cells and enteropathy-associated T-cell lymphoma. In the differentiation of low-grade B-cell from T-cell lymphomas, the approximated test analysis figures for UCHL1 are as follows: sensitivity 95%, specificity 95%, accuracy 95%. In contrast, in high-grade lymphomas, the same parameters are 80%, 85% and 83% for UCHL1 are as follows: sensitivity 95%, specificity 95%, accuracy 95%. In contrast, in high-grade lymphomas, the same parameters are 80%, 85% and 83% respectively. Stem cells giving rise to both erythroid and myeloid cells as well as primitive erythroid colony-forming cells express the 180 kD isoform of the CD 45 protein recognized by CD 45RO but more mature erythroid forms lack CD 45 expression. Most granulopoietic colony-forming cells are CD 45RO- while mature monocytes or macrophages and myeloid cells are generally CD 45RO+. These latter cells do not stain with the antibody OPD4, the difference in reactivity possibly being due to a difference in the carbohydrate structure of the epitope presented on these cells (Chittal et al, 1988; Norton & Isaacson, 1989). The OPD4 antibody is not, as originally claimed, specific for CD 4+ T cells. It reacts very similarly to clone UCHL1 and differs only in having a low sensitivity for T-cell lymphoma and is not reactive with monocytic cells (Poppema et al, 1991).

•Browne G, Tobin B, Carney DN, Dervan PA 1991 Aberrant MT2 positivity distinguishes follicular lymphoma from reactive follicular hyperplasia in B5 and formalin-fixed paraffin sections. American Journal of Clinical Pathology 96: 90-4.

•Chittal SM, Caveriviere P, Schwarting R et al 1988. Monoclonal antibodies in the diagnosis of Hodgkin's disease. The search for a rational panel. American Journal of Surgical Pathology 12:9-21.

•Chott A, Kaserer K, Augustin I et al 1990 Ki-1 positive large cell lymphoma. A clinicopathologic study of 41 cases. American Journal of Surgical Pathology 14:539-48.

•Norton AJ, Isaacson PG 1989 Lymphoma phenotyping in formalin-fixed and paraffin wax embedded tissues. II. Profiles of reactivity in the various tumour types. Histopathology 14:557-579.

•Poppema S, Lai R, Visser L 1991 Monoclonal antibody OPD4 is reactive with CD 45RO but differs from UCHL1 by the absence of monocyte activity. American Journal of Pathology 139: 725-729.

•Trowbridge IS, Ostergaard HL, Johnson P 1991 CD 45 - a leucocyte-specific member of the protein tyrosine phosphatase family. Biochemica et Biophysica Acta 1095:46-56.

•Weiss LM, Arber DA, Chang KL 1993 CD 45. A review. Applied Immunohistochemistry 1: 166-181.

Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.