Jacqueline L. Cordell

Diagnosis of Human Lymphoma with Monoclonal Antileukocyte Antibodies

Two monoclonal antibodies have been produced that react with antigens present on human white cells. These reagents differ from other monoclonal antibodies of similar specificity in that the antigens they recognize are resistant to conventional tissue-fixation and embedding procedures. These reagents can therefore be used in immunocytochemical staining of paraffin-embedded tissue sections. We assessed the practical usefulness of this technique in the histopathological diagnosis of human lymphoid neoplasms by staining a wide range of routine surgical biopsy specimens of normal and neoplastic tissue (gathered from five institutions), using an indirect immunoperoxidase technique. In all 40 cases of non-Hodgkins lymphoma, positive labeling of neoplastic cells was obtained with one or both antibodies. In contrast, no staining of neoplastic cells was observed in 60 samples of nonlymphoid neoplasms. We conclude that many of the difficulties encountered by histopathologists in distinguishing between lymphoid and nonlymphoid neoplasms may be overcome by immunohistologic labeling with monoclonal antibodies such as the ones we have studied.

A human macrophage-associated antigen (CD68) detected by six different monoclonal antibodies

Summary. Antibodies grouped together by the Third Workshop on Leucocyte Differentiation Antigens on the basis of pan‐macrophage reactivity on tissue sections were analysed in immunoprecipitation experiments. Antibodies Y2/131. EBM11, Ki‐M6 and Ki‐M7 all precipitated antigens of Mr 110000 which were shown to be identical by preclearing experiments. In addition a recently produced antibody, KP1, which identifies macrophages in paraffin‐embedded tissue, was shown to recognize the same antigen. The antibodies were tested on murine cells transfected with two clones, which had been isolated by screening a cDNA library with antibodies Y1/82A and EBM11. Cells transfected with the longer cDNA clone, coding for a molecule of Mr 110000, reacted with antibodies Y2/131. EBM11. Y1/82A and Ki‐M6, whilst the shorter clone, encoding a molecule of Mr 70000 gave the same result except that it did not induce expression of the Ki‐M6 epitope. KP1 antibody did not recognize any transfectants, possibly because of differences in glycosylation by the transfected cell line compared with human tissue. Five of the six antibodies appear to recognize different epitopes (the sixth, Ki‐M7, not having been evaluated in this way). It was concluded that these six antibodies react with a macrophage‐associated antigen for which the gene has been cloned. This group of antibodies has recently been designated CD68 by the Fourth Workshop on Human Leucocyte Differentiation Antigens.

The immunohistological detection of platelets, megakaryocytes and thrombi in routinely processed specimens

The production and characterization of a new monoclonal antibody, Y2/51, against platelet glycoprotein IIIa is described. A useful feature of this antibody is its ability to recognize platelets and megakaryocytes in formalin‐fixed routinely processed material. It could also be used to reveal platelets both in thrombi in large vessels and in microthrombi too small to be readily apparent on conventional microscopic examination. For this purpose it was helpful to use the antibody in conjunction with a new monoclonal reagent (Ret40f) against red cell sialoglycoprotein β‐which detects red cells and their precursors in routinely processed tissue. The use of these antibodies should be valuable for the detection of thrombi in a variety of situations such as renal transplant rejection, coronary artery disease and vasculitis.

Immunohistological analysis of human bone marrow trephine biopsies using monoclonal antibodies.

Summary. This paper describes the use of a recently developed immuno‐alkaline phosphatase method (the ‘APAAP’technique) for labelling frozen sections of undecalcified bone marrow biopsies with monoclonal antibodies, including reagents reactive with T cells and their subsets, B cells, glycophorin, HLA‐DR antigen, common ALL antigen, epithelial cells and megakaryocytes. Use of an immuno‐alkaline phosphatase technique avoids problems due to endogenous enzyme activity encountered when staining bone marrow by immunoperoxidase procedures. Immunohistological labelling of frozen trephine biopsies is of particular value when it is impossible to aspirate marrow particles and for identifying cells which do not readily enter suspension (e.g. dendritic reticulum cells or stromal cells). Details are given of cases in which immunohistological analysis was used for the phenotyping of acute leukaemias, for the differential diagnosis of intramedullary T and B cell proliferations, and for identifying bone marrow metastases.

The expression of the B‐cell marker mb‐1 (CD79a) in Hodgkin's disease

Recent evidence indicates that membrane‐bound immunoglobulin on B lymphocytes is associated with a molecule which comprises the products of the mb‐1 and B29 genes. This molecule is a highly specific marker for B‐cells, presumably because of its central functional role in antigen triggering, and has recently been clustered as CD79a at the 5th Leucocyte Workshop. Recently there has been controversy surrounding reports of B‐cell antigen expression by Reed–Sternberg and related cells, and we have therefore studied 108 cases of Hodgkins disease immunohistochemically using a novel antibody which detects mb‐1 protein in paraffin sections. The results were compared with those achieved using antibody L26 to detect CD20. The mb‐1 protein was present in the neoplastic cells in all 14 cases of lymphocyte predominance Hodgkins disease studied, and CD20 immunoreactivity was also found in seven of the eight cases of this subtype studied. Of the non‐lymphocyte predominance cases, 20% (19/94) expressed mb‐1 and 30% (20/67) CD20 in the Reed–Sternberg cells, but the cells positive for either of these two markers usually constituted only a very small proportion of the neoplastic population. However, in occasional cases (one of 94 for mb‐1 and five of 67 for CD20), more than 50% of the neoplastic cells expressed one or both B‐cell antigens. These results confirm the B‐cell origin of the neoplastic cells in lymphocyte predominance Hodgkins disease, but they also indicate that, contrary to our previous study, mb‐1 expression may occasionally be found in what appears, on histological grounds, to be other types of Hodgkins disease.

Description of a sequential staining procedure for double immunoenzymatic staining of pairs of antigens using monoclonal antibodies

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H2O2 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations

The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein overexpression in most follicular lymphomas. However the expression of BCL2 is not always homogeneous and may demonstrate a variable degree of heterogeneity. This study analysed BCL2 protein expression pattern in 33 cases of t(14;18)‐positive follicular lymphomas using antibodies against two different epitopes (i.e. the widely used antibody BCL2/124 and an alternative antibody E17). 16/33 (49%) cases demonstrated strong BCL2 expression. In 10/33 (30%) cases, BCL2 expression was heterogeneous and in some of these, its loss appeared to be correlated with cell proliferation, as indicated by Ki67 expression. Double immunofluorescence labelling confirmed an inverse BCL2/Ki67 relationship, where in 24/28 (86%) cases cellular expression of BCL2 and Ki67 was mutually exclusive. In addition, seven BCL2 ‘pseudo‐negative’ cases were identified in which immunostaining was negative with antibody BCL2/124, but positive with antibody E17. Genomic DNA sequencing of these ‘pseudo‐negative’ cases demonstrated eleven mutations in four cases and nine of these were missense mutations. It can be concluded that in follicular lymphomas, despite carrying the t(14;18) translocations, BCL2 protein expression may be heterogeneous and loss of BCL2 could be related to cell proliferation. Secondly, mutations in translocated BCL2 genes appear to be common and may cause BCL2 pseudo‐negative immunostaining.

Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1α localization in erythroblasts

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1(gt/gt) homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.

Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia

Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.

Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann's thrombasthenia

A patient is described with a disturbance of platelet function comparable to that in Glanzmanns thrombasthenia. Platelet aggregation and binding of fibrinogen to the patients platelets were defective and thrombin‐induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced. As measured by monoclonal antibody binding in the presence of divalent cations, the platelets contained about 15% of the normal amount of GPIIb and GPIIIa and only 6% of the normal amount of intact GPIIb/IIIa complex. The residual GPIIb/IIIa complex exhibited a decreased stability as shown by the lack of binding of a complex‐dependent anti‐GPIIb/IIIa antibody to platelets incubated with ethylene diamine tetraacetic acid (EDTA) at 22°C. Crossed immunoelectrophoresis (CIE) in the presence of divalent cations showed partial dissociation of GPIIb/IIIa as well as the presence of two forms of the residual intact GPIIb/IIIa complex. In addition, both CIE in the presence of the EDTA and two‐dimensional sodium dodecyl sulphate (SDS) gel electrophoresis showed the presence of two forms of GPIIb. This form of thrombasthenia is characterized by a defective platelet function, a marked reduction of GPIIb and GPIIIa, decreased stability of the residual GPIIb/IIIa complex and structural heterogeneity of GPIIb.