Deficiency of glycosyl-phosphatidylinositol-linked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic cytofluorometric assay.

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease that affects not only red cells, but other blood cells as well. The common defect is supposed to be an acquired deficiency of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins, which may be present already at the hematopoietic stem cell level. Recently, a panel of monoclonal antibodies (MoAbs) has become available directed against various GPI-linked membrane proteins. This makes it possible to study various cell lineages for the deficiency of such proteins in PNH in more detail. Using cytofluorography, we could show that the granulocytes of 20 different PNH patients miss not only GPI-linked FcRIII (CD16 antigen), but also three other GPI-linked proteins, ie, CD24 antigen, CD67 antigen and a granulocyte-specific 50 to 80 Kd antigen. The affected granulocytes were not only neutrophils but also eosinophils, as was found in a more detailed analysis of three patients. Moreover, in all 10 PNH patients tested, the monocytes were found to be deficient for the GPI-linked CD14 antigen, and we found with CD24 and CD55 (DAF) antibodies that lymphocytes may be involved as well. However, abnormal B and T lymphocytes were detected only in a subset of patients (2 of 10 tested). The uniform deficiency of GPI-linked proteins of granulocytes allows the introduction of a new diagnostic cytofluorometric assay for PNH with MoAbs against GPI-linked granulocytic antigens. This test was positive in all PNH patients studied and not in a group of 40 control patients or 50 normal donors, with the exception of three of 16 aplastic anemia (AA) patients. In the three AA patients, subpopulations (10% to 20%) of PNH granulocytes could be detected, whereas these patients had a negative acidified serum (Ham) test. This indicates that the new test is more sensitive than the Ham test and allows the early diagnosis of PNH in AA. An advantage of the neutrophil assay is that, in contrast to the Ham test, it is not influenced by recent red-cell transfusions. Moreover, it is possible to quantify the number of affected cells by single cell analysis.

Monoclonal antibodies against myeloperoxidase are valuable immunological reagents for the diagnosis of acute myeloid leukaemia.

Whereas the diagnosis of acute lymphoid leukaemia greatly depends on immunophenotyping on the leukaemic cells, the diagnosis of acute myeloid leukaemia (AML) is still only based on morphological and cytochemical criteria. Here we describe that with a monoclonal antibody, directed against myeloperoxidase (MPO), the immunological diagnosis of AML is possible in most cases. A monoclonal antibody against lactoferrin (LF) was used to detect more mature myeloperoxidase-containing cells. Of the cell samples tested from 206 different patients with AML, 95% were found to express myeloperoxidase in more than 15% of lactoferrin-negative cells. Compared with other myeloid-reactive monoclonal antibodies (VIM2, anti-CD13, anti-CD14, anti-CD15 and anti-CD33), a higher diagnostic sensitivity and specificity for AML was found. No significant correlation with the FAB classification was found. In most patients, more MPO-positive cells were detected by the monoclonal antibody than by the cytochemical staining. This could be due to the recognition of enzymatically inactive precursor forms of myeloperoxidase by the antibody. The use of anti-myeloperoxidase monoclonal antibodies for the diagnosis of AML has the advantage that objective quantification is possible.

Chronic lymphocytic leukemia. Immunologic markers and functional properties of the leukemic cells.

In 230 cases of chronic lymphocytic leukemia (CLL), marker analysis was performed with rosette techniques and a panel of xeno-antisera. A monoclonal B-cell proliferation was found in the majority of cases (94%). In most cases, the B-cells carried IgM, with or without IgD. Cytoplasmic immunoglobulin-inclusion bodies were seen in 7% of the cases of B-CLL. The number of patients with non-B/non-T-CLL was small (2%) in this series. In eight patients (4%), a proliferation of T-cells was established. These patients had a different clinical presentation and marker analysis of the lymphocytes, together with functional studies, showed that this group represented a mixture of different disease processes. Functional analysis of the B-CLL cells in 19 cases showed a poor or absent mitogen response and in nine cases the absence of the capacity to differentiate in vitro into plasma cells and/or to produce immunoglobulins.

Cooperative effects in mitogen- and antigen-induced responses of human peripheral blood lymphocyte subpopulations.

Human peripheral blood lymphocytes were separated into T cell-enriched and T cell-depleted fractions by E rosette sedimentation. These two fractions, as well as the unseparated lymphocyte suspension, were tested for their responsiveness to the mitogens phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) and to the antigens PPD (purified protein derivative of tuberculin) and tetanus toxoid. The response to PHA, ConA and the antigens was found to be confined to the purified T cell fraction; PWM could stimulate both purified T and non-T cells. However, the T cell response to ConA, PPD and tetanus toxoid was always decreased by 50-70%, when compared to the unseparated lymphocytes. Addition of monocytes could restore the T cell response. In the response to PHA and tetanus toxoid, the (primarily unresponsive) non-T cell fraction could be recruited into proliferation by gamma-irradiated T cells. Moreover, in the response to tetanus toxoid, lymphocytes (T as well as non-T) from a nonimmune individual could be recruited into proliferation by gamma-irradiated immune T cells.

Quantification of antigen-reactive cells among human T lymphocytes.

The number of antigen-reactive cells among human peripheral blood T lymphocytes was estimated by a limiting dilution analysis. Antigen-induced lymphocyte activation was measured by means of incorporation of tritiated thymidine [3H]dThd. We have studied the frequency of memory T cells for the bacterial antigens tuberculin PPD and tetanus toxoid in immune donors, as well as the frequency of alloantigen-reactive T cells. In 11 different donors, the observed frequencies of the antigen-reactive T cell ranged between 1:300 and 1:16 000 for PPD; for tetanus toxoid values, between 1:750 and 1:11 500 were obtained in 5 different donors. The frequency of alloantigen-reactive T cells was found to be higher: between 1:200 and 1:600 (n = 10). For 3 donors, the estimated frequencies proved to be reproducible over a period of several months. Finally, a correlation could be demonstrated between the frequency of PPD-reactive T cells and the [3H]dThd incorporation of 4 X 10(4) PPD-stimulated lymphocytes.