Richard A. Jones

Circulating plasma cells in multiple myeloma: characterization and correlation with disease stage.

The aim of this study was to develop a flow cytometric test to quantitate low levels of circulating myeloma plasma cells, and to determine the relationship of these cells with disease stage. Cells were characterized using five‐parameter flow cytometric analysis with a panel of antibodies, and results were evaluated by comparison with fluorescent consensus‐primer IgH‐PCR.

Immunophenotypic and DNA Genotypic Analysis of T-Cell and NK-Cell Subpopulations in Patients with B-Cell Chronic Lymphocytic Leukaemia (B-CLL)

Absolute numbers and distributions of peripheral blood T-cells and NK cells were immunophenotypically determined in 21 patients with B-CLL and compared with those obtained from a series of 13 elderly normal controls with an age range of 60-87 years. For absolute CD3+, CD4+ and CD8+ T-cell, and CD16+ NK subpopulation numbers, there were no consistent differences between the normal and B-CLL groups although some individual patient variation was seen. Immunophenotypic analyses did however reveal that CD3+ T-cells in almost half (10/21) of the B-CLL patients were Ia+ (defined as > 20% positive cells), compared to 0/13 of the elderly control group (p < 0.001), and that the proportions of CD4+ and CD8+ cells expressing membrane CD45RO were significantly increased compared to the control group. Subdivision of the B-CLL cases into those with low (< 20%) and high (> 20%) proportions of CD3+ T-cells co-expressing Ia further showed that CD45RO expression by CD4+ fractions was particularly prominent in the Ia+ subgroup, and that the relative increase of CD4+CD45RO+ cells was primarily a consequence of decreased absolute numbers of CD4+CD45RA+ lymphocytes. This study also examined extracted DNA from enriched CD3+ T-cell fractions (obtained by immunomagnetic bead selection in 9 of the B-CLL cases) by PCR analysis with two primers for the T-cell gamma gene locus. With the V gamma C (consensus) primer, 8/9 cases were polyclonal and the remaining case was oligoclonal. For comparison, 7/9 CD3+ fractions were oligoclonal with the V gamma 9 primer with the other two cases being polyclonal. No monoclonal CD3+ components were found. It is suggested that the observed increased Ia expression by CD3+ cells and the predominance of CD4+ cells expressing membrane CD45RO in patients with B-CLL may be of potential relevance to understanding the pathogenesis and patterns of disease progression.

Outreach monitoring service for patients with indolent B-cell and plasma cell disorders: a UK experience.

Disease progression occurs in over 1% of monoclonal gammopathy of undetermined significance, monoclonal B‐cell lymphocytosis and early stage chronic lymphocytic leukaemia patients every year therefore regular monitoring is indicated. We assessed the efficacy of an outreach service to replace clinic monitoring using local phlebotomy with central haematologist review of laboratory parameters and symptoms identified by a patient self‐assessment questionnaire. The service was used by 299 patients for 2 years and provided accurate monitoring, improved patient satisfaction, support for primary care and reduced the burden on haematology clinics without an increase in inter‐assessment admissions due to disease progression.

Diagnostic differentiation of chronic B‐cell malignancies using monoclonal antibody L161 (CD1c)

The expression of membrane CD1c, as defined by monoclonal antibody L161, was examined on malignant lymphoid cells from 191 cases of chronic lymphoproliferative disease and on eight ‘normal’ enriched tonsil B‐cell extracts. Of 79 cases of chronic lymphocytic leukaemia (CLL) studied, 77 showed low (<20% positive cells) CD1c expression whereas 63/71 (89%) cases of B‐PLL, HCL and B‐NHL showed increased CD1c+ (but not CD1a or CD1b) components. In contrast, malignancies corresponding to terminal stages of B‐cell differentiation (immunocytoma and myeloma) generally showed low CD1c expression as did lymphoid cells from 10 cases of post‐thymic malignancy. Although there was some correlation between the expression of membrane CD1c and immunoglobulin (SIg) light chain densities (P<0.001), it is relevant in diagnostic terms that seven cases of B‐NHL, with low SIg staining intensities more typically associated with CLL were CD1c+. CD1c expression was not, however, correlated with the presence of CD23 or FMC7 determinants but did show a similar pattern of expression to that previously reported for beta‐2 microglobulin. Determination of cellular CD1c by APAAP immunocyto‐chemistry confirmed the presence of higher antigen densities in malignant B‐cells at intermediate/late stages of differentiation and this interpretation was further supported by the finding that the majority of phenotypically mature tonsil B‐cells were also CD1c+. The determination of CD1c expression by malignant B‐cells may therefore be of particular value in the diagnostic differentiation of chronic lymphoproliferative disorders.

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: I, Diverse Patterns of Expression in Mature (Post-Thymic) T-Cell Proliferations

By simultaneous two- and three-colour flow cytometry, this study analysed the expression of membrane CD45RA (2H4) and CD45RO (UCHL1) determinants by normal thymocytes (n = 5) and peripheral blood lymphocyte subpopulations (CD4(+), n = 21; CD8(+), n = 12; CD8(dim+), n = 12) and compared these patterns with those of T-cells from representative CD4(+)CD8(-) (n = 8), CD4(+)CD8(+) (n = 2), CD4(-)CD8(+) (n = 10) and CD4(-)CD8(-) (n = 1) proliferations. These comprised cases of prolymphocytic leukaemia (T-PLL, n = 5), adult T-cell leukaemia-lymphoma (ATLL, n = 2), Sezary Syndrome (SS, n = 4), chronic lymphocytic leukaemia (T-CLL, n = 4), and lymphoproliferative disease of granular lymphocytes (LDGL, n = 5). Normal thymocyte fractions, of which a mean of 85% cells co-expressed membrane CD4 and CD8, were predominantly (mean 89%) 2H4(-)UCHL1(+) with the remaining cells consisting of 2H4(int)UCHL1(+) and 2H4(+)UCHL1(-) components. Further analysis showed that virtually all CDla(+) thymocytes were UCHL1(+) whereas the CD1a(-) fraction comprised similar proportions of both UCHL1(-) and UCHL1(+) subpopulations. Similarly, normal blood CD4(+), CD8(+) and CD8(dim+) lymphocytes showed reciprocal CD45RA/CD45RO expression and could be phenotypically grouped into 2H4(+)UCHL1(-) 2H4(int)UCHL1(+) and 2H4(-)UCHL1(+) subpopulations. Mean proportions of 48% and 68%, for CD4(+) and CD8(+) lymphocytes respectively, showed a composite 2H4(+)UCHL1(-) phenotype, whereas the percentage of NK-associated CD8(dim+) cells with this phenotypic pattern was considerably higher (mean, 85%). Normal lymphocyte subpopulations lacking both determinants (2H4(-)UCHL1(-)) were only rarely noted. Comparing normal patterns of CD45RA/CD45RO expression with those of the T-cell proliferations revealed diverse and abnormal patterns of staining for 3/6 of the CD4(+)CD8(-) SS and ATLL, and for 5/5 of the T-PLL (CD4(+)CD8(-), n = 2; CD4(+)CD8(+), n = 2; and CD4(-)CD8(+), n = 1) cases studied. In contrast, the nine cases of CD4(-)CD8(+) T-CLL and LDGL all showed CD45RA/CD45RO staining patterns similar to that of normal CD8(+)/CD8(dim+) blood lymphocytes (i.e. a predominance of 2H4(+)UCHL1(-) cells). Although the variant CD45RA/CD45RO pattern types of the CD4(+) proliferations did not appear to be related to either the diagnostic category or other phenotypic characteristics, the high proportion of abnormal patterns within this case group suggests that recognition of these abnormalities may be potentially relevant to the differentiation of benign and malignant CD4(+) proliferations and, in addition, may be of aetiological importance with respect to the diverse acquired defects in immunity commonly seen in patients with such disorders.

Expression of MHC class I and class I-like gene products on the cell membrane of mature and immature T cells

Beta 2-microglobulin (beta 2m) constitutes the 12 kD light chain of the MHC-encoded 43-kD glycoprotein HLA-ABC molecules, which are expressed on most nucleated cells. The T6 (CD1a) antigen is expressed on thymocytes in reciprocal manner to HLA-ABC, having a similar structure to the HLA-ABC molecule. We have determined the expression of beta 2m, HLA-ABC and the T6 antigen on the cell-surface of CD1a+ and CD1a- thymocytes (defined by NA1/34 expression), and have shown that despite immunophenotypic differences between these two stages of thymic maturation, the quantity of beta 2m-associated molecules expressed was not significantly different. The nature of the heavy (alpha) chain associated with beta 2m, however, differed since HLA-ABC had largely replaced T6 on the surface of CD1a- thymocytes. We also determined the expression of beta 2m and HLA-ABC on the surface of peripheral blood CD4+ and CD8+ T-cells, and showed that the CD8+ population expressed higher levels of these antigens than CD4+ T-cells, with no detectable excess beta 2m over HLA-ABC for either subpopulation. In addition, thymocytes expressed fewer beta 2m-associated determinants than peripheral blood T-cells. These results indicate an increase in the expression of beta 2m-associated molecules with differentiation from thymic to peripheral T-cell, with a further increase in such molecules expressed on the CD8+ compared to the CD4+ peripheral T-cell subpopulation.

Patterns of Membrane Antigen Expression by AML Blasts: Quantitation and Histogram Analysis

Leukaemic myeloid blasts from non-monocytic (M1-M3, n = 36) and monocytic (M4 and M5, n = 21) AML cases were examined for the expression of 12 different membrane determinants by flow cytometry. Data analyses for each antigen included the determination of (a) the mean fluorescence intensity for the whole blast cell population, (b) the relative levels of membrane fluorescence for individual events (cells), and (c) a conventional assessment of the proportion of cells staining positively (i.e. exceeding a pre-defined level of fluorescence). Three main types of staining histogram were observed and, of these, the most commonly seen (348/432 and 176/252 of non-monocytic and monocytic AML histograms respectively) was characterised by an homogenous distribution of staining intensities which did not exceed two log decades of fluorescence (S-type). The second staining pattern was characterised by a continuous spectrum of fluorescence which exceeded two log decades of fluorescence (SE-type), and the third pattern showed evidence of two leukaemic populations with different levels of fluorescent staining (BI-type). With the exception of occasional AML cases which expressed CD7 or CD19 with low staining intensity, the expression of lymphoid-associated membrane CD3, CD10, and CD22 by AML blasts was insignificant. For comparison, analysing the histogram patterns of expression for the myeloid and non-lineage associated membrane determinants revealed that CD11c, CD13, CD14, and CD38 were mainly of S- or SE-type for the non-monocytic AML variants, with a minor but significant proportion of such cases expressing CD33 (7/36), CD34 (6/36) and HLA-Dr (6/36) with a BI-type staining pattern. Similarly, histogram patterns for CD13, CD33, CD34 and CD38 expression by the monocytic AML variants were predominantly of S- or SE- type, with minor proportions of cases expressing CD11c (7/21), CD14 (10/21), and HLA-Dr with BI-type staining. Comparisons between the mean fluorescence staining intensities for the whole blast cell population and conventional positive versus negative delineations for each antigen studied further suggested that semi-quantitative measurements of fluorescent staining were more informative and potentially of greater relevance to the study and diagnostic assessment of acute myeloid leukaemia subtypes.