The posterior bone block procedure in posterior shoulder instability: a long-term follow-up study.

We present the long-term outcome, at a median of 18 years (12.8 to 23.5) of open posterior bone block stabilisation for recurrent posterior instability of the shoulder in a heterogenous group of 11 patients previously reported on in 2001 at a median follow-up of six years. We found that five (45%) would not have chosen the operation again, and that four (36%) had further posterior dislocation. Clinical outcome was significantly worse after 18 years than after six years of follow-up (median Rowe score of 60 versus 90 (p = 0.027)). The median Western Ontario Shoulder Index was 60% (37% to 100%) at 18 years' follow-up, which is a moderate score. At the time of surgery four (36%) had glenohumeral radiological osteoarthritis, which was present in all after 18 years. This study showed poor long-term results of the posterior bone block procedure for posterior instability and a high rate of glenohumeral osteoarthritis although three patients with post-traumatic instability were pleased with the result of their operations.

Gating of the so-called 'lymphocytic' cell population for the quantification of natural killer cells (CD16+) by flow cytometry causes loss of CD16 positive cells.

Natural killer cells can phenotypically be identified as CD16 positive with a specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics. In this paper we demonstrate that a substantial part of the NK cell population is situated outside this gate in the total mononuclear cell population. We therefore recommend that the number of CD16+ cells is determined in the total mononuclear cell population. However, in the total mononuclear cell population, a group of dimly CD16 positive cells, probably monocytes, interferes with a clear separation of cells with a positive and negative fluorescence. We describe two methods to overcome this problem.

Natural killer cell function is not diminished in the healthy aged and is proportional to the number of NK cells in the peripheral blood.

We studied natural killer (NK) cell subsets and NK function in young (25-35 years) and aged (75-84 years) persons by means of the single-cell assay. The subjects admitted to the study all fulfilled the SENIEUR health criteria in order to avoid confounding factors such as underlying disease or the influence of medication. We found no significant difference in the NK function between healthy young and aged persons on a per cell basis. A new application of the two-wavelength immunofluorescence technique during the single cell assay made it possible to define the phenotypes of the conjugate-forming cells responsible for the natural killer function. Most of the conjugate-forming cells were CD 16-positive, and half of these were also positive for Leu 7. The CD 16 antigen disappeared from the cell surface during the effector:target interaction. T-cell markers were found on some of the conjugate-forming cells but not on the strongly bound effector cells. The NK cell function was directly proportional to the number of NK (CD 16) cells in the peripheral blood.

The expanded null cell compartment in ageing: increase in the number of natural killer cells and changes in T-cell and NK-cell subsets in human blood.

Analysis of the subpopulations of mononuclear cells in human blood in ageing has revealed a striking increase in the number of null cells, defined as non-T, non-B, non-monocyte cells, and a decrease in the number of T and B cells. By using recently developed monoclonal antibodies against natural killer cells in combination with T-cell markers in two-wavelength immunofluorescence, we were able to define 13 subpopulations of mononuclear cells and compare them in two groups of persons, respectively aged 25-34 and 75-84 years, all fulfilling the stringent admission criteria for immunogerontological studies described in the SENIEUR protocol, and thus all to be considered as optimally healthy and immunologically uncompromised. We found that the increased null cell population in the aged is a result of an increase in the numbers of NK cells, mostly the CD16+Leu7+ subset. The number of CD8+ suppressor/cytotoxic cells is decreased. This is due to a decrease of the number of CD8+Leu7- cells. All NK and T-cell subsets bearing the Leu7 antigen, namely CD16+ Leu7+, CD4+Leu7+ and CD8+Leu7+, are increased. These changes can be due to defects of the ageing immune system, but they can also represent the optimal state of the immune system in the healthy aged and may be linked to survival. These values can be used as reference values for the 75-84 years age group and serve to monitor attempts to reconstitute the immune defects in ageing.

Intracellular immunoglobulin G 'pseudocrystals' in a patient with chronic B-cell leukemia.

A patient with chronic B-cell leukemia in whom the malignant lymphocytes showed intracellular inclusions of immunoglobulin (Ig) G kappa molecules is described. Electron microscopy revealed filamentous material in the nuclear envelopes and in the cisternae of the rough endoplasmic reticulum. These in vivo surface Ig-negative, nonexcreting cells could be stimulated in vitro to excrete immunoglobulin-free light chain molecules into the supernatant, which were not found in the cytoplasm after stimulation.

Subpopulations of mononuclear cells in ageing: expansion of the null cell compartment and decrease in the number of T and B cells in human blood.

Study of the immune system in ageing has yielded conflicting results. These controversies are mainly due to the selection of the subjects studied. We investigated the mononuclear cell subpopulations in the peripheral blood of subjects fulfilling strict admission criteria meant to exclude persons with diseases that influence the immune system. These criteria are described in the SENIEUR protocol devised by a working group in the framework of EURAGE, the Concerted Action Programme on Ageing of the European Community. We compared two groups of volunteers aged 25-34 years, and 75-84 years. Mononuclear cells were investigated by two-wavelength immunofluorescence combined with phase-contrast microscopy. We found a striking increase in the number of 'null' cells (non-T, non-B, non-monocyte) in the blood of the aged persons. The number of T cells was decreased, especially in the suppressor/cytotoxic subset. The number of B cells was slightly, but significantly, decreased; the number of monocytes did not change. The changes in these cell populations may be related to functional changes, and their quantification could be used to monitor attempts to reconstitute the immune defects in ageing. These findings can also serve as reference values in the study of aged persons not fulfilling the SENIEUR criteria, which, in turn, can contribute to the dissection of the influence of disease versus age on the immune system.

Chronic B-cell leukemias: relation between morphological and immunological features.

To better understand the heterogeneity of chronic B-cell leukemias we correlated morphological and immunological features by studying the peripheral blood from 80 patients with a panel of anti-immunoglobulin and fourteen monoclonal antibodies, which hitherto were studied separately or with respect to one single morphological entity only. Of these the surface immunoglobulins (sIg) and monoclonal antibodies (McAb) BA-1, BA-2, FMC7, OKM1, and anti-T65 allowed a fair distinction between five cytological subtypes: chronic lymphocytic (CLL), "lymphoplasmacytoid" (LPL), centrocytic (CL), prolymphocytic (PLL), and hairy cell leukemia (HCL). In that order the sIg showed a decreasing number of cases of mu +/- delta class and an increase of alpha or gamma positivity. The number of BA-1-positive cases was decreased in PLL and HCL. There was a decline of BA-2- and anti-T65-positive cases in the order mentioned, while this was accompanied by an increase of FMC7 and OKM1 positivity. A significant mutual exclusion between anti-T65 and FMC7 was observed and the same was true for FMC7 and BA-2. The antibodies FMC7 and OKM1, and anti-T65 and BA-2 were linked to each other. Also FMC7 positivity was related to sIg of the alpha and gamma classes. On the basis of this unique combination of markers a differentiation scheme of B lymphocytes is proposed, in which prolymphocytic leukemia and hairy cell leukemia seem to represent a maturation arrest at a more advanced stage than chronic lymphocytic or "lymphoplasmacytoid" leukemia.

Hairy cell leukemia: its place among the chronic B cell leukemias.

Hairy cell leukemia is a chronic B cell leukemia. The presence of surface Ig (SIg) of gamma or multiple isotypes on the cells locates HCL at a rather mature stage of B cell differentiation. The reactivity of HC with McAb is in accordance with this concept (T65-, OKM1+, FMC7+, BA-1-). To relate HCL to other chronic B cell leukemias, a morphologic classification was developed that distinguishes, besides HCL, five subtypes of chronic B cell leukemia. CLL showed weak staining for SIg of mu +/- delta class. The phenotype with McAb was T65+, OKM1-, FMC7-, BA-1+. Prolymphocytic transformation of CLL had essentially the same membrane phenotype. LPL often had brighter SIg of mu +/- delta class with gamma or multiple isotypes in about half of the cases. McAb gave a T65-or+, OKM1-or+, FMC7-or+, BA-1+ phenotype. The same surface-marker profile was found in CL. Finally, PLL showed bright SIg of gamma or multiple isotypes in the majority of cases and reactivity with McAb according to a T65-or+, OKM1+, FMC7+, BA-1+ pattern. The various immunologic phenotypes of the morphologic subtypes showed a considerable overlap. The various chronic B cell leukemias should be located in the scheme of B cell differentiation in the sequence CLL-LPL/CL-PLL-HCL.

Surface bound or cytoplasmic immunoglobulins: interpretation of the immunofluorescence observed in cytocentrifuge slides of human lymphocytes.

Results of immunofluorescence observations in the study of normal and malignant blood lymphocytes are described. Data which support the proposition that most membrane bound immunoglobulin molecules are stable enough to remain intact during cytocentrifuge slide preparation are presented. Therefore not all positive cells in a fixed cytocentrifuge slide should be considered as containing cytoplasmic immunoglobulins. A correct interpretation is essential because of its bearing on our concepts of B lymphocyte differentiation.

Prognostic significance of immunologic phenotype in hairy cell leukemia.

Hairy cell leukemia (HCL) is a usually chronic B cell lymphoproliferative disorder. To evaluate the prognostic significance of the various heavy and light chain determinants of the surface immunoglobulins (slg), we analyzed the clinical data and immunologic phenotype of 64 patients with HCL. Sixty-two of the 64 patients showed slg, which was invariably of only one light chain type (kappa 33, lambda 29). The actuarial survival of the cases expressing kappa-light chains was significantly better than those with lambda-light chains (p less than 0.002). This difference persisted when only cases with gamma or alpha gamma heavy chains were considered. No differences between the kappa and lambda-subgroups were discovered with respect to parameters of clinical importance. The various heavy chain classes of slg did not correlate significantly with the survival time. These results suggest that the immunologic phenotype, in particular the light chain type, may be a prognostic factor in patients with HCL.

Paraproteinaemia plus osteolytic lesions in typical hairy-cell leukaemia.

Most cases of hairy-cell leukaemia (HCL) involve proliferations of neoplastic B lymphocytes. In rare cases, M-proteins or osteolytic lesions have been documented in patients with HCL. In this study two patients with typical HCL are reported in whom both paraproteinaemia and osteolytic lesions of the femoral neck developed. In one of the patients the production of the M-protein by hairy cells could be established. In the other patient, at autopsy no signs of myeloma were found. The hairy cells from inside the osteolytic lesion had the same immunological phenotype as hairy cells from the peripheral blood, the spleen, and other parts of the bone marrow. These cases once more confirm the B-cell nature of many cases of HCL, and show that hairy cells can have functional capacities usually attributed to much more mature B lymphocytes, i.e. plasma cells.

Primary plasma cell leukaemia. A case report and a review of the literature.

A case history of a patient with primary plasma cell leukaemia is presented. Analysis of serum showed an IgD lambda paraprotein, and lambda-light-chains were found in the urine. Immunofluorescence studies of a bone marrow aspirate revealed intracytoplasmatic IgD of lambda-type in plasma cells. Moreover J-chain could be demonstrated in these plasma cells. A complete remission of the disease, with disappearance of the paraprotein, was obtained following treatment with a combination of cyclophosphamide, vincristine and prednisone. 12 months later, the patient developed multiple extramedullary plasmocytoma lesions in the skin. After treatment with cyclophosphamide, vincristine, doxorubicin and prednisone, another remission was achieved. The literature on the clinical features and the response to chemotherapy of primary plasma cell leukemia is reviewed.

Cell markers in hairy cell leukemia studied in cells from 51 patients.

To determine the maturation arrest of the neoplastic cells of hairy-cell leukemia (HCL) and the spectrum of the surface markers on these cells, a series of 51 patients with this disease was studied. The cells of all but two of the patients showed monoclonal surface Ig with respect to light chains. In about one-third of the cases, only gamma heavy chain determinants were present on the cells; the majority carried multiple heavy chain determinants as documented by the application of different fluorochromes. Two patients each showed two different clones of cells, both of the same light chain type. In one of these two patients, two paraproteins were present in the serum. Intracytoplasmic Ig was found in only 4 of 39 cases, in all instances being IgM. All cases studied concerned cells with FclgG receptors; however, the density of this receptor varied. FcIgM receptors also showed a spectrum of density, with some cases showing very few FcIgM-positive cells. Receptors C3 were not observed on the hairy cells. Serum immunoglobulin levels were normal or increased. Paraproteins were found in the sera of 4 of 38 patients. These data suggest that HCL is a neoplasm of B lymphocytes. The neoplastic cells are probably arrested at a more mature stage than the cells of chronic lymphocytic leukemia. The multiple isotypes on the cells indicate a block at the "switch" phase from the small micro-carrying lymphocyte to the larger Ig-producing lymphocyte or plasma cell.

Multiple heavy chain isotypes on the membrane of the small B lymphocytes in human blood.

Small lymphocytes from adult human blood were examined for the presence of membrane-associated alpha, gamma, delta and mu Ig isotypes by means of a direct immunofluorescence technique. Since less than 10% of the small lymphocytes in blood are B cells as defined by positive reactivity with an anti-Fab conjugate, our experiments were performed on a T cell-depleted fraction in which about 80% of the small lymphocytes were B cells. With the two-wavelength immunofluorescence method, all of the double-isotype combinations were found. The percentage of cells bearing more than two isotypes was deduced. The delta mu and the alpha delta mu combinations were the most common, as previously found on tonsillar lymphocytes. In contrast with the tonsils, no lymphocytes bearing only delta were observed and the proportions of alpha gamma- and alpha gamma mu-bearing lymphocytes were very small. The presence of lymphocytes bearing four isotypes could practically be excluded.

The specificity of commercial conjugates to human immunoglobulins: results of performance tests on monoclonal and polyclonal plasma cells and lymphocytes.

The activity and the specificity of 29 fluorochrome conjugated antisera against human immunoglobulin heavy and light chains were evaluated by performance testing with the direct technique of immunofluorescence using plasma cells and lymphocytes as biological substrates. Fifteen conjugates gave satisfactory results in the detection and classification of cytoplasmic and surface bound immunoglobulins and were therefore considered specific. Fourteen conjugates did not meet the required standards. The usefulness of the biological substrates for quality control tests is discussed.

Identification of mononuclear cells in human blood. I. Qualitative and quantitative data on surface markers after formaldehyde fixation of the cells.

The technical details of a fixation procedure with formaldehyde which was applied in a direct membrane immunofluorescence technique to mononuclear cells from normal human blood are described. After separation of the cells with Ficoll--Isopaque according to Böyum (1963) they were washed and fixed with 0 . 04% formaldehyde in PBS for 10 min and washed again. This cell suspension can be stored at 4 degrees C for at least 24 hr and the slides prepared from them at -20 degrees C for at least some months. In practice, this fixation procedure not only appeared to be effective in the preservation of cells but also showed a number of additional advantages, such as the short handling period, including the fixation procedure and the avoidance of loss of cells. Moreover, true B lymphocytes, as defined by the synthesis of immunoglobulins and the incorporation of these molecules into their cell membrane, are recognized convincingly.

Identification of mononuclear cells in human blood. II. Evaluation of morphological and immunological aspects of native and formaldehyde-fixed cell populations.

The presence of surface-associated immunoglobulins and Fc receptors on mononuclear cells from normal human blood was investigated by the direct immunofluorescence technique combined with phase-contrast microscopy. Formaldehyde-fixed cells were compared to unfixed cells and to cells preincubated at 37 degrees C. In the unfixed samples a separate population which showed Fc receptors in an immunofluorescence technique using a labelled antigen--antibody complex was detected. This cell population showed an atypical, i.e. not clearly membrane-associated, pattern of fluorescence with anti-Fab conjugates. This interaction most probably is due to autologous IgG molecules taken up by these cells from the donor serum. Using phase-contrast microscopy, these cells were morphologically distinct from lymphocytes and mature monocytes. They will be referred to as 'undefined mononuclear cells' (UMC). After formaldehyde fixation or preincubation at 37 degrees C the interaction of the UMC with anti-Fab conjugates could no longer be demonstrated. Mature monocytes show the same atypical fluorescence pattern with anti-Fab conjugates, but in contrast to the UMC the interaction persists after formaldehyde fixation or preincubation at 37 degrees C. No evidence was found for passive uptake of labelled IgG from conjugates by any mononuclear cell F(ab')2 fragments of IgG from antisera gave results similar to those obtained with intact IgG fractions. The morphology of the different cell subpopulations is described and their relative numbers in normal blood are given. Formaldehyde fixation proved to be a simple and useful procedure, especially for the determination of the number of B lymphocytes, because the Fc receptor of the undefined mononuclear cell does not give rise to confusion.

Presence of multiple isotypes on the surface of human tonsillar lymphocytes.

Formaldehyde-fixed small lymphocytes from human tonsils were investigated for the presence of the Ig heavy chain class isotypes alpha, gamma, delta and mu on their membranes by means of a direct immunofluorescence technique. About 50% of the small lymphocytes were defined as B cells with an anti-Fab antiserum. Most of the B lymphocytes carried more than one isotype on their membrane, and all combinations of two isotypes were observed. The existence of three or four isotypes on the membrane of individual lymphocytes was deduced. The sIgD, sIgM and sIgA, sIgD, sIgM combinations were the most common. The isotypes were present as separate molecules since they showed independent distribution patients undeesent as separate molecules since they showed independent distribution patterns under capping conditions.

Distinct subtype within the spectrum of hairy cell leukemia.

Most cases of hairy cell leukemia represent malignancies of B cells. However, recent findings suggest that there is a spectrum of functional capacities within the entity hairy cell leukemia. Two patients with hairy cell leukemia, whose malignant cells in the peripheral blood showed both T- and B-cell features, are reported. The malignant cells of the spleens showed only B-cell characteristics. The hairy cells of both patients did not adhere to glass and lacked the la antigen. Both patients showed pronounced polyclonal hypergammaglobulinemia and developed frank leukemic blood pictures after splenectomy. Within the spectrum of hairy cell leukemia, these two cases probably represent a distinct subtype.