A Scanning Electron Microscopy and Immunological Study of 84 Cases of Lymphacytic Leukaemia and Related Lymphoproliferative Disorders

The surface features of cells from 84 cases of lymphocytic leukaemia, and related lymphoproliferative disorders are described as seen by scanning electron microscopy (SEM). Most of the 46 cases of CLL were shown to be B-derived, but rare cases of mixed B and T cell leukaemia and leukaemia with cells bearing both B and T markers were also encountered. Despite the existence of a spectrum of cell surface morphology, it was possible in many cases to identify a dominant cell type. Cells from cases of B derived malignancies were most frequently of the ‘predominantly villous’ type while a smaller proportion of cases were of the predominantly ‘smoother’ or ‘mixed villous and smooth’ type. Variations in surface morphology also occurred with progression of the disease. In most cases of acute lymphoblastic leukaemia (ALL) ‘smoother’ cells predominated. However, more cases of ALL and T derived leukaemia need to be examined before definite conclusions can be drawn concerning the surface of these cell types. This study also illustrates the importance of examining large numbers of cases of leukaemia, before conclusions are drawn concerning their surface features and indicates that SEM cannot consistently distinguish between leukaemic B and T cells. It will be of interest to determine whether the surface architecture of the leukaemic cell is related to the degree of cell differentiation and eventual prognosis in these cases.     

Scanning electron microscopic study of leukemic human B lymphocytes.

Peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL), lymphoplasmacytoid lymphoma, centrocytic lymphoma and hairy cell leukemia were studied by scanning electron microscopy (SEM). In general, SEM revealed rather homogenous cell populations. Most lymphocytes displayed a moderately villous surface architecture, although smooth surfaces predominated in 3 cases with CLL and in 1 case with lymphoplasmacytoid lymphoma. Hairy cells showed surface features of both lymphocytes and monocytes. The results indicate that leukemic B and T lymphocytes cannot be distinguished by SEM alone.     

Hairy cell leukaemia: effects of phorbol ester on cell morphology and adherence and comparison with other B-cell neoplasias

This report documents phorbol ester-induced changes in cell morphology in 9 patients with hairy cell leukaemia (HCL) and contrasts them with those obtained in cells from 20 patients with B-type chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma in leukaemic phase (NHL). The phorbol ester caused hairy cells to adhere strongly and produce thin elongated cytoplasmic extensions. Other cells showed marked surface ruffling, and contained increased numbers of phagolysosomes, although cells still retained ribosomal lamellar complexes. This in vitro growth pattern differed from that observed in B-CLL and NHL cells, which readily detached in clumps with minimal adherence. CLL and NHL-cells did not show macrophage features but developed plasmacytoid and hairy cell-like features. It is suggested that these different growth patterns may aid in distinguishing HCL from other B-cell neoplasias.     

Prolymphocytic Leukaemia: an Ultrastructural Study of 22 Cases

. Twenty-two cases of prolymphocytic leukaemia (PLL) have been studied by transmission electron microscopy (TEM); 17 had B-cell surface markers (B-PLL) and five had T-cell characteristics (T-PLL). The predominant cell, the prolymphocyte, has distinct features which were common to all cases: it is a relatively large lymphoid cell with a prominent nucleolus, well condensed peripheral nuclear chromatin and a variable amount of heterochromatin in intranuclear clumps.
Eight cases of PLL (seven B-PLL, one T-PLL) showed minor morphological deviations from typical PLL which may present diagnostic difficulties when studied only at light microscopy: the cells in four of these had some features in common with those of chronic lymphocytic leukaemia (CLL), namely round regular nuclei and heavy chromatin condensation, but the nucleoli were larger in the PLL cases. In four other cases nuclear clefting was a prominent feature at TEM.
B-PLL can be distinguished by ultrastructural features from other B-cell malignancies, e.g. follicular lymphoma, B-CLL and B-acute lymphoblastic leukaemia.     

Surface features of Sezary cells: A scanning electron microscopy study of 5 cases.

The surface features of circulating cells from 5 patients with typical Sezary's Syndrome (SS) are described using scanning electron microscopy (SEM). Sezary cells prepared by different methods, with and without prior fixation in cell suspension, showed similar surface architectures. SS cells were mostly spherical and moderate to markedly villous in appearance, and in this respect, resembled the majority of circulating lymphocytes from patients with chronic lymphocytic leukaemia (CLL). A proportion of cells were larger and more irregular in shape while others had small extensions of cytoplasm resembling small uropods with clusters of polarised microvilli. Despite the latter findings, most SS cells cannot be distinguished from CLL cells on the basis of their surface architecture under the SEM.     

Surface Features of Sezary Cells: A Scanning Electron Microscopy Study of 5 Cases

The surface features of circulating cells from 5 patients with typical Sezary's Syndrome (SS) are described using scanning electron microscopy (SEM). Sezary cells prepared by different methods, with and without prior fixation in cell suspension, showed similar surface architectures. SS cells were mostly spherical and moderate to markedly villous in appearance, and in this respect, resembled the majority of circulating lymphocytes from patients with chronic lymphocytic leukaemia (CLL). A proportion of cells were larger and more irregular in shape while others had small extensions of cytoplasm resembling small uropods with clusters of polarised microvilli. Despite the latter findings, most SS cells cannot be distinguished from CLL cells on the basis of their surface architecture under the SEM.     

''Hairy'' Cell Leukaemia (Leukaemic Reticuloendotheliosis): a Scanning Electron Microscopic Study of Eight Cases

Eight cases of 'hairy' cell leukaemia (leukaemic reticuloendotheliosis) were evaluated by scanning electron microscopy. The surface of critical-point-dried 'hairy' cells was characterized by prominent and exaggerated broad-based, ruffled membranes and scattered small clusters of stub-like microvilli. The surface morphology resembled that of normal and leukaemic monocytes, but differed from that of normal and leukaemic lymphocytes. Some cells with features of both lymphocytes and monocytes were difficult to categorize; the overall impression of the surface architecture of most 'hairy' cells suggests, however, that they are related to the monocytic series. From the examination of these cases it is evident that scanning electron microscopy may be used as a means of distinguishing chronic lymphocytic leukaemia from 'hairy' cell leukaemia on the basis of surface ultrastructure.     

Acute lymphoblastic leukemia: A study of 25 cases by scanning electron microscopy

Summary Cells from 25 cases of acute lymphoblastic leukemia (ALL) were studied under the scanning electron microscope (SEM). In 24 of the cases, the vast majority of circulating leukaemic cells had few microvilli. Villous cells were rarely encountered and prominent ridge-like profiles and ruffled membranes were not seen. Only six cases were studied by immunological techniques and four of the cases were of the null type while in two the cells bore detectable T-markers. It seems that ALL is almost always associated with the presence of cells with few microvilli in the peripheral circulation, differing in this respect from most cases of CLL. Although circulating leukaemic lymphocytes with few microvilli are sometimes seen in CLL, the most frequent cell type encountered is a more villous lymphocyte.Differences between leukaemic cells from patients with ALL, CLL and non-lymphoblastic leukaemias are discussed. It appears that SEM may help to distinguish lymphoblastic and nonlymphoblastic leukaemic cells in many instances and can be used as a useful adjunct to other modes of microscopy in the diagnosis of acute leukaemia.

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Zusammenfassung Bei 25 Patienten mit akuter Lymphoblasten-Leukämie (ALL) wurden die Leukämiezellen mit Hilfe des Raster-Elektronenmikroskops untersucht. Bei 24 Patienten fanden sich überwiegend glatte Zellen mit nur wenigen Mikrovilli. Bei diesen Patienten wurden nur wenige zottige Zellen gefunden; Zellen mit prominenten Streifenprofilen und gefalteten Membranen wurden nicht beobachtet. 6 Fälle wurden zusätzlich mit immunologischen Techniken untersucht; in 2 Fällen waren T-Zell-Marker nachzuweisen, 4 gehörten zur sogenannten Null-Fraktion. Die ALL zeigt also fast immer glatte Zellen, im Gegensatz zu den meisten Fällen von chronisch-lymphatischer Leukämie (CLL). Bei der letzteren sind zwar gelegentlich auch glatte Zellen zu sehen; häufigster Zelltyp ist jedoch eine stärker mit Zotten versehene Zelle.Die Unterschiede der Zelloberfläche bei CLL, ALL und anderen Leukämien werden besprochen. Die Raster-Elektronenmikroskopie erlaubt in vielen Fällen eine Unterscheidung zwischen lymphatischen und nicht-lymphatischen Zellen und ist eine wertvolle Ergätnzung zu anderen mikroskopischen Methoden bei der Leukämie-Diagnose.

     

Effects of Prednisolone in 6 Cases of Glucocorticoid Receptor-Positive CLL

The effect of treatment with prednisolone on clinical and laboratory variables was studied in 6 patients with chronic lymphocytic leukaemia (CLL). The laboratory variables analyzed on the leukaemic cells were surface immunoglobulin (S-Ig), sheep red blood cell receptors (SRBC), proliferative activity (PI) and glucocorticoid receptor (GR). In all cases the CLL was of B-cell type and the leukaemic cells contained a significant amount of GR. 4 out of 6 patients had a progressive disease with increased PI. On treatment they went into clinical remission, which was paralleled by a reduction in the leukaemic B-cell number and in PI. The amount of GR was unaffected. In 2 patients with nonprogressive disease, prednisolone produced no clearcut effect on clinical or laboratory variables.     

The relationship between chronic lymphocytic leukaemia and prolymphocytic leukaemia

The peripheral blood WBC size distribution was assessed by morphological and volume measurements in 73 patients with B-cell chronic lymphocytic leukaemia (CLL) and prolymphocytic leukaemia (PLL). Patients with typical CLL, with less than or equal to 10% prolymphocytes, had a homogeneous major population of small cells which could be recognized both by morphology and volume (median volume 211.5 +/- 32.5 fl). In PLL, the volume of the main cell component was significantly larger than in CLL: in two-thirds of cases the major cell population was distributed in a first lognormal fitted curve of the volume histogram, with median volume 281.8 +/- 38.0 fl; in the remaining cases the main cell component showed a larger median volume (353.5 +/- 71.9 fl) contained within the second lognormal curve which was preceded by a minor peak. CLL patients with 11-55% prolymphocytes (CLL/PL) had characteristic cell volume histograms in which two lognormal curves could always be fitted: in 80% of cases the main cell component was located in a first curve with median volume of 257.9 +/- 28.6 fl; in the remaining cases the major population was represented by cells with median volume of 349.0 +/- 83.9 fl distributed in a second peak. Although in both CLL and CLL/PL the majority of cells was defined morphologically as small, the median volume of these lymphocytes was significantly larger in CLL/PL. The degree of concordance in the assessment of cell size between morphology and volume measurements was high in CLL, whereas in CLL/PL and PLL morphology underestimated the cell size of the major population, compared with its actual volume, in over 50% of cases. We conclude that the identification of prolymphocytes as larger cells in blood films may be hampered by distortions and artefacts of spreading. Volume measurements may provide a more objective indicator of the cell populations in this group of disorders.     

Altered expression of metabolic pathways in CLL detected by unlabelled quantitative mass spectrometry analysis

Chronic lymphocytic leukaemia (CLL) remains the most common incurable malignancy of B cells in the western world. Patient outcomes are heterogeneous and can be difficult to predict with current prognostic markers. Here, we used a quantitative label-free proteomic technique to ascertain differences in the B-cell proteome from healthy donors and CLL patients with either mutated (M-CLL) or unmutated (UM-CLL) IGHV to identify new prognostic markers. In peripheral B-CLL cells, 349 (22%) proteins were differentially expressed between normal B cells and B-CLL cells and 189 (12%) were differentially expressed between M-CLL and UM-CLL. We also examined the proteome of proliferating CLL cells in the lymph nodes, and identified 76 (~8%) differentially expressed proteins between healthy and CLL lymph nodes. B-CLL cells show over-expression of proteins involved in lipid and cholesterol metabolism. A comprehensive lipidomic analysis highlighted large differences in glycolipids and sphingolipids. A shift was observed from the pro-apoptotic lipid ceramide towards the anti-apoptotic/chemoresistant lipid, glucosylceramide, which was more evident in patients with aggressive disease (UM-CLL). This study details a novel quantitative proteomic technique applied for the first time to primary patient samples in CLL and highlights that primary CLL lymphocytes display markers of a metabolic shift towards lipid synthesis and breakdown.     

A preliminary study of low-dose splenic irradiation for the treatment of chronic lymphocytic and prolymphocytic leukaemias

21 patients, 18 with chronic lymphocytic leukaemia (CLL) and 3 with prolymphocytic leukaemia (PLL), all B-lymphocyte origin and with progressive disease, were treated with a regime of low-dose splenic irradiation (SI). All patients experienced a rapid relief of disease-related symptoms. Following SI the total lymphocyte count (TLC) was markedly reduced in 18 patients. Partial to complete regression of splenomegaly occurred in 9 patients. Pre-existing anaemia of production failure type improved in 6 patients and as a result the haemoglobin rose to normal or near normal levels. SI caused the loss of T-, B- and Null-lymphocytes, but the loss of B-lymphocytes predominated. CLL/PLL became quiescent for long periods in 9 patients (CLL = 8; PLL = 1) but remained progressive in the other 9, all CLL. SI had no demonstrable effects on TLC, splenomegaly or anaemia in the 3 remaining patients (2 CLL, 1 PLL). The treatment was well tolerated by all, and side effects were almost absent. Transient reduction of neutrophils and platelets occurred commonly. No patient with initially normal neutrophil and platelet counts developed irreversible neutropenia or thrombocytopenia. In view of these effects, the ease of administration and the lack of side effects, further evaluation of low-dose SI, particularly in comparison with other regimes, seems worthwhile.     

The relationship between chronic lymphocytic leukaemia and prolymphocytic leukaemia

The clinical and laboratory features of 300 patients with chronic lymphocytic leukaemia (CLL) and prolymphocytic leukaemia (PLL) of B-cell type were studied in order to investigate the relationship between these two diseases. Statistical analysis demonstrated that more than 55% circulating prolymphocytes (PROL) was a defining criterion for PLL, disorder characterized by marked splenomegaly without lymph-node enlargement, cells with high density of membrane-immunoglobulin (SmIg), low mouse-rosettes (M-rosettes) and strong reactivity with the monoclonal antibody FMC7. Patients with typical CLL, defined as having less than 10% PROL, were on average 10 years younger than those with PLL and showed preferential lymph-node to spleen involvement. Characteristic markers of CLL were weak SmIg, high M-rosettes and low reactivity with FMC7. Patients with 11-55% PROL, group designated as CLL/PL, were found to have intermediate features between CLL and PLL: the degree of splenomegaly was disproportionate to the lymph-node enlargement, the number of cases with strong SmIg was closer to that found in PLL, but the other markers were not significantly different from CLL. The CLL/PL group appeared to be heterogeneous and includes at least two types of CLL, one with increased proportions of PROL but otherwise typical disease, and another in 'prolymphocytoid' transformation. Our study suggests that although PLL cannot be considered as the extreme end of a continuous spectrum from typical CLL, the spleen may be the source of PROL both in PLL and in CLL/PL.     

Monoclonal antibodies against the chronic lymphatic leukemia antigen cCLLa: characterization and reactivity

Monoclonal antibodies (MoAbs) were developed against the cCLLa, a 69-kilodalton leukemia-associated antigen expressed on malignant cells of B-type chronic lymphatic leukemia (B-CLL) and its variants: prolymphocytic (PLL) and hairy cell leukemias (HCL). Two hybridomas yielded approximately 2 and approximately 7.5 mg/mL of IgG2a kappa and IgM kappa, respectively. Monoclonal surface immunoglobulin-bearing cells of all B-CLL patients studied (n = 30) reacted with the MoAbs (r greater than .99) regardless of stage or lymphocyte count. This suggests that the malignant clone in CLL can be identified and its size monitored by using our MoAbs. In contrast, normal B lymphocytes, a large panel of normal, reactive and neoplastic cells, and malignant cell lines failed to react with either MoAb as judged by indirect immunofluorescence and by flow cytometry. Only two patients (one with non-Hodgkin's lymphoma, the other with acute myeloblastic leukemia) exhibited a small cell subset reactive with the MoAbs. cCLLa specificity was suggested by selective target cell reactivity and competitive inhibition-absorption and confirmed by immunoprecipitation. MoAbs IgG2a kappa and IgM kappa appeared to share antigenic determinants and were moderate and avid complement binders inducing 100% and 40% target cell lysis, respectively. cCLLa density on malignant CLL and HCL cells was estimated by equilibrium binding studies using the IgG2a kappa MoAb at 1.7 and 9 X 10(6)/cell, respectively. The restricted expression of the cCLLa and the specificity and cytolytic activity of the anti-cCLLa MoAbs support these antibodies as probes for the classification of lymphoproliferative diseases and for the specific diagnosis and treatment of B-CLL and its variants.     

The relationship between chronic lymphocytic leukaemia and prolymphocytic leukaemia

A series of 55 patients with B-cell chronic lymphocytic leukaemia (CLL) and prolymphocytic leukaemia (PLL) was studied in order to investigate the evolution of prolymphocytoid transformation of CLL. Peripheral blood films were assessed for the percentages of prolymphocytes (%PROL) during a followup period of 2 months to 24 years. The majority of patients with typical CLL (less than or equal to 10% PROL) had minor variations or transient increases in %PROL throughout the course of the disease, but in one third of them a steady rise in the proportion of these cells was documented. Patients presenting with 11-55% PROL, referred to as CLL/PL, exhibited three patterns of evolution: half of them showed stability of the PROL counts, one third had unsustained increases in the %PROL and 18% showed definite progression to a PLL-like blood picture. Patients diagnosed as PLL (greater than 55% PROL) had maintained a high %PROL during the period of observation. Serial marker studies in some CLL and CLL/PL cases showed that the percentage of M-rosettes and the intensity of SmIg remained at the same initial levels in all but two cases. These two cases in the CLL/PL group showed a significant decrease in the percentage of M-rosettes and stronger SmIg staining associated with progressive prolymphocytoid transformation. Patients with CLL and CLL/PL who had a sustained rise in the %PROL responded poorly to treatment compared with those with stable disease.     

Reactivity of the monoclonal antibody RFB1 in chronic B-cell leukaemias

The monoclonal antibody RFB1, which reacts with early haemopoietic precursors in human bone marrow and peripheral T-lymphocytes, but not with pre-B cells and mature peripheral blood and marrow B lymphocytes, was tested in blood from 60 patients with chronic B cell leukaemias. All 37 cases of B chronic lymphocytic leukaemia (B-CLL) and 9 of hairy cell leukaemia (HCL) were positive. The antibody showed particularly strong reaction in HCL. On the other hand, RFB1 did not react with peripheral blood and B lymphocytes from 9 patients with non-Hodgkin lymphoma (NHL) in leukaemic phase and was negative or weakly expressed in 5 with prolymphocytic leukaemia (B-PLL). These findings may be significant for investigations on the cell origin of B-CLL and HCL. The reactivity of RFB1 was different from that of two anti-T1 monoclonal antibodies. The combined use of these reagents gave distinct patterns in B-CLL (RFB1+, T1+), HCL (RFB1++, T1-) and NHL and B-PLL (RFB1-/±, T1-/+), suggesting they may be of value in the differential diagnosis of these B-lymphoproliferative disorders.     

Immunophenotyping of low-grade B-cell lymphoma in blood and bone marrow: poor correlation between immunophenotype and cytological/histological classification

Results of immunophenotypic examinations of peripheral blood and/or bone marrow (BM), involved in low-grade B-cell non-Hodgkin's lymphomas, were compared with the results of cytomorphological and histopathological examinations in 133 adult patients. 69 cases of chronic B-lymphocytic leukaemia (B-CLL), 16 centrocytic (CC) lymphomas, 14 centroblastic-centrocytic (CB/CC) lymphomas, 15 immunocytomas (IC), 10 cases of hairy cell leukaemia (HCL), four prolymphocytic leukaemias (PLL), two B-CLL in transformation, one splenic lymphoma with villous lymphocytes (SLVL), one hairy cell leukaemia variant (HCL-V), and one lymphocytic lymphoma (LC) were classified according to the Kiel and /or FAB classification. Leukaemic disease was found in 105 cases. The following markers were used for immunocytology (APAAP technique) of blood and/or BM smears: CD 19, CD 5, CD 10, CD 11c, CD 14, CD 21, CD 22, CD 23, CD 25, CD 38 and TdT. All cases tested showed CD 19, but no TdT expression. Every case of HCL had a distinct phenotype with expression of CD 11c, CD 22 and CD 25 and the lack of CD 5 and CD 23 antigens. In all other NHL cases a very heterogenous expression of CD-antigens with no significant correlations to the cytomorphological subtypes was found. The expression of CD 5 is a frequent but inconstant finding in lymphoproliferative diseases other than B-CLL, so 50% of CB/CC, 75% of CC and 80% of IC were CD 5 positive. Our results indicate that, with the exception of HCL, the diagnostic relevance of immunophenotyping for the classification of cytomorphologically and histopathologically defined subtypes in blood and/or BM is of very limited value.     

Non-radioactive in situ hybridization for the detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukaemia

Non-radioactive in situ hybridization (NISH) with a chromosome 12-specific alpha satellite probe was performed on 20 patients with chronic lymphocytic leukaemia (CLL) with normal karyotype (15 cases) or with inadequate mitotic yield (5 cases) from mitogen-stimulated cultures. All patients had over 70% lymphocytes coexpressing the CD5/CD23 antigens. While less than 1% interphase nuclei showed three fluorescent spots in 16/20 patients, evidence of trisomy 12 in 15-25% interphase cells was detected in four patients. According to the FAB classification the diagnosis in these patients was typical B-CLL, stage III (Rai's staging system) in one case, CLL/PLL, stage II and III in two cases, PLL, stage III in one case. In order to confirm these results, NISH was repeated after 1 month in one patient and after 2 years in three patients. All patients had been treated with chemotherapy in the period between the two NISH experiments. In all cases a 1.8-3-fold increase of percentage of trisomic interphase cells was detected. These findings suggest that in B-CLL clones with trisomy 12 may have proliferative advantage over clonal B-lymphocyte without +12 and, possibly, that they may be more resistant to chemotherapy. We conclude that NISH is a sensitive technique allowing for the detection and monitoring of trisomy 12 in a fraction of B-CLL patients with normal karyotype or with no analysable mitoses despite employment of polyclonal B-cell mitogens.     

Quantitative analysis of CD79b, CD5 and CD19 in mature B-cell lymphoproliferative disorders.

BACKGROUND AND OBJECTIVE: Distinction between B-cell chronic leukemias can be difficult due to overlap in cell morphology and immunologic features. We investigated, by quantitative flow cytometry, the expression of CD79b, CD5 and CD19 in cells from a variety of B-cell disorders to see whether this analysis adds further information useful to the diagnosis and characterization of these diseases. DESIGN AND METHODS: Peripheral blood cells from 6 normal individuals were used as reference controls. The diseases of the 63 patients investigated comprised: 29 chronic lymphocytic leukemia (CLL), six of them with atypical morphology, 6 B-cell prolymphocytic leukemia (PLL), 12 splenic lymphoma with villous lymphocytes (SLVL) and 16 mantle-cell (Mc) lymphoma in leukemic phase. The study was carried out by triple immunostaining with directly conjugated monoclonal antibodies (MoAb) against CD79b, CD5 and CD19 and quantitative estimation of the antigens per cell assessed with standard microbeads (Quantum Simply Cellular). RESULTS: Compared to normal B-cells, the number of CD19 molecules was significantly lower in cells from all of the B-cell disorders except PLL. The intensity of CD5 in leukemic B-cells was significantly higher in CLL cells, including atypical cases, and Mc lymphoma than in normal B-cells, whilst PLL and SLVL had values similar to those of normal B-lymphocytes. CD79b was expressed at lower levels in all types of leukemic cells compared to normal B-lymphocytes but differences were statistically significant in CLL, Mc lymphoma and SLVL. The number of CD79b molecules per cell was significantly lower in typical CLL than in the remaining B-cell diseases whilst the comparison of CD5 and CD19 intensity between CLL and non-CLL samples failed to show any statistically significant difference. INTERPRETATION AND CONCLUSIONS: Distinct antigen density patterns for the various conditions emerged from this analysis: Typical CLL was characterized by moderate CD5 and weak or negative CD79b expression. Mc lymphoma showed an homogeneous pattern, characterized by similar expression of CD5 than CLL but significantly stronger expression of CD79b whilst PLL and SLVL had weak CD5 and moderate CD79b expression. Atypical CLL had an intermediate pattern of CD79b antigen expression ranging from weak to moderate with bright CD5. Unlike CD5 and CD79b, CD19 did not discriminate the various B-cell disorders but only between normal and leukemic cells.