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Six thymocyte suspensions, 10 normal blood CD4(+) CD8(-) lymphocyte-enriched fractions and leukaemic cells from 24 patients with CD4(+) mature T-cell lymphoid malignancy (five Sezary Syndrome, six adult T-cell leukaemia-lymphoma and 13 cases of T-cell prolymphocytic leukaemia) were examined in this study for the expression of membrane HLA-ABC by CD45RA (2H4) and CD45RO (UCHL1) subpopulations. These analyses showed that the main increase in HLA-ABC expression by normal CD4(+) CD8(-) blood lymphocytes (mean 490 to 760 FITC units) paralleled the loss of membrane 2H4 whilst the acquisition of UCHL1 was not associated with any significant change in HLA-ABC staining intensity. The sequence of 2H4 differentiation by normal thymocytes, based on the observed increasing levels of HLA-ABC staining intensity appeared to be (a) CD1a (+) 2H4(-) UCHL1(+) (25 HLA-ABC fluorescent units), (b)CD1a(-)2H4(int)UCHL1(+) (134 units), and (c) CD 1a(-) 2H4 (+) UCHL1 (-) (197 units). Quantitative estimates of membrane HLA-ABC expression by leukaemic T-cells revealed marked heterogeneity between individual cases irrespective of diagnostic subgroup. Based on the lower observed limits for normal CD4(+) 2H4(+) (318 units) and CD4 (+) 2H4(-) (478 units) fractions, 14% and 38% respectively of the leukaemic 2H4(+) and 2H4(-) components examined showed reduced HLA-ABC expression. Two cases showed very low membrane HLA-ABC levels that were within the range observed for normal CD1a(-) thymocytes. In contrast, HLA-ABC staining intensities exceeding that of corresponding normal CD4(+) 2H4(+) (710 units) and CD4(+) 2H4(-) (1286 units) subpopulations were seen in a high proportion (65%) of leukaemic 2H4 (+) components, with only 14% of 2H4(-) fractions showing raised levels and, in two cases, these staining intensities exceeded three times the normal observed limits. In addition to the quantitative differences in HLA-ABC expression, a remarkably consistent (81% of evaluable cases) feature of the leukaemic T-cells was that the 2H4(-)UCHL1(+) subpopulation in CD4(+) malignancies had a lower HLA-ABC level than the 2H4(+)UCHL1 subpopulation. This was in marked contrast to normal post-thymic T-cells where increasing HLA-ABC expression was seen with increasing UCHL1 (or decreasing 2H4) staining. These results suggest that leukaemic T-cells have an aberrant intra-thymic and post-thymic sequence of 2H4/UCHL1 expression which has become 'uncoupled' from CD1a/HLA-ABC expression.

This communication reports a case of T-cell acute lymphoblastic leukaemia (T-ALL) which, after treatment and remission induction, relapsed 17 months after apparent disease-free remission as acute myeloblastic leukaemia (AML). Extensive immunophenotypic, analysis an initial presentation revealed a typical T-ALL phenotype (HLA-Dr(-), TdT(+), CD2(+), CD3(+), CD4(-), CD5(+), CD7(+), CD8(-), CD19(-), CD13/33(-)) that was unusual in that the majority of blasts expressed membrane TCR γδ chains but not TCR αβ chains. Similarly, the morphological (Auer rods +), cytochemical (MPO(+) and SBB(+)) and phenotypic characteristics at relapse were unequivocably consistent with a diagnosis of AML. In contrast to previous case studies of "phenotypic switch" or "metachronous bilineal leukaernia", Southern blot analysiis of TCR and Ig gene rearrangements at presentation and relapse were able, because of an unusual genotypic pattern (TCR β +, TCR γ +, TCR δ + and JH), to confirm the common clonal origin of the two leukaemias. It is suggested that the transition from T-ALL to AML may not be a "random" phenomenon but may rather reflect subclonal domination by mycloid blasts with relative drug resistance (i.e. synchronous bilineal differentiation with an undetectable myeloid component), or therapy-induced alterations in the balance of (myelosuppressive) factors.

Expression of peripheral blood neutrophil (PBN) C3b receptors, as assessed by rosette formation with C3b-coated ox erythrocytes, was examined and compared with neutrophil alkaline phosphatase (NAP) activities in both normal and haematologically abnormal conditions. The results indicate that a small percentage of normal PBN are apparently C3b receptor-negative and that these neutrophils do not appear to differ with respect to age from those with detectable C3b receptors. Examination of PBN C3b receptors from 154 cases of various haematological disorders revealed a significant proportion of cases with increased numbers of C3b receptor-negative neutrophils. These abnormalities did not appear to be related to peripheral leucocyte counts, NAP activities or serum lysozyme concentrations and it is suggested that the increased numbers of C3b receptor-negative PBN may be related to intravascular factors such as immune complexes.

The myelodysplastic syndromes (MDS) are disorders in which the abnormalities are thought to be confined to cells of the myeloid series. However, examination of peripheral blood from 56 patients with MDS showed that the majority had low lymphocyte counts. In a detailed study of 25 patients, using OKT3, OKT4 and OKT8 monoclonal antisera together with SRBC rosettes, it was shown that there is a consistent and significant reduction in peripheral blood T lymphocytes. This decrease was primarily confined to the OKT4-defined helper subpopulation. There is a consequent relative increase in the OKT8-defined subset but in absolute numbers however, suppressor cells are reduced compared to normal particularly in those patients with lymphopenia. No correlations between T cell abnormalities and the different morphological groups of MDS were found. The possible implications of the reversed helper/suppressor ratio in the pathogenesis of MDS are discussed.

This study evaluated the extended use of a haematology analyser (Abbott Cell-Dyn CD4000) for the immunofluorescent enumeration of foeto-maternal haemorrhage (FMH) with fluorescein isothiocyanate-labelled monoclonal anti-RhD. Method performance was assessed with artificial FMH standards, and a series of 44 clinical samples. Within run precision was

Laboratory tests for malaria are only performed if there is clinical suspicion of the disease, and a missed diagnosis contributes substantially to morbidity and mortality. Malaria parasites produce haemozoin, which is able to depolarize light and this allows the automated detection of malaria during routine complete blood count analysis (CBC) with some Abbott Cell-Dyn instruments. In this study, we evaluated the Cell-Dyn CD4000 with 831 blood samples submitted for malaria investigations. Samples were categorized as malaria negative (n = 417), convalescent malaria (n = 64) or malaria positive (n = 350) by reference to thin/thick film microscopy, 'rapid test' procedures, polymerase chain reaction analysis and clinical history. With regard to CD4000 depolarization analysis, a malaria positive CD4000 pattern was ascribed to samples that showed one or more abnormal depolarizing purple events, which corresponded to monocytes containing ingested malaria pigment (haemozoin). Positive CD4000 patterns were observed in 11 of 417, 50 of 64 and 281 of 350 of malaria negative, convalescent malaria and malaria positive samples respectively. The specificity and positive predictive values for malaria (active and convalescent) were very high (97.4 and 96.8%, respectively), while sensitivity and negative predictive values were 80.0 and 83.0% respectively. Depolarization analysis was particularly effective for Plasmodium falciparum malaria but there was lower detection sensitivity for White compared with Black African patients. CD4000 90 degrees depolarization vs 0 degrees analysis revealed a proportion of samples with small nonleucocyte-associated depolarizing particles. Appearance of such events in the form of a discrete cluster was associated with P. vivax rather than P. falciparum infection.

Platelet counts and automated detection of platelet clumps were evaluated by optical analysis with the Abbott CD4000 analyser (Abbott Diagnostics, Santa Clara, CA, USA) in this South African study of 828 samples referred for malaria investigations. Based on microscopy (Micro) and rapid tests (RT) for HRP2 protein and parasite-associated LDH, malaria negative samples (n = 417) were defined as Micro-, RT-. Convalescent cases (n = 64) were Micro-, RT+ and had a recent record of positive microscopy. Malaria positive cases were subdivided into Micro+ (n = 315) and Micro-, RT+, PCR+ (polymerase chain reaction) (n = 32) subgroups. The mean platelet count for Micro+ cases (89.7 x 10(9)/l) was significantly lower than both the malaria negative (mean 212.6 x 10(9)/l) and convalescent malaria (mean 152.8 x 10(9)/l) groups; 89% of microscopy positive cases were thrombocytopenic (< 150 x 10(9)/l) and 30% had severe thrombocytopenia (< 50 x 10(9)/l). For comparison, 32% of the 417 malaria negative samples were thrombocytopenic and 6% of these were severe. Two thirds of samples with parasitaemia above 10% had platelet counts of < 50 x 10(9)/l while the counts were largely independent of parasite numbers when the parasitaemia was below 10%. Thirty percent of samples with microscopically detectable parasites had a PltClmp flag compared to 13% of the malaria negative group but, when the actual platelet count was taken into account, it became apparent that appearance of the flag was primarily associated with thrombocytopenia per se rather than malaria status. In most samples with a PltClmp flag, the CD4000 optical platelet clump 'signature' was indicative of small platelet aggregates and giant platelets. Morphological examination confirmed the presence of varying numbers of small platelet aggregates (3-12 individual platelets), often together with increased giant platelets, in many samples with a PltClmp flag. The observations suggest that while patients with malaria may be predisposed to the development of thrombocytopenia, a reduced platelet count in some patients may also be due in part to pseudo-thrombocytopenia.

This study of Plasmodium falciparum malaria evaluated patterns of fluorescent reticulocyte measurements as determined with the Abbott Cell-Dyn® CD4000. The parasitaemia of positive samples (n = 180) ranged from 0.04% to 25.5%, with those (19/180) showing gametocytes having lower parasitaemia levels (mean 0.31%, median 0.2%) compared to those that did not (mean 2.59%, median 0.8%). There was a reasonable association (R 2 = 0.60) between parasitaemia level and CD4000 reticulocyte percentages, although there was overall a small statistical bias towards higher parasitaemia estimates determined microscopically. Consistently high immature reticulocyte fraction (IRF) values of >0.5 were observed in cases with a parasitaemia exceeding 5%, while samples with lower parasitaemia levels showed more variable IRF values. Visual examination of CD4000 reticulocyte histograms revealed that 81/100 malaria-positive samples with an IRF above 0.5 showed the presence of a fluorescent population 'spike' consistent with the staining of intracellular malaria parasites. Only three of the 80 malaria-positive samples with an IRF below 0.5, and none of the 237 malaria-negative samples, showed this histogram pattern. These observations indicate that samples with malaria parasites give erroneously high CD4000 reticulocyte estimates that essentially comprise the sum total of true reticulocytes and parasite-infected red cells (pseudo-reticulocytes). This limitation is common to all automated reticulocyte procedures but recognizing the differences between homogenous staining parasitized red cells and heterogeneous staining reticulocytes has potential applications in monitoring parasitaemia levels both at patient presentation and during subsequent treatment.

Revision of the current decision point for prophylactic platelet transfusion in thrombocytopenic patients requires the availability of a method that is able to provide accurate platelet counts to as low as 1 x 109/l. This study is the first to evaluate the immunoplatelet method (CD61-Imm) of the haematological analyser Cell-Dyn 4000 in direct comparison with the flow cytometric procedure. Additionally CD61-Imm results were compared with CD4000 optical (Plto) counts in the ranges 20-547 x 109/l (n = 127) and 1-35 x 109/l (n = 107). The immunoplatelet and Plto results were in good agreement between 20 x 109/l and 547 x 109/l, but for samples of < 25 x 109/l the Plto tended to overestimate the counts. We determined the limits of detection (LD) and quantification (LLQ) for all three methods using standard statistical procedures. The LD for the flow cytometric CD41a method was 0.02 x 109/l compared with 0.009 x 109/l and 1.73 x 109/l for the CD61-Imm and Plto methods respectively. The LLQCV = 15% for the CD41a method was 1.8 x 109/l compared with 1.6 x 109/l and 18.0 x 109/l for the CD61-Imm and Plto procedures. In conclusion, (i) the CD61-Imm method performance is at least equivalent to the reference flow cytometric method, and (ii) in severe thrombocytopenia the CD61-Imm count is superior to the Plto count.

This study compared CD4 and CD8 lymphocyte counts obtained by microvolume fluorimetry (MVF) with those derived by flow cytometry (FC). Samples from 192 patients with known or suspected HIV were analysed, and the distribution of CD4 counts for these samples ranged from 0 and 1,279/microl, with 142/192 (74%) of the samples having CD4 values of less than 400/microl. Good agreement between FC and MVF CD4 counts was found (MVF = 0.98 x FC + 7.30) although there was a minor constant inter-method bias of approximately +7 cells/microl for the MVF data. For CD8 counts there was a constant bias between the two methods of approximately +23 cells/microl for FC. Most outliers were associated with higher FC CD8 counts. Supplementary analyses showed a high level of agreement between FC and MVF methods for the CD4:CD8 ratios (MVF = 0.98 x FC). This suggests that observed discrepancies between FC and MVF methods were almost certainly a result of the influence of the absolute lymphocyte counts obtained from the haematology analyser. The results confirm that the IMAGN 2000 microvolume fluorimeter system can be used as an alternative to conventional flow cytometry for the enumeration of CD4 and CD8 counts.

This paper reports an evaluation of the Abbott CD35OO instrument in the haematological analysis of blood samples from 277 thoroughbred horses and foals. The age range studied was 20 days to more than 4 years, and CD35OO data were compared to those obtained from both the Coulter S88O and manual determinations of the leucocyte differential. The results showed excellent agreement (p>0.965) between the CD35OO and the S880 instruments for haemoglobin concentration, red blood cell (RBC) count, mean cell volume (MCV) and haematocrit (Hct). For the RBC count and MCV, however, the CD35OO gave slightly lower values than the S880 in the lower range of values and higher counts in the higher range of values. Correlations between the two automated platelet counts were acceptable although there was poorer agreement for platelet counts at higher (>250×109/1) values. In addition, some equine samples showed a higher CD35OO optical (WOC) compared to impedance (WIC) white cell count. This was shown to be due to the presence of lyse-resistant red cells, and further studies confirmed that the CD3500 resistant red cell mode was appropriate for analysis in such circumstances. Correlations between the CD3500 automated and manual differentials were also very acceptable with good correlations being obtained for granulocytes and eosinophils. Absolute CD3500 lymphocyte and monocyte numbers were however respectively lower and higher than determined by the reference procedure even though the total mononuclear cell (lymphocytes plus monocytes) count compared very well. Examination of age-associated variation for the RBC indices in this series of equine samples showed that the red cell count tended to be lowest in the older horses. In addition, there was also a clear trend for increasing MCV and decreasing red blood cell distribution width (RDW) values with progressive age in horses aged one year or older, with the values of these two indices being inversely related. Interestingly, this relationship was reversed in foals whereby during the first part of life, horses showed a decreasing MCV and an increasing RDW with progressive ageing up one year. These results confirm that the CD3500 analyser is well-suited to the analysis of blood samples from both foals and adult thoroughbred horses, and that the automated determination of leucocyte differentials in particular represents a significant advance in veterinary laboratory haematology.

Twenty-one cases of acute promyelocytic leukaemia (FAB M3) demonstrating t(15,17) chromosomal translocation were studied in detail by immunocytochemical techniques using a panel of monoclonal antibodies. A characteristic myeloid phenotype of the leukaemic cells, co-expression of CD9 and CD68 antigens and absence of HLA-DR, was noted in all cases. Although the cellular morphology of acute promyelocytic leukaemia provides the most rapid means for diagnosis, this unique leukaemic cell phenotype provides confirmatory diagnostic evidence. In view of the new therapeutic options and prognosis in acute promyelocytic leukaemia, the detection of cases with atypical morphology based on this unique phenotype would be of value. In addition, the phenotype of acute promyelocytic leukaemia is similar to that of basophils and mast cells and raises the possibility that the leukaemic cells may have undergone a degree of basophilic differentiation.

This communication reports the clinical and cellular features of five elderly female patients with persistent moderate to severe neutropenia and concomitant relative expansions of CD3+TCRγδ+ (n = 4) or CD3+TCRαβ+CD4−CD8− (n = 1) lymphocyte populations. In clinical terms, severe neutropenia was the main contributing factor to patient symptoms although two additionally had long-standing histories of rheumatoid arthritis. The absolute lymphocyte counts did not exceed the normal upper limit in these patients, and morphologically the lymphocytes were not typically of large granular lymphocyte (LGL) type although LGL-associated BLT-esterase staining was consistently increased. Expression of NK-associated (NKa) membrane determinants (CD16, CD56 and CD57) were variable but there was an apparent correlation between weak membrane CD8 and CD 16 expression. DNA genotypic studies confirmed that the four CD3+TCRγδ+ cases were clonal in nature and add further support to an emerging impression that expansions of these ly...

The classification of acute leukaemias is now widely based on a combined morphological, cytochemical and immunophenotyping approach. Difficulties are frequently encountered however in reaching an acceptable degree of diagnostic concordance between different laboratories because of variations in the techniques used (in terms of methodologies, reagents and equipment) and diagnostic interpretation. The International Council for Standardization in Haematology (ICSH) convened an expert panel to consider currently available diagnostic techniques with the aim of defining a minimum cytochemical and immunological diagnostic panel that could be used as core components for the classification of acute leukaemia. The proposed ICSH scheme, which attempts to balance the basic requirement for providing precise and informative diagnostic information without limiting its use to only those laboratories with sophisticated facilities, is based on three sequential levels of investigation; primary cytochemistry, intracellular phenotyping and membrane immunophenotyping. The minimum ICSH recommended cytochemistries comprise myeloperoxidase (MPO), chloroacetate esterase (ChlorE) and alpha-naphthyl acetate esterase (ANAE), and standardised methods for these cytochemistries are detailed in this communication. For cases of acute leukaemia that remain unclassified by primary cytochemistry, subsequent immunological analyses for cytoplasmic CD3, CD22, MPO and nuclear TdT are recommended. The ICSH panel considers that the use of these minimum primary cytochemical and intracellular phenotyping procedures will lead to the consistent classification of most acute leukaemias, and that the third level of investigation (membrane immunophenotyping) should be used for the purposes of confirmation, diagnostic clarification of atypical leukaemias, and the subtyping of acute lymphoblastic leukaemias (ALL). The ICSH panel also recognised that there are a number of additional technologies which can provide definitive diagnostic information, such as cytogenetics and DNA genotyping, but these were excluded from the minimum panel because of their restricted availability. While many specialised laboratories, particularly in the areas of diagnostic research, will continue to use individual investigatory protocols, it is considered that the inclusion of the ICSH scheme as core components would lead to greater consistency when comparing independent studies of acute leukaemia.

Summary Cerebrospinal fluid (CSF) samples (n = 50) from patients with neurological disease (bacterial infection, viral infection, neuroborreliosis and multiple sclerosis) were analysed to characterize cell populations by fluorescent immunocytometry with the CD-Sapphire haematology analyser. Reagent combinations applied to all CSF samples comprised CD3/CD19/HLA-DR and CD4/CD8, with some being further analysed using CD3/CD4, CD3/CD16 and CD3/CD25 protocols. Of the 50 samples, 11 were excluded because of high proportions of nonviable cells (n = 2) or insufficient cell numbers (n = 9). Apart from bacterial infection with granulocytosis, all diagnostic groups showed high proportions (51.4–77.0%) of CD3+ T cells. There was a modest association between T-cell and B-cell counts, but absolute B-cell numbers exceeded 5 cells/μl in only 7/39 cases (neuroborreliosis, n = 6; bacterial meningitis, n = 1). CD3/Ia antigen (activation) co-expression was low and only exceeded 5% in 7/39 samples with no diagnostic correlation. Primary CD4+ and CD8+ T-cell subsets showed similar quantitative trends and CD4/CD8 co-analysis revealed the presence in all diagnostic groups (neuroborreliosis and multiple sclerosis in particular) of a CD4+ CD8int fraction that was predominantly CD3+ and CD16− and had a morphological profile consistent with small lymphoid cells. Supplementary CD-Sapphire cellular immunological analysis of most CSF samples is feasible using the procedure detailed in this communication.

Phenotypic characteristics, and correlations between the expression of membrane NK-associated (NKa) determinants (CD11b, CD16, CD56 and CD57) and T cell receptor (TCR) genotypic patterns, were examined in 25 patients with persistent (greater than 6 months) expansions of CD3+WT31+NKa+ (CD8+ and CD8dim+) lymphocytes. These studies showed that distinct NKa phenotypic profiles were restricted to cases with rearranged TCR configurations and that clonal CD3+NKa+ components could be predicted in most cases by assessing relationships between membrane CD16 and CD56 expression. For all normal NKa subpopulations, there was a high correlation (P less than 0.0001; n = 31) between the expression of these two membrane determinants. Markedly increased CD16 expression by CD3+NKa+ cells, in relation to CD56 (i.e. a high CD16:CD56 ratio), was found exclusively in cases with rearranged TCR (13/16 cases); 2/3 of the remaining cases showing significantly reduced CD16:CD56 ratios and high (greater than 2.0) CD3+CD56+ absolute numbers. In contrast, 7/9 of the germline TCR cases had a normal CD16:CD56 ratio and 2/9 a decreased ratio with low (less than 1.0) CD3+CD56+ absolute numbers. A high ratio of CD16:CD56 expression by CD3+NKa+ lymphocytes was therefore informative for 82% of TCR rearrangements in this series; and analysis of CD16 and CD56 expression was predictive for germline and rearranged TCR configurations in 24/25 persistent CD3+NKa+ expansions.

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.

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.

The platelet count has a primary role in the diagnosis and treatment of idiopathic thrombocytopenic purpura (ITP). This study analysed the accuracy of ITP patient platelet counts determined by Abbott CD-Sapphire (impedance/optical) and Bayer Advia 120 (optical) analyses, compared with a reference immunoplatelet method. Instrument platelet estimates showed broad equivalence in the higher range of observed values, but significant discrepancies against the immunoplatelet count were seen when platelet counts were10 x 10(9)/l. CD-Sapphire mean platelet volume (MPV) results revealed increased (12 fl) platelet volumes in eight of eight ITP patients with counts of20 x 10(9)/l compared with 6/6 and 5/13 patients with platelet counts of 20-50 and50 x 10(9)/l. In contrast, Bayer Advia MPV values showed no relationship with the platelet count. Increased reticulated platelets were associated with an increasing CD-Sapphire MPV (R(2) = 0.61) and a decreasing platelet count. High (40%) reticulated platelet values were seen in 9/9 patients with immunoplatelet counts of20 x 10(9)/l compared with 0/19 patients with platelet counts above 20 x 10(9)/l. There may be a need for caution in the interpretation of platelet counts in ITP patients obtained with conventional instrument methods, and therapeutic decisions should ideally be validated by reference immunoplatelet procedures.

Using single and multiple colour flow cytometry (FACSCAN), this study has examined the expression of NK-associated (NKa) CD11b, CD16, CD56 and CD57 membrane antigens by normal lymphocyte subpopulations. In addition to determining the normal proportions and absolute numbers of NKa(+) cells, this investigation also related the expression of these four NKa markers to the presence of; (a) morphologically-defined large granular lymphocytes, (LGL); and (b) membrane CD2, CD3, CD8 and TCR chain expression. This was achieved by preparing highly enriched normal blood CD4-CD8(+)/CD4 CD8(+) and CD4(-)CD8(-) lymphocyte fractions (n = 6) by immunomagnetic depletion of CD4(+)/CD14(+)/CD19(+) or CD4(+)/CD8(+)/CD14(+)/CD19(+) components respectively. Morphological assessment of these fractions showed that the sequential depletion procedures resulted in the enrichment of LGL, from a mean of 9% for the "whole" lymphocyte (pre-depletion) fraction, to 35% following removal of CD4(+), CD14(+) and CD19(+) cells and to 80%) following the additional removal of CD8(+) cells. FACS analysis revealed the presence of three CD8 subgroups (CD8(-), CD8(dim+) and CD8(+)), defined by differences in membrane staining intensity, and subsequent three-colour (CD2/CD3/CD8) studies indicated that the main composite CD2/CD3 phenotypes for these CD8 groups were (relative frequencies in parenthesis); (a) CD8(+), CD2(+)CD3(+) (94%); (b) CD8(dim+), CD2(+) CD3(+) (31%) and CD2(+)CD3(-) (52%); and (c) CDS(-), CD2(+)CD3(+) (20%), CD2(+)CD3(-) (58%) and CD2(-)CD3(-) (22%). Analysis of paired NKa marker expression (CD11b and CD56; CD11b and CD57; CD16 and CD57), when correlated with CD3 and CD8, showed that (a) the predominant NKa phenotype of CD3(+)CD4(-)CD8(+) cells was CD11b(-)CD16(-)CD56(-)CD57(-); (b) CD3(+)CD4(-)CD8(dim+) and CD3(+)CD4(-)CD8(-) subpopulations showed CD11b(-)CD16(-)CD56(-)CD57(±) and CD11b(±)CD16(-)CD56(-)CD57(-) composite phenotypes respectively (where - and ± denote20% and 21-60%0 NKa(+) positive cells); and (c) the highest proportions of NKa(+) cells were associated with CD3- subpopulations, with composite phenotypes for CD3-CD4-CD8(dim+) and CD3(-)CD4(-)CD8(-) components being CD11b(-)CD16(±)CD56(+)CD57(±) and CD11b(+)CDl6(+)CD56(+)CD57(±) (where + denotes60% NKa(+) cells). Expression of NKa determinants by normal CD4(-) lymphoid subpopulations was thus shown to increase in the order CD3(+)CD8(+)CD3(+) CD8(dim+) or CD3(+)CD8(-)CD3(-)CD8(dim+)CD3(-)CD8(-). Studies of TCR chain expression by NKa(+) (CD16/CD56) and NKa(-) subgroups of the CD4(-)CD8(-) fraction additionally showed that the presence of membrane TCRαδ (mean 18%) or TCRγδ (mean 35%) chains were primarily associated with the NKa(-) component and that virtually all (93%) NKa(+) cells were TCRαβ-TCRγδ. Examination of CD4(-)CD8(-)NKa(+) cells for the expression of other T-cell associated markers (CD5, CD7 and CD38) also indicated that this NKa(+) LGL-rich fraction could be further subdivided into CD2(+)CD3-CD5(-)CD7(+)CD38(+) and CD2-CD3(-)CD5(-)-CD7(+)CD38(+) subpopulations.

Using previously described procedures, this study quantified T-cell, T-cell subset, B-cell and NK-cell populations with the CD-Sapphire haematology analyser in a series of patients with mild to moderate lymphocytosis. Lymphocyte counts ranged from 6.0 to 14.9 x 10(9)/l, with 86/97 being10.0 x 10(9)/l. Immunophenotyping (CD3/CD19/HLA-DR, CD4/CD8 and CD16/CD56 combinations) was performed using EDTA-anticoagulated blood, automated CD-Sapphire analysis and subsequent software processing. Of 35 samples from younger (12 years) patients, 22 (63%) had nonspecific lymphocyte changes, 4 (11%) showed specific increases in nonreactive T-Helper or T-Suppressor cells, and five showed a reactive T-cell lymphocytosis. The remaining four were classified as 'Transient/Persistent NK-associated (NKa) Expansion' (n = 3) and specific B-cell lymphocytosis (n = 1). For older patients (n = 59), 15 (25%) had an increase (1.5 x 10(9)/l) in B-cells, and seven investigated for surface immunoglobulin expression were all found to be clonal. The remaining samples were categorized as 'Transient/Persistent NK-associated (NKa) Expansion' (13/59), Reactive Lymphocytosis (5/59), 'Reactive Lymphocytosis or Transient/Persistent NKa Expansion' (8/59), specific T-Helper cell (n = 8) or T-Suppressor cell (n = 3) lymphocytosis, and 'Lymphocytosis of Undetermined Significance' (n = 7). This study has demonstrated the feasibility of applying limited immunophenotyping protocols to the investigation of patients with abnormal lymphocyte counts in routine haematology. By using commercially purchased liquid monoclonal reagents to determine lymphocyte subpopulation profiles, haematology laboratories can provide more definitive information of potential clinical importance.

This communication details a method for the quantitative and qualitative analysis of blood T-, B- and NK-cell populations using the Abbott Cell-Dyn CD4000 haematology analyser. A series of 66 ethylenediaminetetraacetic acid (EDTA)-anticoagulated samples with lymphocyte counts between 0.2 and 33.3 x 10(9)/l were selected and analysed with CD3, CD19, Ia and CD56 monoclonal reagents. The flow cytometry reference method utilized a lymphocyte gate defined by optical scatter, with phenotypic analyses referencing to this gate and the absolute lymphocyte count. The CD4000 method analysed all leucocyte events, set primary gates for specific immunophenotypic fractions, and then determined population counts by reference to the white blood cell (WBC) count. Comparisons of CD3+ T-cell and CD19+ B-cell numbers showed high coefficients of correlation (R(2) > 0.95) and agreement (y = 1.01x) between the CD4000 and flow cytometry reference methods. Lower coefficients of correlation were obtained for CD3-CD56+ (R(2) = 0.52) and CD3+CD56+ (R(2) = 0.83) components. No major discrepancies were observed, and the CD4000 procedures additionally provided qualitative insights into the possibility of T-cell activation. The potential to undertake immediate analysis of EDTA-anticoagulated blood samples to determine the nature of abnormal lymphocyte morphology or numbers represents a considerable advance in the capability of haematology laboratories.

Background and Objectives The blood platelet content (in numbers) of platelet concentrates is required for production quality control and to predict clinical responses. Materials and Methods This study compared the performance of automated counting from impedance and optical instruments to data from immunoplatelet reference analysis. Results All methods showed good linearity with evidence of significant instrument-specific deviations from the line of agreement. Relational formulae largely corrected bias, but did not resolve platelet count variability. A second confounding factor, related to the proportion of small (activated) platelets, was also shown to contribute to intermethod discrepancies. Conclusions Blood processing centres should establish correction factors for each instrument compared to reference methods, such as the immunoplatelet count.

During a 7-month period a prospective study of 71 anaemic patients (29 males and 42 females) over the age of 50 was undertaken in order to identify patients with myelodysplastic syndrome (MDS). The mean values of mean corpuscular volume (MCV), serum ferritin, folate, vitamin B12 and red cell folate (RCF) of patients grouped according to the diagnosis were compared to those observed in age-matched blood donors. Forty-four of the 71 elderly patients showed macrocytic anaemia: 21 of them had gastric disease and the remaining 23 MDS. Two further patients with MDS showed microcytic anaemia. The 25 patients diagnosed with MDS were subclassified according to the FAB nomenclature: 9 had a refractory anaemia with excess of blasts and 16 refractory anaemia. The mean values of MCV, serum folate, ferritin, vitamin B12 and RCF were statistically different between patients with macrocytic anaemia due to gastric disease and patients with MDS. Among patients with MDS, the RCF level pathologically high was inversely correlated to the haemoglobin level (r=-0.39; p

Immunophenotypic changes in the T-cell compartment in B-CLL are well recognised, although the functional significance is less well established. In this study we examined the immunoregulatory capacity of CD45RO+ T-cells to modulate in vitro IgG and IgM production by B-CLL cells in comparison to normal PB B-cells. Removal of CD45RO+ T-cells from normal PB lymphocyte cultures was associated with a 2.3-fold reduction in IgM production and a 7.9-fold reduction in IgG production. Activation of the T-cell component by alpha CD3 stimulation enhanced IgG and IgM production by factors of 1.85 and 3.4 respectively. Removal of CD45RO+ T-cells from alpha CD3-stimulated cultures reduced IgG production 3.7-fold, whereas no significant change in IgM production occurred. Supplementing T- and NK-depleted B-cell fractions with purified autologous CD45RO+ T-cells produced a positive correlation between Ig concentration and the CD45RO:CD19 ratio for IgG production but not for IgM. Collectively, these results suggest that: 1) 'resting' CD45RO+ ('primed' or memory) T-cells drive mainly the IgG response; 2) activation of these T-cells enhances this response; 3) activated CD45RO+ T-cells derived from the recent transformation of the CD45RA+ ('virgin' or naive) population drives mainly the IgM response. In B-CLL cultures however, the pattern of Ig production in response to alpha CD3 stimulation is more typical of regulation by CD45RO+ T-cells derived from the recent activation of virgin CD45RA+ T-cells. We believe this challenges the view that T-cells in B-CLL are largely memory cells.

This study reports the clinical, haematological and immunophenotypic features of a series of 25 patients with clonal expansions of large granular lymphocytes (LGL)/NK-associated (NKa) cells. These showed a male predominance (16:9) with a median age of 67 (range 38-91) years; four had a documented history of rheumatoid arthritis, a further 18 had diverse clinical disorders, and the remaining three were clinically well. Mild anaemia was found in approximately half the patients and a lymphocytosis (seen in approximately 70% of the cases) was usually modest (< 10.0 x 10(9)/l). Neutropenia was the most frequently observed feature, and this was typically persistent in nature. Serum studies revealed few consistent features although positive rheumatoid factor and increased soluble CD8 levels were noted in 67% and 87% of those cases tested. Phenotypically, all cases were CD2+CD3+CD8+ and expressed membrane TCR alpha beta chains; most (17/22) were additionally CD5+ and (19/22) CD7+. The staining intensities of CD5 and CD7 antigens were however lower than that of normal CD4+ and CD8+ blood lymphocytes. Expression of NKa antigens was variable although 16/22 cases were CD16+CD56- and 19/22 were CD57+. Clonal CD3+CD8+ LGL/NKa expansions with a CD16+CD56+ composite phenotype were not seen in this patient series. Analyses of 'activation' antigens showed a consistent lack of CD25 expression by CD3+ cells, but increased CD3/Ia co-expression was found in a high proportion (19/25) of cases. Studies of CD45R isoform expression by CD8+ LGL/NKa cell fractions revealed a consistent CD45RA+RO- profile for all cases tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary. This communication reports a patient with concomitant expansions of CD4+ and CD8+ large granular lymphocytes. Immunological analyses revealed that the abnomally increased CD4+ LGL fraction was phenotypically similar to other reported persistent CD4+ LGL expansions, whereas the phenotypic profile for the CD8+ LGL component was unusual. of particular note was the finding that both the CD4+ and CD8+ LGL fractions showed high membrane the CD45RO isoform expression, thus suggesting their ‘primed’ status. Molecular biology studies of immunomagnetically fractionated cells using a Tγ9 TCR gamma gene primer further revealed that the CD4+ and CD8+ components were both clonal but showed different patterns of rearrangement It is suggested that the simultaneous presence of CD4+ and CD8+ clonal populations are unlikely to have been derived from a common progenitor and that they reflect expansions of functionally restricted subpopulations

Literature trends indicate an increasing awareness regarding the frequency, nature and clinical associations of abnormal and persistent expansions of lymphocytes with cytoplasmic granulation. These particular cells, which represent a minor normal lymphoid subpopulation and are widely referred to as large granular lymphocytes (LGL), generally (but not invariably) express monoclonal antibody-defined membrane NK-associated (NKa) determinants and appear to functionally correspond to those populations involved in cellular cytotoxicity. Increased proportions or absolute numbers of blood lymphocytes with LGL morphology and/or NKa+ phenotypes are associated with a diverse spectrum of clinical (haematological and non-haematological) disorders and may be broadly viewed as secondary (acute and chronic reactive) or primary in nature. Both primary and secondary LGL/NKa+ expansions may be persistent in type and the clinical distinction between the two may be difficult. A number of investigators have proposed schemes for the classification of these disorders but, because of their diversity, abnormal LGL/NKa+ expansions often defy rigid compartmentalisation. This communication examines the general basis of these classifications and illustrates their limitations by reviewing the data for 97 patients recorded in the largest (Yorkshire Leukaemia Group) survey to date of persistent LGL/NKa+ expansions.

Natural killer (NK) cells are the subject of great current interest because of their possible (in vivo) role in tumour cell surveillance and killing, and because of the potential application of cytokine-modulated NK cells in cancer immunotherapy. In addition, clonal proliferations of NK-associated (NKa) cell populations represent a high proportion of chronic (non-B) lymphoid malignancies and abnormal (both clonal and non-clonal) NKa components are being increasingly reported in association with diverse clinical pictures such as autoimmune disease. This communication extensively reviews what is presently known regarding normal and leukaemic NKa phenotypic diversity, the mechanisms of NK-mediated cytolysis, the role of NK cells in malignancy, and the diagnostic and cellular aspects of malignant NKa proliferations.

Blast cells from 26 cases of acute myeloid leukaemia (AML) were examined, by single and "two-colour" flow cytometry, for relationships between membrane CD11b (monoclonal antibody OKM1), CD11c (KB90) and CD14 (Leu-M3). Increased expression of all three determinants was associated with myelomonocytic leukaemias, with their relative diagnostic value in discriminating monocytic (M4 and M5) from non-monocytic (M1, M2 and M3) subtypes being CD14 greater than CD11c greater than CD11b. However, the results also indicated, because of the heterogenous expression of CD11c in particular, and to a lesser extent CD11b, that the patterns or histograms of fluorescent staining were potentially more informative than an empirical subdivision of blasts into positive and negative subpopulations. In addition, analysis of phenotypic correlations by simultaneous two-colour fluorescence showed that the expression of CD11b and CD11c determinants by leukaemic myeloid blasts was highly correlated, in contrast to the expression of CD14 and CD11c which were relatively independent. Consequently, CD11c+ myeloid blasts almost always coexpressed CD11b whereas CD14+ cases of AML often comprised CD14+ CD11c+ and CD14+ CD11c- subpopulations. It is concluded from these observations that CD11c immunophenotyping is a useful supplementary investigation, particularly in CD14- cases of myelomonocytic leukaemia. However, it is also apparent that the presence of membrane CD11c per se is not lineage-specific and that the level of expression is perhaps a more discriminatory factor.

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.

Serum concentrations of lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBD), phosphohexose isomerase (PHI), leucine aminopeptidase (LAP), beta-glucuronidase (beta-gluc) and angiotensin-converting enzyme (ACE), were measured in 107 cases of untreated acute myeloid leukaemia which were classified by morphological, immunological and cytochemical criteria. The results show that serum LDH was increased in most cases, irrespective of leukaemic subtype, serum PHI and LAP were significantly higher in monocytic variants and serum beta-gluc and ACE levels were generally within normal limits. In addition, significant (p less than 0.05) relationships were found between serum LDH, PHI, LAP and beta-gluc concentrations and the numbers of circulating leucocytes.

Using a standardized rosetting technique with IgG-coated ox erythrocytes, avid IgG (Fc) receptors were demonstrated on red cell precursors. The proportion of receptor-positive cells in normal marrows was highest in early precursors and appeared to be lost with maturation. In megaloblastosis, the absolute percentage of early precursors increase, but there is an even greater increase in the proportion of receptor-positive cells. It is proposed that this reflects the degree of maturation arrest. The specificity of the receptor was confirmed by inhibition studies with aggregated human IgG. In contrast, the expression of the C3b “immune adherence” receptor, assessed by IgM-C3b-coated ox erythrocytes, was seen to increase with erythroid maturation. Early megaloblasts, especially in severe megaloblastosis, showed a marked decrease in C3b receptor activity, again in proportion to the level of maturation arrest. The significance and possible function of these receptors is discussed.

The expression of Fc(IgG) and C3b membrane receptors by granulocytes and their precursors was examined in 23 cases of megaloblastic anaemia which were graded I-III according to morphological severity. Fractionated bone marrow and peripheral blood granulocyte receptors were assessed by rosette formation with optimally sensitized ox erythrocytes and the results compared with those found in 14 normal marrows. Promyelocyte Fc and C3b receptor activities in megaloblastic anaemia did not differ from normal whilst the number of Fc receptor positive myelocytes and later cells showed a significant increase (P less than 0.05 in Grade I, P less than 0.01 in Grades II and III) proportional to the severity of megaloblastosis. An increase in the number of C3b receptor positive granulocytes was seen in early megaloblastic anaemias and, in contrast to Fc receptor expression, showed the highest receptor activities in the Grade II cases. The most significant changes in receptor expression were seen at the metamyelocyte stage and appear to be related to the numbers of these cells found in the megaloblastic marrows. It is suggested that these alterations are primarily related to asynchronous granulocyte maturation and the application of these findings to the study of granulocytic disorders is discussed.

Peripheral blood cells from nine patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate). Like the phorbol ester 12- 0-tetradecanoyl-phorbol 13-acetate (TPA), bryostatin 1 activates protein kinase C (PKC), which plays a central role in the phosphatidylinositol signal transduction pathway. The effects of bryostatin 1 alone and in combination with TPA or with the calcium mobilizing ionophore A23187 were assessed by morphological appearance, cell adherence and aggregation, RNA and DNA synthesis, and immunoglobulin (Ig) production. While eight of nine B-CLL cultures remained proliferatively inert, bryostatin 1 could effectively trigger activation and differentiation of B-CLL cells in all cases as inferred by the induction of morphological changes, RNA synthesis, and monotypic Ig production. Addition of calcium ionophore A23187 to bryostatin 1- exposed cells resulted in significantly increased values for RNA synthesis and Ig production and in the acquisition of plasmacytoid morphology. Bryostatin 1 and the dual signal of bryostatin 1 plus A23187 mimicked the stimulatory action of TPA and the combination of TPA plus A23187, respectively. Overall, bryostatin 1 was less active than equivalent concentrations of TPA. This lesser efficacy may, however, reflect a quantitative rather than qualitative difference. Bryostatin 1 partially antagonized TPA-mediated effects on B-CLL cells suggesting different modes of action by the two activators. These studies indicate that bryostatin 1 has effective differentiation- inducing properties on B-CLL cells that can differentiation-inducing properties on B-CLL cells that can be accentuated by a calcium ionophore.

Leukaemic myeloid and lymphoid cell N-acetyl β-d glucosaminidase (hexosaminidase) enzymes were fractionated by Fast Protein Liquid Chromatography (FPLC) using high-resolution ion exchange (Mono-S and Mono-Q), gel filtration (Superose-6) and chromatofocusing (Mono-P) columns. Although only one molecular weight species was detected in haemopoietic cells, with an apparent mass of 129 kD, “isoenzyme” variants defined by differences in molecular charge were considerably more diverse than previously thought. Separation of the major Hex forms (A and B) by chromatofocusing indicated that intermediate (I) components were present in most acute leukaemias irrespective of lineage supporting the concept that the I form is a nonspecific marker of haemopoietic immaturity. Substrate and inhibitor studies further revealed that leukaemic cell hexosaminidases hydrolyse galactopyranosides at significantly lower rates than glucopyranosides and that the hydrolysis of N-acetyl β-d glucosamine is inhibited by both glucosamine and galactosamine products.

Of 417 cases of chronic lymphocytic leukaemia (CLL) examined morphologically, 346 (83%) showed low (less than 10%) numbers of cells with prolymphocytoid (PLC) morphology. Immunological studies in 267 of these cases, with particular reference to membrane surface immunoglobulin (SIg) densities and FMC7 expression, revealed that of 223 cases with insignificant prolymphocytoid components, 185 were phenotypically consistent with typical CLL whilst the other 38 cases showed significant (greater than 20% positive cells) FMC7 expression and/or increased SIg density. In contrast, 40 of the 44 cases with greater than 10% PLC showed atypical immunophenotypic features even though the expression of individual membrane components associated with B-cell differentiation appeared to be unrelated. MRBC receptor expression showed little correlation with the degree of prolymphocytoid change although all TU1- cases showed greater than 10% PLC. The results suggest that abnormal phenotypic features, similar to those observed in "prolymphocytoid" transformation, may be found in cases of CLL in the absence of morphological change.

The expression of membrane CD11c by leukaemic blast cells was examined (indirect immunorosetting) in 75 cases of acute leukaemia (myeloid, n = 60; lymphoid, n = 15) and evaluated as a potential marker for the diagnostic discrimination between monocytic (AMML-M4 and AMoL-M5) and non-monocytic (M1, M2 and M3) AML subtypes. Preliminary studies of normal bone marrow cells indicated that CD11c expression was not restricted to cells of monocytic lineage but was also present, with apparent lower density, on significant proportions of mature and immature granulocytes. Examination of acute myeloid leukaemia (AML) subtypes revealed that the non-monocytic leukaemias (n = 33) were CD11c-, defined as less than 30% positive cells, whereas all but one of the AMML-M4 (n = 13) and AMoL-M5 (n = 14) cases were CD11c+. All 15 cases of lymphoblastic leukaemia (ALL) showed less than 5% CD11c+ blasts. Membrane CD11c expression was also compared to the more widely used markers of monocytic differentiation; cytoplasmic alpha-naphthyl acetate esterase (ANAE) and membrane CD14 expression. This analysis showed that all 13 AMML-M4 leukaemias studied, including seven cases that were CD14- and eight that were ANAE-, were CD11c+. In addition, the AMoL-M5 cases (all of which were ANAE+) could be phenotypically subdivided into CD11c+ CD14+ (n = 9), CD11c+ CD14- (n = 4) and CD11c- CD14- (n = 1) subgroups. The study also confirmed that the discriminitive ability and sensitivity of the immunorosetting procedure for the detection of membrane CD11c compared favourably to immunofluorescent staining intensities as measured by flow cytometry.

Using a standardized rosetting technique with IgG-coated ox erythrocytes, avid IgG (Fc) receptors were demonstrated on red cell precursors. The proportion of receptor-positive cells in normal marrows was highest in early precursors and appeared to be lost with maturation. In megaloblastosis, the absolute percentage of early precursors increase, but there is an even greater increase in the proportion of receptor-positive cells. It is proposed that this reflects the degree of maturation arrest. The specificity of the receptor was confirmed by inhibition studies with aggregated human IgG. In contrast, the expression of the C3b “immune adherence” receptor, assessed by IgM-C3b-coated ox erythrocytes, was seen to increase with erythroid maturation. Early megaloblasts, especially in severe megaloblastosis, showed a marked decrease in C3b receptor activity, again in proportion to the level of maturation arrest. The significance and possible function of these receptors is discussed.

Alpha-naphthyl acetate esterases (ANAE) were examined by cytochemical and isoelectric focusing (IEF) techniques in 48 cases of acute myeloid leukemia that were classified by conventional morphological criteria. Four main types of ANAE isoenzyme patterns were found by IEF, and comparisons with the expression of membrane receptors (Fc-IgG and C3b) and monocyte-specific antigens (UCHM1, UCHALF, and E11) suggest relationships between ANAE isoenzyme synthesis and distinct myeloid maturational stages. The results further indicate that the blast cells of acute myelomonocytic leukemia (AMML) may represent an immature variant of monocytic leukemia (AMoL) and that morphological examination alone is inadequate in the assessment of monocytic differentiation in acute myeloid leukemias. Inhibition studies of cytochemical ANAE activity with sodium fluoride (NaF) show that the presence of NaF- sensitive or NaF-resistant ANAE enzymes is often unrelated to the diagnostic category of acute leukemia. The results of this study are examined in relation to current concepts of myeloid differentiation, and the application of these findings to the subclassification of acute myeloid leukemias is discussed.

Absolute T cell counts and the relative distributions of antibody-defined helper and cytotoxic/suppressor T lymphocytes are reported in 26 patients with chronic idiopathic thrombocytopenic purpura (ITP). T cell numbers are generally normal in ITP and alterations in relative helper (OKT4 positive) and suppressor (OKT8 positive) T cell numbers show no consistent patterns, although 35 % of the cases examined showed inverted distributions. The combined percentages of helper and suppressor T cells consistently exceeded 100 % in ITP indicating the presence of increased OKT4+8+ cells in these patients. A cell population, previously defined as SRBC positive and OKT3 negative, was found in the normal group and in ITP patients following splenectomy but was absent in those patients with intact spleens. The role of these changes in the pathogenesis of ITP is discussed.

A total of 412 cases of acute leukaemia were examined for the presence of nuclear terminal deoxynucleotidyl transferase (TdT) by indirect immunofluorescence. Of the 129 cases of acute myeloblastic leukaemia (AML FAB groups M1/M2) examined, 18% (n = 23) had significant proportions (greater than 10%) of TdT-positive blasts. Although most of these AML cases (n = 18) were of poorly differentiated (M1) type; 5 cases of AML showing features of granulocytic differentiation (M2) were also found to be TdT-positive. Even though TdT was generally more strongly expressed in the M1 group and associated with other markers of myeloid immaturity (Ia positive and lack of chloroacetate esterase), there was no inverse relationship with Sudan black or myeloperoxidase activity. In addition, although the proportion of AML-M1 cases with increased TdT-positive cells was slightly higher (18/95, 19%) than for the AML-M2 group (5/34, 15%) the results suggest that the presence of nuclear TdT in leukaemic myeloblasts may not only reflect cellular immaturity but may also be due to maturational asynchrony in otherwise well-differentiated blasts.

The clinical, morphological and immunological features of 22 cases of chronic lymphocytic leukaemia in 'prolymphocytoid' transformation (CLL-Pro) are reported. Immunophenotypic patterns in CLL-Pro differ from CLL by the appearance of significant (greater than 15%) FMC7-positive components and/or increased SIg densities in most cases. Membrane TU1 and MRBC receptor expression was similar to that found in typical CLL. Morphologically, all cases showed a mixture of small lymphocytes and larger nucleolated 'prolymphocytes' although the degree of prolymphocytoid change was unrelated to immunological patterns. Clinically, the cases behaved in a very heterogeneous fashion, with some patients dying rapidly following transformation despite treatment, while others even if untreated had a long, stable and relatively benign course. It was not possible to predict which patients would do badly from immunological or morphological features but the presence of more than one involved lymph node site and the occurrence of B-symptoms appeared to identify a group that did badly. Immunological assessments were however important in therapeutic terms, drawing distinction between CLL-Pro variants and prolymphocytic leukaemia (PLL).

Acid alpha naphthyl acetate esterase (ANAE) and combined ANAE-chloroacetate esterase cytochemistry was performed on 121 bone marrow aspirates from primary myelodysplastic syndromes (MDS) and a secondary dysplasia-megaloblastic anaemia (MA). The investigation demonstrated the presence of abnormal ANAE positive granulocyte populations in a significant proportion of cases. These cells, in which the staining patterns were characterized by atypical granular ANAE positivity and double ANAE-chloroacetate reactions, were shown immunologically to lack the receptor and antigenic characteristics of monocytes and morphologically to be granulocytes. Isoelectric focusing, however, indicated that the atypical esterase cytochemistry of these granulocytes was due to the presence of markedly increased concentrations of ANAE isoenzymes usually found in monocytes. Atypical ANAE-staining granulocytes were particularly evident in MDS marrows showing sideroblastic erythroid changes, whilst in MA they were mainly seen in cases of intermediate severity. It is suggested that these cells are associated with dysmyelopoietic changes in both malignant and non-malignant conditions.

Summary. Immunological and cytochemical findings are presented from 12 cases of morphologically unequivocal acute monocytic leukaemia (AMoL). The results indicate considerable heterogeneity and three main non-morphological subgroups were identified. The blast cells from half the patients were positive for the presence of both cytoplasmic alpha naphthyl acetate esterase (ANAE) and monocyte-associated membrane determinants whereas the cells from three cases lacked detectable monocytic antigens despite the presence of strong cytochemical ANAE activity. A further three cases expressed monocytic antigens but were cytochemically unreac-tive for ANAE. These cytochemical results, which were extended by electrophoretic studies of ANAE isoenzymes, suggest that the absence of significant cytoplasmic ANAE activity does not preclude the diagnosis of AMoL and that serum lysozyme estimations may be of value in the recognition of irnmunocytochemically-atypical cases.

The alpha naphthyl acetate esterase (ANAE) cytochemical staining patterns were examined in 40 cases of acute myeloblastic leukaemia (AML: FAB groups M1 and M2) classified by morphological and immunological criteria. The blast cells in most cases (62%) were ANAE-negative with the remainder showing diffuse, granular or focal reactions of varying intensity. The nature of cytoplasmic ANAE enzymes was further characterised in 20 cases by isoelectrophoretic analysis of ANAE isoenzymes. The results suggest that the presence of significant cytoplasmic ANAE reactivity in leukaemic myeloblasts is not due to the presence of monocyte-associated isoenzymes, in otherwise well-defined myeloblasts, but may reflect abnormally increased synthesis or atypical localisation of normally-occurring ANAE isoenzymes. In particular, the results of this study indicate the lack of discriminatory value of ANAE cytochemistry in the differentiation of AML from other acute leukaemias of non-monocytic type.