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To determine whether skin puncture blood can be used reliably for CD4 lymphocyte counts, the numbers of the major subsets of lymphocytes were assessed in paired venous and skin puncture blood samples from 22 children and 10 adults. Paired values were highly correlated, with skin puncture values being about 7% higher than venous values for each cell type. Differences were of borderline statistical significance for total lymphocytes and for each subset except CD3+ CD8+ T lymphocytes. Nevertheless, the magnitude of the differences was small and unlikely to be of clinical importance, and it seems that skin puncture samples may be preferable for CD4 counts in children or adults with difficult venous access.

Aims: Children in a United Kingdom national trial for relapsed non-B lymphoblastic leukaemia (ALL) had their diagnostic and relapse marrow cytomorphology compared to see what changes occur during the evolution of the disease., Methods: Each relapse slide was assessed blindly for French American British (FAB) type and other morphological features by a panel of three independent microscopists without reference to each other or any diagnostic material. Diagnostic slides had been assessed by the same panel on an earlier occasion., Results: A total of 134 consecutive children was studied. Six (5%) were classified as FAB type L2 at diagnosis, compared with 18 (13%) at relapse (a difference of 9%). Twenty two (16%) changed their FAB type, 17 (13%) from L1 to L2 and five (4%) from L2 to L1. The FAB score fell at relapse in 34 children and rose in 14, a difference of 14%. Cell size was the commonest feature to change (increasing in 22 and diminishing in nine) followed by prominent nucleoli (appearing in 21 and disappearing in six). Forty four (33%) children had vacuolated blasts at diagnosis, compared with 48 (36%) at relapse. Twenty five changed their vacuole score substantially, 14 gaining > 10% and 11 falling < 10%., Conclusions: These findings reflect the variability of lymphoblast cytomorphology, but also show a trend for cells to have more prominent nucleoli and greater size at relapse. Factors controlling these features of the FAB type are unknown, but they may simply be related to the growth fraction of a particular disease and not to any lineage specific biological feature.

A study was carried out to determine the frequency of combined iron deficiency and beta-thalassaemia trait in a cohort of British Asian children to see whether the trait protects iron status. Of 470 consecutive children with red cell microcytosis, 77 had beta-thalassaemia trait and 26 (34%) of these also had evidence of iron deficiency. It was most common and profound in children under five years of age where the prevalence was 16 in 33 (48.5%). This suggests that iron deficiency is no less common in Asian children with beta-thalassaemia trait than in those without. It should not be presumed that the trait protects iron status or that the two are in any way mutually exclusive, at least in the early years.

Aims: To assess the prevalence and degree of periodic acid Schiff (PAS) positivity in blast cells from children with lymphoblastic leukaemia (ALL); its association with other disease characteristics; and its clinical importance in predicting the outcome of treatment., Methods: Marrow slides from entrants to a large United Kingdom multicentre ALL trial (UKALL X) were batch processed and assessed blind for PAS positivity by one morphologist. Patients were classified into groups A, B, and C, corresponding to less than 1% PAS positive cells, 1-10%, and over 10%, respectively. Their PAS pattern was then compared with other clinical and pathological features of ALL and with treatment outcome., Results: Slides from 921 children were examined of which 371 (40%) were categorised as group A, 324 (35%) as group B, and 226 (25%) as group C. There was a clear association between the presence of blast cell vacuoles on Romanowsky staining and PAS positivity. Group A (PAS negative) patients included a disproportionate excess of those with L2 morphology, those under 2 or over 6 years of age, those with an initial white cell count over 50 x 10(9)/l, those with a T or null cell immunophenotype, and those with chromosomal abnormalities other than "high hyperdiploidy". Four years from diagnosis, group C patients had an 8% disease free survival advantage over those in group A (2p = 0.01). This was irrespective of initial white cell count, but not of immunophenotype or the presence of vacuoles., Conclusions: Strong PAS positivity is a feature of "common" ALL and is particularly associated with blast cell vacuoles. It does occasionally occur in other disease subtypes with or without vacuoles. It predicts a better response to current treatment, but not independently of other cell characteristics.

Aims: To evaluate the reliability of fluorescence in situ hybridisation (FISH) in the retrospective cytogenetic assessment of old bone marrow smears stored for periods of up to 20 years., Methods: A series of bone marrow smears either Romanowsky stained, or frozen and unstained, and aged from one month to 20 years were hybridised with biotin labelled probes specific for the centromeric regions of human chromosomes X, 6, and 18. Sites of hybridisation were detected with fluoresceinated avidin. One hundred to 400 cells from each preparation were examined and the number of signals observed was recorded., Results: All smears exhibited signals in most cells examined. In cytogenetically normal cases, an average 67.6% of cells (range 36%-90%) demonstrated the appropriate number of X centromere signals. In those samples known to contain extra chromosomes X, 6, or 18 the presence of cells with the abnormal copy number was clearly detected in each case., Conclusion: When applied in the way described, FISH can give consistent and accurate results with a variety of archival bone marrow smears, including aged prestained material. This will permit retrospective assessment of specific cytogenetic abnormalities in patients with leukaemia using their initial diagnostic slides even where these are several years old.

To define further the clinical importance of cytogenetic analysis in acute lymphoblastic leukaemia (ALL) a prospective study was performed on 139 unselected children. Analyses were considered adequate in 104, of whom 35 were normal and 69 had clonal abnormalities. Abnormalities were categorised according to banded chromosome analysis as well as chromosome count. Karyotypes were correlated with clinical and laboratory features at diagnosis and with survival. Of the successful analyses, thirty five (34%) children had no abnormalities; this group contained an excess of T cell disease. Twenty five (24%) had a "characteristic" hyperdiploid karyotype and as a group had lower presenting white counts, a tendency to CD10, and periodic acid schiff positivity of the blast cells and smaller spleens. None was an infant and only one was over 10 years old. Seven (7%) children with t(9; 22), t(8; 14), or t(4; 11) translocations were grouped together as "specific" translocations. Collectively they had a significantly worse prognosis than the remainder. Nine children developed central nervous system relapse, six of whom had either t(4; 11) or abnormalities of 9p or 19p. A descriptive classification taking into account chromosome bonding pattern is cytogenetically more appropriate and may be more clinically useful than grouping children simply by chromosome number. As knowledge and techniques improve, the classification of cytogenetic abnormalities in ALL will need to be kept under frequent review.

A method was developed for the measurement of factor VIII inhibitors by the Bethesda technique using small volumes of skin puncture blood. Human and porcine inhibitor concentrations on paired venous and skin puncture samples measured on 10 occasions were highly correlated, with no significant difference between them. Assays on four inhibitor negative haemophiliac patients were also negative by the skin puncture method. Sample dilution at the collection stage results in loss of sensitivity to inhibitor concentrations below 1 unit/ml, but the technique will be of value in monitoring higher concentrations when venous access is difficult and must be saved for parenteral treatment.

Blast cell morphology was assessed at diagnosis and subsequent bone marrow relapse in 33 unselected patients with lymphoblastic leukemia (ALL). Each marrow was classified 'blind' according to the French-American-British (FAB) criteria, and it was found that 19 of 24 (79%) patients initially typed as FAB L1 changed to FAB L2 during the course of their disease, but no patient made the reverse morphological change (p is less than 0.001). Five patients retained FAB L1 appearances; these included three of the four who had T-cell markers. One patient typed as FAB L3 did so consistently. This study indicates that FAB L2 ALL frequently emerges as a treatment-resistant offshoot of FAB L1 and provides further evidence that this marks a more aggressive form of the disease.

Diagnostic bone marrow smears from 132 patients with acute lymphoblastic leukaemia, (ALL) were stained simultaneously by the periodic acid-Schiff (PAS) reaction, and the blast cell positivity was assessed quantitatively. The patients fell naturally into two unequal groups: those with more than 20% PAS-positive blast cells (44 patients) and those with less (88 patients). There was no relation between the degree of positivity and age, sex, or presenting leucocyte count. Actuarial survival studies showed that the group with more than 20% PAS-positive blast cells survived longer, but that this difference assumed statistical significance only after the exclusion of patients over 14 years old and those with high white cell counts at the time of diagnosis. It appears that the PAS reaction can identify long survivors among patients with ALL, but not in the absence of features strongly associated with a poor prognosis.

Three hundred and twenty consecutive children with lymphoblastic leukaemia (ALL), treated on the Medical Research Council UKALL VIII schedule, had their Romanowsky stained diagnostic marrows reviewed for the presence of azurophil granules in blast cell cytoplasm. Twenty patients (7%) had greater than 5% blasts showing this feature; 19 had the cell phenotype of "common ALL." Male children and those with French-American-British (FAB) L2 morphology predominantly showed this feature. There was also a strong correlation between granularity and non-diffuse acid phosphate positivity, but no obvious difference between the 20 patients in their response to treatment emerged during a minimum follow up of 15 months. The "granular" variant occurs in around 7% of children with ALL, but has no clear prognostic importance. Morphologists should be aware of its existence and incidence to avoid confusion with acute myeloid leukaemia.

As part of the Medical Research Council Leukaemia Trial UKALL VIII, 738 unselected children with acute lymphoblastic leukaemia (ALL) had the morphology of their marrow blast cells reviewed by a panel of three haematologists. Ninety four (13%) showed appearances classifiable as type L2 by the French American and British (FAB) cooperative group's criteria, five (0.7%) were typed L3, and the remaining 639 (86%) as L1. Disregarding the patients classified as L3, those with the L2 variant showed an inferior disease free survival to that of the remainder (p less than 0.01), and more of them failed to remit after receiving "standard" remission induction treatment (p less than 0.01). They included an excess of older children (p less than 0.01) with less profound marrow failure at diagnosis, and fewer of them expressed the common ALL antigen (p = 0.05). There was no association between L2 morphology and the diagnostic white cell count, sex, or the presence of a mediastinal mass. These findings confirm earlier reports that FAB L2 ALL is associated with a poor prognosis and that it occurs more commonly in older children. The high remission failure rate is a recent observation and indicates that alternative early treatment may be appropriate for such patients.

Mouse erythrocyte (M) rosette formation and colchicine sensitivity were compared for their ability to differentiate chronic lymphocytic leukaemia (CLL) from B cell non-Hodgkin's lymphoma (NHL) with overspill. Twenty-two cases of CLL and eight of NHL were studied along with 31 normal adults. Results from the patients in both tests differed significantly from the controls but colchicine sensitivity failed to differentiate them further. M Rosettes, on the other hand, while increased in some patients with NHL were, without overlap, much more numerous in those with CLL, and clearly distinguished the two conditions. A significant autolymphocytotoxic effect of plasma from both study groups was also noted which was not found in the controls.

A semiquantitative assessment of blast cell acid phosphatase activity, expressed as a score, was made in 41 unselected children with newly diagnosed and untreated non-T acute lymphoblastic leukaemia (ALL). Despite a wide range of enzyme activity in both sexes boys had significantly higher scores than girls, and, in view of the known association between males and T ALL on the one hand, and between acid phosphatase and T ALL on the other, these findings raise the possibility that boys may have a predisposition to a type of pre-T ALL which could contribute to the as yet unexplained difference in prognosis between the sexes.