734 Correspondence

These results confirm the results of Preisler et al (1983, 1984). Day 6 bone marrow examination could be a simple method to predict treatment outcome and to offer the opportunity of early treatment changes.

Department of Haematology, Leiden University Medical Centre, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands

REFERENCES

W. G. PETERS R. WILLEMZE L. P. COLLY L. COLLA

PREISLER, H.D., EPSTEIN, J., BARCOS, M., PRIORE, R., RAZA, A., BROWMAN, G.P., VOGLER, R., WINTON, E., GRUNWALD, H., RAI, K., BRENNAN, J,, BEN- NETT, J., GOLDBERG, J., GOTTLIEB, A., CHERVENICK, P., JOYCE, R., MILLER, K., LARSON, R., D.’ARRIGO, P., DOEBLIN, T., STEIN, M., BLOOM, M., STEELE, R. & LEE, H. (1 984) Prediction of response of acute nonlymphocytic leukaemia to therapy with

‘high dose’ cytosine arabinoside. British Journal of Haematology, 58, 19-32.

PREISLER, H., BARCOS, M.. REESE, P., PRIORE, R.L. & POTHIER, L. (1983) Recognition of drug resis- tance during remission induction therapy for acute non-lymphocytic leukemia: utility of day 6 bone marrow biopsy. Leukemia Research, 7, 67-75.

DETECTION OF ABNORMAL HAEMOGLOBINS BY THE TECHNICON H6000 AUTOMATED CELL COUNTER

We report the detection of a number of unsuspected haemoglobinopathies due to the presence of an aberrant differential leucocyte count on the Technicon H6000 automated cell counting system. In the majority of cases there were no clinical or other indications of a haemoglobinopathy and all red cell parameters were normal. Samples came from a population of primarily British descent in which the finding of abnormal haemoglobins had previously been an infrequent occurrence. Over a 10 month period, with 29000 blood samples, the H6000 results led to the detection of 18 abnormal haemoglobins.

The Technicon H6000 system is designed to provide a full blood cell count, including platelet and red cell parameters. In addition, it performs a differential leucocyte count, using two channels, one of which involves lysis of the red cells in a sodium dodecylsulphate solution prior to cytochemical staining of the leucocytes for peroxidase activity. Measurements of absorption (enzyme activity) and light scatter (cell size) by each cell passing through the flow cells are represented on an X-Y display (Fig la) . This ‘scattergram’ represents data from 10 000 cells. Once thresholds are set for each of the parameters, 10 categories are defined in which all cells can be differentiated and classified.

The problem of incomplete red cell lysis in the peroxidase channel has been known to occur when there is an elevated blood urea concentration, and leads to a falsely elevated leucocyte count. However, we have found that incomplete lysis may also occur due to the presence of an abnormal haemoglobin. It has been described specifically in the case of erythrocytes containing HbC (Booth & Mead, 1983), but it appears that this is a more widely occurring phenomenon associated with a variety of haemoglobin variants.

Correspondence 7 3 5

.

. .

LLypphocytes . I . , . . . 0 . . .*

5 . ) , . Red,cell debris

Fig 1. (a) A section of the H6000 scattergram of ,a normal blood sample. There is a clear area between the cloud of red cell debris and the lymphocye cloud. (b) The typical pattern obtained from samples containing abnormal haemoglobins, showing the merging of red cell debris with lymphocytes. (c) This occurs to a gross degree with HbC.

In the 10 months during which the H4000 analyser has been in routine use, 29 000 samples have been tested. Of 20 samples producing atypical scattergram patterns due to poor red cell lysis, 18 were due to abnormal h(aemog1obins. The remaining two samples had elevated urea concentrations. The first abnormal haemoglobin was detected in a patient with normal red cell indices and morphology. Hlowever, the H6000 white blood cell count was grossly elevated (33.1 x 109/1) and the differential count indicated 9% neutrophils and 85% lymphocytes. The scattergram (Fig 1 b) clearny demonstrates a spread of the cloud of cells from the area of red cell debris at the extreme bottom left of the graph, into the lymphocyte area. A manual white cell count was 8.7 x 109/1 anld the discrepancy was explained by failure of red cell lysis in the peroxidase channel. The blood urea level was normal. Further studies of the

736 Correspondence

Table I. Haemoglobin variants detected in the H6000 analyser, and identified by tryptic peptide mapping

Haemoglobin Variant No. found

Heterozygous HbS 8 Heterozygous HbE 3 Homozygous HbE 1. Heterozygous HbE Saskatoon 3 Heterozygous HbD 2 Heterozygous HbC 1

patient’s blood sample revealed the presence of an electrophoretically abnormal haemoglobin which, on cellulose acetate at alkaline pH, migrated in the same position as HbE. This abnormal component, which represented 43% of the total haemoglobin. was isolated and identified by tryptic peptide mapping as HbE Saskatoon (p22 Glu-+Lys). Similar findings led to the identification of other abnormal haemoglobins as shown in Table I.

The scattergram patterns obtained from patients with haemoglobinopathies appear to be variable, even in individuals tested on several occasions, and presumably this is related to differences in the degree of red cell lysis. We are unable, therefore, to distinguish between haemoglobin variants, with the possible exception of HbC, in which a particularly dramatic picture was obtained with the red cell cloud spread extensively into the lymphocyte cloud (Fig lc). Furthermore, although the aberrant pattern may be evidence of a haemoglobinopathy, a normal pattern does not exclude the presence of an abnormal haemoglobin.

Nevertheless, we believe that abnormal scattergram results from the H6000 analyser provide a useful indication, in the clinical laboratory, of an abnormal haemoglobin.

Department of Haematology, Medical Laboratory, Dunedin, New Zealand

Department of Clinical Biochemistry, Christchurch Hospital, Christchurch, New Zealand

REFERENCE

J. REES

D. WILLIAMSON R. W. CARRELL

BOOTH, F. & MEAD, S.V. (1983) Resistance to lysis system. Journal of Clinical Pathology, 36, of erythrocytes containing haemoglobin 8 16-8 18. C-detected in a differential white cell counting