I 0 Correspondence

KRANTZ S.B. & RAO V. (1967) Studies on pure red cells aplasia I: Demonstration of a plasma inhibitor to haem synthesis and an antibody to erythroblast nuclei. Proc. Nar. Acad. Sci., USA. 58,493-500

J. Wright M. Greaves

Department of’ Haernatology Royal Hallamshire Hospital Shefield S I O 21F

Clostridium perfringens in the immunocomprised

Sir; In their recent report, Ifthikaruddin & Holmes (Clin. Lab. Haemat. 14) describe a fatal case of Clostridium perfringens septi- caemia with intravascular haemolysis following an autologous bone marrow trans- plant. In contrast, we describe a patient who survived 2 episodes of Clostridial septicaemia within 6 weeks despite, being markedly immunosuppressed.

A 52-year-old man with a history of rheu- matoid arthritis became pancytopenic with myelodysplasia (RAEBt) diagnosed in November 1991. By December 1991, the disorder had transformed to acute myeloid leukaemia (M6) and he conimenced standard induction therapy. In mid-January 1992, following his second course of chemotherapy (DAT 2+8), he became pyrexial with diar- rhoea and rectal discomfort (white cell count 0.3 x 10y/I). Treatment with both intravenous ceftazadime and vancomycin was initiated. Faecal cultures showed no growth but Clostridium tertium was grown from initial blood cultures. His temperature settled, symp- toms resolved and he was discharged.

He was carefully monitored over the following weeks during which time his renal and liver function were normal and his blood count recovered to a neutrophil count of 1.5 x 1OY/1, platelets 158 x 109/1, haemoglobin 9.4 g/dl. He was therefore readmitted one week later in early March for the next course of chemotherapy. On admission he was febrile (38.6”C) but asymptomatic. His haemoglobin had fallen to 7.7 g/dl, white cell count 0.4 x 109/1, platelets 24 x 10’/1 with micro- spherocytes and no neutrophils seen in the blood film. His coagulation screen showed an INR of 1.3, a normal PTTK and FDPs of

2000ng/ml. He was commenced on pipera- cillin and gentamicin and blood and platelets were given. By the following morning, he had become jaundiced and his bilirubin rose from 56mmol/l on the day of admission to 156 mmol/l the following day with no change in liver enzymes but with macroscopic haemoglobinuria and slight deterioration in renal function (creatinine 156 mmol/l, urea 15.5 mmol/l). During the first three days of hospitalization, he required 6 units of blood with haemoglobin reaching a nadir of 6.9 g/dl on the second day of admission. Initial blood cultures grew Clostridium perfringens sensitive to penicillin G and metronidazole. He made a good rccovery following this episode of septi- caemia and has subsequently received another 2 courses of chemotherapy without further Clostridial infection.

The initial Clostridium septicaemia in our patient appeared to be due to a rectal abscess. The second episode with associated intravas- cular haemolysis shows some similarities with the case described by Ifthikaruddin & Holmes (1992) as both patients had no gut signs or symptoms at presentation. Ifthikaruddin & Holmes conclude that Clostridium septicaemia in the immunocompromised has a poor prog- nosis even if antibiotic therapy is commenced promptly. In contrast, our patient remained clinically stable throughout both septicaemiac episodes and responded well to antibiotics.

References

IFTHIKARUDDIN, J.J. & HOLMES, J.A. (1992) Clostridium perfringens septicaemia and massive intravascular haemolysis as a complication of autologous bone marrow transplant. Clin. lab. Huemat. 14, 159-161

P. Murphy A. Parker S. Pavord

J. K. Wood Department of Haernatology Leicester Royal Infirmary Leicester LEI SWW, UK

Automated differential count rejections and in vitro platelet aggregation in blood from patients taking warfarin

Sir: It was noted in this laboratory that there seemed to be a preponderance of white cell

Corresporidence 7 1

differential rejections by the Coulter S plus IV when processing fresh blood specimens from patients on oral anticoagulant therapy (OAT) compared with fresh specimens from other patients. A retrospective survey of 397 blood samples processed through the Coulter supported this vicw. Of 57 OAT patients, 16 (28%) had been rejected compared with only 12 (3.5%) from the remaining 340 samples. Notwithstanding the disparity in group sizes, samples from patients on warfarin appeared to have undergone differential rejections more frequently than control groups and this difference was very highly significant (P < 0.001).

Patients attending the anticoagulant out- patient clinic participated in a further study, all were maintained on warfarin therapy. Controls came from other out-patients. Blood was taken from the antecubital vein by clean venepuncture and immediately added to 1.4 mg/ml dry dipotassium EDTA anti- coagulant. These samples were cycled through the Coulter S Plus IV immediately, (despite Coulter Electronics recommendation not to sample within 15 min of collection), to mini- mize the possibility of EDTA induced changes taking place, and once again after an hour’s incubation at room temperature. A blood smear was made directly from the non-anti- coagulated blood in the syringe and from the EDTA anticoagulated blood after incubation, fixed in methanol and stained in a Romanowsky stain. Using light microscopy the number of aggregates consisting of three or more platelets was counted in 20 fields of the stained film at a 40 times magnification.

When the fresh blood samples were processed regardless of patient group, all leucocyte counts were within normal limits of 4-11 x 109/1 and all differential counts were

Table 1. Rejected differential counts in blood samples from control patients and those taking warfarin as oral anticoagulant therapy.

OAT Controls

Number of samples 37 39 Differential rejection 12 (32%) 5 (12.8%) Differential acceptance 25 (68%) 34 (87.2%)

The difference in incidence of rejection was very highly significant using the two sample t test ( P < 0,001).

Table 2. The incidence of differential leucocyte rejections in blood samples with demonstrable platelet aggregates

OAT Control Patient samples (n = 21) (n = 17)

~ ~

Differential rejection 9 (42.9%) 3 (17.6%) Differential acceptance 12 (57.1%) 14 (82.3%)

There was no significant difference between the groups.

normal with no precursor or atypical cells present nor nucleated or abnormal erythro- cytes as any of these could cause differential rejection. The numbers of differential rejec- tions by the Coulter are shown in Table 1. The size of the patient groups was similar and more than twice as many rejections occurred in samples from OAT patients, the difference being statistically significant using a two sample t test (P < 0.05).

An attempt was made to try to correlate incidence of aggregation with a rejected differ- ential. There were 38 samples in which aggre- gates had occurred, 68% of which had an accepted differential and were evenly spread across the two patient groups. Of the samples causing a differential rejection, three times as many came from the anticoagulated patients (Table 2) but no statistically significant differ- ences were found when the data was analysed using a Mann-Whitney U test. Thus the pre- sence of platelet aggregates per se could not be considered responsible for the rejections of the differential count.

Table 3. Differential leucocyte rejections set against numbers of platelet aggregates occurring in corresponding blood films

Mean aggregate count f SD

OAT Control Patient samples (n = 21) (n = 17)

Differential rejections 54k62.9 14.3 17.4

Differential acceptance 64.4 1 13 38.8 + 47.8 (n = 9) (n = 3)

(n = 12) ( n = 14)

Samples came from anticoagulated and control patients. In both cases there was a large scatter of results about the mean.

12 Correspondence

The samples were further analysed to see if the actual numbers of aggregates formed influenced the Coulter in rejecting the differen- tial count. This did not appear to be the case, in fact there tended to be more aggregates formed in the samples where the differential was accepted whichever group they had come from. The figures suggest that whether or not a differential count was rejected, more aggre- gates were found in the blood of patients on warfarin than the controls, the means being 60.1 and 34.3 per film respectively. However, when the Mann-Whitney U test was applied the difference was not significant due in part no doubt to the extreme variability as can be seen in Table 3.

When aggregates were counted in 24 samples before and after they had been incu- bated in EDTA for 1 h at room temperature, it was found that the numbers were highly significantly less ( P < 0.01), the mean values being 0.08 0.3 post incubation and 77 & 105 in the corresponding fresh film.

Despite the apparent equal incidence of aggregates in both groups the actual numbers found in the respective films was quite different, though a large scatter of results makes interpretation difficult. As the films were made directly from the syringe two possible conclusions may be drawn. Firstly the platelet aggregates could have been present in the circulation prior to blood being taken, as was suggested by Wu & Hoak (1974) who demonstrated the presence of platelet aggre- gates in diabetics, Secondly the process of blood taking may itself have caused the aggre- gation, as suggested by Kohanna (1984). If the platelets had clumped in contact with the glass slide (Dacie & Lewis 1975) it is unlikely that the actual number of aggregates would show much disparity. It would seem reasonable to suppose that platelets from patients suffering from a thromboembolic disorder might be hyperactive and thus more susceptible to physical trauma.

References DACIE J.V.C. LEWIS S.M. (1975) Practical

Haematology, 5th edn. Churchill Livingstone, Edinburgh

KOHANNA F.H., SMITH F.H. & SALZMAN E.W. (1984) Do patients with thromboembolc disease have circulating platelet aggregates? Blood 64, 205-209

Wu K.K. & HOAK J.C. (1974) A new method for the quantitative detection of platelet aggregates in patients with arterial insufficiency. Lance! ii, 924

W.P. Davis, MPhil, FIMLS

Department of Haematology Eastbourne District General Hospital Eastbourne BN21 2UD

Pamela Graham, BSc, DPhil

John Harris Clinical Pharmacy Unit University of Brighton Brighton, E. Sussex

Red cell distribution width (RDW) in sickle cell disease

Sir: We read with interest the article by Thame er al. (1991) (Clin. lab. Haemat. 13, 229-237). We have also assessed the value of the RDW in sickle cell disease and can confirm the value of an elevated RDW as an aid for detection of sickle cell related disorders (Hammersley et al. 1981). Blood samples from 78 patients with homozygous sickle cell (SS), 12 patients with sickle cell haemoglobin C (SC), 1 sickle cell hereditary persistence of foetal haemoglobin (all in steady state) and 44 patients with sickle cell trait (AS) were analysed on a Sysmex E5000 Blood Counter using the SD option for reporting RDW. A further 20 samples from patients with homozygous SS were in run on a Coulter Counter S Plus IV blood analyser. Of the latter group, 5 had blood counts repeated during an acute vaso-occlusive crisis.

All of the patients with HbSS had an increased RDW (59k8.3 fl, reference range 43.0k5.0 fl), 10 out of 12 patients with HBSC disease had increased RDW. Of note, the 2 HBSC patients with normal RDW and one SHPFH had a paucity of sickle cells on blood film per 1000 cell count. All patients with HbAS (sickle cell trait) had normal RDW values. There was no correlation between RDW values measured during acute sickling episodes and those in the same individuals in steady state and it is therefore of no predictive value to disease severity. These results were reproducible with both Coulter S Plus IV and Sysmex E5000 analysers.

In contrast, we could not demonstrate any direct relationship between RDW value and