complications and prevention of haemorrhage is the main explanation for the improvement of results in AML in the elderly rather than the employment of more efficient antileukaemic drugs. ‘H6pitul Haut-kvique, 33604 Pessac, *Department of Hematology. H6pital Purpan, 3 1 0 6 9 Toulouse, ’lnstitut Paoli Calmettes. 1 3 2 7 3 Marseille Ceder 9, 4Department of Hematology. H6pital de la Tronche, 38000 Grenoble, Tuboratoire Farmitalia Carlo Erba. BP 2 0 3 , 9 2 5 0 2 Rueil Malmaison


F. HUCUET~ A. M. STOPPA’ M. M I C H A L L E ~ ~ P. HIJRTEtoUP5 for the BGMT Group

REFERENCES Fstey. E.H..Keating. M.J..McCredie,K.B.. Bodey,G.P.&Freireich.E.J. (1982) Causes of initial remission induction failure in acute myelogenous leukemia. Blood. 60, 309-31 5. Hutchinson. R.H. & Winfield.D.A. (1992)Mitozantrone and cytosine arabinoside as first-line therapy in elderly patients with acute myeloid leukaemia. British Journal of Haernatology. 80, 41 6-41 7. Liu Yin, J.A.,johnson, P.R.E.. Davies. J.M., Flanagan. N.G.. Gorst, D.W. &Lewis, M.J. (1991)Mitozantrone and cytosine arabinoside as first-line therapy in elderly patients with acute myeloid leukaemia. British Journal of Haernatology. 79, 41 5 4 2 0 . Reiffem J.. Rigal-Huguet, F.. Stoppa. A.M.. Michallet. M.. Marit. G., Attal. M.. Gastaut. J.A., Corront. B., Lepeu. G . . Routy. M.. ConyMakhou. P.. Pris. J., Hollard, D., Maraninchi, D..Mercier. M.. Hurteloup, P., for the B M T Group (1992) Prospective study comparing idarubicin and daunorubicin in elderly patients with acute rnyeloid leukaemia. Haernatology and Blood Transfusion, 34, 628-631.

The results of Professor Reiffer’s group confirm that it is now possible to achieve high CR rates in elderly patients with AML. I agree with their view that better supportive care is the

most important factor responsible for the improved results reported in this group of patients. It is also worth reiterating that physicians who undertake to treat elderly AML patients with intensive chemotherapy should be prepared to provide maximal supportive care as they would for younger patients. Whilst good results can be achieved with the use of mitozantrone. it is equally true that similar results can be obtained with daunorubicin or the newer anthracycline, idarubicin. Agents like mitozantrone or idarubicine may, however, be particularly suitable for elderly patients, by virtue of their lower cardiotoxicity, but their future role in induction chemotherapy must await the results of large randomized studies such as the Medical Research Council AML 11 trial or European co-operative study groups. Department of Clinical Haernatology, The Royal Infirmary, Manchester M I 3 9 W L



I was pleased to see the letter from Professor Reiffers and think his points are very valid. The use of platelets of course vanes according to the criteria for their use, i.e. treatment of a platelet count below 20 or if there are frank haemorrhagic signs. I am glad that a comparison between ida- and daunarubicin has been carried out. The AML XI trial will show if mitozantrone/cytosine is any better in terms of remission and toxicity than daunarubicin. I would certainly agree that supportive care is better now that we have become experienced in handling AML XIII, X and XI and that is why toxicity is lower and probably the CR rate is higher compared with the AML IX date.

Department of Haematology, The Leicester Royal Infirmary. Leicester LEI 5 WW


FUNCTION OF ACID-TREATED PLATELETS FOR TRANSFUSION Shanwell et a1 (1991) reported a patient with platelet refractoriness due to H L A alloimmunization who had improved responses after transfusion of random donor platelets which had been treated with acid at O°C to ‘peel’ HLA antigens. Their serological studies suggested that this treatment was effective in removing HLA antigens from platelets, and platelet recovery and survival of acid-treated platelets was similar to untreated platelets in two control subjects. However, no comment was made on the possible effect of this treatment on platelet function, although platelets are damaged by exposure to low pH and low temperature (Murphy, 1985). Blumberg et a1 (1984) first suggested the use of platelets ‘stripped’of HLA antigens for the treatment of alloimmunized patients with platelet refractoriness. However, they doubted whether the treated platelets would be haemostatically

effective. Chloroquine treatment later became an established technique in platelet serology for the differentiation of HLA and cell-specific antibodies (Nordhagen & Flaathen, 1985) and acid treatment was found to be an effective alternative (Sugawara et al, 1987: Kurata et al, 1990). However, little attention was given to the potential therapeutic use of ‘stripped’ platelets, presumably because of the known antiplatelet effect of hydroxychloroquine. We have carried out preliminary studies on the platelet function and HLA antigen expression of acid-treated platelets. Platelets were obtained from a platelet concentrate within 2 h of its preparation by apheresis. Platelets were either: (a)left untreated at room temperature (RT);(b)treated with acid solution, pH 2.8. for 25 min at O°C at approximately 600 x lo9platelets/l2 ml acid, similar to the technique described by Shanwell et at (1991); (b) treated with chloro-


17 7

Table I. Studies of (i) platelet aggregation, and (ii) reactions in the MAIPA of multispecific HLA antibodies kom five mukitransfused patients with platelets which were (a) untreated, (b) treated with a low volume of acid similar to the method of Shanwell et a1 (1991). (c) treated with a low volume of CQ (see text), (d) acid-treated, and (e) CQ-treated, as used in platelet serology. ~~

Apheresis platelets (C)




(4 High-CQ

(i) Platelet aggregation (%) (results of tests with two donors) Aggregation with ADP (9.9 PM) 23, 16 8, 11




Aggregation with collagen (4 pg/ml)

73, 75

0, 11

0, 0



(ii) MAIPA (absorbance values) Negative control*






Sera with HLA antibodies 1 2 3 4 5

0.526 0.556 0.888 0.410 0.479

0.486 0.339 0.714 0.174 0.251

0.407 0.507 0.782 0.185 0.374

0.159 0.084 0.101 0.025 0.025

0.125 0.048 0.056 0.029 0.012

(a) Untreated

(b) Low-acid

* Pooled AB serum. ND=not done.

quine solution (CQ) (chloroquine diphosphate 200 mg/ml, pH 5.0) under the same conditions as (b); (d) treated with acid solution, pH 3.0, for 10 min at 0°C at 6 x lo8platelets/ 0.5 ml acid (i.e. 40 times more acid per platelet than used in (b), as recommended for platelet serology by Kurata et a1 (1990): (e) treated with CQ for 2 h at RT at 6 x 108platelets/ 0.5 ml CQ, as used in platelet serology (Nordhagen & Flaathen, 1985). After treatment, platelets were either washed and resuspended in plasma for between 2 and 3 h prior to platelet function tests or washed and resuspended in PBS/EDTA for serological investigations. Platelet function was assessed by platelet aggregation, using a Payton 1015B aggregometer. Platelet aggregation was measured as the percentage of the maximal response after addition of ADP (9.9 p ~ and ) collagen (4 pg/ml). Serological studies were carried out using the MAIPA technique (Kiefel et al, 1987). Five multispecific HLA antibodies were tested against platelets treated as in (a) to (e) above. Platelet aggregation was reduced in response to ADP and collagen when platelets were treated with low concentrations of acid or chloroquine (Table I). Under these conditions, both acid-treated and CQ-treated platelets showed only small reductions in reactions with HLA antibodies (Table I). Thus, although both treatments inhibited platelet function they were not rigorous enough to prevent reactions with HLA antibodies using the MAIPA technique. In contrast, reactions with HLA antibodies were reduced when platelets were treated with the higher concentrations of acid and CQ used in platelet serology (Table I).

The findings of our study extend the observations made by Shanwell et ul (1991). Although they found that acid-treated platelets survived normally after transfusion, our studies suggest that the function of the treated platelets may be compromised. In addition, we were unable to confirm the effectivenessof low concentrations of acid in removing HLA antigens from platelets. Further studies are required to determine whether a balance can be found between effective removal of €€LA antigens from platelets and preservation of platelet function. Department of HuemutoZogy, St Bartholomew's Hospital and Medical College, London E C I A 7BE


REFERENCES Blumberg, N., Masel, D., Mayer, T., Horan, P. & Heal, J. (1984) Removal of HLA-A, B antigens from platelets. Blood, 63,448-450. Kiefel, V., Santoso, S., Weisheit, M. & Mueller-Eckhardt, C. (1987) Monoclonal antibody-specific imrnobilisation of platelet antigens (MAIPA): a new tool for the identication of platelet-reactive antibodies. Blood, 70, 1722-1726. Kurata. Y., Oshida, M., Take, H., Furubayashi, T., Mizutani, H., Tomiyama, Y.. Yonezawa, T. & Tarui, S. (1990) Acid treatment of platelets as a simple procedure for distinguishing platelet-specific antibodies from anti-HLA antibodies: comparison with chloroquine treatment. Vox Sunguinis, 59, 106-1 11. Murphy, S. (1985) Platelet storage for transfusion. Seminars in Huernatology, 12, 165-1 77. Nordhagen, R. & Flaathen, S.T. (1985) Chloroquine removal ofHLA



antigens from platelets for the platelet immunofluorescencetest. Vor Sanguinis. 48, 156-1 59, Sugawara, S.. Abo. T. & Kumagai. K. (1987) A simple method to eliminate the antigenicity of surface class I molecules from the membrane of viable cells by acid treatment at pH 3. Journal of

Shanwell. A.. Sallander. S.. Olsson. I., Gulliksson. H.. Pedajas. I. & Lerner. R. (19 9 1 ) An alloimmunized. thrombocytopenic patient successfully transfused with acid-treated,random-donor platelets. British Journal of Haernatology. 79, 462-465.

Immunological Methods. 100, 8 3-90.

EXERCISE-INDUCED VARIATIONS OF LYMPHOCYTOSIS IN THE LYMPHOPROLIFERATIVE DISEASE OF LARGE GRANULAR LYMPHOCYTES The lymphoproliferative disorder of large granular lymphocytes (LDLGL) is a recently recognized disease entity, whose cytological, biological and clinical features may vary within fairly wide boundaries (Bassan et al, 1986).Although it has been usually regarded as a rare condition, some recent reports point to an overall frequency higher than previously suspected (Scott et al, 199 1). Since the clinical course of LDLGL ranges from an apparently benign, possibly reactive lymphocytosis to a n aggressive, openly malignant cell proliferation (Semenzato et al, 198 7 ) ,the availability of reliable prognostic indices would be of undoubted interest. An extensive multicentre study (Pandolfi et al. 1990),based on the retrospective evaluation of 151 patients, has suggested that ( a )fever at diagnosis, (b)a low percentage of circulating lymphocytes positive for the HNK-I (CD57) monoclonal antibody, and (c)a relatively low ( 70%) of lymphocytes with the typical morphological features of LGLs. A fluorocytometric study of cell membrane antigens revealed different patterns in LGLs from the two cases: respectively CD2 , CD3 -, CD4 -, CD8+. CD16+. CD56+. CD57- and CD2+, CD3+, CD4+. CD8+, CD16+, CD56+, CD57-. Both patients did not complain of any distressing symptoms, and no other abnormal clinical or laboratory findings were detected. In particular a n association with chronic infectious, autoimmune or neoplastic disorders could be ruled out, and no anaemia, neutropenia or thrombocytopenia coexisted. This situation has remained substantially unchanged, and no treatment whatsoever has been administered up to the present time, with LGL lymphocytosis persisting over a follow-up period of 19 and 21 months respectively. Graded exercise testing was carried out on a bicycle under supervised conditions, according to current methods (Sheffield, 1984).In the first patient the test was repeated on two separate occasions, about 3 months apart. Blood samples were obtained immediately before the start (sample 1).1rnin after reaching the target heart rate (sample 2), and 10 rnin after completion of the exercise test (sample 3). On all samples the absolute numbers of neutrophils, lymphocytes and LGLs (the latter identified on the basis of expression of the NK-associated antigens. CD16 and CD56) were evaluated. The results are summarized in Fig l(a). In all instances





_. _ _ .




L -

8 7


-I y1


, 4 t

.i I



Patient #I’ Test 2

Patient Y 1 Test 1

? --c.

Function of acid-treated platelets for transfusion.

176 Correspondence complications and prevention of haemorrhage is the main explanation for the improvement of results in AML in the elderly rather t...
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