Scand J Haematol( l983) 31,283-286

The Effects of Cisplatin on Normal Human Erythrocytes in Vitro

MARGARET DAWSON, ETHER A. BROWN & RONALD D. BARR

Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada

Therapeutic administration of cisplatin is often followed by anaemia which may not be due entirely or even largely to myelosuppression but rather to drug-induced haemolysis. Similar experience with other cytotoxic agents prompted earlier investigations which yielded evi- dence of drug-induced changes in RBC shape in vitro. Current studies of cisplatin and bleomycin, which have been used together in clinical practice, revealed a significant alteration in the profile of RBC morphology; specifically a reduction in the number of ‘bowl’ shaped cells. However, this finding is in contrast to the increase in ‘bowls’ which was observed previously with other drugs. So it appears that direct erythrocytotoxicity cannot explain the occurrence of anaemia in patients who receive cisplatin. An alternative mechanism, similar to that of penicillin induced haemolysis, is suggested by immunoradiometric assays of surface IgG on red cells.

K e y words: cisplatin - erythrocyte - shape

Accepted for publication March 1, 1983

Correspondence to: Dr. Ronald D. Barr, Room 3N27D, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario L8N 325, Canada

ABBREVIATIONS Bleo = bleomycin CDDP = cis-diamminedichloroplatinum DAGT = direct antiglobulin test PBS = phosphate-buffered saline VBL = vinblastine

Anaemia has been reported to develop commonly after the administration of cis- diamminedichloroplatinum I1 (cisplatin =

CDDP) as part of combination cancer chemo- therapy ( l ) , the incidence varying greatly between different studies (2-5). It has been suggested that the mechanism is one of non-immune haemolysis (6), which is sup- ported by early onset, poikilocytosis and a rate of fall in haematocrit greater than that

produced by complete cessation of erythro- poiesis. However, other studies have shown that CDDP is myelosuppressive (1, 5), and there have also been reports of delayed onset anaemia associated with a positive direct anti-globulin test (7-9).

The possibility of direct, drug-induced erythrocytotoxicity was investigated because of prior experience that other chemother- apeutic agents produce a change in red cell shape in vitro at therapeutic concentrations (10). CDDP, Bleo and VBL were chosen for the study since administration of these drugs in combination has been associated frequently with the development of anae- mia (11). In none of the reported cases of anaemia has CDDP been the only chemo-

284 DAWSON, BROWN & BARR

therapeutic agent used, so the drugs were examined alone as well as in combination. The results of these investigations form the basis of this report.

MATERIAL AND METHODS

30 ml of venous blood were collected from each of 10 normal adult volunteers using 21 g needles and plastic syringes, and transferred to plastic tubes containing 1000 U heparin. Plastic ware was used throughout to avoid the echinocytogenic effect of glass on erythro- cytes (12, 13). Only whole blood was analyzed since CDDP is 90 70 protein-bound in plasma (12). The red cell concentration was measured by a Coulter Counter Model S Plus and adjusted to 4 x 1012/1 by the addition of isotonic phosphate-buffered saline (PBS) at pH 7.4.

Stock solutions of CDDP - 150 mg/l, Bleo - 6 mg/l and VBL 45 mg/l were made in PBS, then 1 in 1.5 and 1 in 3 dilutions were prepared and identified as solu- tions 1, 2 and 3 respectively for each drug. 8 tubes each containing 0.25 ml drug(s) or PBS, were labelled A-H as in Table 1. A 2.5 ml aliquot of the adjusted red cell

TABLE 1 Stock operating solutions of drugs

Aliquot Drlig additions

A B C D E F G

H (control) Standard

250 p1 CDDP-3

250 UI VBL-3 250 pl Bleo-3

125 GI CDDP-2 + 125 pl Bleo-2 125 UI CDDP-2 + 125 UI VBL-2 125 bl Bleo-2 + 125 pl’VBL-2 83 pl each of CDDP-1, Bleo-1,

and VBL-1

None 250 pl PBS

TABLE 2 Peak plasma level of drugs

Drug Plasma level Reference

Cisplatin 5 mg/l ( 14) Bleomycin 0.2 mg/l (15) Vinblastine 1.5 mg/l (16)

suspension was added to each tube so that the final concentration of each drug was equal to its peak thera- peutic concentration in vivo (Table 2). After incubation at 37°C for 1 h in a water bath, 45 pI aliquots were fixed and examined by the glutaraldehyde-glycerol technique as described by Neville et al (17).

100 cells were counted per slide using the criteria of Bessis (18) and Zipursky et al (19). Only those cells were included which could be seen completely in 3- dimensions. Cells were classified into 1 of 3 groups: discocyte; bowl i.e. stomatocyte, 1-11 cell (transition phase between discocyte and stomatocyte) and hemi- sphere; and ‘non-disc, non-bowl’. This is a modified form of the classification used by Neville et al (17). Due to the wide variation in the relative proportions of discocytes and bowls in normal subjects, each subject acted as his own control. The results were evaluated statistically by one-way analysis of variance and paired t tests.

RESULTS

A significant difference ( P < 0.001) in the number of bowls was revealed by one way analysis of variance, but there was no such difference in the number of discs or non- disc, non-bowl cells. The proportion of bowls was then examined by paired t tests. Taking P = 0.02 as the arbitrary level of biological significance, differences were ob- served between standards and CDDP alone ( P < 0.005) and between standards and Bleo alone ( P < 0.02), but not between standards and VBL alone or between stan- dards and controls. There were also signi- ficant differences between the standards and those samples containing CDDP or Bleo in combination, the largest t value (4.40) being obtained when all 3 drugs were combined. In each instance, however, the change in distribution of red cell shapes was in the opposite direction to that observed pre- viously with other chemotherapeutic agents (lo), i.e. CDDP and Bleo produced a reduc- tion in the proportion of bowls.

CISPLASTIN EFFECTS ON RED CELLS 285

TABLE 3 Immunoradiometric assay

Surface IgG Aliquot Drug/Saline (molecules/

RBC)

1 (control) 133 p1 PBS 34 2 133 PI CDDP-1 41 3 (control) 664 p1 PBS 42 4 664 PI CDDP-1* 52 5 (control) 3.32 rnl PBS 39 6 3.32 ml CDDP-1 39

Normal upper limit for surface IgC is 134 molecules per red cell (mean + 3 SD). * Final concentration 5 mg/l.

In an immunoradiometric assay, con- ducted by Dr. John Kelton, on 20 ml aliquots of whole blood obtained from 1 normal subject, the amount of IgG bound to red cells in the presence of CDDP at therapeutic, 5 x therapeutic and x ther- apeutic concentrations was measured. The results are shown in Table 3. There was no significant difference from concurrent con- trols nor from the normal range established for this assay. By contrast, in a single sam- ple of whole blood, collected from a patient who had received several courses of CDDP therapy, an elevated value of 155 molecules of IgG per red cell was obtained in the presence of the drug.

DISCUSSION

Cisplatin binds avidly to red cell mem- branes (20) although, in a recent examina- tion of erythrocyte morphology, Najarian et a1 (6) found no evidence of any alteration induced by this drug in vitro. However, these investigators gave no details of the experimental methods they employed, and a significant change in the distribution of red cell shapes was observed in the current

studies using therapeutic concentrations of the agent in whole blood. A similar profile was observed with Bleo, in contrast to VBL which produced no significant effect. The latter finding confirms the earlier results re- ported by Neville et al (17). combination of all 3 cytotoxic agents was associated with the greatest alteration in red cell shape.

Superficially, these findings may suggest a possible pathogenetic mechanism for the development of anaemia in patients who receive CDDP, Bleo and VBL in a single therapeutic protocol, as in the Einhorn re- gimen (11). However, the change in the profile of red cell shapes was in the opposite direction to that obtained with cytosine- arabinoside and thioguanine, the use of which may well be accompanied by direct erythrocytotoxicity and consequent haemo- lytic anaemia (10). Furthermore, CDDP is myelosuppressive, yet Bleo, which is not, produces the same change in red cell shape in vitro but does not cause anaemia in clin- ical practice. In addition, anaemia may occur only after several courses of CDDP, accompanied by a positive direct anti-glo- bulin test (7) and even a response to the administration of corticosteroids (8).

Immunoradiometric assays indicate that the occurrence of a positive DAGT in these circumstances may not be due to non-spe- cific binding of Ig to red cells. Rather, CDDP may cause a specific antibody-mediated haemolytic anaemia of the penicillin type, as has been proposed (7). This may not occur in all subjects in contrast to the anaemia of myelosuppression which is more clearly dose- related.

ACKNOWLEDGEMENT

We wish to thank Dr. J. G. Kelton for undertaking the immunoradiometric assays.

286 DAWSON, BROWN & BARR

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