Psychopharmacology (1996) 124:201-204 © Springer-Verlag 1996

Martina H u m m e r • Barbara Sperner-Unterweger Georg Kemmler • Markus F a l k . Martin Kurz Harald Oberbauer • W. Wolfgang Fleischhacker

Does eosinophilia predict clozapine induced neutropenia?

Received: 18 September 1995 / Final version: 9 November 1995

Abstract The atypical antipsychotic clozapine carries a high risk of inducing agranulocytosis. We attempted to investigate whether eosinophilia during clozapine treat- ment has predictive value for subsequent neutrope- nia/agranulocytosis. One hundred and seventy-seven pa- tients were studied in a prospective naturalistic design using haloperidol as the reference compound. Clozapine was found to differ from haloperidol in respect to their influence on neutrophit granulocytes. In the clozapine group patients with eosinophilia showed a decrease in neutrophil count (less than 2000/mm 3 neutrophil granu- locytes) significantly more often than patients without eosinophilia.

Key words Clozapine • Haloperidol • Eosinophilia • Neutropenia - Agranulocytosis

Introduction

The therapy of treatment resistant schizophrenia with clozapine is hampered by its propensity to cause agranu- locytosis (Krupp and Barnes 1992). The incidence of clozapine induced agranulocytosis is 0.8% at 1 year and 0.9% at 1.5 years (Alvir et al. 1993). Despite intensive research, investigators have so far failed to find a mecha- nism underlying clozapine induced agranulocytosis. Studies indicate both immunologic (Lieberman et al. 1988; Pfister et al. 1992; Safferman et al. 1992; Pisciotta et al. 1992) and toxic (Fischer et al. 1991) mechanisms.

M. Hummer ([5~) • B. Sperner-Unterweger • M. Kurz W.W. Fleischhacker Department of Biological Psychiatry, Innsbruck University Clinics, Anichstrasse 35, A-6020 Innsbruck, Austria

M. Falk Department of Biostatitics, Innsbruck University, A-Innsbruck, Austria

G. Kemmler • H. Oherbauer Department of General Psychiatry, Innsbruck University Clinics, A-6020 Innsbruck, Austria

ttoffbrand (1992a) suggested that both mechanisms could welt be implicated together. Another question which is often discussed in the context of clozapine in- duced agranulocytosis is whether it possible to predict patients who are at a higher risk for this side effect. Alvir et al. (1993) found that older patients and females were at greater risk. An immunogenetic basis for vulnerability to develop agranulocytosis with clozapine is also dis- cussed. Some authors suggest that HLA typing of poten- tial clozapine candidates is useful in predicting the risk (Hoffbrand 1992b; Pfister et al. 1992) but the results of these investigations are controversial. While Yunis et al. (1992) and Pfister et al. (1992) found an association of major histocompatibility complex with clozapine in- duced agranulocytosis, the studies of Claas et al. (1992) and Abt et al. (1992) do not support this finding. Regular white blood count monitoring remains to be the only rel- evant way to minimize the risk of agranulocytosis. It al- lows an early detection of granulocytopenia before the onset of the infectious complications of agranulocytosis.

In the last 3 years, two cases of agranulocytosis were observed in our department (Hummer et al. 1994). Both patients showed an increase of eosinophil granulocytes 3 weeks before neutropenia occurred. Since until then eo- sinophilia had only been reported in connection with leu- cocytosis (Stricker and Tielens 1991; Frankenburg and Kando 1992; Tiihonen and Paanila 1992), we started to study the role of eosinophilia prospectively to find out whether our intitial observation hold up in a larger series of patients treated with clozapine.

Materials and methods

Our department has an ongoing clozapine drug monitoring pro- gram since 1989. In accordance with the guidelines of Sandoz Ba- sel and the Austrian Ministry of Health, all patients with clozapine prescriptions are seen weekly in the first 18 weeks of treatment and monthly thereafter. We do differential white blood counts at all of these visits. Additionally, we monitor patients treated with haloperido! following the same guidelines. All patients that are started on either clozapine or haloperidol on one of our inpatient units enter this program.

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The following hypotheses were investigated: 1) Do haloperidol and clozapine cause different effects on eosinophil and neutrophil granulocyte counts? 2) Is there a relationship between neutrophil and eosinophil granulocytes? 3) Does eosinophilia increase the risk of neutropenia? Only patients with normal eosinophil and neutrophil count at baseline were included in our calculations. Neutropenia was defined as a neutrophil count of less than 1500/mm 3, eosinophilia as an eosinophil count of more than 4% of the white blood count. When calculating sensitivity, specificity, positive predictive value and negative predictive value, a neutro- phil granulocyte count of less than 2000/mm 3 was chosen to en- large the sample. We decided to calculate neutrophil count in ab- solute values in accordance with the clinical guidelines of Sandoz Basel (Krupp and Barnes 1992).

Statistical methods

To compare the two treatment groups with respect to continuous variables, such as age and blood counts, Mann-Whitney U-tests were performed. Chi-square tests were used for comparisons with respect to discrete variables. The relation between neutrophil and eosinophil blood counts was studied using Pearson correlation co- efficients.

To compare the two treatment groups with respect to the corre- lation between eosinophil and neutrophil count, a standard test for comparing correlation coefficients was used (Steel and Torrie 1981), {>Levels of P<_0.05 were defined as significant. _<-Levels of P_>0.1 were labelled non-significant (NS).

Results

We report data on 177 patients, in which treatment with either clozapine (n=107) or haloperidol (n=70) was initi- ated in our inpatient units. Descriptive statistics are sum-

marized in Table 1. There is no significant difference in sex distribution between the two treatment groups. The patients in the clozapine group are slightly older than the patients in the haloperidol group. The mean clozapine dose was 234 mg/day+132, the mean haloperidol dose was 18.8 mg/day+17.9. During treatment 61 patients (57%) in the clozapine group developed eosinophilia and three patients (2.8%) showed neutropenia (<1500 neutro- phil granulocytes/mm3). In the haloperidot group 31 pa- tients (44%) developed eosinophilia. There was no case of neutropenia. No alternative explanations (e.g. con- comitant medication, illness, etc.) for eosinophilia or neutropenia were found in both groups.

Table 2 shows eosinophil and neutrophil counts dur- ing the first 6 weeks o f treatment. During weeks 0, 1 and 2, 5 and 6 no difference of eosinophil counts was found between the clozapine and haloperidol group. During the third and the fourth week of treatment we found a signif- icantly higher eosinophil count in the clozapine group. A compar ison of max imum eosinophil counts showed no significant difference. In the clozapine group the maxi- mum eosinophil count occurred in the fourth week of treatment on average, in the haloperidol group in the fifth week. A compar ison of neutrophil counts did not show a difference before treatment and during weeks 3, 4, 5 and 6. The neutrophil count was significantly higher in the clozapine group during weeks 1 and 2. There was no difference in min imum neutrophil counts and time of occurrence between groups.

Furthermore, we found the min imum neutrophil count significantly later than max imum eosinophit count in the

Table 1 Sample description

Mann-Whitney U-test b Pearson's chi-square test

Clozapine Haloperidol P-value

Number of cases 107 70 Age (mean_+SD) 31. I +_ 11.8 27.7± 1 0 . 1 (P=0.062) a

range 18-67 17-60 Sex female 46 (43.0%) 18 (25.8%) n.s. b

male 61 (57.0%) 52 (74.2%) Daily dose in mg/day M e a n _ + S D 234+132 18.8±17.9

Median 211.7 13.4 Plasma level in ng/ml Mean+SD 165_+149 12.8±18.4

Median 115.6 8.2

Table 2 Eosinophil and neu- trophil counts during the first 6 weeks of treatment. Eosinophil and neutrophil counts are indi- cated as % of the total white blood counts

Week Clozapine Italoperidol Comparison Mean±SD M e a n _ + S D (Mann-Whitney)

(U-test)

Eosinophils (%) 0 2.1+1.1 2.0+1.1 n.s. 1-2 3.3+1.9 2.9-+1.7 n.s. 3---4 3.8+_2.4 2.7+1.5 P=0.004 5-6 4.3_+4.2 3.0_+2.2 n.s.

Maximum Count 6.8+4.5 4.6_+2.2 (P=0.093) Week 4.2_+2.6 5.1_+3.1 n.s.

Neutrophils (%) 0 58.7+10.5 58.7+11.2 n.s. 1-2 60.5-+9.9 54.9-+9.0 P=0.001 3-4 58.0_+8.7 55.7+9.0 (P=0.087) 5-6 55.9_+ 11.0 56.7_+ 10.3 n.s,

Minimum Count 47.0_+9.8 50.1-+8.3 n.s. Week 5,7-+3.2 4.1 _+3.3 (P=0.097)

Table 3 Correlation between eosinophil and neutrophil counts (Spearman correlation coefficient)

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Week Clozapine P n H~opefidol P n Comparison of treatmentgroups

1-2 -0.35 <0.001 107 -0.15 n.s. 70 n.s. 3-4 -0.25 0.029 78 -0,09 n.s. 50 n.s. 5-6 -0.59 <0.001 55 -0,22 n.s. 32 (P=0,055)

TaMe 4 Eosinophilia and tow neutrophil count (clozapine group). Patients with eosinophilia show a minimum neutrophil count of <2000 significantly more often than patients without eosinophilia (P=0.050 chis-square test)

Eosinophilia during first 12 weeks

"Ibtal n Minimum neutrophil count (weeks 1-12)

> 2 0 0 0 1500-2000 <1500

Yes 61 51 (84%) 8 (13%) 2 (3%) No 46 44 (96%) 1 (2%) 1 (2%) Total 107 95 (89%) 9 (8%) 3 (3%)

clozapine group (Wilcoxon Signed Rank Test, P<0.003). The mean time to reach peak eosinophil counts was 4 weeks, while it took a mean time of 6 weeks to reach the minimal neutrophil count. We did not find this phenome- non in the haloperidol group (see Table 2). Calculating the correlation between eosinophil and neutrophil granu- locyte counts during the first 6 weeks of treatment a signficant negative correlation was found in the cloza- pine group (see Table 3). In the haloperidol group this correlation did not reach statistical significance. A com- parison of treatment groups with respect to the correla- tion between eosinophil and neutrophil counts showed a significant difference in weeks 5-6.

When analyzing the value of eosinophilia as a poten- tial predictor for subsequent neutropenia, the following results were found. Two of the three patients developing neutropenia (<1500/mm 3 neutrophil granulocytes) showed an eosinophilia in the forefield of neutropenia. One patient did not show eosinophilia (Table 4). We also investigated nine patients with a neutrophil count less than 2000/mm 3. Eight patients had developed eosinophil- ia prior to neutropenia in this larger sample. Patients with eosinophilia more often show a minimum neutro- phil count <2000 than patients without eosinophilia (P<0.050 chi-square test). Eosinophilia prediced neutro- penia with a sensitivity of 83.3% (95% confidence inter- va1=51.6%- 97%) and a specificity of 46.3% (95% con- fidence interval=36.0-56.8%). We found a positive pre- dictive value of 16.4% (95% confidence inter- val=8.1-28.1%) and a negative predictive value of 95.7% (95% confidence interval=85.2-99.5%).

Discussion

The results of this study imply both theoretical and prac- tical considerations. By doing regular differential blood counts it is possible to detect neutropenia or agranulocy- tosis before the development of secondary infections.

Usually the mode of onset is gradual and it takes several weeks before the manifestation of agranulocytosis, but cases with a sudden onset have also been observed (Lie- berman et all. 1988). It would therefore be of major clini- cal relevance to find early indicators of incipient agranu- locytosis. So far, two cases of agranulocytosis have oc- curred in our hospital. Both patients showed an eosino- philia in the forefield. The results of our study point in the same direction. Eosinophilia can be a first warning sign of a decrease in neutrophils. At first, we investigat- ed if there are different effects of clozapine and haloperi- dol on eosinophil and neutrophil counts; secondly, the relationship between eosinophil and neutrophil counts. In the clozapine group we found the maximum eosino- phil count significantly earlier than the minimum neutro- phil counts. Furthermore, there was a significant correla- tion between eosinophil and neutrophil count. No case of neutropenia was found in the haloperidol group. In a third step we investigated the predictive value of eosino- philia in the clozapine group. Patients with a ueutrophil count less than 1500/mm 3 as well as patients with a neu- trophil count less than 2000/mm 3 were investigated. In the former group two patients developed clear eosino- philia in the forefield of neutropenia. The third patient showed a maximum eosinophil count of 4%. Analyzing the patients with a neutrophil count of less than 2000/mm 3 comparable results were found. The sensitivi- ty of 83.3% shows that eosinophilia during clozapine- treatment should direct our attention more intensively to the neutrophil counts of the following weeks. Our results show that the probability to develop neutropenia with a negative test result (no eosinophilia during the treatment with clozapine) is only about 4%. On the other hand, due to high incidence rate of eosinophilia we found a poor positive predictive value, meaning that its occurrence does not reliably predict subsequent neutropenia. The fact that many patients showing eosinophilia do not de- velop neutropenia could indicate that these patients are able to compensate for hypothetical adverse effects of clozapine or haloperidol on granulopoesis.

Although the actual mechanisms causing blood dyscr- asias in clozapine-treated patients are still unknown, re- cent studies present more and more evidence that a my- eloid precursor cell might be the target for the pathoge- netic process (Gerson and Meltzer 1992; Pisciotta et al. 1992). Without regard to other etiological factors like cu- mulative dose effect, toxicity of clozapine and its active metabolites and/or primary or secondary immune reac- tions (Bender et al. 1987; Ruef and Coleman 1991; Veys et al. 1992), the observed selective depletion of granulo- cyte precursors from bone marrow as reported by Lieber-

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man et al. (1988) is also suggestive of an injury to a stem cell compartment committed for differentiation into neu- trophil granulocytes. This putative affection of a myeloid precursor cell by clozapine would in turn represent a he- matopoetic stress situation which is followed by com- pensatory mechanisms. The various differentiation steps of the hematopoietic system are controlled by a complex network of interacting cytokines (Bender et al. 1987). The majority of precursor cells committed for granulo- monocytopoiesis are primarily controlled by granulo- cyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) (Bend- er et al. 1987). In order to compensate for disturbances in granulomonocytopoietic maturation, elevated GM- and G-CSF levels have been reported in serum (Ruef and Coleman 1991). Assuming that the neutrophil differenti- ation is impaired by a pathogenetic mechanism, the com- pensatory G- and GM-CSF release might cause a stimu- lation in eosinophilic maturation while no being suffi- cient for the differentiation of neutrophil granulocytes. The corresponding laboratory feature for this compensa- tory regulation would show eosinophilia followed by neutropenia with a time delay of a few days.

Consequently, the question arises why some patients respond to the putative causative factor with only tran- sient neutropenia (Hummer et al. 1992) while others go forward to a progressive disorder. One speculative expla- nation focuses on successful compensatory mechanisms by cytokines which produce sufficient stimulation of granulomonocytopoiesis. In such patients increased eosi- nophil counts would be followed by a transient or no ef- fect on neutrophil granulocytes. On the other hand, if the cytokine compensation is insufficient, neutropenia will become apparent and at times progressive.

Additional pathophysiological considerations, includ- ing the role of prostaglandin E as discussed by Banov et al. (1993), have to be mentioned. In contrast to the re- sults of Banov et al. (1993), we did not find an increased incidence of eosinophilia in female patients.

The available data and considerations outlined above add evidence to a stem cell impairment hypothesis of clozapine induced blood dyscrasias. Pharmacogenetic properties as discussed by Pfister et al. (1992) could be the explanation why some patients seem to be "immune" against these effects, while others show pathological re- sponses in qualitatively and quantitatively different ways (Hummer et al. 1992). The question whether clozapine- induced blood dyscrasias are immune mediated or toxic phenomena remains unresolved, although the involve- ment of eosinophil granulocytes would seem to favour an immunological explanation.

It is difficult to draw definitive conclusions from this study, which needs to be replicated in a larger sample. Nevertheless, our results appear to indicate that eosino- philic patients should be given more attention than pa- tients without eosinophilia where the development of subsequent neutropenia is less likely.

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