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American Journal of Hematology 67:147149 (2001)

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FL 33612 to permit rapid consideration for publication.

Chlorambucil/Prednisone-Induced Seizures in a Patient

With Non-Hodgkins Lymphoma
To the Editor: Chlorambucil is an oral alkylant agent that is widely used for
the treatment of patients with chronic lymphocytic leukemia, nonHodgkins lymphoma, and Hodgkins disease. Myelosuppression is the
most common adverse effect. Seizures are rarely reported as another form
of acute toxicity. We report a case that highlights this potential complication when chlorambucil is associated with prednisone.

ticonvulsant therapy with valproic acid was given. The patient remained
free of seizures and on valproic acid at the time of last follow-up in May

The neurotoxicity of chlorambucil is suggested by three arguments: (i)
After oral administration, chlorambucil is metabolized to phenylacetic acid
mustard. The monohydroxy and dihydroxy hydrolysis products of both
chlorambucil and its alkylating metabolite may yield to chloroacetaldehyde, which is also produced by ifosfamide metabolism [1]. This substance
is thought to be neurotoxic because of its structural similarity to metabolites of ethanol (acetaldehyde) and chloral hydrate (trichloroacetaldehyde)
[2]. (ii) Experimental data in animals suggest that high-dose chlorambucil
may induce lethal seizures [3,4]. (iii) Even if they are few in number,
several clinical reports enhance the relationship between chlorambucil and
seizure. Salloum et al. reviewed these cases in 1997 [5], and just one
additional case has been published since then [6].
Analysis of the literature shows that most cases of chlorambucil-induced
seizures occurred in children with nephrotic syndrome. In this situation it
is not clear whether seizures were due to an enhanced sensitivity in childhood or because of altered pharmacokinetics of chlorambucil as a result of
nephrotic syndrome. In adults, most of the patients received high-dose
chlorambucil (in preparative regimens for bone marrow transplantation or
in Phase III studies for the treatment of non-small-cell lung carcinoma and
other solid tumors) or had a medical history of seizures. Our case demonstrates that neurotoxic side effects of chlorambucil may occur in standard
situation of prescription, such as pulse therapy. The role of corticosteroids
associated with chlorambucil in this case and in the others reported in
adults treated for Hodgkins disease or non-Hodgkins lymphoma may be
stressed. Corticosteroids, because of their own action on the cortical neuron
excitability level, decrease the load of neurotoxics (as chlorambucil metabolites) that are able to start up seizures [7].


A 56-year-old woman was referred to our center in October 1999 for the
diagnosis of a subcutaneous tumefaction of the left lumbar fossa. She had
a medical history of cervical spine fracture, with residual pain treated with
tramadol and clonazepam. Physical examination was normal except for the
tumefaction. A biopsy of the lesion showed a B-cell non-Hodgkins lymphoma of the marginal zone. Staging evaluation, including chest and abdominal computed tomographic (CT) scans and bone marrow biopsy, was
normal. Cytologic examination of cerebrospinal fluid was not performed at
diagnosis. Treatment with chlorambucil 12 mg daily for 5 days associated
with prednisone 50 mg daily for 5 days was planned for 3 courses every 4
weeks. On day 13 of the first course she had a syncopal episode with
generalized tonicclonic seizures. She was brought to the hospital where
she had similar seizures, witnessed by medical personnel and confirmed by
electroencephalogram (EEG). Seizures were controlled by intravenous infusion of clonazepam, which was discontinued in a few days. No evidence
of cerebral localization of her lymphoma was found in cerebrospinal fluid
examination; cerebral CT scan and encephalic magnetic resonance imaging
(MRI) were normal. Although chlorambucil was held, the patient took her
second course of chemotherapy as had been planned initially. On Day 3 of
treatment she was again noted to have generalized tonicclonic seizures
while at home. Seizures discontinued spontaneously, and prophylactic an-

2001 Wiley-Liss, Inc.

Service de Medecine Interne B, Hopital Caremeau, Nmes, France

Centre Regional de Pharmacovigilance, CHU Montpellier, Montpellier,
Service de Medecine Interne B, Hopital Caremeau, Nmes, France

1. Chabner B, Longo D. Cancer chemotherapy and biotherapy. Principles and practice. 2nd edition. Baltimore: Lippincott Williams & Wilkins; 1996.
2. Goren MP, Wright RK, Pratt CB, Pell FE. Dechloroethylation of ifosfamide and
neurotoxicity. Lancet 1986;2(8517):12191220.
3. Hagen E, Hurwitz L, Davis K, Jenner P. Toxic effects of chlorambucil (CB1348)
in animals. Fed Proc 1957;16:304.
4. Pradhan S, Ajmono Marsan C. Chlorambucil toxicity and EEG centrencephalic
patterns. Epilepsia 1963;4:114.
5. Salloum E, Khan KK, Cooper DL. Chlorambucil-induced seizures. Cancer
6. Apaydin S, Ozaras R, Erek E, Tahan V, Altiparmak MR, Celik Y, Cetinkaya A.
Chlorambucil-induced seizure in a patient with nephrotic syndrome. Nephron
7. Sapolsky RM. Stress, glucocorticoids, and damage to the nervous system: the
current state of confusion. Stress 1996;1(1):119.


Letters and Correspondence

Apoptotic Cells in a Peripheral Blood Smear in the

Context of EBV Infection
To the Editor: Epstein-Barr virus (EBV) infection is usually detected on a
blood smear by lymphocytosis and atypical T-cell cells so inappropriately
called atypical mononuclear cells. Apoptosis refers to the morphologic
features of programmed cell death, which is characterized by cell shrinkage, nuclear condensation, membrane blebbing, and fragmentation into
membrane bound apoptotic bodies [1]. Some viral infections have been
characterized by increased death of T-cells (for example, induced by the
human immunodeficiency virus). We present here a case report, stressing
the association between apoptotic lymphocytes and EBV infection.
A 1-year-old infant presented with fever, hepatosplenomegaly, and moderate thrombopenia. Bacterial investigations proved to be negative. The
diagnosis of EBV infection was made on the basis of clinical findings, the
presence of heterophil antibodies, and atypical pleomorphic lymphocytes
in the blood smear. Apoptotic lymphocytes were found (Figs. 1 and 2)
comprising 20% of the white blood cells. The cells showed condensed
acidophilic cytoplasms and fragmented nuclei with intense basophilic chromatin, sometimes collapsing into the vacuoles. Dense, round, pyknotic

nuclei were also observed. Treatment was supportive, and symptoms resolved in 3 weeks. Follow-up after 1 month was normal.
Visual inspection of scattergrams generated by some automated hematology analyzers can be useful for the detection of apoptotic lymphocytes
in the peripheral blood [2]. As similar changes could occur if the blood was
left at room temperature for a long time, an in vitro necrobiotic artifact
should be rule out [3]. Apoptotic lymphocytes have been transiently found
in blood smears from patients with suspected infections mononucleosis
affecting up to 20% of the lymphoid cells [4]. Among patients presenting
a reactive change in lymphocytes with lymphoid apoptotic cells and a viral
infection, 82% had a real EBV infection [4]. Apoptotic cells have been
suggested to be EBV infected T-cells or EBV transformed B-cells, dying
after Fas ligand-induced apoptosis. The presence of apoptotic lymphoid
cells has been associated with a prolonged and severe clinical course of
infectious mononucleosis [4].
As reported here, the detection of apoptotic lymphoid cells in the context
of suspected infectious mononucleosis is an additional argument in favor of
EBV infection.

Laboratoires dHematologie, CHU Nancy & Caen, France

Figs. 1a and 1b. Apoptotic lymphoid cell with atypical mononuclear cell (May-Grunwald-Giemsa, original magnification
1000). [Color figure can be viewed in the online issue, which is available at]

Figs. 2a and 2b. Apoptotic lymphoic cell (May-Grunwald-Giemsa, original magnification 1000). [Color figure can be
viewed in the online issue, which is available at]

Letters and Correspondence


1. Andreoli TE. The apoptotic syndromes. Am J Med 1999;107:488506.
2. Taga K, Yoshida M, Kaneko M, Asada M, Okada M, Taniho M, Tosato
G. Contribution of automated hematology analysis to the detection of
apoptosis in peripheral blood lymphocytes. Cytometry 2000;42:209
3. Bain BJ. Blood cells. A practical guide. 2nd edition. Oxford: Blackwell
Science; 1995. 326 p.
4. Lach-Szyrma V, Brito-Babapulle F. The clinical significance of apoptotic cells in peripheral blood smears. Clin Lab Haematol 1999;21:

Detection of a Rare Splice Acceptor Site Mutation (IVS I nt

130 GC) of the Globin Gene in 3 Patients of
Eastern India
To the Editor: Twenty-nine mutations of the globin gene have been
reported so far to occur in Thalassemia patients of India. Only 5 or 6
mutations seem to occur at an appreciable frequency, the most common
one being IVS I nt 5 (GC) [1]. Others occur at different frequencies at
different regions of the country, reflecting the composite nature of Indian
population. A few cases have remained uncharacterized in all studies, and
the frequency of uncharacterized mutations is highest (9%) in Bengal [1],
indicating presence of hitherto unidentified mutations present in population
of this region. We report here the presence of a rare splice acceptor site
mutation IVS I nt 130 (GC) in three patients of West Bengal. This
mutation, previously reported in Japan and Turkey, has not been detected
in Indian population so far.
The first subject is a 10-year-old Bengalee HbE -thalassemia patient
with hematological features of Hb 8.5 g/dL, PCV 28.5, MCV 72.4, MCH
21.7, MCHC 29.9, HbF 4%, and E + 19.2%. The boy is regularly
transfused. His mother is a carrier of HbE mutation. None of the globin
mutations commonly occurring in Indian population could be detected in
the boys or his fathers DNA. So a 499-base pair fragment, from the 1st
nucleotide of the 19th codon to the 72nd nucleotide of the second intron
was amplified by polymerase chain reaction (PCR) and subjected to conformation-sensitive gel electrophoresis analysis (CSGE) [2]. Heteroduplex
formation was indicated in both the boys and his fathers DNA. This
fragment did not carry any sequence polymorphism in the fathers case but
still showed heteroduplex formation. Upon sequencing, using thermal
cycle sequencing protocol, a GC substitution was found in the 130th
position of the first intron (Fig. 1). This change in splice acceptor site
produces a mutation which has not been reported in India so far.
This mutation abolishes a CvnI restriction site in the DNA and can be
detected by the failure of CvnI to digest the 0.5-kb fragment at the mutation
site, so that instead of 88,201- and 211-bp fragments, 211- and 289-bp
fragments are obtained. The exact nucleotide change can be confirmed by
Two other patients also harbored this mutation along with the IVS I nt
5 (GC) mutation.
Of the three chromosomes harboring the IVS I nt 130 (GC) mutation,
2 were subjected to haplotype analysis of the globin gene cluster by
PCR-RFLP, using the sites HindII , HindIII G, HindIII A, HindII
5, HindII 3, and HinfI [3]. One chromosome was associated
with the pattern + +, which is very common in Indian population,
both in normal and thalassemia chromosomes. The other chromosome
carried the haplotype + + + +, which we found also with chromosomes
carrying the Co 30 (GC) mutation [3]. This haplotype has been reported
to be associated with the Co 30 (GC) mutation in Tunisian people [4].
Though the two chromosomes bear different 5 haplotypes, the only
globin site studied has the same status (+) in both, and the difference in
haplotypes might be explained as a result of recombination. This mutation
has also been reported in Turkey and Japan at a very low frequency, but to
the best of our knowledge the haplotypes associated with the chromosomes

Fig. 1. Part of the sequencing plate showing the GC mutation on the coding strand at IVS I nt 130.
have not been published. So it is not possible to decide whether this
mutation originated here or whether its presence in India is a result of the
Muslim invasion of India in the middle ages. Incidentally this is also the
first application of CSGE in the detection of globin gene mutation.

Department of Biophysics, Molecular Biology and Genetics, Calcutta, India
Kothari Medical Centre, Calcutta, India

1. Verma IC, Saxena R, Thomas E, Jain PK. Regional distribution of thalassemia
mutations in India. Hum Genet 1997;100:109113.
2. Ganguly A, Williams C. Detection of mutations in multi-exon genes, comparison
of conformation sensitive gel electrophoresis and sequencing strategies with
respect to cost and time for finding mutations. Hum Mut 1997;9:339
3. Bandyopadhyay A, Bandyopadhyay S, Dutta Chowdhury M, Dasgupta UB. Major
-globin gene mutations in Eastern India and their associated haplotypes. Hum
Hered 1999;49:232235.
4. Chibani J, Vidaud M, Duquesnoy O, Berge Lefran C, Piratsu M, Ellouze F, Rosa
J, Goossens M. The particular spectrum of thalassemia genes in Tunisia. Hum
Genet 1988;78:190192.