LETTERS ANDCORRESPONDENCE

Letters and correspondence submitted for possible publication mustbe identified as such. Text length must not exceed 500 words andfive bibliographic references. A single concise figure or table may beincluded if it is essential to support the communication. Letters nottyped double-spaced will not be considered for publication. Letters notmeeting these specifications will not be returned to authors. Letters tothe Editor are utilized to communicate a single novel observation orfinding. Correspondence is to be used to supplement or constructivelycomment on the contents of a publication in the journal and cannotexceed the restrictions for Letters to the Editor. The Editor reservesthe right to shorten text, delete objectional comments, and makeother changes to comply with the style of the journal. Permission forpublication must be appended as a postscript. Submissions must besent to Paul Chervenick, M.D., Editor of Brief Reports/Letters toEditors, American Journal of Hematology, H. Lee Moffitt CancerCenter, University of South Florida, 12902 Magnolia Drive, Tampa,FL 33612 to permit rapid consideration for publication.

Chlorambucil/Prednisone-Induced Seizures in a PatientWith Non-Hodgkins Lymphoma

To the Editor: Chlorambucil is an oral alkylant agent that is widely used forthe treatment of patients with chronic lymphocytic leukemia, non-Hodgkins lymphoma, and Hodgkins disease. Myelosuppression is themost common adverse effect. Seizures are rarely reported as another formof acute toxicity. We report a case that highlights this potential complica-tion when chlorambucil is associated with prednisone.

OBSERVATION

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

ticonvulsant therapy with valproic acid was given. The patient remainedfree of seizures and on valproic acid at the time of last follow-up in May2000.

DISCUSSION

The neurotoxicity of chlorambucil is suggested by three arguments: (i)After oral administration, chlorambucil is metabolized to phenylacetic acidmustard. The monohydroxy and dihydroxy hydrolysis products of bothchlorambucil and its alkylating metabolite may yield to chloroacetalde-hyde, which is also produced by ifosfamide metabolism [1]. This substanceis thought to be neurotoxic because of its structural similarity to metabo-lites of ethanol (acetaldehyde) and chloral hydrate (trichloroacetaldehyde)[2]. (ii) Experimental data in animals suggest that high-dose chlorambucilmay induce lethal seizures [3,4]. (iii) Even if they are few in number,several clinical reports enhance the relationship between chlorambucil andseizure. Salloum et al. reviewed these cases in 1997 [5], and just oneadditional case has been published since then [6].

Analysis of the literature shows that most cases of chlorambucil-inducedseizures occurred in children with nephrotic syndrome. In this situation itis not clear whether seizures were due to an enhanced sensitivity in child-hood or because of altered pharmacokinetics of chlorambucil as a result ofnephrotic syndrome. In adults, most of the patients received high-dosechlorambucil (in preparative regimens for bone marrow transplantation orin Phase III studies for the treatment of non-small-cell lung carcinoma andother solid tumors) or had a medical history of seizures. Our case demon-strates that neurotoxic side effects of chlorambucil may occur in standardsituation of prescription, such as pulse therapy. The role of corticosteroidsassociated with chlorambucil in this case and in the others reported inadults treated for Hodgkins disease or non-Hodgkins lymphoma may bestressed. Corticosteroids, because of their own action on the cortical neuronexcitability level, decrease the load of neurotoxics (as chlorambucil me-tabolites) that are able to start up seizures [7].

E. JOURDAND. TOPART

Service de Medecine Interne B, Hopital Caremeau, Nmes, FranceV. PINZANI

Centre Regional de Pharmacovigilance, CHU Montpellier, Montpellier,France

J. JOURDANService de Medecine Interne B, Hopital Caremeau, Nmes, France

REFERENCES1. Chabner B, Longo D. Cancer chemotherapy and biotherapy. Principles and prac-

tice. 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

1997;79(5):10091013.6. Apaydin S, Ozaras R, Erek E, Tahan V, Altiparmak MR, Celik Y, Cetinkaya A.

Chlorambucil-induced seizure in a patient with nephrotic syndrome. Nephron1999;82(4):368.

7. Sapolsky RM. Stress, glucocorticoids, and damage to the nervous system: thecurrent state of confusion. Stress 1996;1(1):119.

American Journal of Hematology 67:147149 (2001)

2001 Wiley-Liss, Inc.

Apoptotic Cells in a Peripheral Blood Smear in theContext of EBV InfectionTo the Editor: Epstein-Barr virus (EBV) infection is usually detected on ablood smear by lymphocytosis and atypical T-cell cells so inappropriatelycalled atypical mononuclear cells. Apoptosis refers to the morphologicfeatures of programmed cell death, which is characterized by cell shrink-age, nuclear condensation, membrane blebbing, and fragmentation intomembrane bound apoptotic bodies [1]. Some viral infections have beencharacterized by increased death of T-cells (for example, induced by thehuman immunodeficiency virus). We present here a case report, stressingthe association between apoptotic lymphocytes and EBV infection.

A 1-year-old infant presented with fever, hepatosplenomegaly, and mod-erate thrombopenia. Bacterial investigations proved to be negative. Thediagnosis of EBV infection was made on the basis of clinical findings, thepresence of heterophil antibodies, and atypical pleomorphic lymphocytesin the blood smear. Apoptotic lymphocytes were found (Figs. 1 and 2)comprising 20% of the white blood cells. The cells showed condensedacidophilic cytoplasms and fragmented nuclei with intense basophilic chro-matin, sometimes collapsing into the vacuoles. Dense, round, pyknotic

nuclei were also observed. Treatment was supportive, and symptoms re-solved in 3 weeks. Follow-up after 1 month was normal.

Visual inspection of scattergrams generated by some automated hema-tology analyzers can be useful for the detection of apoptotic lymphocytesin the peripheral blood [2]. As similar changes could occur if the blood wasleft at room temperature for a long time, an in vitro necrobiotic artifactshould be rule out [3]. Apoptotic lymphocytes have been transiently foundin blood smears from patients with suspected infections mononucleosisaffecting up to 20% of the lymphoid cells [4]. Among patients presentinga reactive change in lymphocytes with lymphoid apoptotic cells and a viralinfection, 82% had a real EBV infection [4]. Apoptotic cells have beensuggested to be EBV infected T-cells or EBV transformed B-cells, dyingafter Fas ligand-induced apoptosis. The presence of apoptotic lymphoidcells has been associated with a prolonged and severe clinical course ofinfectious mononucleosis [4].

As reported here, the detection of apoptotic lymphoid cells in the contextof suspected infectious mononucleosis is an additional argument in favor ofEBV infection.

J.F. LESESVEX. TROUSSARD

Laboratoires dHematologie, CHU Nancy & Caen, France

Figs. 1a and 1b. Apoptotic lymphoid cell with atypical mononuclear cell (May-Grunwald-Giemsa, original magnification1000). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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

148 Letters and Correspondence

REFERENCES1. 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 ofapoptosis in peripheral blood lymphocytes. Cytometry 2000;42:209214.

3. Bain BJ. Blood cells. A practical guide. 2nd edition. Oxford: BlackwellScience; 1995. 326 p.

4. Lach-Szyrma V, Brito-Babapulle F. The clinical significance of apop-totic cells in peripheral blood smears. Clin Lab Haematol 1999;21:277280.

Detection of a Rare Splice Acceptor Site Mutation (IVS I nt130 Gfi C) of the b Globin Gene in 3 Patients ofEastern IndiaTo the Editor: Twenty-nine mutations of the b globin gene have beenreported so far to occur in Thalassemia patients of India. Only 5 or 6mutations seem to occur at an appreciable frequency, the most commonone being IVS I nt 5 (Gfi C) [1]. Others occur at different frequencies atdifferent regions of the country, reflecting the composite nature of Indianpopulation. A few cases have remained uncharacterized in all studies, andthe frequency of uncharacterized mutations is highest (~ 9%) in Bengal [1],indicating presence of hitherto unidentified mutations present in populationof this region. We report here the presence of a rare splice acceptor sitemutation IVS I nt 130 (Gfi C) in three patients of West Bengal. Thismutation, previously reported in Japan and Turkey, has not been detectedin Indian population so far.

The first subject is a 10-year-old Bengalee HbE b-thalassemia patientwith hematological features of Hb 8.5 g/dL, PCV 28.5, MCV 72.4, MCH21.7, MCHC 29.9, HbF 4%, and bE + d 19.2%. The boy is regularlytransfused. His mother is a carrier of HbE mutation. None of the b globinmutations commonly occurring in Indian population could be detected inthe boys or his fathers DNA. So a 499-base pair fragment, from the 1stnucleotide of the 19th codon to the 72nd nucleotide of the second intronwas amplified by polymerase chain reaction (PCR) and subjected to con-formation-sensitive gel electrophoresis analysis (CSGE) [2]. Heteroduplexformation was indicated in both the boys and his fathers DNA. Thisfragment did not carry any sequence polymorphism in the fathers case butstill showed heteroduplex formation. Upon sequencing, using thermalcycle sequencing protocol, a Gfi C substitution was found in the 130thposition of the first intron (Fig. 1). This change in splice acceptor siteproduces a b mutation which has not been reported in India so far.

This mutation abolishes a CvnI restriction site in the DNA and can bedetected by the failure of CvnI to digest the 0.5-kb fragment at the mutationsite, so that instead of 88,201- and 211-bp fragments, 211- and 289-bpfragments are obtained. The exact nucleotide change can be confirmed bysequencing.

Two other patients also harbored this mutation along with the IVS I nt5 (Gfi C) mutation.

Of the three chromosomes harboring the IVS I nt 130 (Gfi C) mutation,2 were subjected to haplotype analysis of the b globin gene cluster byPCR-RFLP, using the sites HindII e, HindIII Gg, HindIII Ag, HindII58Cb, HindII 38Cb, and HinfI b [3]. One chromosome was associatedwith the pattern + +, which is very common in Indian population,both in normal and b thalassemia chromosomes. The other chromosomecarried the haplotype + + + +, which we found also with chromosomescarrying the Co 30 (Gfi C) mutation [3]. This haplotype has been reportedto be associated with the Co 30 (Gfi C) mutation in Tunisian people [4].

Though the two chromosomes bear different 58 haplotypes, the only bglobin site studied has the same status (+) in both, and the difference inhaplotypes might be explained as a result of recombination. This mutationhas also been reported in Turkey and Japan at a very low frequency, but tothe best of our knowledge the haplotypes associated with the chromosomes

have not been published. So it is not possible to decide whether thismutation originated here or whether its presence in India is a result of theMuslim invasion of India in the middle ages. Incidentally this is also thefirst application of CSGE in the detection of b globin gene mutation.

A. BANDYOPADHYAYS. BANDYOPADHYAY

U.B. DASGUPTADepartment of Biophysics, Molecular Biology and Genetics, Calcutta, India

S. CHANDRAM.K. DAS

Kothari Medical Centre, Calcutta, India

REFERENCES1. Verma IC, Saxena R, Thomas E, Jain PK. Regional distribution of b 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 withrespect to cost and time for finding mutations. Hum Mut 1997;9:339343.

3. Bandyopadhyay A, Bandyopadhyay S, Dutta Chowdhury M, Dasgupta UB. Majorb-globin gene mutations in Eastern India and their associated haplotypes. HumHered 1999;49:232235.

4. Chibani J, Vidaud M, Duquesnoy O, Berge Lefran C, Piratsu M, Ellouze F, RosaJ, Goossens M. The particular spectrum of b thalassemia genes in Tunisia. HumGenet 1988;78:190192.

Fig. 1. Part of the sequencing plate showing the G fi C mu-tation on the coding strand at IVS I nt 130.

Letters and Correspondence 149