Correspondence

TRANSFER OF NUCLEATED MATERNAL CELLS INTO FETAL CIRCULATIONDURING THE SECOND TRIMESTER OF PREGNANCY

We read with interest the report by Petit et al (1997)demonstrating the transfer of maternal cells into the fetalcirculation during the third trimester of pregnancy. Theauthors were unable to detect maternal DNA sequencesin their single second-trimester sample at a gestation age of20 weeks.

In order to investigate whether there is passage ofmaternal nucleated cells into the fetal circulation in thesecond trimester of pregnancy, we studied nine womenundergoing elective termination of pregnancy for socialindications at the Fetal Medicine Unit, Homerton Hospital,London, U.K. Informed consent was obtained in each case.Ethics approval was obtained from the Ethics Committee atHomerton Hospital. Maternal blood samples were collectedfrom the antecubital fossae of the patients prior to anyinvasive procedure. Fetal blood sampling was performedunder ultrasound guidance immediately before terminationof pregnancy as previously described (Abbas et al, 1993). 2–5 ml of amniotic fluid was taken prior to fetal blood samplingas a control for maternal bleeding during the procedure.

Extraction of DNA from fetal and maternal blood samples(Lo et al, 1996) and amniotic fluid samples (Rebello et al,1991) was as previously described. Three polymorphicsystems were used for the detection of maternal DNAsequences in fetal blood, based on the detection of

polymorphisms in a region 50 to the human delta-globingene (the RT region), the glutathione S-transferase M1locus (GSTM1) and the angiotensin-converting enzyme(ACE) gene (Lo et al, 1996). These polymorphic systemswere all bi-allelic: the R and T alleles for the RT region, thepresence (GSTþ) or absence (GST¹) of the gene for theGSTM1 system, and the insertion (I) and deletion (D) of a287 bp fragment in intron 16 of the ACE gene. To beinformative for the detection of maternal DNA sequences infetal blood, the fetus should be homozygous for at least one ofthese polymorphisms and the mother heterozygous for thesame polymorphism. Detection of maternal DNA was thenperformed by amplifying the maternal allele which was notpossessed by the fetus.

Of the nine cases, RT, GSTM1 and ACE genotypingrevealed five informative scenarios (Table I: cases 1, 2, 5, 7and 9). Maternal DNA sequence was detected in the fetalblood sample obtained from case 7 at a gestation age of 13weeks. The PCR analysis was repeated with the same result.The amniotic fluid sample taken before fetal blood samplingin this case was negative for maternal DNA sequences, thusdemonstrating that the result was not an artefact ofmaternal bleeding during the invasive procedure. Othernegative controls, including water blanks and controlDNA samples not containing the amplified allele, wereconsistently negative.

These results demonstrate that the prenatal transfer ofnucleated maternal cells into the fetal circulation can occuras early as the 13th week of gestation. This represents theearliest documented case of maternofetal cell transfer inhumans. This prenatal cell trafficking is a potentialmechanism for the mother to influence the development ofthe immune system of the fetus. This may be one of theexplanations why certain individuals exhibit a degree oftolerance to the noninherited maternal HLA antigens(Claas et al, 1988). It also provides a possible route for theprenatal transmission of infectious agents from the motherto the fetus.

ACKNOWLEDGMENT

Y.M.D.L. and N.M.H. are supported by the Hong KongResearch Grant Council.

University of Oxford, E. S. F. L O

Nuffield Department ofClinical Biochemistry,

John Radcliffe Hospital,Oxford OX3 9DU, U.K.

British Journal of Haematology, 1998, 100, 605–618

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Table I. Prenatal nucleated cell transfer from mother to fetus.

Maternal cellsInformative Informative in fetal blood

Gestation maternal fetal (detectionCase age (weeks) genotype genotype system used)

1 19 ID DD ¹(I)2 14 ID DD ¹(I)3 15 NI NI NI4 16 NI NI NI5 15 RT TT ¹(R)6 16 NI NI NI7 13 RT RR þ(T)8 13 NI NI NI9 16 RT TT ¹(R)

NI ¼ not informative. R ¼ R allele of the RT polymorphism;T ¼ T allele of the RT polymorphism; I ¼ insertion allele of theangiotensin-converting enzyme gene polymorphism; D ¼ deletionallele of the angiotensin-converting enzyme gene polymorphism. þ,presence of PCR signal; ¹, absence of PCR signal. The system usedfor detecting maternal DNA in fetal blood is indicated in parentheses.

Department of Chemical Pathology, Y. M. D. L O

The Chinese University N. M. H J E L M

of Hong Kong,Prince of Wales Hospital,Shatin, N.T., Hong Kong

Fetal Medicine Unit, B. T H I L AG A NAT H A N

Homerton Hospital,London, U.K.

REFERENCES

Abbas, A., Thilaganathan, B., Buggins, A.G.S., Layton, D.M. &Nicolaides, K.H. (1993) Fetal plasma interferon gamma concen-tration in normal pregnancy. American Journal of Obstetrics andGynecology, 168, 1414–1416.

Claas, F.H.J., Gijbels, Y., Van der Velden-De Munck, J. & Van Rood, J.J.(1988) Induction of B cell unresponsiveness to noninheritedmaternal HLA antigens during fetal life. Science, 241, 1815–1817.

Lo, Y.M.D., Lo, E.S.F., Watson, N., Noakes, L., Sargent, I.L.,Thilaganathan, B. & Wainscoat, J.S. (1996) Two-way cell trafficbetween mother and fetus: biologic and clinical implications.Blood, 88, 4390–4395.

Petit, T., Dommergues, M., Socie, G., Domez, Y., Gluckman, E. &Brison, O. (1997) Detection of maternal cells in human fetal bloodduring the third trimester of pregnancy using allele-specific PCRamplification. British Journal of Haematology, 98, 767–771.

Rebello, M.T., Hackett, G., Smith, J., Loeffler, F.E., Robson, S.,MacLachlan, N., Beard, R.W., Rodeck, C.H., Williamson, R.,Coleman, D.V. & Williams, C. (1991) Extraction of DNA fromamniotic fluid cells for the early prenatal diagnosis of geneticdisease. Prenatal Diagnosis, 11, 41–6.

We found the results reported by Lo et al very interestingsince their work constitutes a useful complement to ourpreviously published study (Petit et al, 1997). In the latter we

were able to detect maternal cells in 8/8 fetal blood samplescollected during the third trimester of pregnancy but wecould not detect them in the single second-trimester samplethat was tested. Lo et al have searched for the presence ofmaternal cells in five second-trimester samples using allele-specific PCR amplification. Although the authors did notmention the sensitivity limit of their assay, it should becomparable to ours since we both used similar methods.They detected maternal cells in one sample out of five. Webelieve that their result (1/5) was not significantly differentfrom ours (0/1). Altogether, the results of both studiesindicate that transfer of maternal cells into the fetalcirculation is probably a rare event during the secondtrimester of pregnancy but is quite frequent during the thirdtrimester. It might be worth extending the study to a largersample in order to obtain a statistically significant assess-ment of the frequency of the phenomenon. We do agreewith the conclusion of Lo et al that ‘this prenatal celltrafficking is a potential mechanism for the mother toinfluence the development of the immune system of thefetus’.

Laboratoire de Genetique T H I E R RY P E T I T

Oncologique, O L I V I E R B R I S O N

URA 1967 CNRS,Institut Gustavc-Roussy, Villejuif

Service de Gynecologie-Obstetrique, M A RC D O M M E RG U E S

Maternite Port-Royal, Paris Y V E S D U M E Z

Service d’Hematologie-Greffe G E R A R D S O C I E

de Moelle, E L I A N E G L U C K M A N

Hopital Saint-Louis, Paris, France

Keywords: maternal cells, fetomaternal cell trafficking,polymerase chain reaction, fetal blood, immune tolerance.

MENINGEAL RELAPSE IN A PATIENT WITH ACUTE PROMYELOCYTIC LEUKAEMIATREATED WITH ALL-TRANS RETINOIC ACID

We would like to extend the interesting observation of Evanset al (1997) concerning central nervous system (CNS)relapse in acute promyelocytic leukaemia (APL). We recentlyexperienced the occurrence of meningeal relapse in a patientaffected with APL in complete clinical-haematologicalremission after all-trans-retinoic (ATRA) and idarubicininduction therapy according to the AIDA GIMEMA study(Avvisati et al, 1996).

The patient, a 50-year-old man, was diagnosed with APLin March 1997. Bone marrow examination showed classicmorphological APL. The phenotype was typical for APLexcept for CD56 positivity (77% of cells). The WBC count was7·5 × 109/l. The t(15;17) was demonstrated, with a bcr3PML breakpoint at the molecular level. In April 1997, afterinduction chemotherapy, a complete morphological remis-sion was obtained. 5 months later the patient complained ofheadache, vomiting and visual disturbances. The cerebro-spinal fluid was infiltrated with 0·7 × 109/l blasts which

showed a bcr3 PML breakpoint; CNS MNR was normal. Bonemarrow examination showed a morphological completeremission status with persistence of the same molecularlesion. The patient received intrathecal methotrexate andARA-C and systemic chemotherapy with high-dose ARA-Cfollowed by mitoxantrone, VP 16 and standard-dose ARA-C.He is now in CNS and bone marrow remission.

The present report raises the question whether the use ofATRA therapy increases the incidence of either CNSinvolvement or unusual sites of relapse in APL. In additionto those of Evans et al (1997) and this patient, three othercases have so far been reported (Weiss & Warrel, 1994;Molero et al, 1997). It has been claimed that theserecurrences could be related to the enhancement of adhesionmechanisms, possibly mediated by ATRA, through the over-expression of endothelial adhesion molecules and increasedhoming for CNS (Di Noto et al, 1996). Moreover, the peculiarimmunophenotype of our case, namely the CD56 marker

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expression, deserves comment. So far, no association of thismarker with molecularly confirmed APL has been found;in our experience, this was the only CD56þ case out of 16consecutive APL patients. It has been reported that, amongseveral cell-surface antigens studied, the expression ofneural-cell adhesion molecule CD56 in acute myeloidleukaemia seems to be a potential risk factor for extra-medullary involvement (Byrd et al, 1995). Recently, CD56expression was significantly associated with shorter remis-sion duration and survival in AML with t(8;21) (Baer et al,1997). Three of these cases relapsed as granulocyticsarcoma.

In conclusion, we would like to emphasize the need for theanalysis of a large series in order to confirm the possibleimpact of ATRA therapy in CNS and extramedullary disease.Moreover, the presence of neural-cell adhesion moleculeCD56, together with an unusual molecular lesion, couldcontribute to the identification of APL patients with a highrisk for extramedullary localization, susceptible to specifictherapeutic approaches.

Divisione di Ematologia, B RU N O M A RT I N O

Dipartimento di Emato-Oncologia, I O L A N DA V I N C E L L I

Azienda Ospedaliera A N T O N I O M A R I N O

‘Bianchi-Melacrino-Morelli’, M A RG H E R I TA C O M I S

89100 Reggio Calabria, F R A N C E S C A RO N C O

Italy F R A N C E S C O N O B I L E

REFERENCES

Avvisati, G., Lo Coco, F., Diverio, D., Falda, M., Ferrara, F., Lazzarino,M., Russo, D., Petti, M.C. & Mandelli, F. (1996) AIDA (all-trans-retinoic acidþidarubicin) in newly diagnosed acute promyelocyticleukemia: a Gruppo Italiano Malattie Ematologiche Malignedell’Adulto (GIMEMA) pilot study. Blood, 88, 1390–1398.

Baer, M.R., Stewart, C.C., Lawrence, D., Arthur, D.C., Byrd, J.C.,Davey, F.R., Schiffer, C.A. & Bloomfield, C.D. (1997) Expression ofthe neural cell adhesion molecule CD56 is associated with shorterremission duration and survival in acute myeloid leukemia witht(8;21)(q22;q22). Blood, 90, 1643–1648.

Byrd, J.C., Edenfield, J., Shields, D.J. & Dawson, N.A. (1995)Extramedullary myeloid cell tumors in acute nonlymphocyticleukemia: a clinical review. Journal of Clinical Oncology, 13, 1800–1816.

Di Noto, R., Schiavone, E.M., Ferrara, F., Manzo, C., Lopardo, C. & DelVecchio, V. (1996) All-trans retinoic acid promotes a differentialregulation of adhesion molecules on acute myeloid blast cells.British Journal of Haematology, 88, 247–255.

Evans, G., Grimwade, D., Prentice, H.G. & Simpson, N. (1997)Central nervous system relapse in acute promyelocytic leukaemiain patients treated with all-trans retinoic acid. British Journal ofHaematology, 98, 437–439.

Molero, T., Valencia, J.M. & Gomez-Casales, M.T. (1997) Centralnervous system (CNS) infiltration in a case of promyelocyticleukemia. Haematologica, 82, 637.

Weiss, M.A. & Warrel, R.P. (1994) Two cases of extramedullaryacute promyelocytic leukemia. Cancer, 74, 1882–1886.

Keywords: APL, ATRA, meningeal relapse, CD56, bcr3 PMLbreakpoint.

EOSINOPHILS AND SERUM EOSINOPHILIC CATIONIC PROTEINSIN INTERLEUKIN-2-BASED IMMUNOTHERAPY FOR CANCER

We read with interest the recent short report by Trulson et al(1997) showing a significant increase in eosinophiliccationic proteins in renal cancer patients before andduring IL-2 immunotherapy, when compared with healthycontrols. Comparing the serum levels of the three proteinsbefore and after treatment, only eosinophilic protein X (EPX)increase was statistically significant. The tendency ofeosinophils to degranulate, liberating their highly cytotoxiccationic proteins, was interpreted by the authors as thereason of the proposed cytotoxicity of eosinophils againsttumour cells (Trulson et al, 1997).

Several studies suggest that eosinophils may play a majorrole, in vivo, in the vast and complex anticancer activity ofthe immune system activated by IL-2 administration.Particularly, eosinophils may effect direct (Huland &Huland, 1992) or antibody-dependent (Rivoltini et al,1993) cancer lysis, or they may interact with CD4þ

lymphocytes serving as antigen-presenting cells, sinceHLA-DR expression seems to be inducible on eosinophils.

However, the potential mechanisms by which eosinophilsare reported to effect direct cytolysis remain debatable.Thus, even though some products of eosinophil oxidativemetabolism, such as superoxide anions, hydroxyl radicalsand singlet oxygen, can damage several cell types, and

eosinophilic free radicals are therefore likely to participate inthe supposed anticancer cytotoxicity of eosinophils, greatattention has also been paid to eosinophilic cationic proteins,which can damage surrounding tissues.

Using a sensitive radioimmunologic assay (Confalonieriet al, 1995), our group has recently found that eosinophiliccationic protein (ECP) and EPX increase significantly duringIL-2 immunotherapy according to Buzio’s schedule (Buzioet al, 1997) relative to pretreatment values in 12 renalcarcinoma patients enrolled in a multicentre adjuvantprotocol (Table I).

Our data are in disagreement with the Swedish results(Trulson et al, 1997) because baseline ECP and EPX values inrenal carcinoma patients do not differ from those of age- andsex-matched healthy controls. A possible explanation is thatthe Swedish patients presented a metastatic disease whichthe immune system (and therefore eosinophils) was acti-vated against, whereas our patients had undergone primarylesion resection and their immune system was less active,maybe also because of surgical immunosuppression. More-over, immunotherapy caused a marked increase in botheosinophilic proteins in our patients, unlike those ofTrulson et al (1997).

Finally, the role of eosinophilic cationic proteins in IL-2-

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induced anticancer activity as suggested by Trulson et al(1997) partially clashes with our in vitro observations thatthe cationic protein-rich supernatant of cultured eosinophilsfrom IL-2-treated patients cannot exert any cytotoxic effecton tumour cell lines (Moroni et al, 1997). On the other hand,the direct contact of eosinophils with cancer cells, with theirconsequent degranulation on cancer cells, may be necessaryfor the cytotoxicity of eosinophilic proteins to be effected(Huland & Huland, 1992).

Such contradictions will be solved only by accuratestatistical analyses of the influence of eosinophils andeosinophilic cationic proteins on the survival of large cohortsof IL-2-treated cancer patients.

Dipartimento di Medicina Interna C A M I L L O P O RTA

e Terapie Medica, M AU RO M O RO N I

Sezione di Medicina Interna e Nefrologia, M A R A D E A M I C I *and *Istituto di Clinica Pediatrica,Universita degli Studi di Pavia,I.R.C.C.S. Policlinico San Matteo,Pavia, Italy

REFERENCES

Buzio, C., De Palma, G., Passalacqua, R., Potenzoni, D., Cattabiani,M.A., Manenti, L. & Borghetti, A. (1997) Immunotherapy inadvanced renal cell cancer: repeated cycles at very-low doses.British Journal of Cancer, 76, 541–544.

Confalonieri, M., Tacconi, F., De Amici, M., Maccario, R., Porta, C.,Gioglio, L. & Bobbio-Pallavicini, E. (1995) Benign idiopathichypereosinophilia: a feeble masquerader or a smoldering form ofthe hypereosinophilic syndrome? Haematologica, 80, 50–53.

Huland, E. & Huland, H. (1992) Tumor-associated eosinophilia ininterleukin-2-treated patients: evidence of toxic eosinophil de-granulation on bladder cancer cells. Journal of Cancer Research andClinical Oncology, 118, 463–467.

Moroni, M., Porta, C., Gritti, D., De Amici, M., Giacobbe, O., Bobbio-Pallavicini, E. & Notario, A. (1997) Cationic protein-rich super-natants of cultured eosinophils from IL-2-treated patients have nocytotoxic activity on human renal cell carcinoma and melanomacells: a preliminary report. Annals of the New York Academy ofSciences, 832, (in press).

Rivoltini, L., Viggiano, V., Spinazze, S., Santoro, A., Colombo, M.P.,Takatsu, K. & Parmiani, G. (1993) In vitro anti-tumor activity ofeosinophils from cancer patients treated with subcutaneous

administration of interleukin-2: role of interleukin.-5. Inter-national Journal of Cancer 54, 8–15.

Trulson, A., Nilsson, S. & Venge, P. (1997) The eosinophil granuleproteins in serum, but not the oxidative matabolism of the bloodeosinophils, are increased in cancer. British Journal of Haematology,98, 312–314.

We read with interest the letter by Porta and co-workers inwhich they report a disagreement between our and theirdata on eosinophil proteins in serum in patients with renalcarcinoma, as they could not demonstrate any elevationsbefore treatment with IL-2. This was in spite of the fact thatwe and Porta’s group used the same methods of measuringthese eosinophil proteins, i.e. the ones we have developed incollaboration with Pharmacia (Peterson et al, 1991). Aspointed out in the letter, the difference in the results may bedue to the fact that our patients had metastases, whereas thepatients of Porta et al did not. This would therefore indicatethat activation of eosinophils is dependent on an activeimmune system present in patients with a metastatic disease.In agreement with Porta et al, we also found a tendencytowards higher eosinophil protein levels in serum aftertherapy with IL-2. However, the modest elevations in ourpatients may due to simultaneous treatment with a-interferon and the fact that the eosinophilopoietic activitieswere already operative.

The role of the eosinophil in cancer is still obscure, but ourfindings again emphasize a potential role in cancer cellkilling, since we (unpublished observations) and others(Roberts et al, 1990) have clearly shown that eosinophils arevery potent cancer cell killers either by means of oxygenradical production or by means of the cytotoxic granuleproteins. The reason for the lack of any cytotoxic activity insupernatants of cultured eosinophils as found by Porta et alcannot be judged from the data presented in the letter, butmay have several explanations, one of which is offered by theauthors themselves.

The main message of our brief paper was to draw attentionto a potential role of eosinophils in cancer, since we havefound clear evidence of the activation of the eosinophilpopulation in cancer. Studies are now underway which willanswer some of the questions raised by Porta et al.

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Table I. Serum ECP and EPX titres (mean 6 standard deviation) in 12 IL-2-treatedrenal cell cancer patients, before and after treatment, and in 12 age- and sex-matchedhealthy controls. Both ECP and EPX titres after treatment are significantly increasedwhen compared with pretreatment titres (P ¼ 0·0005 by paired Student’s t-test in bothcases). To the contrary, pretreatment levels in patients do not differ from those ofhealthy subjects. ECP and EPX were titred only after the first treatment cycle, to avoid apossible prolonged effect of the previous cycle of immunotherapy on the titrations ofthe following one.

ECP (mg/ml) EPX (mg/ml)

Healthy control subjects 10·50 6 0·75 19·48 6 1·21Kidney cancer patients before IL-2 treatment 10·80 6 1·03 19·15 6 1·21Kidney cancer patients after IL-2 treatment 33·19 6 2·83 79·76 6 8·94

Uppsala Universitet, AG N E TA T RU L S O N

Institutionen for Kliniska S T E N N I L S S O N

Laboratorievetenskaper, P E R V E N G E

Avdelningen for Klinisk Kemi,Akademiska Sjukhuset,751 85 Uppsala, Sweden

REFERENCES

Peterson, C.G., Enander, I., Nystrand, J., Andersson, A.S., Nilsson, L.

& Venge, P. (1991) Radioimmunoassay of human eosinophilcationic protein (ECP) by an improved method: established ofnormal levels in serum and turnover in vivo. Clinical andExperimental Allergy, 21, 561–565.

Roberts, R.L., Ank, B.A. & Stiehm, E.R. (1990) Human eosinophilsare more toxic than neutrophils in antibody-independent killing.Journal of Allergy and Clinical Immunology, 87, 1105–1115.

Keywords: eosinophils, ECP, EPX, cancer, IL-2 immuno-therapy.

SPLENIC LYMPHOMA WITH VILLOUS LYMPHOCYTES (SLVL)RESPONDING TO 2-CHLORODEOXYADENOSINE (2-CdA)

We read with interest the recent report by Bolam et al (1997)concerning their experience in the use of fludarabine in thetreatment of four patients with SLVL, all of whom achieved acomplete remission. Here we describe a case of SLVLresponding to 2-CdA.

In September 1994 a 58-year-old female presented with awhite cell count of 68·4 × 109/l found incidentally at thetime of a urinary tract infection. Haemoglobin and plateletcount were both normal, as was physical examination andCT body scan. Morphology of the peripheral blood revealedabnormal lymphoid cells with plentiful cytoplasm and hairyprojections. Immunophenotype analysis by APAAP con-firmed them to be clonal B cells (l restricted) and they wereCD25 negative. Bone marrow aspirate confirmed infiltrationwith similar cells and in trephine there was a diffuse excess oflymphoid cells forming vague aggregates in some areas. Themorphology and immunophenotype of these lymphoid cellswere reviewed and a diagnosis of SLVL was confirmed (D.Catovsky, personal communication). In particular, the cellswere negative for the cell surface markers CD11c, CD25,HC2 and Bly7 (CD103) which was consistent with thediagnosis (Matutes et al, 1994a, b).

Her disease progressed so that by October 1995 her whitecell count had risen to 143 × 109/l with mild thrombo-cytopenia (122 × 109/l) and she had marked hepatospleno-megaly. She was initially treated with two courses ofchlorambucil followed by six courses of weekly cyclophos-phamide but she did not respond. In March 1996 sheproceeded to splenectomy, prior to which her white cellcount was 177·5 × 109/l and platelets were 114 × 109/l.Surgery was uneventful and although her platelet countnormalized her white count remained unchanged. Histologyof the spleen showed diffuse expansion of the white pulp witheffacement of the normal architecture which is consistentwith SLVL.

Following splenectomy we treated her with four courses of2-CdA 0·1 mg/kg/d for 7 d between April and July 1996,which was well tolerated without any significant toxicity.A good partial remission was obtained with normalizationof her white count and a low level of residual bone marrowinfiltration. To date, her disease remains stable and she iswell and asymptomatic.

As pointed out by Bolam et al (1997), apart from

splenectomy, which is not feasible for all patients withSLVL, conventional therapy such as the use of alkylatingagents is unsatisfactory (Mulligan et al, 1991; Troussard et al,1996). 2-CdA, a purine nucleoside analogue like fludar-abine, is well tolerated with its main toxicity beinghaemopoietic suppression and prolonged immunosuppres-sion due to its effect on CD4þ lymphocyte (Delannoy, 1996).We and others (Delannoy, 1996) have seen good responses,and feel therefore that 2-CdA should be considered along-side fludarabine in the treatment of SLVL, particularly assecond-line therapy.

Department of Haematology, A N D R E S V I RC H I S

The Royal Free Hospital, AT U L M E H TA

London NW3 2QG

REFERENCES

Bolam, S., Orchard, J. & Oscier, D. (1997) Fludarabine is effective inthe treatment of splenic lymphoma with villous lymphocytes.British Journal of Haematology, 99, 158–161.

Delannoy, A. (1996) 2-Chloro-20-deoxyadenoside: clinical applica-tions in haematology. Blood Reviews, 10, 148–166.

Matutes, E., Morilla, R., Owusu-Ankomah, K., Houlihan, A. &Catovsky, D. (1994a) The immunophenotype of spleniclymphoma with villous lymphocytes and its relevance to thedifferential diagnosis with other B-cell disorders. Blood, 83, 1558–1562.

Matutes, E., Morilla, R., Owusu-Ankomah, K., Houlihan, A., Meeus,P. & Catovsky, D. (1994b) The immunophenotype of hairy cellleukaemia (HCL): proposal for a scoring system to distinguish HCLfrom B-cell disorders with hairy or villous lymphocytes. Leukaemiaand Lymphoma, 14, (Suppl. 1), 55–61.

Mulligan, S.P., Matutes, E., Dearden, C. & Catovsky, D. (1991) Spleniclymphoma with villous lymphocytes: natural history and responseto therapy in 50 cases. British Journal of Haematology, 78, 206–209.

Troussard, X., Valensi, F., Duchayne, E., Garand, R., Felman, P.,Tulliez, M., Henry-Amar, M., Bryon, P.A. & Flandrin, G. (1996)Splenic lymphoma with villous lymphocytes: clinical presentation,biology and prognostic factors in a series of 100 patients. BritishJournal of Haematology, 93, 731–736.

Keywords: splenic lymphoma with villous lymphocytes,2-chlorodeoxyadenosine.

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RETINOIC ACID SYNDROME DURING THE TREATMENT OF ACUTE MYELOMONOCYTIC LEUKAEMIAWITH ALL-TRANS-RETINOIC ACID AND LOW-DOSE CYTOSINE ARABINOSIDE

All-trans-retinoic acid (ATRA) yields a high rate of completeremission (CR) in patients with acute promyelocyticleukaemia (APL) through the induction of differentiation ofthe leukaemic cells (Degos et al, 1995). One of the major side-effects of ATRA is retinoic acid syndrome (RAS) consisting offever, respiratory distress, weight gain, lower extremityoedema, pleural and pericardial effusions, and hypotension(Frankel et al, 1992).

A 46-year-old Japanese male was diagnosed as havingacute myelomonocytic leukaemia (AMMoL) (FAB classifica-tion M4) in April 1995. His haemoglobin was 6·3 g/dl, whiteblood cell count 98·2 × 109/l (92% blasts), and plateletcount 19 × 109/l. A bone marrow aspirate, showing theproliferation of blasts, had a normal karyotype and wasnegative for PML/RAR-a by reverse transcriptase polymer-ase chain reaction. After obtaining CR with the inductiontherapy of idarubicin (IDR) 12 mg/m2/d i.v. for 5 d andcytosine arabinoside (AraC) 100 mg/m2 by continuous i.v.over 24 h for 10 consecutive days, he received two courses ofintermediate-dose AraC consolidation therapy. Then heunderwent autologous peripheral blood stem cell transplan-tation in September 1995. He relapsed in November 1995.Because of his extensive previous therapy and early relapse,a conventional re-induction therapy was not attempted.Instead, treatment with ATRA (45 mg/m2/d, p.o.) and low-dose AraC (10 mg/m2 × 2/d, s.c.) was started (Venditti et al,1993). 2 weeks later his white blood cell (WBC) count beganto increase with the maturation tendency of leukaemic cells

as evidenced by the emergence of bizarre but differentiatedmonocytes. To lower the WBC count, aclarubicin (15 mg/m2/d) was started on 8 December. However, the WBC countcontinued to increase, and on 10 December he developedfever, respiratory distress (PaO2 of 5·60 kPa on room air),weight gain, lower extremity oedema, and hypotension.Chest computed tomography showed bilateral interstitialinfiltrates with pleural and pericardial effusions. A diagnosisof RAS was made. ATRA was discontinued and high-dosemethyl-prednisolone together with IDR (10 mg/m2 i.v. for 5consecutive days) and standard-dose AraC (200 mg/m2 bycontinuous i.v. for 5 consecutive days) were administered.With these therapies the WBC count decreased and hisrespiratory distress resolved until 14 December. On 15December, melphalan 100 mg/m2 was intravenously admi-nistered. 3 d later, previously collected autologous peripheralblood stem cells (PBSC) were infused. On day 1 after thePBSC infusion, granulocyte-colony stimulating factor wasstarted to promote granulocyte recovery. On day 13 hisgranulocyte count exceeded 0·5 × 109/l. Bone marrowexamination on day 21 showed the disappearance ofleukaemic cells. However, AMMoL relapsed on day 51, andthe patient died of AMMoL progression on day 161 (Fig 1).

The pathophysiology of RAS is poorly understood. Severalpossibilities have been suggested, including a stimulatoryeffect of ATRA on the expression of cytokines by APL cellsand a change in the deformability of APL cells allowing theirrelease from the bone marrow (Degos et al, 1995). ATRA has

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Fig 1. The treatment with ATRA and low-dose AraC induced leukaemic differentiation and increased the WBC count. Afterwards the patientdeveloped retinoic acid syndrome (RAS) consisting of fever, respiratory distress, weight gain, lower extremity oedema, hypotension, pleural andpericardial effusions. RAS resolved by the administration of steroids and anti-leukaemic agents. ACR, aclarubicin; IDR, idarubicin.

differentiation-inducing potential in vitro for various leukae-mic cells other than APL (Koeffler, 1983). Furthermore,leucocytosis associated with ATRA therapy in metastaticnon-small cell lung cancer has been reported (Kahn et al,1992).

The clinical course of our patient is peculiar in that ATRAinduced differentiation of leukaemic cells other than APL invivo, and typical RAS developed. To our knowledge, this isthe first case of RAS in a non-APL patient.

Recently, ATRA has been investigated as a treatmentmodality for non-APL haematological disorders such as non-APL AML (Venditti et al, 1993), myelodysplastic syndrome,and cutaneous T-cell lymphoma (Tallman, 1994).

Patients with haematological malignancies other thanAPL who are treated with ATRA must be observed carefullybecause of the possibility that they may develop RAS.

Department of Haematology, KO J I NAG A F U J I

Hara Sanshin General Hospital, T E T S U YA E T O

and *First Department of Internal YA S U N O B U T O K U NA G A

Medicine, S H I N H AYA S H I

Faculty of Medicine, YO S H I Y U K I N I H O *Kyushu University,Fakuoka, Japan

REFERENCES

Degos, L., Dombret, H., Chomienne, C., Daniel, M.T., Miclea, J.M.,Chastang, C., Castaigne, S. & Fenaux, P. (1995) All-trans-retinoicacid as differentiating agent in the treatment of acute promyelo-cytic leukemia. Blood, 85, 2643–2653.

Frankel, S.R., Eardley, A., Lauwers, G., Weiss, M. & Warrell, R.P., Jr(1992) The ‘retinoic acid syndrome’ in acute promyelocyticleukemia. Annals of Internal Medicine, 117, 292–296.

Kahn, M.J., Luginbuhl, W., Gaines, L., Bratschi, J., Bavaria, J., Kaiser,L. & Treat, J. (1992) Leukocytosis associated with all-trans-retinoic acid therapy in metastatic non-small-cell lung cancer.Journal of the National Cancer Institute, 84, 1699–1671.

Koeffler, H.P. (1983) Induction of differentiation of human acutemyelogenous leukemia: therapeutic implications. Blood, 62, 709.

Tallman, M.S. (1994) All-trans-retinoic acid in acute promyelocyticleukemia and its potential in other hematologic malignancies.Seminars in Hematology, 31, 38–48.

Venditti, A., Stasi, R., Copetelli, U., Poera, G.D., Caravita, M.M.T.,Tribalto, M. & Papa, G. (1993) All-trans-retinoic acid: effectivetherapy even for ANLL other than APL? Evaluation of associationwith low-dose cytosine arabinoside. (Abstract). Blood, 82, (Suppl.1), 2144.

Keywords: retinoic acid syndrome, ATRA, acute myelo-monocytic leukaemia.

SPLENIC SIZE IN HOMOZYGOUS aþ THALASSAEMIA

The aþ thalassaemias are the commonest known humangenetic disorders. Clinically silent, these conditions generallyresult from the deletion of one of the paired a-globin geneson chromosome 16 and can occur in heterozygous (¹a/aa)or homozygous (¹a/¹a) forms. They are thought to havereached their current population frequencies throughselection by, and protection from, malaria (Flint et al,1986); we were therefore surprised to observe in a recentstudy that, paradoxically, a thalassaemia was associatedwith an increased susceptibility to uncomplicated malaria inearly childhood, a phenomenon that may result in protectionthrough improved immune priming (Williams et al, 1996).Our conclusions were supported by a further observation:that splenomegaly, an index of malaria infection, was alsomore common in such children (relative risk of splenomegalyin ¹a/¹a compared to normal children 1·5; 95% c.i. 1·2–1·9; P ¼ 0·004).

Although no data are available on red cell survival in theaþ thalassaemias, like b thalassaemia they are probablyassociated with some degree of ineffective erythropoiesis andreduced red cell survival. This seemed a possible alternativeexplanation for the raised prevalence of splenomegaly inhomozygous children. However, this was neither supportedby published data nor by our own observations: althoughsplenomegaly was associated with the presence of malariaparasites in our study, it was not independently associatedwith aþ thalassaemia on regression analysis. Nevertheless,we could not exclude the possibility that splenic size is abovenormal in such children and that enlargement to the point ofpalpability is more likely during malaria episodes. We have

therefore undertaken a study to assess splenic size inhomozygous aþ thalassaemic children living in a non-malarious region.

We measured splenic size by ultrasound examination of19 children (12 ¹a/¹a; seven aa/aa) aged between 92·4and 97·8 months who were living on the Polynesian islandof Tahiti. Like Santo, the most common aþ thalassaemiamutation in this population is the ¹a3·7III deletion(Philippan et al, 1995). These children were part of a birthcohort recruited to study the haematology of aþ thalassae-mia during childhood (Philippon et al, 1995); genotypes,determined by the Southern blot procedure, were thereforeavailable for all children. Each child was examined in therecumbant position and the spleen visualized sonographi-cally by standard methods (Rosenburg et al, 1991). Thechildren’s genotypes were not known by the radiologist atthe time of the examination.

No significant difference was found in splenic size betweenaþ thalassaemic and normal children; the mean (SD) spleniclength in homozygous children was 7·5 cm compared to7·2 cm in normals (P ¼ 0·5, n/s). The groups were wellmatched for age [95·3 (2·0) v 95·8 (1·9) monthsrespectively; P ¼ 0·5, n/s]. Although we cannot excludethe possibility that the spleen is inherently more reactive inaþ thalassaemia, and therefore more prone to enlargementduring malaria episodes, splenomegaly per se does notappear to be a feature of this condition. We conclude thatsplenomegaly, if found in such individuals, should not beattributed to aþ thalassaemia alone but should be investi-gated along standard lines.

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ACKNOWLEDGMENTS

We thank the parents and children who co-operated withour studies and the Wellcome Trust, U.K., for financialsupport.

MRC Molecular Haematology Unit, T. N. W I L L I A M S

Institute of Molecular Medicine, K. M A I T L A N D

John Radcliffe Hospital, P. M. V. M A RT I N *Oxford OX3 9DS, and D. J. W E AT H E R A L L

*Institute Malarde, J. B. C L E G G

BP 30 Papeete, Tahiti

REFERENCES

Flint, J., Hill, A.V.S., Bowden, D.K., Oppenheimer, S.J., Sill, P.R.,Serjeantson, S.W., Banakoiri, J., Bhatia, K., Alpers, M.P., Boyce,

A.J., Weatherall, D.J. & Clegg, J.B. (1986) High frequencies ofalpha-thalassaemia are the result of natural selection by malaria.Nature, 321, 744–750.

Philippon, G., Martinson, J.J., Rugless, M.J., Moulia-Pelat, J.P.,Plichart, R., Roux, J-F., Martin, P.M.V. & Clegg, J.B. (1995)Alpha thalassaemia and globin gene rearrangements in FrenchPolynesia. European Journal of Haematology, 55, 171–177.

Rosenburg, H.K., Markowitz, R.I., Kolgerg, M., Prk, C., Hubbard, A.& Bellah, R.D. (1991) Normal splenic size in infants: sonographicmeasurements. American Journal of Radiology, 157, 119–121.

Williams, T.N., Maitland, K., Bennett, S., Ganczakowski, M., Peto,T.E.A., Newbold, C.I., Bowden, D. K., Weatherall, D.J. & Clegg, J.B.(1996) High incidence of malaria in alpha thalassaemic children.Nature, 383, 522–525.

Keywords: a thalassaemia, malaria, spleen, Tahiti, spleno-megaly.

HIGH-DOSE ETOPOSIDE ENABLES THE COLLECTION OF PERIPHERAL BLOOD STEM CELLSIN PATIENTS WHO FAILED CYCLOPHOSPHAMIDE-INDUCED MOBILIZATION

We read with interest the paper by McQuaker et al (1997)demonstrating that an ifosphamide, etoposide and epirubicin(IVE) mobilization regimen plus granulocyte colony-stimu-lating factors (G-CSF) gave a significantly higher medianyield of CD34þ cells compared with cyclophosphamide (CTX)plus G-CSF in patients affected with resistant lymphomas.In particular, out of the 66 cases analysed, three patientsunderwent successfully a peripheral blood stem cell (PBSC)mobilization with IVE regimen following initial failedmobilization with CTX.

We would like to report our experience of a high-doseetoposide (VP16) 2 g/m2 plus G-CSF mobilization regimen inthree patients who failed to mobilize with CTX 4 g/m2 plusG-CSF. One acute non-lymphoid leukaemia (ANLL), one non-Hodgkin lymphoma (NHL) and one chronic lymphocyticleukaemia (CLL) were evaluated. Previous chemotherapyconsisted of ifosphamide, Aracytin and VP16 (ICE) for theANLL case, three lines of treatment for the NHL, and high-dose chlorambucil and fludarabine for the CLL patient. Allpatients were mobilized by CTX 2·5–3 months from the last

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Table I. Stem cell collection analysis comparing cyclophosphamide (CTX) andetoposide (VP16) plus G-CSF mobilization regimens.

Apheresis CD34þ cells × 106/Apheresis no. CD34þ cells (%) leukapheresis/kg

CTX G-CSFANLL 1 0·6 0·7

2 0·4 0·4

NHL 1 0·8 0·92 0·8 1·23 0·5 0·5

CLL 1 0·8 0·52 0·5 1·03 0·9 1·74 0·5 0·95 0·4 0·9

VP16 G-CSFANLL 1 2·8 7·8

2 1·4 2·0

NHL 1 3·3 3·02 3·3 13·8

CLL 1 1·1 1·12 1·2 2·23 1·6 3·8

chemotherapy, and the intervals between the two mobiliza-tion regimens were 5, 1·5 and 1 months for the ANLL, NHLand CLL patients, respectively. Apheretic procedures wasperformed by an AS 104 Fresenius cell separator using aC4Y device (Biofil, Mo, Italy).

Table I shows the comparison between the two mobiliza-tion regimens in terms of stem cell collection. The number ofCD34þ cells collected with CTX plus G-CSF 10 mg/kg waslower in all cases as compared with the amount of progenitorcells collected after high-dose VIP16 plus the growth factor.

In conclusion, our results, together with those ofMcQuaker et al (1997), suggest that high-dose VP16 orVP16-containing regimen may be considered a suitablealternative for those patients who fail CTX-inducedmobilization.

Centro Trapianti di Midollo Osseo G. P U C C I

e Terapia Sovramassimale G. I R R E R A

Emato-Oncologica ‘A Neri’, M. M A RT I N O

Dipartimento di Emato-Oncologia, G. M E S S I N A

Azienda Ospedaliera G. C O N S O L E

Bianchi-Melacrino-Morelli, F. M O R A B I T O

Reggio Calabria, Italy P. I A C O P I N O

REFERENCE

McQuaker, I.G., Haynes, A.P., Stainer, C., Anderson, S. & Russell,N.H. (1997) Stem cell mobilization in resistant or relapsedlymphoma: superior yield of progenitor cells following a salvageregimen comprising ifosphamide, etoposide and epirubicin com-pared to intermediate-dose cyclophosphamide. British Journal ofHaematology, 98, 228–233.

Keywords: CD34þ cells, stem cell mobilization.

THE PLATELET AND ENDOTHELIUM IN HIV INFECTION

Although Bain’s excellent recent review (Bain, 1997) of thehaematological aspects of human immunodeficiency virus(HIV) infection provided a thorough overview, there isincreasing evidence that the physiology of the endothelium isinfluenced by this condition. Endothelial cells have animportant role in haemostasis, with the capacity for influencinganticoagulant, procoagulant and fibrinolytic pathways (Jaffe,1987). Vallance et al (1997) have recently proposed mechan-isms by which infection and inflammation influence the correctfunctioning of the endothelium. The frequent co-infection ofHIV patients by other viruses such as cytomegalovirus andherpes simplex may also be detrimental to the endothelium.

Endothelial function may be assessed in vitro by measuringlevels of plasma markers such as von Willebrand factor,soluble thrombomodulin and soluble E-selectin (Blann &Taberner, 1995). All three of these molecules are raised inHIV infection, with levels of von Willebrand factor beinghighest (at a mean of 60% greater than controls) andcorrelating (inversely) most exactly with levels of CD4þvelymphocytes (r ¼ ¹0·485) (Seigneur et al, 1997). Inaddition, levels of von Willebrand factor and solublethrombomodulin correlated inversely with levels of theHIV p24 antigen (a marker of viral replication). Weinterpret these findings as evidence of a link between viralinfection and endothelial cell damage above and beyondthat which may be due to that of a simple inflammatoryresponse to the virus. Bain (1997) has also drawn ourattention to platelet abnormalities (e.g. thrombocytopenia)in HIV infection; our additional contribution is todemonstrate that, despite this thrombocytopenia, patientsinfected with HIV have raised levels of soluble P-selectin,a likely marker of platelet activation (Blann et al, 1997).

The consequences of these adverse changes to both types ofcell are likely to contribute to the excess risk of thrombosis inthese patients. Endothelial cell dysfunction may contribute to aprocoagulant profile (i.e. with raised levels of von Willebrand

factor, which has the capacity to promote platelet aggregationand adhesion, and changes in levels of molecules such as tissueplasminogen activator and its inhibitor). This may besynergistic with raised levels of activated or pre-activatedplatelets, and so may provide a further opportunity for thedevelopment of coagulopathy. We believe that this aspect of thepathogenesis of HIV should not be overlooked.

Haemostasis, Thrombosis A N D R E W B L A N N

and Vascular Biology Unit,University Department of

Medicine, City Hospital,Birmingham B18 7QH, U.K.

Service de Medecine J O E L C O N S TA N S

Interne et PathologieVasculaire,

Hospitalier Saint-Andre,Bordeaux, France

Laboratoire d’Hematologie F R A N C O I S E D I G NAT-G E O RG E

et Immunologie,UFR de Pharmacie,Marseille, France

Laboratoire Universitaire M A RT I N E S E I G N E U R

d’Hematologie,Universite de Bordeaux II,Bordeaux, France

REFERENCES

Bain, B.J. (1997) The haematological features of HIV infection.British Journal of Haematology, 99, 1–8.

Blann, A.D., Seigneur, M., Constans, J. & Pellegrin, J.L. (1997)Soluble P-selectin, thrombocytopenia and von Willebrand factor inHIV infected patients. Thrombosis and Haemostasis, 77, 1221–1222.

Blann, A.D. & Taberner, D.A. (1995) A reliable marker of endothelialcell dysfunction: does it exist? British Journal of Haematology, 90,244–248.

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Jaffe, E.A. (1987) Cell biology of endothelial cells. Human Pathology,18, 234–239.

Seigneur, M., Constans, J., Blann, A., Renard, M., Pellegrin, J.L.,Amiral, J., Boisseau, M. & Conri, C. (1997) Soluble adhesionmolecules and endothelial cell damage in HIV infected patients.Thrombosis and Haemostasis, 77, 646–649.

Vallance, P., Collier, J. & Bhagat, K. (1997) Infection, inflammationand infarction. Does acute endothelial dysfunction provide a link?Lancet, 349, 1391–1392.

Keywords: HIV, endothelial cell damage.

URINARY METHYLMALONIC ACID/CREATININE RATIODEFINES TRUE TISSUE COBALAMIN DEFICIENCY

I read with interest the recent annotation (Chanarin & Metz,1997) noting limitation on commonly used methods forcobalamin (Cbl) deficiency diagnosis. There may be benefitsin using the random spot urinary methylmalonic (MMA)assay by gas chromatography mass spectrometry (GC/MS).

The advantages of using the urinary MMA test by GC/MSare greater sensitivity and specificity, functional tissue Cblassessment, and non-invasive and convenient testing. Fig 1illustrates a plot of urinary MMA levels versus serum Cblresults for 35/36 subjects identified after screening 809elderly individuals for elevated urinary MMA (Norman &Morrison, 1993). Of the 35 individuals depicted, 28 hadconcurrent urinary MMA and serum Cbl tests. The otherseven had a follow-up serum Cbl test: four with low serumCbl, two with low-normal serum Cbl (212, 242 pg/ml), andone with normal serum Cbl (406 pg/ml). Two subjects, uponfollow-up testing, had normal MMA; oral Cbl supplementscould not be ruled out. As previously described, all subjects(n ¼ 16) treated with Cbl injections showed normal MMAlevels with follow-up testing. High MMA results with normalserum Cbl values represent functional Cbl deficiency at thetissue level. Other investigators have also reported studiesfinding elevated serum MMA levels in non-anaemic elderly

individuals having normal serum Cbl levels (Yao et al, 1992;Pennypacker et al, 1992; Lindenbaum et al, 1994; Naurathet al, 1995).

More recent studies demonstrating the high sensitivity ofthe urinary MMA assay involved a total (strict) vegetariangroup (Crane et al, 1997). Serum Cbl was <200 pg/ml in 8/9(the remaining one was 331), serum MMA was elevated in5/8 (all except one normalized with Cbl therapy) and urinaryMMA was elevated in 7/8 (all normalized). Combining serumCbl and urinary MMA tests, all nine subjects showedevidence of Cbl deficiency.

The urinary MMA assay by GC/MS is highly specific for Cbldeficiency. The serum MMA assay may give falsely highresults in conditions of renal insufficiency, thyroid disease,pregnancy, small bowel bacterial overgrowth, haemocon-centration, and for unexplained reasons (reviewed inNorman & Cronin, 1996). As noted (Chanarin & Metz,1997), the specificity of the serum MMA assay has not beenadequately evaluated. The greater specificity of the urinaryMMA assay over the serum MMA test could be related to thenormalization of urine MMA to urine creatinine and urineMMA being 40 times more concentrated than serum MMA,thereby impeding falsely-high values. Problems with false

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Fig 1. Urinary MMA levels versus serum Cblvalues in elderly subjects. Due to the axesbreaks, detailed urinary MMA levels andserum cobalamin levels are given inparentheses for five patients. X, Normal range180–900 pg/ml; B, normal range 200–900 pg/ml.

positive results have not been reported for the urinary MMAtest (Matchar et al, 1987; Norman & Morrison, 1993;McCann et al, 1996). Upon re-evaluation of the data fromthe previously mentioned screening study using the currentnormal value for urinary MMA (< 4·0), 6·7% (54/809) ofindividuals tested had elevated urinary MMA.

A major advantage of the urinary MMA test over theserum Cbl assay is that MMA levels are directly related to aCbl-dependent metabolic pathway giving a measurement ofCbl function at the tissue level. Analogously, when assessingautomobile velocity would you rather rely upon a speed-ometer or a petrol gauge?

The serum Cbl assay has very poor specificity. Thepresence of low serum Cbl levels without tissue deficiencymay be partially explained by the fact that <20% of the totalserum Cbl is bound to transcobalamin II, the carrier proteinneeded for delivery of Cbl to marrow and nerve cells(Norman & Morrison, 1993). One study determined thepositive predictive value of a low Cbl level to be 22%(Matchar et al, 1994) and another evaluation of patientswith low serum Cbl found only 22% (109/504) withclinically important disease (Cooper et al, 1986). The firststudy had a 5-year follow-up period to ensure low resultswere not incipient deficiency. The poor specificity for theserum Cbl in no way compares with the 99% specificityreported for the urinary MMA test (Matchar et al, 1987).

Norman Clinical Laboratory Inc., E R I C J. N O R M A N

Cincinnati, Ohio, U.S.A.

REFERENCES

Chanarin, L. & Metz, J. (1997) Diagnosis of cobalamin deficiency: theold and the new. British Journal of Haematology, 97, 695–700.

Cooper, B.A., Fehedy, V. & Blanshay, P. (1986) Recognition of deficiencyof vitamin B12 using measurement of serum concentration. Journalof Laboratory and Clinical Medicine, 107, 447–452.

Crane, M.G., Register, U.D. & Lukens, R.H. (1997) Cobalamin (CBL)studies on two total vegetarian (vegan) families. In: Proceedings of

the Third International Congress on Vegetarian Nutrition. School ofPublic Health, Loma Linda, Calif., U.S.A.

Lindenbaum, J., Rosenberg, I.H., Wilson, P.W.F., Stabler, S. & Allen,R.H. (1994) Prevalence of cobalamin deficiency in the Framing-ham elderly population. American Journal of Clinical Nutrition, 60,2–11.

Matchar, D.B., Feussner, J.R., Millington, D.S., Wilkinson, R.H.,Warson, D.J. & Gale, D. (1987) Isotope-dilution assay for urinarymethylmalonic acid in the diagnosis of vitamin B12 deficiency.Annals of Internal Medicine, 106, 707–710.

Matchar, D.B., McCrory, D.C., Millington, D.S. & Feussner, J.R. (1994)Performance of the serum cobalamin assay for diagnosis ofcobalamin deficiency. American Journal of Medical Science, 308,276–283.

McCann, M.T., Thompson, M.M., Gueron, I.C., Lemieux, B., Giguere,R. & Tuchman, M. (1996) Methylmalonic acid quantification bystable isotope dilution gas chromatography–mass spectrometryfrom filter paper urine samples. Clinical Chemistry, 42, 910–914.

Naurath, H.J., Joosten, E., Riezler, R., Stabler, S.P., Allen, R.H. &Lindenbaum, J. (1995) Effects of vitamin B12, folate, and vitaminB6 supplements in elderly people with normal serum vitaminconcentrations. Lancet, 346, 85–89.

Norman, E.J. & Cronin, C. (1996) Cobalamin deficiency. Neurology,47, 310–311.

Norman, E.J. & Morrison, J.A. (1993) Screening elderly populationsfor cobalamin (vitamin B12) deficiency using the urinarymethylmalonic acid assay by gas chromatography mass spectro-metry. American Journal of Medicine, 94, 589–594.

Pennypacker, L.C., Allen, R.H., Kelly, J.P., Matthews, L.M., Grigsby, J.,Kaye, K., Lindenbaum, J. & Stabler, S.P. (1992) High prevalence ofcobalamin deficiency in elderly outpatients. Journal of the

American Geriatric Society, 40, 1197–1204.Yao, Y., Yao, S.-L., Yao, S.-S., Yao, G.Y. & Lou, W. (1992) Prevalence

of vitamin B12 deficiency among geriatric outpatients. Journal ofFamily Practice, 35, 524–528.

Keywords: cobalamin, urinary methylmalonic acid, GC/MS(gas chromatography–mass spectrometry), stable isotope,urine creatinine.

METHYLMALONIC ACID AS A MARKER FOR COBALAMIN DEFICIENCY: FACT OR FANTASY?ELUCIDATIONS FROM THE COBALAMIN-DEFICIENT RAT

It was with considerable interest that we read the annotation‘Diagnosis of cobalamin deficiency: the old and the new’(Chanarin & Metz, 1997). We would like to raise some pointsfor discussion, mainly concerning the validity of somebiochemical markers (methylmalonic acid (MMA) andhomocysteine (HCYS)) for cobalamin (Cbl) deficiency. Werealize that the annotation was addressed mainly tohaematologists, but we think that the question as to thereliability of ascertaining a Cbl-deficient status concerns notonly haematologists but also neurologists, gastroenterolo-gists and even oncologists and therefore is of considerableimportance. Over the last few years (Scalabrino et al, 1990,1995) we have worked with a new experimental model, thetotally gastrectomized (TGX) rat, for reproducing a perma-nent Cbl-deficient status, which eventually brings about a

severe anaemia and a severe spongiosus myeloneuropathy,similar to the human subacute combined degeneration(SCD). We have also carried out similar studies on rats whichhad been chronically fed a Cbl-deficient (Cbl-D) diet(Scalabrino et al, 1990, 1995).

More recently we have collaborated with Dr Lindenbaumand his group in investigating whether or not the bio-chemical markers for Cbl deficiency could also be meaningfulin the TGX-rat, paying particular attention to a possibleinvolvement of MMA in the pathogenesis of SCD (Allen et al,1993). Although we realize that it is hazardous toextrapolate from the rat to humans and/or vice versawe can conclude from published data (Scalabrino et al,1997) that MMA may be a much more reliable markerthan HCYS for Cbl deficiency, in agreement with that

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demonstrated in human patients (Carmel et al, 1996). Tofurther support this statement, we now report previouslyunpublished data (obtained in collaboration with DrLindenbaum) on serum levels of MMA and HCYS of ratschronically fed a Cbl-D diet (see Table I). In this case, serumMMA levels increased in a constant manner, but when theexperimental period was prolonged, the magnitude ofincrease did not alter.

Furthermore, in TGX-rats we found: (a) increased MMAlevels even in some organs having a L-methlymalonilCoA(MMCoA) mutase (E.C. 5.4.99.2) activity; (b) a fall in serumlevels of MMA and HYCS after repeated Cbl injections(Scalabrino et al, 1997). These responses to postoperative Cbltreatment (Scalabrino et al, 1997) were not ‘partial’ andcertainly not ‘trivial’, as Drs Chanarin and Metz judged theMMA levels to Cbl therapy to be. Furthermore, we canspecify that among the TGX-rats only one out of 28 and onlytwo of the rats chronically fed a Cbl-D diet had a low serumCbl level together with a serum MMA level within the normalrange of values.

We did not ignore the well-known possibility of a rise inserum MMA levels not specifically linked to the Cbl deficiency(Lindenbaum et al, 1990). We demonstrated that increasesin serum MMA levels of TGX-rats are partially due to anoverproduction of propionic acid by gut flora. Indeed, thepostoperative treatment of TGX-rats with lincomycin mark-edly decreased serum MMA levels, which, however, stillremained significantly higher than those observed in thecontrols (Scalabrino et al, 1997). In keeping with this is thefact that the magnitude of the increase of serum MMA levelsin TGX-rats (in whose intestine a bacterial overgrowth isknown to occur) (Scalabrino et al, 1997) is far greater thanthat observed in rats chronically fed a Cbl-D diet (where abacterial overgrowth in the intestine can be reasonablyexcluded) (see Table I).

Finally it is quite surprising that Drs Chanarin and Metz donot consider the behaviour of serum MMA level in the Cbl-deficient patients before and after Cbl therapy as ‘evidence of aCbl deficiency’. If this were so, we should rediscuss theprinciple of causality in epistemology and in biology! This byno means necessarily signifies that MMA enhancement is dueonly to Cbl deficiency and that it might not occur in otherdiseases without Cbl deficiency (Fenton & Rosenberg, 1995).It is just that we think one cannot deny that Cbl deficiencybrings about an impairment in L-MMCoA mutase reaction,which leads to an enhancement in MMA levels in physiological

fluids, such as serum, urine and cerebrospinal fluid (Allenet al, 1993). This should not lead to the conclusion thatconsidering serum MMA levels alone constitutes a definitivetest, but simply that it can be one of the tests for Cbl deficiency,in agreement with the final statement in the annotation byDrs Chanarin and Metz.

Institute of General Pathology, and G. S C A L A B R I N O

*Institute of Human Anatomy, F. R. BU C C E L L AT O

University of Milan, G. T R E D I C I *Via Mangiagalli 31,20133 Milano, Italy

REFERENCES

Allen, R.H., Stabler, S.P., Savage, D.G. & Lindenbaum, J. (1993)Metabolic abnormalities in cobalamin (vitamin B12) and folatedeficiency. FASEB Journal, 7, 1344–1353.

Carmel, R., Rasmussen, K., Jacobsen, D.W. & Green, R. (1996)Comparison of the deoxyuridine suppression test with serum levelsof methylmalonic acid and homocysteine in mild cobalamindeficiency. British Journal of Haematology, 91, 311–318.

Chanarin, I. & Metz, J. (1997) Diagnosis of cobalamin deficiency: theold and the new. British Journal of Haematology, 97, 695–700.

Fenton, W.A. & Rosenberg, L.E. (1995) Disorders of propionate andmethylmalonate metabolism. The Metabolic and Molecular Bases ofInherited Disease (ed. by C. R. Scriver, A. L. Beaudet, W. S. Sly and D.Valle), Vol. 1, chap. 41, pp. 1423–1449. McGraw-Hill, New York.

Lindenbaum, J., Savage, D.G., Stabler, S.P. & Allen, R.H. (1990)Diagnosis of cobalamin deficiency. II. Relative sensitivities ofserum cobalamin, methylmalonic acid, and total homocysteineconcentrations. American Journal of Hematology, 34, 99–107.

Scalabrino, G., Buccellato, F.R., Tredici, G., Morabito, A., Lorenzini,E.C., Allen, R.H. & Lindenbaum, J. (1997) Enhanced levels ofbiochemical markers for cobalamin deficiency in totally gastrec-tomized rats: uncoupling of the enhancement from the severity ofspongy vacuolation in spinal cord. Experimental Neurology, 144,258–265.

Scalabrino, G., Lorenzini, E.C., Monzio-Compagnoni, B., Colombi,R.P., Chiodini, E. & Buccellato, F.R. (1995) Subacute combineddegeneration in the spinal cords of totally gastrectomized rats:ornithine decarboxylase induction, cobalamin status, and astro-glial reaction. Laboratory Investigation, 72, 114–123.

Scalabrino, G., Monzio-Compagnoni, B., Ferioli, M.E., Lorenzini,E.C., Chiodini, E. & Candiani, R. (1990) Subacute combineddegeneration and induction of ornithine decarboxylase in spinalcords of totally gastrectomixed rats. Laboratory Investigation, 62,297–304.

Keywords: cobalamin deficiency, biochemical markers.

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Table I. Effect of chronic feeding a Cbl-D diet on levels of Cbl, MMA and HCYS in rat serum.

Treatment Cbl (pg/ml) MMA (nmol/l) HCYS (mmol/l)

None 744·2 6 67·1 (6) 682·2 6 55·2 (6) 9·2 6 0·9 (6)3 months on Cbl-D diet 102·0 6 14·4 (4)** 991·5 6 90·4 (4)* 8·2 6 0·9 (4)5 months on Cbl-D diet 111·4 6 15·3 (6)** 957·5 6 87·5 (6)* 12·0 6 1·1 (6)7 months on Cbl-D diet 117·5 6 11·4 (4)** 947·5 6 95·3 (4)* 8·5 6 0·7 (4)

Values are means 6 SEM; number of rats in parentheses. Cbl, cobalamin; Cbl-D, cobalamindeficient; HCYS, homocysteine; MMA, methylmalonic acid.

**P < 0·01, *P < 0·05 versus controls (Dunnett’s test).

DIAGNOSIS OF COBALAMIN DEFICIENCY: A REPLY

We did not discuss urinary MMA in any detail in ourannotation (Chanarin & Metz, 1977) but are glad that DrNorman has raised the matter. He claims a 100% sensitivityfor his urinary MMA test based on 11 patients with possibleCbl deficiency (Matchar et al, 1987; McCann et al, 1996).Although Dr Norman claims 99% specificity, one of hisreports showed five of 35 patients with elevated urinaryMMA for no apparent reason (Norman & Morrison, 1993).The figure in Dr Norman’s letter shows that most of thesubjects with normal serum Cbl levels, which excludes Cbldeficiency, also have raised urinary MMA suggesting a poorspecificity.

Data reviewing serum Cbl and MMA levels in 24 h urinesamples in 110 patients, including 59 with megaloblasticanaemia due to Cbl deficiency, showed that all the Cbl-deficient patients had low serum Cbl levels and most, but notall, had raised urinary MMA levels (Fig 1). Further, all thepatients with normal serum Cbl had normal levels of MMA(Charanin, 1969). This suggests that MMA measurement asperformed 30 years ago (ether extraction and gas chromato-graphy) is a specific and relatively sensitive method fordiagnosis of Cbl deficiency but current methods of MMAassay are too non-specific to be of value.

Dr Scalabrino and colleagues appear to have misinter-preted our comments on the reduction of MMA levelsfollowing Cbl therapy. We expect patients with clinical and/or haematological evidence of Cbl deficiency to have raisedlevels of MMA and have no problem with the return of suchlevels to normal within a few days of Cbl treatment. It is insubjects where raised MMA is an isolated finding that webelieve a reduction in MMA level following Cbl therapy is notnecessarily evidence of Cbl deficiency.

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REFERENCES

Chanarin, I. (1969) The Megaloblastic Anaemias, p. 383, Fig. 14.3.Blackwell Scientific Publications, Oxford.

Chanarin, I. & Metz, J. (1997) Diagnosis of cobalamin deficiency: theold and the new. British Journal of Haematology, 97, 695–700.

Matchar, D., Feussner, J.R. Millington, D.S., Wilkinson, R.H.,Warson, D.J. & Gale, D. (1987) Isotope-dilution assay for urinary

617Correspondence

q 1998 Blackwell Science Ltd, British Journal of Haematology 100: 605–618

Fig 1. A comparison of the serum Cbl (B12) level and MMA in 24 h urine collections in 110 patients, including 59 with megaloblastic anaemia,due to Cbl deficiency. Controls generally have <4 mg MMA in a 24 h urine collection (interrupted line) but a few may excrete up to 15 mg. (Takenfrom Megaloblastic Anaemia, 1969, with permission of Blackwell Science.)

methylmalonic acid in the diagnosis of vitamin B12 deficiency.Annals of Internal Medicine, 106, 707–710.

McCann, M.T., Thompson, M.M., Gueron, I.C., Lemieux, B., Giguere,R. & Tuchman, M. (1996) Methylmalonic acid quantification bystable isotope dilution gas chromatography–mass spectrometryfrom filter paper urine samples. Clinical Chemistry, 42, 910–914.

Norman, E.J. & Morrison, J.A. (1993) Screening elderly populationsfor cobalamin (vitamin B12) deficiency using the methylmalonicacid assay by gas chromatography mass spectrometry. AmericanJournal of Medicine, 94, 589–594.

Keywords: cobalamin deficiency, urinary MMA.

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q 1998 Blackwell Science Ltd, British Journal of Haematology 100: 605–618