1285

Manning (1957b) and Reedy et al. (1957) showed thatremoval of staphylococcus antibody from pooled humanglobulins deprived them of their effect in staphylococcalinfections.

In the present case the infecting organism producedpenicillinase; in-vitro growth was inhibited for 48 hoursby approximately 3 units penicillin per ml. It was

thought that large doses of penicillin might have a

therapeutic effect; and it is likely, though by no meanscertain, that the final recovery of the patient was duelargely to penicillin. At the time methicillin was notavailable.

SummaryA case of severe septicaemia caused by a penicillin-

resistant Staphylococcus aureus is described.Staphylococcal antitoxin produced shortlived improve-

ment. Treatment with 36,000,000 units of penicillin dailywas successful. The patient showed an initial y-globulindeficiency, and on two occasions received humany-globulins.

REFERENCES

Blair, J. E. (1958) in Bacterial and Mycotic Infections of Man (edited byR. J. Dubos); p. 325. London.

Harris, J. R., Schick, B. (1954) J. Mt Sinai Hosp. 21, 148.Fisher, M. W., Manning, M. C. (1957a) Antibiot. Chemother. 7, 315.

— — (1957b) Antibiot. Annu. 1957-58; p. 572.Knouf, E. G. (1957) ibid. p. 585.Parish, H. J., Cannon, D. A. (1960) Brit. Med. J. i, 743.Reedy, R. J., Oswald, E. J., Wright, W. W. (1957) Antibiot.

Annu. 1957-58; p. 581.Waisbren, B. A. (1957) Antibiot. Chemother. 7, 322.

A RARE ANTIBODY (ANTI-Ge) CAUSINGHÆMOLYTIC DISEASE OF THE NEWBORN

R. BARNESM.B. Lond.

REGISTRAR IN CLINICAL

PATHOLOGY

T. L. T. LEWISM.B. Cantab., F.R.C.S.,

M.R.C.O.G.OBSTETRIC SURGEON

GUY’S HOSPITAL, LONDON, S.E.1

THE study of human red-cell antigens is intimatelyrelated to the isolation of the corresponding antibody.Such an antibody recently led to the discovery by Rosen-field et al. (1960) of the common " public " red-cell

antigen designated Gerbich (Ge). These workers recordedthree cases with anti-Ge. A further case is now presented;and we discuss the problems involved in the managementof the patient and her pregnancy.

Case-historyA 21-year-old Turkish Cypriot primigravida presented

herself on June 7, 1960, when she was 17 weeks pregnant. Shehad had no previous illness of note, no miscarriage, and ncblood-transfusion. Clinical examination and a chest radiographwere normal. Her blood-group was 0, Rh-positive. Onroutine screening of her serum with an R1 R2rr cell mixture at37 °C, using an albumin-addition technique, antibodies weredetected.With the help of the Medical Research Council’s Blood

Group Reference Laboratory, and the South London BloodTransfusion Centre, further blood specimens were examined,with the following results:The husband:

A2 B; cde/cde; NS/Ns; Lu (a-); Pi+++; K-k+Kp (a-b+);Le (a-b-); Fy (a+ b+); Jk (a-b+).

The patient:0; CDe/cde; Ms/Nã; Lu (a-b+); P2; K-k+Kp (a-b+);

Le (a-b+); Fy (a+b-); Jk (a+b-).Furthermore, the patient’s red cells were negative with anti -V,- Jsa, -Dia, -Swa, -Wra, -By, -Mia, -Vw, V, and -Bea. Theywere positive with anti -I, -Tja, and -Vel. The direct Coombstest was weakly positive, the coating being of the cold non-gamma-globulin type, and it was considered of little significance

The antibody worked best as a cold agglutinin, and on testingit was found to agglutinate 160 random 0-cell samples. It alsoreacted with cells of types Vel-negative, Tj (a-), Ko, and Lu(a+b-); but no reaction was detected with the 2 cell samplesthat were Ge-negative, and these cells enabled the presence ofanti-E, -K, -Lea, -Fyb, -S, and -P1 to be excluded. Titrationof the antibody using random cell samples varied between 1/8and 1/64 during the pregnancy, but no tendency for the titreto increase was noted in the later weeks of pregnancy after the

antibody had been isolated.Antenatal Care

Identification of the antibody as anti-Ge raised difficultieswith regard to the antenatal care of the patient. We found thatthere were only three known Ge-negative subjects, all with

antibodies; that over 10,000 blood samples had been examinedin the New York City Rhesus Laboratory, 1000 samples at- theMount Sinai Hospital, and 500 samples at the Blood GroupingLaboratory in Aarhus, Denmark, without revealing a singleGe-negative blood-donor (Rosenfield et al. 1960). We wereanxious to provide Ge-negative blood should either the patientor her baby require transfusion. There were three possiblesources of a Ge-negative donor: first the immediate relativesof the patient, secondly the relatives of the three original cases,and finally the blood-donor panel in the United Kingdom. Allhad to be screened for a suitable Ge-negative donor.

Tracing the patient’s relatives in Cyprus was difficult, butthe search revealed an unmarried sister who, as well as beinggroup 0, Rh-negative, was Ge-negative without antibodies.Cross-matching later showed that her blood was compatibleand could be given to the patient, if required.

In the 8 weeks before the birth the Blood TransfusionService screened 16,000 donors without revealing a singleGe-negative subject; and a further 10,000 have now beenscreened, still without success. But examination of the relativesof the original cases showed that two of the three reported hadeither a brother or sister who was Ge-negative, thus bringingthe total number of Ge-negative subjects to seven; and fourof these carried anti-Ge as well.During the 8 weeks before delivery the patient was bled

several times; the plasma was used by the Blood TransfusionService for their screening programme, and the red cells werereturned to the patient immediately. 6 weeks before labour apint of compatible Ge-negative blood was obtained from thesister in Cyprus in case labour should be premature. 2 weeksbefore the baby was due to be born, because no suitable donorhad been detected, and the blood from Cyprus was nearing itsexpiry date, a pint of blood was taken from the patient. Thecells (group 0, Rh-positive, Ge-negative) were washed free ofantibody, resuspended in fresh AB plasma, and stored in caseeither the mother or her baby should require a blood-transfusion.

Delivery and Postpartum CareBecause the antibody titre did not rise appreciably as

pregnancy progressed, we decided not to induce labourprematurely. Labour began spontaneously at term on Nov. 24,and a healthy boy, weighing 71/2 lb. was born normally after26 hours. The total blood-loss was 5 oz.The cord-blood bilirubin was 2-9 mg. per 100 ml. (indirect

2-1 mg., direct 0-8 mg.) and the haemoglobin 11-8 g. per100 ml. (75%). The direct Coombs test was negative.Nevertheless, the infant’s pallor, slight jaundice, moderateenlargement of the liver and spleen, cord-blood anaemia, andhyperbilirubinsemia suggested that he had mild haemolyticdisease of the newborn. Because an exchange transfusion mightbe required, a further pint of blood was taken from the mother,and the cells were immediately washed free of antibody andresuspended in fresh AB plasma.The serum-bilirubin 4 hours after birth was 4-0 mg. per

100 ml. (all indirect). 20 hours after birth the level had fallento 2-9 mg. per 100 ml. (indirect 2-1 mg., direct 0-8 mg.) and thecapillary Hb was 15-6 g. per 100 ml. (99%); this figure wasthought to be influenced by the normal 1st day physiologicalhoemoconcentration. At this stage it was clearly obvious that

1286

exchange transfusion would not be necessary. The serum-bilirubin levels continued to decrease, and 5 days after birththe infant was clinically normal apart from slight pallor. Onfurther examination of the cord blood, the infant’s blood-groups were found to be:

A,; cde/cde; Pl++; MsfNs; Lu (a-b+); Le (a-b-);K-k+; Fy (a+b-); Jk (a+ b+); Mi (a-); Vw-; Ge-t-; Vel+and no free anti-Ge was detected.The mother’s recovery was uneventful, and the baby pro-

gressed satisfactorily, although 3 months later (when seen atoutpatients), despite iron therapy by mouth, the Hb was8-5 g. per 100 ml. (54%).

Discussion

There are now several well-established antibodies thatreact with the red cells of practically everyone tested. The

case presented above is the fourth where such an antibody-anti-Ge-has been isolated.The three other cases, reported by Rosenfield et al.

(1960), resemble ours in several respects, although thereare some differences:A woman of Italian extraction (" Mrs. Ge ") presented in

the same way as our patient except that antibodies weredetected for the first time in her third pregnancy, her twoprevious children being normal. A normal infant was born,and although jaundice was not apparent the direct Coombstest was strongly positive.A Mexican case presented when a third baby’s cord blood

gave a positive direct Coombs test. Investigation of themother’s serum then revealed an antibody which was anti-Ge.

In Denmark, a woman was found to have anti-Ge during hersecond pregnancy. The direct Coombs test on the cord bloodwas positive, and the baby became slightly jaundiced. A historyof jaundice in her elder child during the lst week of life

unfortunately could not be followed serologically.Our patient, like those of Rosenfield et al., presented

during pregnancy, but unlike the others this was her firstpregnancy. Although hxmolytic disease of the newbornwas likely on clinical grounds (jaundice, pallor, hepato-megaly, splenomegaly), the direct Coombs test was

negative. The Coombs test has been repeated severaltimes, using different reagents, with consistently negativeresults; and this contrasts with the other three cases

which were Coombs-positive although they showed lessevidence of haemolysis. But anaemia and hyperbili-rubinoemia of the cord blood would suggest that the babywas affected apart from the clinical features; and, althoughpreliminary investigations into the prevalence of Ge-

negative subjects imply that this antibody is extremelyrare, hxmolytic disease of the newborn (with or withouta positive Coombs test) may be the result of an immuneanti-Ge.

Including our case, four affected infants have so farbeen reported (three Coombs-positive). None of these hasbeen so severely affected as to necessitate either prematureinduction of labour or exchange transfusion.

Summary .

A case is described in which the anti-Ge antibody wasdetected during pregnancy. The subsequent management,involving the birth of a child with mild haemolytic disease,required neither premature induction of labour nor

exchange blood-transfusion.We are indebted to Dr. T. E. Cleghorn, of the National Blood

Transfusion Service, Sutton, for identification of the antibody andfor the blood-grouping tests on the family; to Dr. F. Kissmeyer-Nielsen of Arrhus, who had two Ge-negative donors standing by;and to the medical authorities in Cyprus for their cooperation.

REFERENCE

Rosenfield, R. E., Haber, G. V., Kissmeyer-Nielsen, F., Jack, J. A., Sanger,R., Race, R. R. (1960) Brit. J. Hœmat. 6, 344.

Preliminary Communications

CLINICAL APPLICATIONS OF

DEHYDROGENASE ISOENZYMESA Simple Method for their Detection

By means of starch-gel electrophoresis it is possible,because of migration differences, to separate certain

enzymes into a number of components-the so-called

isoenzymes 1-which are closely similar in their biologicalactivity but have different pH maxima. Their distributionoften differs from tissue to tissue, so that in certainconditions the isoenzyme distribution pattern of a diseasedtissue may become manifest and recognisable in the

serum,2 presumably by leakage through altered cellmembranes. These changes are especially evident afterinfarctions, and it is now well established that studies ofserum’ isoenzymes of lactic-acid dehydrogenase are par-ticularly useful after cardiac infarction.The quantitative methods of detection are somewhat

cumbersome and require many estimations for eachpatient; but we have developed a simple visualisation

technique whereby serum-dehydrogenase isoenzymes canbe studied very rapidly and in relatively large numbers ofsamples. The results obtained in relation to lactic-aciddehydrogenase are so promising in their clinical applica-tions that the time seems ripe for a preliminarycommunication.The technique employed depends upon the fact that

lactic-acid dehydrogenase oxidises lactic acid, whilst at thesame time it brings about the reduction of nicotinamide-adenine dinucleotide (NAD; formerly referred to as

DPN) to its reduced form (NADH2). The hydrogen of thelatter is then utilised to reduce a tetrazolium salt to aninsoluble coloured formazan. A substance must be usedwhich is capable of carrying the hydrogen and handingit on for this final reduction. We have used methyl-phenazonium methosulphate; the tetrazolium salt we haveemployed is MTT (3-(4, 5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide) which is also used byothers for a similar purpose.34 The areas occupied byeach isoenzyme after electrophoresis appear as purplebands.

METHODS

The isoenzymes were separated by vertical starch-gelelectrophoresis by a technique 5 involving minor modificationsof the method of Smithies. The gels at pH 8-6 were madewith 0-05 M tris-HCl buffer at pH 8-8 and hydrolysed starchobtained from the Connaught Laboratories (Canada). Theywere allowed to set for half an hour at room temperature andone and a half hours at 4°C. Each gel was 25 cm. long x 12 cm.wide x 0-6 cm. thick. A slot-former, as described by Smithies,was used to make insertions one-third of the way along the gel.One to ten slots were made, each of which was filled with0-03-0-1 ml. of human serum or tissue extract, the quantityused being determined by the size of the slot. A voltagegradient of 10-12 volts per cm. was applied for two hours at4°C with a bridge buffer consisting of 0-3 M tris-HCl at pH 8-6.After electrophoresis the gel was sliced with a fine wire alongits whole length into sections approximately 1-5 mm. thick.One of the inner slices was stained for lactic dehydrogenase

by placing it in a solution containing 10 mg. NAD, 5 mg. KCN,4 mg. MTT, 0-25 mg. methylphenazonium methosulphate dis-solved in a mixture of 5 ml. M sodium lactate and 10 ml. of

1. Markert, C. L., Møller, F. Proc. nat. Acad. Sci. 1959, 45, 753.2. Wróblewski, F., Gregory, K. Proceeding of IV International Congress o

Clinical Chemistry. Edinburgh, 1961. 3. van der Helm, H. J. Lancet, 1961, ii, 108.4. Pearse, A. G. E. Histochemistry Theoretical and Applied. London, 19605. Hodson, A. W., Latner, A. L., Raine, L. Clin. Chim. Acta (in the press)6. Smithies, O. Biochem. J. 1959, 71, 585.