LEUCOCYTE-MIGRATION TECHNIQUE

1184 to

various extents

bleaching

are

compared with

and without the

treatment.

Samples consisted of 0-5 ml. of 3H-digoxin in 0-026M potassium phosphate buffer pH 7-4 containing 0-45% bovine serumalbumin and 10% appropriately hxmolysed serum. To each was added either 0-425 ml. distilled water or 0-4 ml. Milton’ bleach, followed 10 minutes later by 0-025 ml. 0-75M acetic acid to bring the pH to about 7. Finally 4-5 ml. of the scintillator cocktail was added. This consisted of 2/1 AnalaR toluene/ scintillation-grade triton X-100, containing 0.7% P.P.o. and

0-035%

P.O.P.O.P.

A further source of error, which is easily forgotten, results from the difficulty of accurately determining the low counts experienced in many radioimmunoassays. With a maximum permissible counting-time of 20 minutes per sample, in this laboratory it has seldom been possible to collect more than 4000 counts from each. Since 4000 counts allows only a 95% certainty limit of 6’4% (±2 standard deviations), on occasion additive errors in the counting of unknown and standard-curve samples may amount to 10% If one has a stable scintillation cocktail this or more. phenomenon may be demonstrated by repeated recycling of samples and recalculation of results. Home Office Central Research

Establishment, Aldermaston, Reading, Berkshire R67 4PN.

A. P. PHILLIPS.

acid mixture (3/1), and slides are made and air-dried. The slides are then placed in jars containing Sorensen buffer (pH 6-8) and incubated at 80° C for 11 hours. They are then stained for 6 minutes in a solution containing 7 ml. Giemsa stock and 20 ml. Sorensen buffer made up to 100 ml. with distilled water. After a brief rinse in distilled All the prowater the slides are dried and mounted.

metaphase spreads (see figure)

are

consistently banded,

with the more condensed chromosomes staining uniformly. We omit the addition of colchicine before harvesting because this causes condensation, making the banding patterns more difficult to see. Similar bands can be obtained from our routine cell suspensions (harvested without colchicine) by dropping the cells on to a hot slide and fiercely, but carefully, heating to dryness. The slides are then stained as above for 6 minutes. This procedure is a simple adaptation of our routine chromosome staining and has been found to be very important in the study of banding patterns of chromosomes. By using the technique described above, we obtained reproducible results, and we found that heating is the essential factor producing the banding patterns-a process termed differential denaturation.5 We have found that not all published techniques are reproducible, but we believe that our technique is simple and produces consistent results. Research Department, Marie Curie Memorial Foundation, The Chart, Oxted,

SIMPLIFIED TECHNIQUE FOR DEMONSTRATING BANDING PATTERNS IN HUMAN CHROMOSOMES

techniques

SIR,-Lately there have been many reports of for demonstrating the banding patterns of mammalian chromosomes.! These have involved various methods of denaturating D.N.A.-e.g., by enzymes, alkalis, and heat treatment.2-5 We have attempted to reproduce these processes but have obtained inconsistent results, except with heat treat-

P. DOYLE N. BISHUN.

Surrey.

HUMAN CHROMOSOME BANDING SiR.—Dr. Scheres (April 15, p. 849) reports a modification of Seabright’s technique for human chromosome banding by treatment of metaphase spread with trypsin.1 In our laboratory, we obtain satisfactory chromosome banding patterns from short-term cultures of whole blood, provided that an interval of 6-8 days elapses from preparation of cell spread to exposure to trypsin. The slides are then treated with 0-25% trypsin solution in isotonic saline for 10 minutes at room-temperature and stained for 10 minutes in Giemsa (pH 7). In this way, the proportion

metaphases with satisfactorily banded chromosomes is

of

near

80%. Cytogenetics Laboratory,

Department of Anatomy, University of Santiago de Compostela,

Spain.

ALICIA ANSEDE.

LEUCOCYTE-MIGRATION TECHNIQUE SIR,-When we tried to reproduce Dr. Clancy’s tech-

Prometaphase spread of a culture leucocyte (without colchicine), demonstrating banding. ment.5 Consistent results are obtained with the following technique, which is a modification of that previously mentioned.5 Chromosomes from human peripheral-blood cultures are harvested by standard procedures, without the addition of colchicine. Chromosomes are fixed in ethanol/acetic1. 2. 3. 4. 5.

Pearson, P. New Scient. 1972, 53, 371. Seabright, M. Lancet, 1971, ii, 971. Schnedl, W. Chromosoma, 1971, 34, 448. Lomholt, B., Mohr, J. Nature New Biol. 1971, 234, 109. Chaudhuri, J. P., Vogel, W., Voiculesch, I., Wolf, U. Humangenetik, 1971, 14, 83.

nique (Jan. 1, p. 6) for demonstrating leucocyte-migration inhibition in patients with chronic idiopathic thrombocytopenic purpura (I.T.P.), using autologous platelets as antigen, we found that the citrate-phosphate buffer (pH 6-0) inhibited leucocyte migration even in the absence of antigen. With only 0-1 ml. of buffer (necessary to suspend platelets to the desired concentration) added to the culture medium (0-9 ml., pH 7-4), the final pH was 6-7. At this pH no cell migration occurred whether the platelet antigen was present or not, and there was no change in the pH after 22 hours’ incubation at 37 °C. Synthesis of migrationinhibition factor is

a

function of viable cells, and the

unphysiological pH of the medium would be detrimental to all the cells participating in migration inhibition. We have got round this difficulty by modifying the Kissmeyer-Nielsen2 method of platelet preparation. The platelets are washed twice with citrate-phosphate buffel 1. 2.

Seabright, M. Lancet, 1971, ii, 971. Kissmeyer-Nielsen, F. Progr. clin. Path. 1969, 2,

161.

1185 and once more with tissue-culture medium With this method we have found significani migration inhibition of the I.T.P. patients’ leucocytes in the presence of autologous platelets in media containing normal human serum. Furthermore, migration inhibition was blocked by the addition of the patients’ own serum.

(pH 6-0) (pH 7-4).

Hektoen Institute for Medical Research, 627 South Wood Street, Chicago, Illinois 60612, U.S.A.

SYAMAL KUMAR DATTA

JOSEPH EIPE NICOLAS COSTEA.

DEPRESSED LYMPHOCYTE RESPONSE TO P.H.A. IN WOMEN TAKING ORAL CONTRACEPTIVES SIR,-Dr. Purtilo and his co-workers (April 8, p. 769) report a depressed lymphocyte response to phytohasmagglutinin (P.H.A.) in pregnant women. They discuss the possibility that this may represent an important mechanism that prevents maternal rejection of the conceptus. Our observations on young women taking oral contraceptives suggest that the depressed lymphocyte response to P.H.A. may be due to a non-specific oestrogen or progesterone action.

Lymphocytes were cultured from nine women aged 22-28 taking oral contraceptives, and nine healthy non-pregnant controls aged 22 to 30. The lymphocytes were separated from whole blood by sedimentation in a gelatin solution,’ washed twice in Hanks’ solution, and cultured for 46 hours in McCoy’s medium supplemented by 30% fetal-calf serum. Three cultures were stimulated by P.H.A. and one was left unstimulated. Transformation was determined by the incorporation of tritiated thymidine into the lymphocytes during the last three hours of culture.2

All the

women were

studied

on at

least

two

separate

occasions, a week or more apart. The results were expressed the average of all values from P.H.A.-stimulated cultures and were as follows:

as

A Mann-Whitney test gave a P value of less than 0-01 in favour of a depressed lymphocyte reaction in women taking oral contraceptives. The diagram of Dr. Purtilo and his co-workers shows that depression of the P.H.A. response is most pronounced during the last weeks of pregnancy, when oestrogen and progesterone production are at a maximum. Medical Dept. F, Copenhagen County Hospital, 2900 Hellerup,

Denmark.

C. HAGEN A. FRØLAND.

G.-6-P.D. deficiency affects 25-35% of the population) the prevalence of the enzyme deficiency was only 13% (p < 0-01). In a population from Liguria (northern Italy) which

settled in the Sardinian island of Carloforte over two centuries ago, the frequency of G.-6-P.D. deficiency is only 3-4%, compared with 25—35% in neighbouring Sardinian populations, and the death-rate from cancer is almost twice as high (15%) as in the populations of Sardinian origin (9%). Sardinia has a lower cancer prevalence than both northern Italy as a whole and the rest of Europe. An explanation for these findings might be found in the block of the metabolic pathway of glucose degradation known as the " pentose shunt ", of which G.-6-P.D. is the key enzyme. This would cause slower formation of ribose and hence of nucleic acids, with a subsequent obstacle to neoplastic proliferation. Carriers would be spared from the neoplastic event, probably because of slow proliferation of This would allow organic defence the various tissues. systems to act as an obstacle to, and destroy, tumour invasion, especially in its early stages. Therefore, another explanation, apart from protection against malaria, seems to be evolving for the persistence of a high frequency of this enzymopathy among some ethnic groups. A. Businco Cancer Regional Hospital, E. SULIS. Cagliari, Sardinia.

GEL-PRECIPITATING ANTIBODIES IN DIAGNOSIS OF E.B. VIRUS INFECTION

SIR,-We read with interest the report by Dr. Nikoskelainen and his colleagues (May 6, p. 1020) on the diagnostic value of tests for gel-precipitating antibodies in E.B.-virus infections. We also have observed these antibodies, and our results, albeit obtained through a less sensitive technique, confirm their late development in infectious mononucleosis and demonstrate their similarity to antibodies developed in patients with Burkitt’s tumour. Sera from patients with Burkitt’s tumour, from patients with infectious mononucleosis, from control patients (mostly with infectious diseases), and from healthy medical students and nurses were procured as described elsewhere.1 The P3J line of Jijoye cells (derived from a patient with Burkitt’s tumour) was used as the source of antigen; this was prepared and gel-precipitation tests were performed by the methods of Old et al.22 446 sera from 372 individuals were tested for gelprecipitating antibodies. These were found in 3 of 9 patients with Burkitt’s tumour and in 4 of 111 patients with infectious mononucleosis. In the latter group, 2 of the positive sera were taken about 20 months after the onset of illness from patients in whom earlier specimens were negative. These sera showed reactions of identity with each other and with sera from patients with Burkitt’s tumour. Of the other 2 positive sera, 1 was taken 2 months after the onset of infectious mononucleosis and 1 was taken at 16 days after onset (a further serum from this patient, taken at 26 days, was negative). Of 109 control patients, 2 had antibodies; 1 was a girl of 19 years with serological evidence of rubella-virus infection and the other was a woman of 45 years, admitted with an unrelated complaint, who had an unusually high E.B.-virus complement-fixing antibody titre and who had a considerably (4-fold) raised level of

G.-6-P.D. DEFICIENCY AND CANCER SiR,-Naik and Anderson 3 and others 4,5 have reported an inverse relationship between the frequency of glucose-6phosphate-dehydrogenase (G.-6-P.D.) deficiency and the incidence of tumours in different populations. In further studies in Sardinia we have noticed that among 320 males with cancer who came from the area near Cagliari (where

serum-y-globulins. Gel-precipitating antibodies were not found in 143 healthy controls. The method which we used is clearly much less sensitive than that used by the Finnish workers and by Stevens

1. Coulson, A. S., Chalmers, D. G. Lancet, 1964, i, 468. 2. Hagen, C., Frøland, A. Br. med. J. 1972, i, 445. 3. Naik, S. N., Anderson, D. E. Lancet, 1970, i, 1060. 4. Beaconsfield, P., Rainsbury, R., Kalton, G. Oncologia, 1965, 19, 11. 5. Sulis, E., Spano, G. Boll. Soc. ital. Biol. sper. 1968, 44, 1246.

Sutton, R. N. P., Marston, S. D., Emond, R. T. D. J. clin. Path. 1971, 24, 801. 2. Old, L. J., Boyse, E. A., Oettgen, H. F., De Harven, E., Geering, G., Williamson, B., Clifford, P. Proc. natn. Acad. Sci. U.S.A. 1966, 56, 1699. 1.