- Email: [email protected]
SOURCES OF ERROR IN RADIOIMMUNOASSAYS
1183
Applying the figures quoted by Dr. Galant, with specific therapy 309,600 (72%) could be well by the time they reach 16, leaving only 120,400 (28%) with asthma. It seems self-evident that more widespread resources for the practice of clinical allergy techniques and for research into how to improve results, using objective methods, would be a very valuable investment. Today’s children are tomorrow’s adults, and it seems ridiculous that a National Health Service which is the best in the world should not treat a condition which is often completely reversible. I am glad to state that in Derby my ptdiatric colleagues are very enlightened, in that they allow me the use of beds for intensive therapy and investigation and ask for help with allergic problems. This degree of cooperation is all too rare, but it is an encouraging beginning. A tremendous amount of research effort and money is devoted to comparative rarities today. Is it not time that a common, often reversible, and even-dare I say itsometimes curable chronic respiratory disease gets the help it deserves, if only from the point of view of conserving our
national resources ?
Derby Chest Clinic, Derwent Hospital, and Children’s Hospital, Derby.
H. MORROW BROWN.
STOKE PARK: THE REMEDIES " 24 SiR,—The discussants after the recent B.B.C. Hours" programme on Stoke Park Hospital for the mentally handicapped are to be congratulated. They managed to bring to the programme some optimism by stressing that, given the resources, even the most bizarrelooking residents of the hospital were capable of learning new, appropriate, and dignified behaviours which replaced their existing inappropriate ones. They pointed out that the present image of mental handicap largely reflects the squalor of the existing hospital provisions. I may have overlooked something during the programme, but I cannot recall the participants stressing the urgent need to solve the problem in this particular hospital, as in many others in the country, by rapidly implementing the policy described in command paper 4683. This would include:
(i) Building, in areas which do not already have a hospital, new hospital facilities which serve total populations of maximum size 250,000. (Children 13 places, adults 55 places, per 100,000 total population.) (ii) Building local-authority homes for sub-areas within the catchment areas. (Children 10 places, adults 60 places, per 100,000 total population-plus foster-care places.) (iii) Provision of school and day-training facilities for ALL mentally retarded people at home as well as in residential care. (Norms defined.) "
cational, and medical services, available to families with a mentally person living at home and to the retarded in residential care. This is to be facilitated by the alignment of existing hospitals, and by the sectorisation of existing wards (paras. 266
handicapped
and 267 of the
white-paper).
Unless there are major objections to the command paper’s aims, would it not be useful if all efforts were to be directed to ensuring that the command paper proposals were implemented well within its own target dates ? If there are objections, is it not important both to air these in detail and urgently to suggest concrete alternative
proposals ? 42 Thornbury Avenue,
ALBERT KUSHLICK.
Southampton.
SOURCES OF ERROR IN
RADIOIMMUNOASSAYS SIR,-With the increasing availability of radioimmunodrugs, it is important to ensure
assays for hormones and
that newcomers to the technique are aware of its inherent limitations. In this laboratory the method for the radioimmunoassay of digoxin in plasma has been modified considerably for use with blood collected post mortem. Some of the data from this specialised study may be used to illustrate the pitfalls typical of radioimmunoassays of
plasma or serum components. Quantitation based on radioisotope counting demands either a knowledge of the counting efficiency of all samples or an assurance
of
a
constant
counting efficiency.
Dr.
"
Since conditions in many existing hospital wards do not, for one reason or another, permit implementation of adequate care for the present residents, transfers of people who have long been in such wards to the new facilities in their own areas will be inevitable. If undertaken with sensitivity, these transfers will also be humane to those who leave the existing overcrowded facilities and to those who remain behind. It also involves and would facilitate:
(iv) Urgent improvement of facilities for the reduced numbers of residents who remain in existing hospitals. (v) Rapid increase of existing grossly inadequate staffing establishment levels-in particular, of caring and domestic staff on overcrowded wards, and especially on those with the most dependent residents with the most disruptive behaviour. (vi) Provision of management training to staff of all disciplines who have managerial responsibility for the acquisition and distribution of resources and for the implementation of policies. (vii) Development of continuous assessment and reassessment programmes, involving collaboration between the social, edu-
Comparison of 3H-digoxin counts in bleached and untreated sera haemolysed to varying extents.
Huffman and Dr. Azarnoff (March 25, p. 695) implicated haemolysis as a source of error in plasma-digoxin radioColour quenching of the scintillation immunoassays. hasm is, of course, a danger in any radioimmunoprocess by assay where serum or plasma is included in the sample presented to the spectrometer. These correspondents have apparently chosen to calculate the degree of quenching of individual samples. Because of the difficulty of quench correction in counting tritium in triton-emulsion scintillators, which in many respects are to be preferred for aqueous samples, my approach has been to establish a relatively uniform counting efficiency by decolorising traces of hxm with a hypochlorite bleach2 (see figure). Quench correction is then necessary only in cases of severe hxmo-
lysis. In the
figure the
counts
of
3H-digoxin in sera hasmolysed
1. Fox, B. W. in Liquid Scintillation (in the press). 2. Phillips, A. P., Sambrook, C. A. ibid.
Counting; vol.
2. London
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.
Applying the figures quoted by Dr. Galant, with specific therapy 309,600 (72%) could be well by the time they reach 16, leaving only 120,400 (28%) with asthma. It seems self-evident that more widespread resources for the practice of clinical allergy techniques and for research into how to improve results, using objective methods, would be a very valuable investment. Today’s children are tomorrow’s adults, and it seems ridiculous that a National Health Service which is the best in the world should not treat a condition which is often completely reversible. I am glad to state that in Derby my ptdiatric colleagues are very enlightened, in that they allow me the use of beds for intensive therapy and investigation and ask for help with allergic problems. This degree of cooperation is all too rare, but it is an encouraging beginning. A tremendous amount of research effort and money is devoted to comparative rarities today. Is it not time that a common, often reversible, and even-dare I say itsometimes curable chronic respiratory disease gets the help it deserves, if only from the point of view of conserving our
national resources ?
Derby Chest Clinic, Derwent Hospital, and Children’s Hospital, Derby.
H. MORROW BROWN.
STOKE PARK: THE REMEDIES " 24 SiR,—The discussants after the recent B.B.C. Hours" programme on Stoke Park Hospital for the mentally handicapped are to be congratulated. They managed to bring to the programme some optimism by stressing that, given the resources, even the most bizarrelooking residents of the hospital were capable of learning new, appropriate, and dignified behaviours which replaced their existing inappropriate ones. They pointed out that the present image of mental handicap largely reflects the squalor of the existing hospital provisions. I may have overlooked something during the programme, but I cannot recall the participants stressing the urgent need to solve the problem in this particular hospital, as in many others in the country, by rapidly implementing the policy described in command paper 4683. This would include:
(i) Building, in areas which do not already have a hospital, new hospital facilities which serve total populations of maximum size 250,000. (Children 13 places, adults 55 places, per 100,000 total population.) (ii) Building local-authority homes for sub-areas within the catchment areas. (Children 10 places, adults 60 places, per 100,000 total population-plus foster-care places.) (iii) Provision of school and day-training facilities for ALL mentally retarded people at home as well as in residential care. (Norms defined.) "
cational, and medical services, available to families with a mentally person living at home and to the retarded in residential care. This is to be facilitated by the alignment of existing hospitals, and by the sectorisation of existing wards (paras. 266
handicapped
and 267 of the
white-paper).
Unless there are major objections to the command paper’s aims, would it not be useful if all efforts were to be directed to ensuring that the command paper proposals were implemented well within its own target dates ? If there are objections, is it not important both to air these in detail and urgently to suggest concrete alternative
proposals ? 42 Thornbury Avenue,
ALBERT KUSHLICK.
Southampton.
SOURCES OF ERROR IN
RADIOIMMUNOASSAYS SIR,-With the increasing availability of radioimmunodrugs, it is important to ensure
assays for hormones and
that newcomers to the technique are aware of its inherent limitations. In this laboratory the method for the radioimmunoassay of digoxin in plasma has been modified considerably for use with blood collected post mortem. Some of the data from this specialised study may be used to illustrate the pitfalls typical of radioimmunoassays of
plasma or serum components. Quantitation based on radioisotope counting demands either a knowledge of the counting efficiency of all samples or an assurance
of
a
constant
counting efficiency.
Dr.
"
Since conditions in many existing hospital wards do not, for one reason or another, permit implementation of adequate care for the present residents, transfers of people who have long been in such wards to the new facilities in their own areas will be inevitable. If undertaken with sensitivity, these transfers will also be humane to those who leave the existing overcrowded facilities and to those who remain behind. It also involves and would facilitate:
(iv) Urgent improvement of facilities for the reduced numbers of residents who remain in existing hospitals. (v) Rapid increase of existing grossly inadequate staffing establishment levels-in particular, of caring and domestic staff on overcrowded wards, and especially on those with the most dependent residents with the most disruptive behaviour. (vi) Provision of management training to staff of all disciplines who have managerial responsibility for the acquisition and distribution of resources and for the implementation of policies. (vii) Development of continuous assessment and reassessment programmes, involving collaboration between the social, edu-
Comparison of 3H-digoxin counts in bleached and untreated sera haemolysed to varying extents.
Huffman and Dr. Azarnoff (March 25, p. 695) implicated haemolysis as a source of error in plasma-digoxin radioColour quenching of the scintillation immunoassays. hasm is, of course, a danger in any radioimmunoprocess by assay where serum or plasma is included in the sample presented to the spectrometer. These correspondents have apparently chosen to calculate the degree of quenching of individual samples. Because of the difficulty of quench correction in counting tritium in triton-emulsion scintillators, which in many respects are to be preferred for aqueous samples, my approach has been to establish a relatively uniform counting efficiency by decolorising traces of hxm with a hypochlorite bleach2 (see figure). Quench correction is then necessary only in cases of severe hxmo-
lysis. In the
figure the
counts
of
3H-digoxin in sera hasmolysed
1. Fox, B. W. in Liquid Scintillation (in the press). 2. Phillips, A. P., Sambrook, C. A. ibid.
Counting; vol.
2. London
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.