The relatively specific effect of alcohol on magnesium POTENCY AND STABILITY OF COMBINED
excretion, together with the observation that low serum-
PERTUSSIS, DIPHTHERIA, TETANUS, AND
magnesium levels are more frequent and last longer than POLIOMYELITIS (QUADRUPLE) VACCINE
other electrolyte changes in patients with acute alcoholism
(Martin etal. 1959), provide strong circumstantial evidence A. J. BEALE
that the hypomagnesasmia develops in these patients M.D. Lond., Dip. Bact.
principally because an increased excretion of magnesium J. UNGAR
results from the intake of alcohol. The observation that M.D. Prague, M.R.C.S.
administration of alcohol to rats increased their require- OF GLAXO RESEARCH LTD., GREENFORD, MIDDLESEX
ment for dietary magnesium (Gottleib et al. 1959) is also
A VACCINE that combines protection against whooping-
explicable by this hypothesis.
cough, diphtheria, tetanus, and poliomyelitis in one series
Summary of injections has considerable advantages. Combined
The incidence of hypomagnessemia among fifty chronic
pertussis, diphtheria, and tetanus vaccine (triple antigen)
alcoholics was 25%. This depletion was not considered has to be given by injection, and the inclusion of polio-
important in the clinical condition of these patients, and vaccine would mean that in one series of three injections
it was usually corrected within a week of a return to
primary immunisation against four diseases could be
normal diet. achieved. The consequent reduction of the number of
The plasma-magnesium was more sensitive to depletion visits to the clinic or doctor should increase the rate of
than was the erythrocyte concentration. No relation was
acceptance for infant immunisation. Particularly impor-
found between the serum concentrations of magnesium rates of immunisation against diphtheria and
and cholesterol. tetanus should be achieved because of the desire of par-
Administration of alcohol to normal subjects caused a ents to protect their children against poliomyelitis and
large rise in urinary magnesium excretion without produc- whooping-cough. Thus, the combination of this protec-
ing a comparable effect on other cations. This was due, tion in one vaccine would simplify the publicity cam-
predominantly, to variation in the activity of the renal paign for immunisation as well as the record-keeping and
tubules. An increased magnesium excretion after consum- administration.
ing alcohol is believed to be the principal cause of hypo- We review briefly here the methods of ensuring that
magneseemia among patients with chronic alcoholism. the components of a quadruple vaccine produce a satis-
We wish to thank Miss M. Nicholson, Miss J. Carter, Mr. W. B.
Simpson, and Mr. K. Taylor for technical assistance. factory immune response in children. Evidence for
stability of all the components in the vaccine is also
Barnes, B. A., Cope, O., Harrison, T. (1958) J. clin. Invest. 37, 430. presented.
Beckett, A. G., Lewis, J. G. (1959) Clin. Sci. 18, 597. Control of the Potency of the Components
Beroshn, I., Oelofse, P. J. (1957) Lancet, i, 1020.
Charnock, J. S., Casley-Smith, J., Schwartz, C. J. (1959) Aust. J. exp. Biol. The diphtheria, tetanus, and pertussis components are
med. Sci. 37, 509.
Dawson, J. B., Heaton, F. W. (1961) Biochem. J. 80, 99. measured by well-established techniques.
Edwards, K. D. G., Whyte, H. M. (1958) Aust. J. exp. Biol. med. Sci. 36, 383. The toxoids are titrated by the flocculation technique, and
Fitzgerald, M. G., Fourman, P. (1956) Clin. Sci. 15, 635.
Flink, E. B., Stutzman, F. L., Anderson, A. R., Konig, T., Fraser, R. (1954) by demonstrating their efficacy in producing antitoxin in
J. Lab. clin. Med. 43, 169. guineapigs. Twenty-five Lf units of diphtheria toxoid and
Gottleib, L. S., Broitman, S. A., Vitale, J. J., Zamcheck, N. (1959) ibid. 10 Lf units of tetanus toxoid are incorporated into one dose
Heaton, F. W. (1960) J. clin. Path. 13, 358. of quadruple vaccine. The pertussis content of the vaccine is
Jankelson, O. M., Vitale, J. J., Hegsted, D. M. (1959) Amer. J. clin. Nutr. controlled by determining the number of organisms present
Martin, H. E., McCuskey, C., Tupikova, N. (1959) ibid. p. 191. (20 x 109 organisms per dose) and potency is finally compared
McCollister, R. J., Flink, E. B., Doe, R. P. (1960) J. Lab. clin. Med. 55. 98. with that of a standard dried preparation by the mouse-pro-
Prasad, A. S., Doe, R. P., Flink, E. B. (1958) ibid. 52, 928. tection test. The potency of the pertussis component (effec-
Mendelson, J., Wexler, D., Kubzansky, P., Leiderman, P. H., Solomon, P. tive dose, E.D’50) is calculated as the number of organisms
(1959) J. nerv. ment. Dis. 128, 352.
Parsons, R. S., Butler, T., Sellars, E. P. (1959) Med. Proc. 5, 487. required to protect 50% of the animals against a lethal intra-
Rogers, T. A., Mahan, P. E. (1959) Proc. Soc. exp. Biol., N. Y. 100, 235. cerebral challenge with 100 L.D.5o pertussis organisms. Results
Sackett, G. E. (1925) J. biol. Chem. 64, 203. of this test correlate well with protection against whooping-
Sunderman, F. W. (1947) Amer. J. clin. Path. 17, 169.
Suter, C., Klingman, W. O. (1955) Neurology, 5, 691. cough (Medical Research Council 1956).
Toribara, T. Y., Terepeka, A. R., Dewey, P. A. (1957) J. clin. Invest. 36, 738. These antigens, when incorporated into this quadruple vac-
Wacker, W. E. C., Vallee, B. L. (1958) New Engl. J. Med. 259, 475.
cine, have been shown to give satisfactory results in children
-for example, by Dane et al. (1962) and in unpublished
"... Is aclassless’standard of social service possible in a
’ dass-divided ’society ? Social policy cherishes and strengthens studies. The response of children to pertussis vaccine has
the family and also enlarges personal freedom and responsible been assessed by the agglutinin test, though the ability of vac-
cine to produce satisfactory results in the mouse-protection
choice. Can it then deny increasingly affluent citizens the right
test is probably a better guarantee of its ability to prevent
to buy the education or medical care they think best for their
children or themselves ? ... We do not want the operations of whooping-cough (Medical Research Council 1956).
social services to be governed by the purchasing power of The poliovaccine contains specified amounts of D anti-
individual users. But we should be insisting very strongly on gen (the antigen stimulating the production of neutralising
alternative ways of obliging these services to attend to users’ antibodies) for all three types of poliovirus. The tech-
wants, feelings and expectations. In part, we have been bemused nique for performing D-antigen assay (Beale and Mason
by the belief that experts have some simple magic enabling 1962) has been shown to give a valid measure of vaccine
them to know just what education or medical care are best
suited to each child’s or patient’s needs. Even were such needs potency (Beale 1961). As additional evidence in support
of its value, Hummeler et al. (1962) found that neutralising
:eaaly ascertainable by reference to ability, aptitude or clinical
education is more than cultivation of abilities and anti-D sera agglutinated the complete particles seen in
aptitudes, medical care more than treatment of clinical states." electron micrographs, whereas anti-C sera agglutinated
- Prof. FRANCOIS LAFITTE, Social Policy in a Free Society; damaged or empty particles. Our own experience with
=.13. University of Birmingham, 1962. more potent preparations of D antigen has revealed
that the linear for example, contained 200 D units for type 1 and 1 unit each
relationship for types 2 and 3. In unpublished trials Dick and Dane
between the showed that a batch of quadruple vaccine (7C) containing
distance of the 15 units of type-1 D antigen was inadequate for producing a
precipitin line satisfactory immune response in children.
from the anti- Gaisford and Perkins have carried out trials with four
batches of quadruple vaccine. The poliovaccine and the freeze-
serum cup and
dried triple-antigen components of these quadruple vaccines
the concentra- was stored separately, because of doubts expressed in the U.S.A.
tion(LeBouvier about the stability of the individual components. They were
1959, Beale and mixed immediately before inoculation. Gaisford and Perkins
Mason 1962) used a batch of quadruple vaccine 7A which contained 150
holds over only units of type-1 D antigen.
a limited range Preliminary results on children who have had a prim-
(fig. 1). D-anti- ary course of immunisation with these vaccines, begin-
gen assay is ning at one week old, suggest that batch 7A provides an
therefore only adequate antigenic stimulus. This would be expected,
reliable when since Gaisford and Perkins (see Perkins 1962) have shown
the D-antigen that the response of one-week-old children to a concen-
preparations trated purified poliovaccine (Merck, Sharpe & Dohme)
are diluted or Fig. 1-Relationship between poliovirus type-1 was satisfactory. This vaccine has been found to contain
concentrated D-antigen concentration and position of 150 D-antigen units per ml. for type 1; the recommended
so that their dose is 0-5 ml., but Gaisford and Perkins used 1 ml.
precipitin lines fall on the linear part of the curve.
Standard preparations of all three types of poliovirus have
been prepared and assigned a unitage such that a
D line " of 24 mm. in the test described by Beale and
Mason (1962) corresponds to 600 units of D antigen for
each type. The results of ten replicate titrations and
the unitage assigned for 1 ml. of the standard prepara-
tions are shown in table I. These preparations have been
dispensed in 0-5 ml. amounts in ampoules. An ampoule
contains in 0-5 ml. the following amounts of D antigen:
type 1, 440; type 2, 195; type 3, 235. The standard
preparations deposited with the Division of Immuno-
logical Products Control Laboratory, Medical Research
Council Laboratories, Hampstead, are used to control
the potency of the poliovaccine components of the new
quadruple vaccine. The poliovaccine components are
assayed against these standards, and they are included
at such levels that one dose of the vaccine contains not
less than 75 D-antigen units of type 1 and not less than
1 D-antigen unit of each of type 2 and type 3. It should
be noted that the D units defined here are not the same
as the arbitrary units referred to by Beale (1961).
The D-antigen content of several commercial batches of
killed poliovaccines have been tested by our method (Beale
and Mason 1962) and found to contain 1 to 8 units of type-1
D antigen per dose. Such vaccines have been obtained both
from this country and from several countries abroad. Fewer
observations have been made on types 2 and 3, but routine
Fig. 2&agr;—Stability of type-! component of quadruple vaccine at
batches of inactivated poliovaccine made by us have con- 4°C; (b) Stability of type-2 and type-3 components of quadruple
tained about 05 unit of D antigen per dose. Clinical trials vaccine at 4°C.
have demonstrated that when they are incorporated in quad-
ruple vaccine little if any increases are required for types 2 and The potency of the poliovaccine is finally checked in
3 for satisfactory results; by contrast a substantial increase in the standard monkey potency test. Initial experience
type-1 component was required to give a satisfactory response with poliovaccines of increased potency supports the
to this antigen. The vaccine (8A) used by Dane et al. (1962), view expressed by Perkins (1962) about potency testing
of poliovaccines: vaccine that is of marginal potency
TABLE I-DISTANCE OF D PRECIPITIN LINE FROM ANTISERUM CUP FOR (0-5 to 2 D-antigen units for type 1) will highlight the
STANDARD D-ANTIGEN PREPARATIONS
variability of the monkey test. Vaccine containing 75 or
more D-antigen units of type 1 is so potent that the
variability of the monkey test is no longer important if
the same standard of vaccine acceptability is employed.
Early studies showed that the poliovaccine component
of quadruple vaccine is not stable when thiomersal, even
in the presence of sodium edetate, is used as a bacterio-
Fig. 3-Stability of diphtheria and tetanus toxoids in quadruple Fig. 4-Stability of pertussis component of quadruple vaccine
vaccine at 4°C. at 4°C.
stat, The results for a batch of quadruple vaccine 7D shows the detailed results after one year’s storage at 4°C.
containing thiomersal (100 parts per million) and sodium The pertussis component of quadruple vaccine has
edetate (700 p.p.m.) are shown in fig. 2a andband recently been shown by tests done in the United States
compared with those for the same vaccine (7c) without (Massachusetts Department of Health 1960, Pittman
thiomersal but with half the amount of sodium edetate. 1962) to lack stability. Various possible reasons for this
It will be seen that there is a rapid fall in potency of the lack of stability in presence of poliovaccine, despite its
polio antigen, as measured by the antigen-extinction proven stability in triple antigen, have been advanced.
technique in chicks (Beale 1961) for batch 7D, whereas Thiomersal, which has been used as the bacteriostat for
batch 7c is stable for at least one year. This rapid fall in triple antigen, has had to be replaced by other bacterio-
poliovaccine potency when quadruple vaccine contains stats because of its deleterious effect on the poliovaccine
thiomersal and sodium edetate as bacteriostat has been component (fig. 2). Benzethonium chloride, which is
found also in children (Perkins and Gaisford, Butler et al., commonly preferred in quadruple vaccine, has been
personal communications). It has, it will be recalled, been found by Corkill (1962) to destroy the potency of per-
established that plain killed poliovaccine is stable at 4°C tussis vaccine when storage is prolonged or when the
in the presence of thiomersal and sodium edetate for at concentration exceeds 1/20,000. Corkill (1962) has also
least 21 months (Davisson et al. 1956, Perkins and Yetts found in monkey-kidney tissue culture fluid two other
1959). factors that affect the pertussis vaccine. The first, which
The stability of the diphtheria and tetanus toxoids in is destroyed by autoclaving, reduces the opacity of per-
the vaccine has proved satisfactory on storage at +4°C tussis preparations. The second, which in fact destroys
for one year, whether thiomersal was present or not (fig. the pertussis antigen, is heat stable and may be dialysable
3), The results of tests for stability of the pertussis com- because after osmoconcentration with polyethyleneglycol
ponent (fig. 4) are given for five vaccines containing per- it disappears from the vaccine. This heat-stable factor is
tussis (quadruple vaccines batches 7c and 7D, the pertussis neutralised by three treatments:
component made up as fluid triple antigen, the same (a) addition of thiomersal (this cannot be used in quadruple
material freeze-dried, and the British Standard). Table 11 vaccine because of its effect on the poliovaccine compon-
TABLE II-POTENCY OF PERTUSSIS COMPONENT OF VACCINES STORED (b) addition of sodium edetate;
FOR 1 YEAR AT 40c
MOUSE PROTECTION TESTS RESULTS
(c) purification of the poliovaccine component by methanol
CHALLENGE DOSE 30,000 VIABLE PERTUSSIS ORGANISMS (100 L.D.50)
These findings may well be the explanation for the diffi-
culties with quadruple vaccine, particularly the observa-
tion of Pittman (1962) that the vaccine deteriorates more
quickly under market conditions than during storage
strictly at +4°C.
There is one other suggested cause of lack of stability
of the pertussis component in quadruple vaccine. This is
the protease found by Baron and Barnett (1960) in mon-
key-kidney tissue-culture fluid and in some samples of
poliovaccine. The relationship of this factor to those
described by Corkill is unknown, but his heat-sensitive
factor might well be protease. It has been found during
manufacture of inactivated poliovaccine in these labora-
tories that the protease present in the tissue-culture fluid
is almost always removed by the Seitz filtration pads. In
the few samples in which protease has been detected
after filtration, only minute traces remained, requiring
more sensitive techniques for their detection than those
described by Baron and Barnett (1960). Subsequently
our vaccine is purified by adsorption on and elution from
aluminium phosphate (Fantes 1962). None of such puri-
fied samples has been found to contain protease. It is
probable that the aluminium-phosphate purification MANAGEMENT OF TRACHEOTOMY
method would remove Corkill’s heat-stable factor, but no IN INFANTS
work has been done on this question. Finally sodium
edetate (350 p.p.m.), but not thiomersal, is added to our GEORGE FENNELL
quadruple vaccine. It seems that these procedures CONSULTANT EAR, NOSE AND THROAT SURGEON,
adopted in our laboratories remove factors responsible for ROYAL INFIRMARY, STIRLING, SCOTLAND
the degradation of the pertussis potency and are the reason
DIFFICULTIES encountered during and immediately
for the striking stability of the pertussis component of the
after tracheotomy in infants have prompted me to set forth
particular quadruple vaccine we are describing (fig. 4 and the technique for the operation, and some suggestions on
Summary management immediately after operation.
Methods used to measure and control the potency of Anaesthesia
the individual components in a quadruple vaccine are always advisable unless there is an
General anaesthesia is
described, including the production of standard prepara- obviously impassable obstruction, such as a foreign body,
tions of poliovirus D antigen. They make it possible to in the pharynx or larynx. If there is impassable obstruc-
ensure that each batch of quadruple vaccine contains tion, laryngotomy or tracheotomy without anssthesia is
sufficient of all the antigenic components to give protec- probably needed immediately.
tive immunity to children. In the very young infant, perhaps only a few days old,
The stability of all components of the vaccine after the trachea should be intubated as the first step, and
storage for one year at 4°C in the absence of a bacteriostat induction with nitrous oxide and oxygen can then be pro-
has been demonstrated. Reasons are given for believing ceeded with. In older infants, induction should begin
that factors present in some poliovaccine preparations with nitrous oxide and oxygen given through a face mask,
and destructive of pertussis vaccine have been removed and after intubation anaesthesia should be continued with
from the quadruple vaccine described here. the addition of ether. As soon as possible after introduc-
It is concluded that an effective, stable, and safe tion of the endotracheal tube the anaesthetist should apply
quadruple vaccine has been developed. suction through a soft rubber catheter, to remove secre-
Many colleagues have helped in the work on quadruple vaccine; tions from the bronchial tree.
in particular Dr. J. E. Crofts and Mr. I. E. Addison have prepared Position
and carried out many tests on the triple antigen components. Dr.
1. G. S. Furminger has allowed us to refer to his unpublished work The infant is placed supine with a small sandbag
on the protease present in monkey-kidney cultures. Dr. J. Corkill, beneath the shoulders, and the head is gently extended;
of the Connaught Medical Research Laboratories, kindly allowed hyperextension is undesirable as this interferes with the
us to see the manuscript of his paper presented at Prague, which
venous return from the head and neck and produces
is being prepared for publication in a scientific journal. Dr. R. J.
Wilson, also of the Connaught Medical Research Laboratories, excessive bleeding. An assistant keeps the head in the
closely concerned with the development of quadruple vaccine in extended position and is responsible for keeping chin,
Canada, has read our manuscript and made many helpful comments. larynx, and suprasternal notch in a straight line throughout
We especially thank Dr. N. R. Butler, Dr. P. F. Benson, Dr. B. D. R. the operation. When the trachea has been exposed and all
Wilson, Dr. J. A. Dudgeon, and Dr. J. Urquhart; Prof. W. F. Gais-
ford and Dr. F. T. Perkins; and Professor G. W. A. Dick and Dr. bleeding has been controlled, a little further extension of
D. S. Dane for carrying out clinical trials on these newly developed the head will bring the trachea nearer to the surface.
vaccines and for many helpful discussions.
REFERENCES Skin and the placing of towels must leave
Baron, S., Barnett, E. V. (1960) Proc. Soc. exp. Biol. N.Y. 103, 527.
a narrow sterile area from the hyoid bone to the upper
Beale, A. J. (1961) Lancet, ii, 1166.
Mason, P. J. (1962) J. Hyg., Camb. 60, 113.
margin of the sternum. The cricoid cartilage is palpated
Corkill, J. M. (1962) Conference on Pertussis Vaccine, Prague. and an incision is made from this level to just above the
Dane, D. S., Dick, G. W. A., Simpson, D. I. H., Briggs, E. M., McAlister,
J., Nelson, R., Field, C. M. B. (1962) Lancet, i, 939. sternal notch, dividing the skin superficial fascia, and the
Davisson, E. O., Powell, H. M., MacFarlane, J. O., Hodgson, R., Stone, platysma. The investing layer of the deep fascia is next
R. L., Culbertson, C. G. (1956) J. Lab. clin. Med. 47, 8.
Fantes, K. H. (1962) J. Hyg., Camb. 60, 123. divided, and the dissection with blunt scissors is carried
Hiatt, C. W., Kaufman, E., Helprin, J. J., Baron, S. (1960) J. Immunol. deeply in the direction of the trachea. As the smallness
Hummeler, K., Anderson, T. F., Brown, R. A. (1962) Virology, 16, 84. and mobility of the infant trachea are such that it can easily
LeBouvier, G. L. (1959) Brit. J. exp. Path. 40, 452. slip to one side, return must constantly be made to the
Massachusetts Department of Public Health (1960) New Engl. J. Med.
263, 410. landmarks of cricoid and sternal notch. The operator
Medical Research Council (1956) Brit. med. J. ii, 454. should place the retractors because an inexperienced
Perkins, F. T., Yetts, R. (1959) ibid. i, 31.
Perkins, F. T. (1962) Proceedings of 7th International Congress for Micro- assistant may retract the trachea laterally.
biological Standardisation; p. 435. Edinburgh. The various tissue planes from skin to trachea should
Pittman, M. (1962) J. Amer. med. Ass. 181, 25.
not be separated one from the other, but all these struc-
tures should be retracted laterally in one mass to reduce
"... We who study transplantation cannot acquit ourselves of the
the risk of infection spreading into the fascial planes of the
charge of making our ideas known to each other in a terminology
that is etymologically ludicrous and inconsistent with certain older neck and the mediastinum. When the whole length of the
immunological usages. At the very least I suggest that we should incision is deepened, the trachea and thyroid isthmus lie
follow Gorer in substituting allogenic’ for homologous ’, and
in replacingisologous’ by’isogenic ’ or, much better, by ’syn-
exposed. The isthmus is clamped on both sides of the
geneic Incidentallyenhancement’ is another offender. It is midline, divided, and tied. Division of the isthmus does
a word to which Nathan Kaliss has given an exact meaning: ’ enhance- much to overcome the difficulty of replacing a tracheotomy
ment ’ is an abrogation of the homograft reaction mediated through tube which has been coughed out, or is being changed.
the action of specific humoral antibodies. Where humoral antibodies during the early postoperative period.
are not known to be involved, should we not use a non-committal
When the pretracheal fascia has been cleared from the
word likepromotion ’-Flexner’s word, dating from 1907-
instead?"—P. B. MEDAWAR in Ciba Foundation Symposium on third and fourth rings, a small transverse incision is made
Transplantation; p. 2. London, 1962. between them, and a semicircular piece of trachea is