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
                                                                                  tant,   high
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
     53, 433.
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
  7, 23.
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
                    precipitin line.
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
                                                                      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.
                                                                                               Quadruple Vaccine
                                                                        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-
                                                                           ent) ;
TABLE II-POTENCY OF PERTUSSIS COMPONENT OF VACCINES STORED              (b) addition of sodium edetate;
                     FOR 1 YEAR AT 40c
                                                                        (c) purification of the poliovaccine component by methanol
                                                                      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
                                                                                                        L.R.C.P.I., D.L.O.
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
table 11).
                                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
     84, 480.
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

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