Immunoelectrophoretic study of proteins in middle ear effusion A study of secretory otitis media in children By A. L. PAHOR*, N. SOZEN*, R. BEETHAM** and D. N. RAINE** (Birmingham)
otitis media (S.O.M.) is a common disorder in childhood. Harrison and Watson (1969) in a field study of over 1,700 five-year-old school children found an incidence of 3-6 per cent. There is controversy concerning the nature and aetiology of the middle ear fluid (M.E.F.) in cases of S.O.M. Some authors believe it to be a transudate resulting from eustachian tube (E.T.) obstruction, whereas others believe that it is an exudate produced by primary inflammation of the middle ear (M.E.) mucosa. Many studies have been carried out to investigate this point, including examination of the constituents of the M.E.F. for electrolytes, glucose, enzymes, mucopolysaccharides and different proteins including immunoglobulins (Juhn, Huff and Paparella, 1971; Mogi and Honjo, 1972; Palva and Raunio, 1975). To study this further, it was decided to examine the protein content in the M.E.F. by a two-dimensional crossed immunoelectrophoretic method (Clarke and Freeman, 1968).
SECRETORY
Material
M.E.F. specimens were collected from cases of S.O.M. operated on at the Children's Hospital, Birmingham. Twelve specimens were collected from 9 patients, in three of whom both ears were sampled. Table I shows the age, sex, race, clinical presentation, previous surgical history, followup, and the character of the M.E.F. collected. Aspiration of the M.E.F. was carried out under general anaesthesia in sterile conditions and the specimens were kept at —2O°C prior to analysis. Methods
Crossed immunoelectrophoresis was performed using a technique based on that described by Clarke and Freeman (1968). Glass slides (8 cm x 8 cm) were coated with agarose, 10 g/1, (L'lndustrie Biologique Franchise) containing rabbit anti-serum to human serum proteins ORCN yf (Hoechst Pharmaceuticals). Bromophenol blue, 5/^1 of a 1 g/1 solution, was added to dilutions of M.E.F. in water (15 pi of water per 3 mg * Department of Otolaryngology, Birmingham Children's Hospital. ** Department of Clinical Chemistry, Birmingham Children's Hospital. 1033
A. L. Pahor, N. Sozen, R. Beetham and D. N. Raine of M.E.F.). This dye provides a visible indication of the progress of albumin, a fast running protein, during electrophoresis. The volume of diluted M.E.F. applied to the agarose was 2 microlitres, and after electrophoresis the gel was stained with Coomassie Brilliant Blue. Serum from a normal adult was used as a constant reference pattern even though the proteins in the two fluids may be neither quantitatively nor qualitatively the same. Results The immunoelectrophoretic patterns of the M.E.F. analysed are shown in Figure 1. Figure 2 shows the pattern of normal serum. Using a polyvalent antiserum to human serum protein, individual peaks in the electrophoretic patterns can only be identified with confidence when serum is analysed. Analysis of protein from a different source presents problems of identification and indeed it is probable that if M.E.F. contains a protein not present in human serum there will be no corresponding antibody in the polyvalent antiserum used and hence no precipitation line will form. Similarly if different proteins in plasma and M.E.F. crossreact to any extent with the antibody to one, precipitation will occur but may not then be proportional to the quantity of the protein present. Because the M.E.F. has been electrophoresed in one direction before the immune reaction takes place it does not necessarily follow that the crossreactive protein in the latter fluid will be in the same position as would be serum protein with whose antibody the reaction is occurring. Thus, the serum protein electrophoresis is only of value as a reproducible pattern with which the analysis of M.E.F. can be compared: the patterns of different M.E.Fs. can of course be compared with each other. It may also be of special interest where fluid was obtained from both ears in the same patient to compare the findings in these. The original electrophoretic patterns were examined independently by two people and scored for the number of peaks, their height and their intensity which was rated as either strong or weak. The two observers were found to agree in these broad terms and their conclusions are sufficiently definite as to be observable in the reproduction of the patterns in Fig. 1. It can be seen that the patterns show a wide variation in the number and height of the individual peaks. For example Fig. 1-12 shows about 15 peaks whereas Fig. 1-1 shows only 7. The heights too are different in both absolute and relative terms. In three patients (S.D., Figs. 1-10 and 1-11; A.M., Figs. 1-12 and 1-5; and R.S., Figs. 1-6 and 1-3) specimens were obtained from both ears. In all instances the patterns obtained show sufficient differences to suggest that the pathological process is different in its stage of development if not in its nature, and that the two states should be considered independently. 1034
Immunoelectrophoretic study of proteins in middle ear eflfusion Fig. I - I shows the fewest peaks of lowest height and least intensity and it is interesting that this fluid was obtained whilst the patient was being treated with penicillin for an upper respiratory infection. In the light of this it is also of interest that Fig. 1-2 which has the next lowest intensity of peaks was obtained from a patient who had received several courses of antibiotics prior to admission to hospital. Whilst the other patients may have had antibiotics at various times this had not been sufficiently definite or consistent to be recorded in the clinical record.
; \ I I
Discussion The concept that the middle ear fluid in serous otitis media results from E.T. obstruction leading to a vacuum in the M.E. and transudate formation (ex vacuo) was proposed by Politzer, who was the first to describe S.O.M, in 1878, and is still held by some (Juhn, Huff and Paparella, 1971). Tumarkin (1961) stated that the E.T. obstruction may be a purely mechanical phenomenon from adenoid hyperplasia, but was more commonly inflammatory with an element of salpingitis. Buckingham (1970) confirmed the presence of a negative pressure in the tympanum in the presence of serous or glue type middle ear fluid by performing a myringotomy through a few drops of Terra-Cortril (Pfizer) solution, when the fluid was sucked into the M.E. Bluestein, Beery and Andrews (1974) showed a high negative middle ear pressure suggesting E.T. obstruction in cases with serous otitis media. This ex vacuo or 'transudate' theory has been challenged by many investigators who favour an exudative origin of the M.E.F. (Palva and Raunio, 1975). Senturia, Gessert and Carr (1958) showed that bacteria (often dead) and many leukocytes were present in the effusion, indicating an inflammatory aetiology. Sade (1966) showed that the basic histopathology of S.O.M. was an inflammatory hyperplasia of the mucosa and its mucous glands with hypersecretion of mucus. The exudate results from an inflammatory reaction which is attributed to various aetiological factors, namely bacterial (Senturia et al. 1958) viral (Berglund, Salmivalli and Gronroos, 1967) or allergic (McGovern, Haywood, and Fernandez, 1967). Still other investigators compromised between the two theories suggesting that when the M.E.F. is serous in character it is a transudate from plasma whereas the more mucous effusions result from enhanced epithelial secretory activity (Friedmann, 1963 and 1974; Mogi and Honjo, 1974). The present study shows that there is a considerable variation in the number (7-16) and size of protein peaks as revealed by crossed immunoelectrophoresis. Moreover neither the number nor the relative heights of the peaks show any marked correlation with those seen in human serum. This supports the evidence already cited against the view that M.E.F. results only from transudation of plasma across the middle ear mucosa. Another point of interest is that in the case of shortest duration (one 1035
FIG. I - I .
FIG.
1-4.
FIG.
1-5.
•**»
FIG.
1-2.
FIG.
1-3.
FIG.
1-6.
FIG.
1-7.
FIG.
1-8.
FIG.
1-9.
FIG.
I-IO.
FIG!
I-II.
FIG. FIG.
I—12.
1.
Immunoelectrophoretic patterns, using human serum protein antiserum, of 12 samples of M.E.F. from cases of serous otitis media. For patient identity see Table 1. 4B
F
M Caucasian
i2y 8m
6y 2m
6y io m
7y 5m
5y 5m
H (2 E.S. 00
G.F.
A.W.
R.S.
Caucasian
F
Caucasian
Asian
M Caucasian
F
Earache Deafness iy
Deafness im
Nasal obstruction Earache
Deafness
Earache Deafness 2y Earache Deafness
Earache Deafness iy U.R.T.I. Recurrent coughs and colds
Clinical Presentation
Patient details
* Bilateral myringotomy and grommets.
A.Me L R
R
5y 9m
M Caucasian
6y im
M.W. R
L R S.D. L
M Caucasian
3y 5 m
V.D.
M Asian
M Caucasian
7y 2m
N.W.
Race
Sex
Name Age at and side time sampled of op.
TABLE I.
On penicillin for 7 days pre-op P.H. of frequent antibiotic intake
History of antibiotic intake
No previous ops; no recurrence 3y B.M. & GTs*: 2 and iy previously; no recurrence 2jy No previous ops; Recurrence, B.M. 2jy later B.M. & GTs*: 2jy previously; no recurrence 2y No previous ops; no recurrence 3y No previous ops; 3 recurrences, B.M. & GTs.* 1, if & 2y 8m later No previous ops; lost to follow-up after 4m
No previous ops; no recurrence 2jy
No previous ops; no recurrence 2^^y
Previous ear ops. and follow-up
Middle ear effusion
o-34 0-26
0-26
0-26
0-27 0-26 0-28
°-33 0-26 O'29
Glue Glue Glue Glue Glue Glue Glue Glue Glue
0-28
0-28
Glue
Very thick glue Glue
16 IO
12
H
15
IO
9
Weight used Number of Consistency for CIEF (mg) precipitincurves
DETAILS OF PATIENTS AND MIDDLE EAR FLUIDS STUDIED.
1-12
1-10
I-II
1-6 1-3
1-9
1-4
Figure
a>
2
a.
f
to o o
3
tr o
•a
Immunoelectrophoretic study of proteins in middle ear effusion month, Figs, I - I O and I - I I ) the pattern obtained has more numerous and more intense peaks than almost any other. This supports the view that there is an inflammatory mucosal reaction with formation of an exudate containing a high concentration of protein and conflicts with the idea that the high concentration results from selective reabsorption of fluid. No correlation was found between the immunoelectrophoretic pattern of the M.E.F. and the age, sex or race of the patients.
FIG. 2. Immunoelectrophoresis of normal serum using human serum antiserum.
It is a common clinical finding that fluid aspirated from both ears in the same patient may be quite different in appearance and consistency. It is of interest that specimens taken from the two ears of the same patient show differences in the number and size of the protein peaks, and in two pairs these differences are marked.;Thus in bilateral S.O.M. the management of each ear should be determined independently by its state. The consistency of most of the specimens of M.E.F. at the time of collection was described as 'glue' (Table I) however, one was considered to be much more viscous than this. Palva and Raunio (1975) have shown that consistency depends less on protein than on mucopolysaccharides. It is therefore of interest that the most viscous fluid in the present series contained the fewest demonstrable protein peaks. It is also of note that the specimen with the fewest and smallest peaks was from a patient who had been treated with penicillin for seven days prior to aspiration of the fluid analysed. Fig. 1-2 which also has few peaks was from a patient who had had frequent courses of antibiotics prior to operation. The patients studied may be arbitrarily divided into those presenting with earache, and those with deafness and/or nasal obstruction. It can be seen that the former have fewer and less marked peaks than the latter. Expressed in a different way: if the specimens are arranged in order of 1039
A. L. Pahor, N. Sozen, R. Beetham and D. N. Raine increasing number and intensity of peaks, the first seven cases all presented with earache and of the remaining five only one presented in this way. This may reflect the fact that children with earache are more likely to receive antibiotics from the family practitioner. A final point of interest is that those in whom M.E.F. had previously been aspirated show more numerous and intense curves (Fig. 1—7 and 1-9). Moreover, cases in which M.E.F. had recurred (Fig. 1-8, 1-10 and 1-11) also show a heavy pattern. Thus it appears that patients showing the less intense pattern have a better prognosis. Summary
An immunoelectrophoretic study of the proteins of middle ear effusions in children with secretory otitis media is described. The findings support the view that the effusion is an exudate as a result of mucosal reaction. The protein content of the fluid appears to decrease as the condition becomes more chronic, and this seems to be enhanced by antibiotic treatment. In cases of bilateral disease, the ears may be in a quite different pathological phase, thus each ear should be treated independently. Acknowledgement
We are grateful to Mr. N. L. Crabtree, Mr. G. A. Dalton and Mr. R. J. Bennett, whose cases we have studied, for their interest and help with this study. REFERENCES BERGLUND, B., SALMIVALLI, A., and GRONROOS, J. A. (1967) Ada Otolaryngologica
(Stockholm) 63, 445. BLUESTEIN, C. D., BEERY, Q. C , and ANDREWS, W. S. (1974) Annals of Otology,
Rhinology and Laryngology (St. Louis) Supplement 11, 27. BUCKINGHAM, R. A. (1970) Otolaryngologic Clinics of North America, 3, 15. CLARKE, H. G. M., and FREEMAN, T. (1968) Clinical Science, 35, 403. FRIEDMANN, I. (1963) Proceedings of the Royal Society of Medicine, 56, 695. FRIEDMANN, I. (1974) Pathology of the Ear. Blackwell, London, p. 78. HARRISON, K., and WATSON, T. J. (1969) Proceedings of the Royal Society of Medicine, 62, 456. JUHN, S. K., H U F F , J. S., and PAPARELLA, M. M. (1971) Annals of Otology, Rhinology,
and Laryngology (St. Louis) 80, 347. MCGOVERN, J. P., HAYWOOD, T. J., and FERNANDEZ, A. A. (1967) Journal of the
American Medical Association, 200, 124. MOGI, G., and HONJO, S. (1972) Annals of Otology, Rhinology and Laryngology (St. Louis) 81, 99. MOGI, G., HONJO, S., YOSHIDA, T., and WATANABE, N. (1974) Annals of Otology,
Rhinology and Laryngology (St. Louis) 83, 239. PALVA, T., and RAUNIO, V. (1975) Journal of Laryngology and Otology, 89, 491. SADE, J. (1966) Archives of Otolaryngology (Chicago) 84, 297. SENTURIA, B. H., GESSERT, L. F., and CARR, L. D. (1958) Annals of Otology, Rhino-
logy and Laryngology (St. Louis) 67, 440. TUMARKIN, A. (1961) Journal of Laryngology and Otology, 75, 487.
The Children's Hospital, Birmingham, B16 8ET, England. 1040
otitis media (S.O.M.) is a common disorder in childhood. Harrison and Watson (1969) in a field study of over 1,700 five-year-old school children found an incidence of 3-6 per cent. There is controversy concerning the nature and aetiology of the middle ear fluid (M.E.F.) in cases of S.O.M. Some authors believe it to be a transudate resulting from eustachian tube (E.T.) obstruction, whereas others believe that it is an exudate produced by primary inflammation of the middle ear (M.E.) mucosa. Many studies have been carried out to investigate this point, including examination of the constituents of the M.E.F. for electrolytes, glucose, enzymes, mucopolysaccharides and different proteins including immunoglobulins (Juhn, Huff and Paparella, 1971; Mogi and Honjo, 1972; Palva and Raunio, 1975). To study this further, it was decided to examine the protein content in the M.E.F. by a two-dimensional crossed immunoelectrophoretic method (Clarke and Freeman, 1968).
SECRETORY
Material
M.E.F. specimens were collected from cases of S.O.M. operated on at the Children's Hospital, Birmingham. Twelve specimens were collected from 9 patients, in three of whom both ears were sampled. Table I shows the age, sex, race, clinical presentation, previous surgical history, followup, and the character of the M.E.F. collected. Aspiration of the M.E.F. was carried out under general anaesthesia in sterile conditions and the specimens were kept at —2O°C prior to analysis. Methods
Crossed immunoelectrophoresis was performed using a technique based on that described by Clarke and Freeman (1968). Glass slides (8 cm x 8 cm) were coated with agarose, 10 g/1, (L'lndustrie Biologique Franchise) containing rabbit anti-serum to human serum proteins ORCN yf (Hoechst Pharmaceuticals). Bromophenol blue, 5/^1 of a 1 g/1 solution, was added to dilutions of M.E.F. in water (15 pi of water per 3 mg * Department of Otolaryngology, Birmingham Children's Hospital. ** Department of Clinical Chemistry, Birmingham Children's Hospital. 1033
A. L. Pahor, N. Sozen, R. Beetham and D. N. Raine of M.E.F.). This dye provides a visible indication of the progress of albumin, a fast running protein, during electrophoresis. The volume of diluted M.E.F. applied to the agarose was 2 microlitres, and after electrophoresis the gel was stained with Coomassie Brilliant Blue. Serum from a normal adult was used as a constant reference pattern even though the proteins in the two fluids may be neither quantitatively nor qualitatively the same. Results The immunoelectrophoretic patterns of the M.E.F. analysed are shown in Figure 1. Figure 2 shows the pattern of normal serum. Using a polyvalent antiserum to human serum protein, individual peaks in the electrophoretic patterns can only be identified with confidence when serum is analysed. Analysis of protein from a different source presents problems of identification and indeed it is probable that if M.E.F. contains a protein not present in human serum there will be no corresponding antibody in the polyvalent antiserum used and hence no precipitation line will form. Similarly if different proteins in plasma and M.E.F. crossreact to any extent with the antibody to one, precipitation will occur but may not then be proportional to the quantity of the protein present. Because the M.E.F. has been electrophoresed in one direction before the immune reaction takes place it does not necessarily follow that the crossreactive protein in the latter fluid will be in the same position as would be serum protein with whose antibody the reaction is occurring. Thus, the serum protein electrophoresis is only of value as a reproducible pattern with which the analysis of M.E.F. can be compared: the patterns of different M.E.Fs. can of course be compared with each other. It may also be of special interest where fluid was obtained from both ears in the same patient to compare the findings in these. The original electrophoretic patterns were examined independently by two people and scored for the number of peaks, their height and their intensity which was rated as either strong or weak. The two observers were found to agree in these broad terms and their conclusions are sufficiently definite as to be observable in the reproduction of the patterns in Fig. 1. It can be seen that the patterns show a wide variation in the number and height of the individual peaks. For example Fig. 1-12 shows about 15 peaks whereas Fig. 1-1 shows only 7. The heights too are different in both absolute and relative terms. In three patients (S.D., Figs. 1-10 and 1-11; A.M., Figs. 1-12 and 1-5; and R.S., Figs. 1-6 and 1-3) specimens were obtained from both ears. In all instances the patterns obtained show sufficient differences to suggest that the pathological process is different in its stage of development if not in its nature, and that the two states should be considered independently. 1034
Immunoelectrophoretic study of proteins in middle ear eflfusion Fig. I - I shows the fewest peaks of lowest height and least intensity and it is interesting that this fluid was obtained whilst the patient was being treated with penicillin for an upper respiratory infection. In the light of this it is also of interest that Fig. 1-2 which has the next lowest intensity of peaks was obtained from a patient who had received several courses of antibiotics prior to admission to hospital. Whilst the other patients may have had antibiotics at various times this had not been sufficiently definite or consistent to be recorded in the clinical record.
; \ I I
Discussion The concept that the middle ear fluid in serous otitis media results from E.T. obstruction leading to a vacuum in the M.E. and transudate formation (ex vacuo) was proposed by Politzer, who was the first to describe S.O.M, in 1878, and is still held by some (Juhn, Huff and Paparella, 1971). Tumarkin (1961) stated that the E.T. obstruction may be a purely mechanical phenomenon from adenoid hyperplasia, but was more commonly inflammatory with an element of salpingitis. Buckingham (1970) confirmed the presence of a negative pressure in the tympanum in the presence of serous or glue type middle ear fluid by performing a myringotomy through a few drops of Terra-Cortril (Pfizer) solution, when the fluid was sucked into the M.E. Bluestein, Beery and Andrews (1974) showed a high negative middle ear pressure suggesting E.T. obstruction in cases with serous otitis media. This ex vacuo or 'transudate' theory has been challenged by many investigators who favour an exudative origin of the M.E.F. (Palva and Raunio, 1975). Senturia, Gessert and Carr (1958) showed that bacteria (often dead) and many leukocytes were present in the effusion, indicating an inflammatory aetiology. Sade (1966) showed that the basic histopathology of S.O.M. was an inflammatory hyperplasia of the mucosa and its mucous glands with hypersecretion of mucus. The exudate results from an inflammatory reaction which is attributed to various aetiological factors, namely bacterial (Senturia et al. 1958) viral (Berglund, Salmivalli and Gronroos, 1967) or allergic (McGovern, Haywood, and Fernandez, 1967). Still other investigators compromised between the two theories suggesting that when the M.E.F. is serous in character it is a transudate from plasma whereas the more mucous effusions result from enhanced epithelial secretory activity (Friedmann, 1963 and 1974; Mogi and Honjo, 1974). The present study shows that there is a considerable variation in the number (7-16) and size of protein peaks as revealed by crossed immunoelectrophoresis. Moreover neither the number nor the relative heights of the peaks show any marked correlation with those seen in human serum. This supports the evidence already cited against the view that M.E.F. results only from transudation of plasma across the middle ear mucosa. Another point of interest is that in the case of shortest duration (one 1035
FIG. I - I .
FIG.
1-4.
FIG.
1-5.
•**»
FIG.
1-2.
FIG.
1-3.
FIG.
1-6.
FIG.
1-7.
FIG.
1-8.
FIG.
1-9.
FIG.
I-IO.
FIG!
I-II.
FIG. FIG.
I—12.
1.
Immunoelectrophoretic patterns, using human serum protein antiserum, of 12 samples of M.E.F. from cases of serous otitis media. For patient identity see Table 1. 4B
F
M Caucasian
i2y 8m
6y 2m
6y io m
7y 5m
5y 5m
H (2 E.S. 00
G.F.
A.W.
R.S.
Caucasian
F
Caucasian
Asian
M Caucasian
F
Earache Deafness iy
Deafness im
Nasal obstruction Earache
Deafness
Earache Deafness 2y Earache Deafness
Earache Deafness iy U.R.T.I. Recurrent coughs and colds
Clinical Presentation
Patient details
* Bilateral myringotomy and grommets.
A.Me L R
R
5y 9m
M Caucasian
6y im
M.W. R
L R S.D. L
M Caucasian
3y 5 m
V.D.
M Asian
M Caucasian
7y 2m
N.W.
Race
Sex
Name Age at and side time sampled of op.
TABLE I.
On penicillin for 7 days pre-op P.H. of frequent antibiotic intake
History of antibiotic intake
No previous ops; no recurrence 3y B.M. & GTs*: 2 and iy previously; no recurrence 2jy No previous ops; Recurrence, B.M. 2jy later B.M. & GTs*: 2jy previously; no recurrence 2y No previous ops; no recurrence 3y No previous ops; 3 recurrences, B.M. & GTs.* 1, if & 2y 8m later No previous ops; lost to follow-up after 4m
No previous ops; no recurrence 2jy
No previous ops; no recurrence 2^^y
Previous ear ops. and follow-up
Middle ear effusion
o-34 0-26
0-26
0-26
0-27 0-26 0-28
°-33 0-26 O'29
Glue Glue Glue Glue Glue Glue Glue Glue Glue
0-28
0-28
Glue
Very thick glue Glue
16 IO
12
H
15
IO
9
Weight used Number of Consistency for CIEF (mg) precipitincurves
DETAILS OF PATIENTS AND MIDDLE EAR FLUIDS STUDIED.
1-12
1-10
I-II
1-6 1-3
1-9
1-4
Figure
a>
2
a.
f
to o o
3
tr o
•a
Immunoelectrophoretic study of proteins in middle ear effusion month, Figs, I - I O and I - I I ) the pattern obtained has more numerous and more intense peaks than almost any other. This supports the view that there is an inflammatory mucosal reaction with formation of an exudate containing a high concentration of protein and conflicts with the idea that the high concentration results from selective reabsorption of fluid. No correlation was found between the immunoelectrophoretic pattern of the M.E.F. and the age, sex or race of the patients.
FIG. 2. Immunoelectrophoresis of normal serum using human serum antiserum.
It is a common clinical finding that fluid aspirated from both ears in the same patient may be quite different in appearance and consistency. It is of interest that specimens taken from the two ears of the same patient show differences in the number and size of the protein peaks, and in two pairs these differences are marked.;Thus in bilateral S.O.M. the management of each ear should be determined independently by its state. The consistency of most of the specimens of M.E.F. at the time of collection was described as 'glue' (Table I) however, one was considered to be much more viscous than this. Palva and Raunio (1975) have shown that consistency depends less on protein than on mucopolysaccharides. It is therefore of interest that the most viscous fluid in the present series contained the fewest demonstrable protein peaks. It is also of note that the specimen with the fewest and smallest peaks was from a patient who had been treated with penicillin for seven days prior to aspiration of the fluid analysed. Fig. 1-2 which also has few peaks was from a patient who had had frequent courses of antibiotics prior to operation. The patients studied may be arbitrarily divided into those presenting with earache, and those with deafness and/or nasal obstruction. It can be seen that the former have fewer and less marked peaks than the latter. Expressed in a different way: if the specimens are arranged in order of 1039
A. L. Pahor, N. Sozen, R. Beetham and D. N. Raine increasing number and intensity of peaks, the first seven cases all presented with earache and of the remaining five only one presented in this way. This may reflect the fact that children with earache are more likely to receive antibiotics from the family practitioner. A final point of interest is that those in whom M.E.F. had previously been aspirated show more numerous and intense curves (Fig. 1—7 and 1-9). Moreover, cases in which M.E.F. had recurred (Fig. 1-8, 1-10 and 1-11) also show a heavy pattern. Thus it appears that patients showing the less intense pattern have a better prognosis. Summary
An immunoelectrophoretic study of the proteins of middle ear effusions in children with secretory otitis media is described. The findings support the view that the effusion is an exudate as a result of mucosal reaction. The protein content of the fluid appears to decrease as the condition becomes more chronic, and this seems to be enhanced by antibiotic treatment. In cases of bilateral disease, the ears may be in a quite different pathological phase, thus each ear should be treated independently. Acknowledgement
We are grateful to Mr. N. L. Crabtree, Mr. G. A. Dalton and Mr. R. J. Bennett, whose cases we have studied, for their interest and help with this study. REFERENCES BERGLUND, B., SALMIVALLI, A., and GRONROOS, J. A. (1967) Ada Otolaryngologica
(Stockholm) 63, 445. BLUESTEIN, C. D., BEERY, Q. C , and ANDREWS, W. S. (1974) Annals of Otology,
Rhinology and Laryngology (St. Louis) Supplement 11, 27. BUCKINGHAM, R. A. (1970) Otolaryngologic Clinics of North America, 3, 15. CLARKE, H. G. M., and FREEMAN, T. (1968) Clinical Science, 35, 403. FRIEDMANN, I. (1963) Proceedings of the Royal Society of Medicine, 56, 695. FRIEDMANN, I. (1974) Pathology of the Ear. Blackwell, London, p. 78. HARRISON, K., and WATSON, T. J. (1969) Proceedings of the Royal Society of Medicine, 62, 456. JUHN, S. K., H U F F , J. S., and PAPARELLA, M. M. (1971) Annals of Otology, Rhinology,
and Laryngology (St. Louis) 80, 347. MCGOVERN, J. P., HAYWOOD, T. J., and FERNANDEZ, A. A. (1967) Journal of the
American Medical Association, 200, 124. MOGI, G., and HONJO, S. (1972) Annals of Otology, Rhinology and Laryngology (St. Louis) 81, 99. MOGI, G., HONJO, S., YOSHIDA, T., and WATANABE, N. (1974) Annals of Otology,
Rhinology and Laryngology (St. Louis) 83, 239. PALVA, T., and RAUNIO, V. (1975) Journal of Laryngology and Otology, 89, 491. SADE, J. (1966) Archives of Otolaryngology (Chicago) 84, 297. SENTURIA, B. H., GESSERT, L. F., and CARR, L. D. (1958) Annals of Otology, Rhino-
logy and Laryngology (St. Louis) 67, 440. TUMARKIN, A. (1961) Journal of Laryngology and Otology, 75, 487.
The Children's Hospital, Birmingham, B16 8ET, England. 1040
