- Email: [email protected]
NON-INVASIVE SAMPLING OF NASAL CILIA FOR MEASUREMENT OF BEAT FREQUENCY AND STUDY OF ULTRASTRUCTURE
564
Methods and Devices
NON-INVASIVE SAMPLING OF NASAL CILIA FOR MEASUREMENT OF BEAT FREQUENCY AND STUDY OF ULTRASTRUCTURE
Fig.
1-Variation in
CBF 14
JONATHAN RUTLAND
PETER
J. COLE
Host Defence
Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, London SW3 6HP
WE describe a non-invasive method for obtaining nasal ciliated epithelium which is suitable for measurement of ciliary beat frequency (CBF) and examination of ciliary ultrastructure by transmission electron microscopy. No local anaesthesia is required and this technique is particularly suitable for children and in situations where multiple samples are required, such as in-vitro studies of human tissue.
Method
blow his nose gently to clear excess secretions, and an auriscope is introduced into one nostril to allow the inferior nasal turbinate to be seen. A cytology brush 2 mm in diameter is inserted through the auriscope between the inferior turbinate and lateral nasal wall, quickly moved posteriorly for about 1 cm along the mucosal surface, and then moved anteriorly as it is removed. Cellular material adhering to the brush is dislodged by brisk agitation in 2 ml of tissue culture medium (Medium 199 with Earle’s salts; Flow Laboratories). Wetting of the brush with medium before sampling increases the amount of tissue obtained. In subjects in whom insertion of the brush between inferior turbinate and lateral nasal wall is difficult, suitable epithelium may be obtained from the medial surface of the turbinate, but the brush must be advanced more posteriorly to obtain ciliated cells. A sealed preparation for measuring CBF can be made by applying a thin layer of silicon grease around the edges of a coverslip by means of a syringe attached to a modified intravenous cannula. Fragments of nasal epithelium can be transferred from the culture medium with a Pasteur pipette and deposited on the coverslip. A standard microscope slide gently placed over the coverslip seals the preparation which can then be inverted and examined microscopically, coverslip upwards. We measure CBF photometrically.l,2 The specimen is placed on an electronically controlled warm stage (Microtec) mounted on a Leitz Dialux 20 microscope. A Leitz MPV compact microscope photometer, transduces light intensity into an electrical signal. We routinely measure CBF at a temperature of 36±0-5°C. A polystyrene cylinder is placed between slide and objective to reduce heat-loss. Strips of epithelium with beating cilia are easily seen at a magnification of x 320 by bright-field illumination with the condenser at a low aperture. Illumination of the photometer’s field diaphragm allows a small rectangular area (approximately 1-5x35 [im) to be oriented in the long axis of the cilia so that only two or three cilia beat across this area, intermittently preventing light from reaching the photometer. The transduced electrical signal is fed through a lowpass filter with a cut-off above 40 Hz (variable to 400 Hz) into an ultra-violet oscillograph (SE Labs 6150) which provides a permanent record of ciliary activity. Timer markings at 100 msec intervals allow calculation of the CBF (fig. 1). The procedure is repeated 10 times at different areas and a mean and standard deviation (SD) are calculated. Only cells which are attached to basement membrane and have vigorously beating cilia are counted. The
subject
As soon ments, still
is asked
to
counting is complete the larger epithelial fragsuspended in medium, can be transferred with a
as
light intensity caused by 3 beats/sec. Paper speed 100 mm/sec.
cilia
beating.
pipette to fixative for electron microscopy. Alternatively, fragments may be placed into fixative immediately after brushing. Samples of epithelium obtained by this technique are satisfactory for studying ciliary ultrastructure (fig. 2). Pasteur
Results Nasal brushings from a 41-year-old man with progressive bronchiectasis, sinusitis, and azoospermia were examined. CBF was 14-2+0.4 beats/sec (mean ±SD) (fig. 1). CBF for five age-matched normal subjects was 14-8±1-0 beats/sec.
Most of the patient’s cilia had normal ultrastructure (fig. 2a) while 7% of 200 cilia counted were abnormal, a single peripheral microtubule in place of one of the normal double microtubules being the commonest finding (fig. 2b). Of 50 consecutive patients examined in this way, 2 complained of pain (one had severe rhinitis) while most described sensations of a tickle or a desire to sneeze (only 1 did so). To examine the effect of local anaesthetic on CBF determined by this method we measured beat frequency in 10 patients. Samples were obtained from both nasal cavities after one (chosen randomly) had been sprayed with an aerosol of 4% lignocaine (approximately 0.7 ml). Mean CBF for the untreated side was 12.2±2.3 beats/sec while that for the side sprayed with lignocaine was 12-3±2-0 beats/sec. Analysis of variance showed no statistically significant difference between the two groups.
2-Electron micrographs showing cross sections of normal and abnormal cilia obtained from nasal mucosa by brushing.
Fig.
Reduced byfrom x 120 000. (a) Normal cilium showing the 9+2 microtubule pattern, inner and outer dynein arms and, central spokes. (b) A single peripheral microtubule at 2 o’clock. (c) Disorganised cilium. (d) 8+2microtubule pattern.
565 An Introduction to the Psychotherapies
Discussion This technique provides a simple, non-invasive, and painfree method for obtaining ciliated epithelium suitable for measurement of beat frequency and examination of ultrastructure. It is well suited for use in children and, if desired, local anaesthetic (lignocaine) may be used without affecting CBF. In some instances the detail of ciliary ultrastructure found in samples obtained by brushing may be less satisfactory than that seen in nasal biopsy specimens. Nasal brushing may be regarded as a screening method-i.e., if normal ciliary structure is seen there is no need to proceed further. If detail is inadequate a nasal biopsy specimen may be required. This technique allows repeated atraumatic sampling of human ciliated tissue to study the role of cilia in mucociliary clearance mechanisms and the effects of various agents on normal and abnormal function. We thank Theresa Cox and Ann Dewar for electron microscopy and Rehahn for statistical analysis. J.R. is supported by Berk Pharmaceuticals Limited and is in receipt of a travel grant from the Postgraduate Medical Foundation, University of Sydney. This work was supported by the Wellcome Trust.
Margaret
Requests for reprints should be addressed to J.R., Host Defence Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, Fulham Road, London SW3 6HP. REFERENCES
1. Dalhamn T, Rylander R. Frequency of ciliary beat measured with a photosensitive cell. Nature 1962; 196: 592-93. 2. Yager J, Chen T-M, Dulfano MJ. Measurement of frequency of ciliary beats of human respiratory epithelium. Chest 1978; 73: 627-33.
Reviews of Books Real-time Ultrasound in Obstetrics Edited
by M. J. BENNETT, Institute of Obstetrics and Gynxcology, London, and S. CAMPBELL, King’s College Hospital Medical School, London. Oxford: Blackwell. 1980. Pp. 147. 1$. ONE difficulty in a rapidly expanding subject such as clinical ultrasound is to produce textbooks fast enough to keep up with the advances achieved. There has also long been a need for a textbook on real-time ultrasound. This book provides a very satisfactory solution to both problems. The two editors, both well known in this field, and their contributors, many of whom are world figures in clinical ultrasound, have produced an excellent text, which is illustrated by many clear diagrams and tables and good reproductions of photographs. To pick out for special mention chapters from a book which overall is very good is not easy, but that by R. J. Blackwell of basic physics deserves mention. Professor Campbell and D. J. Little have maintained their usual high standard in their discussion of the clinical potential of real-time ultrasound, although perhaps they could have emphasised more firmly the importance of ultrasound control for amniocentesis. Dr Little’s other contribution, on the reproducibility of real-time ultrasound measurements, is very convincing and should help silence the critics. A glimpse into the potential of real-time ultrasound is given in the masterly accounts of the assessment of fetal and neonatal cardiac dynamics, by J. W. Wladimiroff et al., and of fetoscopy, by G. R. Devore and J. C. Hobbins. The only criticism I can make of the book, which is strongly recommended for all ultrasound departments and which also has much that should inter- . est obstetricians, paediatricians, and geneticists is that two chapters out of eleven on fetal respiratory movement is excessive.
Department of Ultrasound, Wythenshawe Hospital, Manchester
JAMES HIGGINBOTTOM
Edited by SIDNEY BLOCH, University of Oxford. Oxford: Oxford University Press 1979. Pp. 224. /;8.SO (hard cover); /;3.95
(paperback). IN the first chapter of this compact book Jerome Franking, writing on "What is Psychotherapy", sets the tone of moderation, historico-cultural perspective, and referral to research literature that pervades the whole of this well-edited volume. The next eight chapters deal with individual long-term psychotherapy, group psychotherapy, crisis intervention, behaviour therapy, marital therapy, sex therapy, family therapy, and supportive psychotherapy. In general the contributors have succeeded in making the concepts of psychotherapy more understandable, of minimising the differerices and emphasising the similarities between schools of psychotherapy, of drawing a relation between research and clinical practice, and of guiding the trainee in his reading. Clarity is achieved at the cost of oversimplification, and the short case-histories in each chapter do not show up the complexities and subtleties of the therapeutic encounter. (More intensive long-term therapy is not discussed, and for this the reader will have to turn to David Malan’s recent monograph Individual Psychotherapy and the Science of Psychodynamics, Butterworth). However, within the remit of the editor and the intentions of the authors, this is a successful introductory book on the specific techniques and aims of modern psychotherapy. Long-term psychotherapy is realistically discussed in terms of the once weekly session, which is what is as much as can usually be afforded under the N.H.S. Though both relief of symptoms and personality change are aimed at, many teachers of psychotherapy would recommend more frequent sessions for these aims to be realisable. The most commonly offered form of therapy is supportive psychotherapy, and the editor, Sidney Bloch, is to be commended on having included this chapter and for having written it himself. The importance of supervision and the learning experience that can be obtained by good supervision needs more emphasis, and some examples of the learning processes which take place under supervision would have been helpful. This book complements the other two important books which have been produced in Britain in the past year. David Malan’s book is already referred to; the other is Introduction to Psychotherapy by D. Brown and J. Pedder, Tavistock Publication. The latter is intended as an introduction to the theories of psychotherapy, and this present book is more directed to practice. This book is strongly recommended to general psychiatrists in training who need to learn about the basic approaches to psychotherapy and to prepare them for their
higher examinations. Tavistock Clinic, London NW3 5BA
MALCOLM PINES
The Clinical Practice of Adolescent Medicine Edited by JEROME T. Y. SHEN, St Louis University School of Medicine. New York: Appleton Century-Croft. Hemel Hempstead : Prentice Hall. 1980. Pp. 727. £23.75.
THIS attractively produced volume has twenty-seven sections comprising sixty-two chapters, of which the editor contributes about a fifth. Most of the writers are paediatricians whose task includes the care of adolescents to the age of 21. It is written for primary-care physicians, paediatricians, and internists (those who are generalists) as a practical, clinically oriented handbook for day-to-day use. I found the concept exciting but the content, in the main, disappointing. Those who, like me, will refer to it from the family doctor’s viewpoint, will be somewhat carried out of their interest and depth by much of the detailed scientific medical knowledge. I kept wondering whether many of the writers needed a platform for demonstration of their expertise-with the excuse that they were describing conditions that occurred in adolescence rather
Methods and Devices
NON-INVASIVE SAMPLING OF NASAL CILIA FOR MEASUREMENT OF BEAT FREQUENCY AND STUDY OF ULTRASTRUCTURE
Fig.
1-Variation in
CBF 14
JONATHAN RUTLAND
PETER
J. COLE
Host Defence
Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, London SW3 6HP
WE describe a non-invasive method for obtaining nasal ciliated epithelium which is suitable for measurement of ciliary beat frequency (CBF) and examination of ciliary ultrastructure by transmission electron microscopy. No local anaesthesia is required and this technique is particularly suitable for children and in situations where multiple samples are required, such as in-vitro studies of human tissue.
Method
blow his nose gently to clear excess secretions, and an auriscope is introduced into one nostril to allow the inferior nasal turbinate to be seen. A cytology brush 2 mm in diameter is inserted through the auriscope between the inferior turbinate and lateral nasal wall, quickly moved posteriorly for about 1 cm along the mucosal surface, and then moved anteriorly as it is removed. Cellular material adhering to the brush is dislodged by brisk agitation in 2 ml of tissue culture medium (Medium 199 with Earle’s salts; Flow Laboratories). Wetting of the brush with medium before sampling increases the amount of tissue obtained. In subjects in whom insertion of the brush between inferior turbinate and lateral nasal wall is difficult, suitable epithelium may be obtained from the medial surface of the turbinate, but the brush must be advanced more posteriorly to obtain ciliated cells. A sealed preparation for measuring CBF can be made by applying a thin layer of silicon grease around the edges of a coverslip by means of a syringe attached to a modified intravenous cannula. Fragments of nasal epithelium can be transferred from the culture medium with a Pasteur pipette and deposited on the coverslip. A standard microscope slide gently placed over the coverslip seals the preparation which can then be inverted and examined microscopically, coverslip upwards. We measure CBF photometrically.l,2 The specimen is placed on an electronically controlled warm stage (Microtec) mounted on a Leitz Dialux 20 microscope. A Leitz MPV compact microscope photometer, transduces light intensity into an electrical signal. We routinely measure CBF at a temperature of 36±0-5°C. A polystyrene cylinder is placed between slide and objective to reduce heat-loss. Strips of epithelium with beating cilia are easily seen at a magnification of x 320 by bright-field illumination with the condenser at a low aperture. Illumination of the photometer’s field diaphragm allows a small rectangular area (approximately 1-5x35 [im) to be oriented in the long axis of the cilia so that only two or three cilia beat across this area, intermittently preventing light from reaching the photometer. The transduced electrical signal is fed through a lowpass filter with a cut-off above 40 Hz (variable to 400 Hz) into an ultra-violet oscillograph (SE Labs 6150) which provides a permanent record of ciliary activity. Timer markings at 100 msec intervals allow calculation of the CBF (fig. 1). The procedure is repeated 10 times at different areas and a mean and standard deviation (SD) are calculated. Only cells which are attached to basement membrane and have vigorously beating cilia are counted. The
subject
As soon ments, still
is asked
to
counting is complete the larger epithelial fragsuspended in medium, can be transferred with a
as
light intensity caused by 3 beats/sec. Paper speed 100 mm/sec.
cilia
beating.
pipette to fixative for electron microscopy. Alternatively, fragments may be placed into fixative immediately after brushing. Samples of epithelium obtained by this technique are satisfactory for studying ciliary ultrastructure (fig. 2). Pasteur
Results Nasal brushings from a 41-year-old man with progressive bronchiectasis, sinusitis, and azoospermia were examined. CBF was 14-2+0.4 beats/sec (mean ±SD) (fig. 1). CBF for five age-matched normal subjects was 14-8±1-0 beats/sec.
Most of the patient’s cilia had normal ultrastructure (fig. 2a) while 7% of 200 cilia counted were abnormal, a single peripheral microtubule in place of one of the normal double microtubules being the commonest finding (fig. 2b). Of 50 consecutive patients examined in this way, 2 complained of pain (one had severe rhinitis) while most described sensations of a tickle or a desire to sneeze (only 1 did so). To examine the effect of local anaesthetic on CBF determined by this method we measured beat frequency in 10 patients. Samples were obtained from both nasal cavities after one (chosen randomly) had been sprayed with an aerosol of 4% lignocaine (approximately 0.7 ml). Mean CBF for the untreated side was 12.2±2.3 beats/sec while that for the side sprayed with lignocaine was 12-3±2-0 beats/sec. Analysis of variance showed no statistically significant difference between the two groups.
2-Electron micrographs showing cross sections of normal and abnormal cilia obtained from nasal mucosa by brushing.
Fig.
Reduced byfrom x 120 000. (a) Normal cilium showing the 9+2 microtubule pattern, inner and outer dynein arms and, central spokes. (b) A single peripheral microtubule at 2 o’clock. (c) Disorganised cilium. (d) 8+2microtubule pattern.
565 An Introduction to the Psychotherapies
Discussion This technique provides a simple, non-invasive, and painfree method for obtaining ciliated epithelium suitable for measurement of beat frequency and examination of ultrastructure. It is well suited for use in children and, if desired, local anaesthetic (lignocaine) may be used without affecting CBF. In some instances the detail of ciliary ultrastructure found in samples obtained by brushing may be less satisfactory than that seen in nasal biopsy specimens. Nasal brushing may be regarded as a screening method-i.e., if normal ciliary structure is seen there is no need to proceed further. If detail is inadequate a nasal biopsy specimen may be required. This technique allows repeated atraumatic sampling of human ciliated tissue to study the role of cilia in mucociliary clearance mechanisms and the effects of various agents on normal and abnormal function. We thank Theresa Cox and Ann Dewar for electron microscopy and Rehahn for statistical analysis. J.R. is supported by Berk Pharmaceuticals Limited and is in receipt of a travel grant from the Postgraduate Medical Foundation, University of Sydney. This work was supported by the Wellcome Trust.
Margaret
Requests for reprints should be addressed to J.R., Host Defence Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, Fulham Road, London SW3 6HP. REFERENCES
1. Dalhamn T, Rylander R. Frequency of ciliary beat measured with a photosensitive cell. Nature 1962; 196: 592-93. 2. Yager J, Chen T-M, Dulfano MJ. Measurement of frequency of ciliary beats of human respiratory epithelium. Chest 1978; 73: 627-33.
Reviews of Books Real-time Ultrasound in Obstetrics Edited
by M. J. BENNETT, Institute of Obstetrics and Gynxcology, London, and S. CAMPBELL, King’s College Hospital Medical School, London. Oxford: Blackwell. 1980. Pp. 147. 1$. ONE difficulty in a rapidly expanding subject such as clinical ultrasound is to produce textbooks fast enough to keep up with the advances achieved. There has also long been a need for a textbook on real-time ultrasound. This book provides a very satisfactory solution to both problems. The two editors, both well known in this field, and their contributors, many of whom are world figures in clinical ultrasound, have produced an excellent text, which is illustrated by many clear diagrams and tables and good reproductions of photographs. To pick out for special mention chapters from a book which overall is very good is not easy, but that by R. J. Blackwell of basic physics deserves mention. Professor Campbell and D. J. Little have maintained their usual high standard in their discussion of the clinical potential of real-time ultrasound, although perhaps they could have emphasised more firmly the importance of ultrasound control for amniocentesis. Dr Little’s other contribution, on the reproducibility of real-time ultrasound measurements, is very convincing and should help silence the critics. A glimpse into the potential of real-time ultrasound is given in the masterly accounts of the assessment of fetal and neonatal cardiac dynamics, by J. W. Wladimiroff et al., and of fetoscopy, by G. R. Devore and J. C. Hobbins. The only criticism I can make of the book, which is strongly recommended for all ultrasound departments and which also has much that should inter- . est obstetricians, paediatricians, and geneticists is that two chapters out of eleven on fetal respiratory movement is excessive.
Department of Ultrasound, Wythenshawe Hospital, Manchester
JAMES HIGGINBOTTOM
Edited by SIDNEY BLOCH, University of Oxford. Oxford: Oxford University Press 1979. Pp. 224. /;8.SO (hard cover); /;3.95
(paperback). IN the first chapter of this compact book Jerome Franking, writing on "What is Psychotherapy", sets the tone of moderation, historico-cultural perspective, and referral to research literature that pervades the whole of this well-edited volume. The next eight chapters deal with individual long-term psychotherapy, group psychotherapy, crisis intervention, behaviour therapy, marital therapy, sex therapy, family therapy, and supportive psychotherapy. In general the contributors have succeeded in making the concepts of psychotherapy more understandable, of minimising the differerices and emphasising the similarities between schools of psychotherapy, of drawing a relation between research and clinical practice, and of guiding the trainee in his reading. Clarity is achieved at the cost of oversimplification, and the short case-histories in each chapter do not show up the complexities and subtleties of the therapeutic encounter. (More intensive long-term therapy is not discussed, and for this the reader will have to turn to David Malan’s recent monograph Individual Psychotherapy and the Science of Psychodynamics, Butterworth). However, within the remit of the editor and the intentions of the authors, this is a successful introductory book on the specific techniques and aims of modern psychotherapy. Long-term psychotherapy is realistically discussed in terms of the once weekly session, which is what is as much as can usually be afforded under the N.H.S. Though both relief of symptoms and personality change are aimed at, many teachers of psychotherapy would recommend more frequent sessions for these aims to be realisable. The most commonly offered form of therapy is supportive psychotherapy, and the editor, Sidney Bloch, is to be commended on having included this chapter and for having written it himself. The importance of supervision and the learning experience that can be obtained by good supervision needs more emphasis, and some examples of the learning processes which take place under supervision would have been helpful. This book complements the other two important books which have been produced in Britain in the past year. David Malan’s book is already referred to; the other is Introduction to Psychotherapy by D. Brown and J. Pedder, Tavistock Publication. The latter is intended as an introduction to the theories of psychotherapy, and this present book is more directed to practice. This book is strongly recommended to general psychiatrists in training who need to learn about the basic approaches to psychotherapy and to prepare them for their
higher examinations. Tavistock Clinic, London NW3 5BA
MALCOLM PINES
The Clinical Practice of Adolescent Medicine Edited by JEROME T. Y. SHEN, St Louis University School of Medicine. New York: Appleton Century-Croft. Hemel Hempstead : Prentice Hall. 1980. Pp. 727. £23.75.
THIS attractively produced volume has twenty-seven sections comprising sixty-two chapters, of which the editor contributes about a fifth. Most of the writers are paediatricians whose task includes the care of adolescents to the age of 21. It is written for primary-care physicians, paediatricians, and internists (those who are generalists) as a practical, clinically oriented handbook for day-to-day use. I found the concept exciting but the content, in the main, disappointing. Those who, like me, will refer to it from the family doctor’s viewpoint, will be somewhat carried out of their interest and depth by much of the detailed scientific medical knowledge. I kept wondering whether many of the writers needed a platform for demonstration of their expertise-with the excuse that they were describing conditions that occurred in adolescence rather