E lig ib le Subjects. F ull-term (> 37 weeks gestational age), healthy, Caucasian babies born by normal vaginal d e liv e ry , w ith birthw eight > 2.5 kg and minimum Apgar scores o f 7 and 9 at 1 and 5 minutes re sp e ctive ly, were e lig ib le fo r studies described in th is th e s is .
Measurements were performed in those in fa n ts whose parents gave w ritte n , informed consent (Appendix E).
Exclusion C r ite r ia . Infants whose mothers had received opiate analgesia during labour, who had assisted v e n tila tio n at d e liv e ry , evidence o f congenital, ca rd io -re s p ira to ry or neuromuscular abnormality, or whose parents did not speak or understand English were excluded.
Ethical Committee Approval. Hospital Ethical Committee approval fo r the study was granted by the C ity & Hackney Committee fo r Ethical Research (approval 10/08/88).
Preparation fo r Measurement Introduction
The measurement protocol was defined from a series o f prelim inary
in ve stig a tio n s in the preparatory stage o f th is th e s is . The aims o f the p ilo t studies were:
a) To define measurement conditions
b) To establish a su itable and p ra c tic a l recruitm ent procedure
c) To assess subject accrual rate
d) To va lid a te the prelim inary proto co l, in terms o f: i . the optimal number and sequence o f occlusions
to perform
i i . the re la tio n s h ip between e n d -in sp ira to ry and end-expiratory occlusions, and
i i i . the optimal number o f breaths pre-occlusion on which to base analysis o f control breathing
Full d e ta ils o f the p ilo t studies are given in Appendix B. Environmental Conditions
A ll measurements were performed e ith e r in a measurement room on the Special Care Baby Unit (SCBU) at Homerton Hospital or in the In fa n t Lung Function Laboratory, Hospital fo r Sick Children, Great Ormond S treet. An ambient temperature o f 23-25*C was maintained using a servo-
co n tro lle d a ir-c o n d itio n in g u n it (D aikin, Japan). Each measurement room was f u lly equipped w ith re su scita tio n equipment, which was checked d a ily p rio r to measurements. Medical cover was provided at a ll times by resident medical s ta ff.
Id e n tific a tio n o f E liq ib le Subjects
Prospective re c ru its were id e n tifie d from in fa n t and maternal d e ta ils obtained from the Register o f B irth s at Homerton H ospital. Mothers and in fa n ts were then located on the postnatal wards, and Hospital records examined to obtain d e ta ils o f health in the immediate postpartum period. Midwives on duty were then contacted and asked fo r permission to
approach the mother o f each suita b le in fa n t. Every e ffo r t was made to ensure th a t s e n s itiv ity to the needs o f each in d ivid u a l was demonstrated at a ll times. Any medical or social co n tra -in d ic a tio n s to recruitm ent, given by ward s ta ff were acknowledged.
Recruitment
Parents o f in fa n ts e lig ib le fo r the study were approached in fo rm a lly , given d e ta ils o f the study and asked i f they would lik e to be involved. A series o f photographs showing equipment, a recording in progress and traces o f data obtained from a 2-day old in fa n t were used to explain what the study involved. Frequently, mothers requested time to consider the matter w ith the in fa n t's fa th e r, and/or fo r the procedure to be explained again in the presence o f the fa th e r. This was undertaken as often as requested. I f parents were w illin g to p a rtic ip a te , w ritte n consent (Appendix E) was obtained at th is stage and arrangements fo r the f i r s t measurement made.
Preparation o f Equipment
The a m p lifie rs were switched on and the pneumotachograph connected to the heating c ir c u it and l e f t to warm up fo r at le a st one hour p rio r to c a lib ra tio n and measurement. The computer u n it was switched on and RASP software was loaded. A unique subject f i l e was created fo r each in fa n t re c ru ite d . The facemask was attached to the sh u tte r and the shutter to the pneumotachograph using a c ry lic connectors. The cable release
mechanism was connected to the sh u tte r. The tubing from one side o f the Pao transducer was attached to the pressure port on the sh u tte r. The tubing on the other side was maintained as a reference to atmospheric pressure.
C a lib ra tio n
C a lib ra tio n was performed on a d a ily basis p rio r to each study. This took place at the s ite o f te s t under the same conditions as measurements themselves. W hilst shielding the transducers against random va ria tio n s in the environment such as draughts, c a lib ra tio n was achieved using the c a lib ra tio n menu w ith in RASP. Known se t-p o in ts were applied to each transducer. P rio r to c a lib ra tio n , the s t a b ilit y o f the a m p lifie rs was checked to ensure th a t there was no o ffs e t or bias, as th is may cause a s h if t in f u l l scale d e fle c tio n , and thereby a lte r the range over which accuracy o f signals can be maintained.
When there were no signal inputs, each d iffe r e n tia l pressure a m p lifie r was adjusted to give zero v o lts output. The voltage signal
corresponding to zero and 100 m l.s '^ flow , and zero and 1.964 kPa were then recorded at appropriate gains.
Flow
Flow c a lib ra tio n was achieved by applying a high and a zero
physical setpoint value to the transducer. When there was no flow through the pneumotachograph, a zero reference point was
established. A known flow rate (100 ml.s was passed through the apparatus via a ca lib ra te d rotameter (RDG) and a high reference point established.
C alibrations were confirmed by d e liv e rin g and recording known signals, o f the same magnitude, and checking th a t the d ig ita l values displayed on the computer were recognised as the exact values (+ 2% ) as those th a t had been delivered. The measured
response was deemed to be acceptable i f zero reference, detected by the computer, f e ll w ith in + 0.02 ml.s~^, and high reference between 99 and 101 m l.s '^ .
Volume
The accuracy o f volume c a lib ra tio n was assessed by connecting a ca lib ra te d syringe (Hans Rudolph) via a length o f non-compressible tubing, to the assembled apparatus. Having recorded a zero
reference p o in t, 60 ml o f a ir were in jected and withdrawn, at a frequency o f approximately 40 cycles per minute, corresponding to the approximate re s p ira to ry frequency o f a newborn in fa n t. Care was taken not to exceed the flow lin e a r it y o f the apparatus. The amplitude o f known volume input was then compared w ith th a t
determined by computerised d ig ita l in te g ra tio n . Accuracy was accepted w ith in + 0.5 ml.
Airway Opening Pressure
The airway opening pressure signal was ca lib ra te d by d e liv e rin g a high and a low reference set point to the transducer. A 3-way tap was used to connect the transducer tubing to a w a te r- fille d
manometer. A zero reference was recorded, w ith the 3-way tap open to atmosphere. A 5 ml a i r - f i l l e d syringe was attached to the tap and the manometer pressurised to 1.964 kPa (20 cmHgO). A high reference was recorded at th is p o in t.
The c a lib ra tio n accuracy was assessed by d e liv e rin g and recording known signals, and checking th a t values displayed on computer were recognised as the exact values th a t had been delivered (+ 2%). Low reference was accepted w ith in + 0.001964 kPa (0.02 cmH^O).
Preparation of the In fan t
Immediately a fte r a feed, in fa n ts and parents were colle cte d from the postnatal ward and brought to the measurement room. An a x illa r y
temperature probe was attached to the in fa n t using s o ft microfoam tape (3M). The in fa n t was then lig h t ly clothed and loosely wrapped in a sheet before being l e f t to f a l l asleep n a tu ra lly . Parents were
encouraged to allow th e ir baby to s e ttle to sleep in th e ir usual manner, which could include s e ttlin g in the parent's arms i f so desired. Once sleeping q u ie tly , the in fa n t was placed supine in a cot or basinette. Soft s a n d -fille d bags were used to support the in fa n t's head in the m id -lin e central p o s itio n . A small foam r o ll beneath the neck was used to maintain a s lig h tly extended p o s itio n .
C la s s ific a tio n o f Sleep State
The time o f sleep onset was recorded, and sleep state c la s s ifie d at one minute in te rv a ls using established behavioural c r it e r ia summarised in Table 2.1 (P re chtl, 1974). Once the in fa n t was in quiet sleep, as judged by the absence o f eye movements, stable posture and regular re s p ira tio n , a th in rin g o f s o ft, s te r ilis a b le therapeutic p u tty was positioned around the in fa n t's nose and mouth. An id e n tic a l rin g o f p u tty was placed around the facemask, to cushion the mask and ensure an a ir t ig h t seal. The facemask, attached to the recording equipment as described previously (Section 2 .3 ), was then gently lowered onto the p u tty around the in fa n t's nose and mouth. Fig. 2.4a shows the apparatus applied to an in fa n t in quiet sleep. With the in fa n t breathing
spontaneously through the apparatus, data were recorded and signal q u a lity assessed.
Assessment o f Signal Q uality
A fte r a minimum o f 10 regular breaths w ith a stable end-expiratory level (EEL) had been recorded, a b r ie f e n d -in sp ira to ry occlusion was
performed. Data c o lle c tio n was terminated a fte r a minimum o f 5 breaths post occlusion, and subsequently inspected fo r evidence o f a leak w ith in the c ir c u it .
TABLE 2.1 Behavioural C rite ria fo r Sleep State C la s sificatio n
C rite ria Active Sleep Quiet Sleep
1. Rapid and/or slow
eye movements +
2. Facial grimaces + 3. Frequent small body
movements +
4. S ta rtle s every 2-3
minutes +
5. Irre g u la r re s p ira tio n +
Stable posture Isolated s ta rtle s only Respiration regular
Adapted from Prechtl (1974)
pm
cable release activated shutter
airway pressure port face mask
Leaks may occur a) between the facemask and the in fa n t (F ig .2 .4b) and b) through the shutter and pneumotachograph (F ig .2 .4c) (Dezateux et a l, 1991) Both are e a s ily recognisable and were corrected by appropriate experimental technique such as mask re p o sitio n in g or shutter
replacement. Once a s a tis fa c to ry seal had been achieved, (Fig.2.4d) the sleep state o f the in fa n t was c la s s ifie d . When the in fa n t was in quiet sleep, in fa n t and ambient temperature were recorded and measurements commenced.
Measurement of the Hering Breuer Reflex
Real-time changes in a irflo w , tid a l volume and Pao were recorded. A fte r a minimum o f 10 breaths had been recorded, an airway occlusion w ith in 15% o f the s ta rt o f e xp ira tio n was maintained u n til the in fa n t made one complete re s p ira to ry e ffo r t against the sh u tte r. A minimum o f 5 breaths were recorded post occlusion. Data were inspected immediately to ensure s a tis fa c to ry signals had been obtained. A fte r a one minute in te rv a l to minimise the p o te n tia l influence o f re fle x h a b itu a tion , th is procedure was repeated u n til a series o f 3 to 5 e n d -in sp ira to ry occlusions had been obtained during quiet sleep. Time between successive occlusions was used to assess signal q u a lity and c la s s ify sleep state during measurements. Occlusions were timed from the tid a l volume d isplay.
I f the in fa n t remained in quiet sleep, 3-5 additional occlusions were performed w ith in 15% o f the end o f e x p ira tio n , fo llo w in g the sequence o u tlined fo r end- in s p ira to ry occlusions. P ilo t studies in 7 in fa n ts had suggested th a t there may be no d iffe re n ce between re s u lts obtained from e n d -in sp ira to ry or end-expiratory occlusion. (Appendix 8, Table 82).
The strength o f the H8R was calculated from the mean percent increase in e xp irato ry time during e nd-inspiratory occlusions, or in s p ira to ry time during end-expiratory occlusions, re sp e ctive ly, as described in section 2.2. Full d e ta ils o f analysis are discussed la te r in th is section.
F ig .2 .4b Real-time recording Showing Evidence o f a Leak between the Facemask and the In fa n t
Pao
Footnote; A step-up in the b aseline of the volume signal and decay in the Pao signal suggests a facemask lea k.
F ig.2.4c Example o f a Leak Occurring through the Pneumotachograph and Shutter
Pao
V
Footnote A f l u t t e r on the flow signal during the occlusion suggests a leak through the pneumotachograph. An apparent pressure plateau may s t i l l occur in the presence of a leak.
F1g.2.4d S a tisfa cto ry Test Occlusion I
ci
Iocclusion
PaoEEL
Measurement o f Total Respiratory System Compliance (Crs)
I f the in fa n t remained in quiet sleep, a number o f a d d itional b r ie f occlusions were performed throughout the f i r s t tw o -th ird s o f e x p ira tio n , to enable measurement o f to ta l re s p ira to ry system compliance (Crs) to be performed as described by Stocks et a l, 1987 and Dezateux et a l, 1991. Each occlusion was held u n til an apparently relaxed pressure plateau had been achieved (F ig .2 .4 e ). A minimum o f 6 breaths were allowed between successive occlusions.
Completion o f Measurements Crown-Heel Length
A fte r completion o f measurements, the crown-heel length o f the in fa n t was measured using a ca lib ra te d stadiometer (Harpendon Infantometer, CMS, London). A small cotton sheet was placed on the stadiometer before placing the unclothed in fa n t on it s base. Two adults were required fo r th is procedure, and usually the in fa n t's mother gave assistance. One adult maintained the in fa n t's head in a central p o s itio n at the top o f the instrument, w h ils t the other gently depressed the in fa n t's knees u n til the legs were f u lly extended. A moving fo o tp la te was then positioned against the soles o f the in fa n t's fe e t. The fo o tp la te was