Archives ofDisease in Childhood 1994; 71: 501-505 501 Surfactant abnormalities in ALTE and SIDS Arch Dis Child: first published as 10.1136/adc.71.6.501 on 1 December 1994. Downloaded from

I B Masters, J Vance, B A Hills

Abstract mechanical stability of the distal airspaces.2 Abnormalities in the relative concentra- Reduced concentrations of surfactant, in tions ofthe components ofsurfactant have particular disaturated phosphatidylcholine been implicated in prolonged expiratory (DPPC) have been described in infants apnoea (PEA) and sudden infant death with PEA and SIDS.3-9 Southall et al have syndrome (SIDS). Controversy has, implicated low concentrations of DPPC and however, surrounded these findings, as postulated mechanic, neurosensory, and they may be secondary to terminal pulmonary vascular mechanisms for these life events. In this study the physical events.5 6 properties of surfactant were measured in Previously we have shown that both an children with recurrent apparent life infant and young child with prolonged threatening events (ALTEs), PEA, and expiratory apnoea had significant quantitative SIDS. Bronchial lavage samples were and qualitative abnormalities in their obtained from 21 children with recurrent surfactant.'I These findings were similar to the ALTEs, two SIDS victims, and 26 control low concentrations of DPPC found in other patients. Lipid components were immedi- studies.2-5 The reliability of such findings, ately elutriated from these samples however, is questionable in both those and with liquid chloroform. The physical our own observations, as the ability to extract properties of the extracted surfactant surfactant with lung washings in a standardised were studied on a Langmuir trough in fashion from the living subject's lung is which the area (A) of the monolayer was difficult. Despite this, there were clear cut cycled continuously as the surface tension abnormalities and, most importantly, the (-y) was measured by the Wilhelmy results were reproducible over time. This method using a platinum 'flag'. The suggests that there may be a sustained investigators performing these tests were abnormality and that it is not greatly unaware of the clinical diagnosis. Twenty influenced by intervening events, such as one of 23 patients displayed abnormal hypoxia. Our previous study also found greatly physical properties while seven of 26 reduced hysteresis in the relationship between controls displayed similar abnormalities. surface tension (y) and surface area (A),

These abnormalities were partially with the normal clockwise -y:A loop actually http://adc.bmj.com/ inverted hysteresis (figure of eight) loops reversing and cycling anticlockwise for the and inverted (anticlockwise) loops that index cases. The reason for these changes is also generally exhibited less hysteresis. Of not clear although inverse hysteresis has the 26 controls 20 exhibited a wide hys- been reported for fatigued monolayers of teresis pattern that cycled in a normal surfactant,'1 while contact angle changes could (clockwise) direction. These differences also produce similar loops.'2 were significantly different. It is con- These initial findings clearly supported the cluded that children with recurrent previous hypotheses of Morley et al 3 and on September 23, 2021 by guest. Protected copyright. ALTEs have definable abnormalities in Southall et al5 6 in that abnormal surfactant the physical properties of surfactant and function may result in altered receptor- that these findings may provide a sensitive controller feedback regulation of functional means of identifying those at risk of residual capacity, thus promoting hypoxia and recurrent ALTEs and SIDS. possibly further disorganising respirations to (Arch Dis Child 1994; 71: 501-505) produce apnoea. While long term prospective studies are being planned, we present our findings from The cause or causes of sudden infant death infants presenting with recurrent or severe syndrome (SIDS) remain a major medical and ALTEs to assess further the likelihood of an scientific dilemma. The National Institutes of association of PEA (prolonged expiratory Health and Human Development cooperative apnoea), ALTEs, and SIDS on the basis of epidemiological study of risk factors suggests surfactant abnormalities. Respiratory Research that up to 7% of SIDS victims have had Unit, Mater prolonged apnoeic episodes or an apparent life Children's Hospital, Annerley Road, threatening event (ALTE) before death.1 Subjects and methods South Brisbane, While the clear inference is that ALTEs may INDEX CASES Queensland 4101, have a causative role in SIDS or represent part All patients were previously investigated by Australia I B Masters of a pathophysiological continuum with SIDS, consultant paediatricians before referral to the J Vance this association is low. respiratory unit for further assessment and B A Hills Anatomical pathology studies in children investigation. The index cases comprised Correspondence to: dying from SIDS implicate disturbance in 23 infants and children admitted to the Dr Masters. micromechanical processes and surfactant Mater Children's Hospital for investigation of Accepted 1 September 1994 function as the major factor responsible for recurrent or severe ALTEs from 1991-3. This 502502~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Masters,Vance, Hills

group represents approximately 30% of primary reason for an anaesthetic. Here the

all ALTE patients referred for respiratory bronchoscopy was performed during the same Arch Dis Child: first published as 10.1136/adc.71.6.501 on 1 December 1994. Downloaded from assessment but would represent a much anaesthetic for procedures such as grommet smaller percentage of infants referred to the placement and replacement, cystoscopy, and Mater Children's Hospital for ALTEs over revision of tracheostomy stoma and the airway the same time period. All were well when before decannulation, or because of associated investigated with a bronchoscopic procedure tracheostomy problems such as granuloma. at an elective time at least two weeks after The bronchoscopy was carried out by one their admission for the ALTE. This did of the authors who is an experienced paediatric not include two infants who died from SIDS bronchoscopist. A halothane gaseous anaes- whose samples were obtained by deep thetic was used, the vocal cords sprayed with bronchial lavage through the endotracheal xylocaine and a Pentax 3-5 mm flexible scope tube during the resuscitation process. Four of was passed and wedged into a lower lobe the infants studied were siblings of SIDS where a bronchial lavage was victims and three of the four had had recurrent performed using 2 ml/kg warm normal saline ALTEs without a definable cause. Two instilled in two aliquots via the wedged had a tracheostomy in situ for 'dynamic' or bronchoscope in the lobar bronchus. Samples 'functional' (non-anatomical) upper airway of the lavage fluid recovered by suction were obstruction. No infant had an acute infective immediately shaken with liquid chloroform to process. There were 12 infants and children in elutriate lipid components of surfactant and whom it was logistically possible to undertake to prevent biochemical degradation. All polysomnographic sleep studies at the time of procedures were carried out with the same hospital investigation for their ALTE. instrument and by the same bronchoscopist. Each sample was assayed by scientists unaware of the diagnosis. DIAGNOSIS OF ALTE The diagnosis of ALTE was defined by a consultant paediatrician in accordance with a CONSENT generally accepted definition' and consisted of Informed parental consent was obtained for a convincing history of the sudden onset of the diagnostic bronchoscopy and broncho- colour change, tone change, and apnoea that alveolar lavage, as well as for the use of these required vigorous stimulation and, as such, investigatory results in the scientific forum. was frightening to the parent or caregiver. As Approval for this study was granted by the most infants were referred for investigation Mater Children's Hospital research and ethics after their initial ALTE, they usually had committee. apnoea alarms in use. Consequently the parental history ofALTE was often augmented those events defined an SURFACTANT ASSAYS: HYSTERESIS TESTS by by apnoea http://adc.bmj.com/ alarm. Other conditions were defined by The lipid and other hydrophobic components clinical assessments and usual investigatory of surfactant in the recovered sample were techniques. extracted from the lavage fluid with chloro- form and stored on ice. The aqueous layer was later decanted and the chloroform evaporated CONTROL CASES to dryness and redissolved in hexane/ethanol Twenty six children acted as controls. Twenty for deposition of the surfactant as a monolayer three had undergone diagnostic bronchoscopy on the saline pool of a Langmuir trough. The on September 23, 2021 by guest. Protected copyright. over the same period of time for disorders such evaluation of physical properties was carried as infantile larynx () or other out by a standard procedure whereby the structural abnormalities of the larynx or surface area (A) of the monolayer was cycled airway. Three children had a tracheostomy in over a ratio of 5:1 while its surface tension (-y) situ for severe upper airway obstruction. 507F- BRONCHOSCOPIC PROCEDURE cn 40 In no cases were the bronchoscopies 0) performed 'just to obtain surfactant'. V 30 Indeed all patients were referred from general C unit for 0 paediatricians to the respiratory CD investigation of the recurrent or severe nature 20 0) of the ALTE. The bronchoscopies were U therefore performed in the index and control t 10 groups to define anatomical and physiological C, normality or abnormality of the nasal space, n III larynx, , bronchi, and secretions. 0 20 40 60 80 100 120 During these assessments bronchoalveolar Surface area (% of maximum) lavage was performed and fluid sent for Figure 1 A typical plot of surface tension (y) versus differential cytology, including lipid laden area (A) of the pool of a Langmuir trough for the first as well as for surfactant assays. cycle of monolayers ofpure DPPC and surfactant extracted macrophages, from a lung lavage sample of a normal infant. Note the Where repeat bronchoscopic procedures were standard clockwise direction of rotation of the y.A loop carried out, the procedure was not the characteristic of normal hysteresis. Surfactant abnormalities in ALTE and SIDS 503

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a Arch Dis Child: first published as 10.1136/adc.71.6.501 on 1 December 1994. Downloaded from a.) 60 o v X b c 50 / n c 40 ~~~~~~~~~~0 'a) n C 0 C 0) 30 >o On 20 (/)tn 0 20 40 60 80 100 120 Surface area (% of maximum) 0 20 40 60 80 100 120 Figure 2 A plot ofsurface tension (y) versus area (A) Surface area (% of maximum) of the pool ofa Langmuir trough for thefirst cycle ofa Figure 4 A repeat plot ofsurface tension (y) versus monolayer ofsurfactant extractedfrom a lung lavage area (A) of the pool ofa Langmuir trough for thefirst cycle sample ofan index case. Note that the y.A loop now cycles ofa monolayer ofsurfactant extractedfrom a lung lavage in an anticlockwise direction with hysteresis inverted sample ofan index case. In thefirst test this in fact gave an (reversed) relative to normal (fig 1). inversion (a-b) ofonly 0-8 dynes/cm at 100% area and was one oftwo such cases classified as normal in the table. In the repeat test it can be seen how the inversion is clearly was measured by the Wilhelmy method using significant with a= 64-7 dynes/cm and b=60-6 dynes/cm a platinum 'flag'. All studies were carried out when these would have been unequivocally classified as at room temperature. Plots of -y against A index cases. normally reveal a wide loop that cycles in the clockwise direction. Any loop that failed median age (range) at which the bronchoscopy to do so either in toto (anticlockwise) or in (test) was performed for index cases and con- part (figure of eight) was classified as abnor- trols was 8 months (range 1-120) versus mal. Representative loops are shown in figs 7-5 months (range 1-120) (p>005, NS). 1-4. The reason for such a disparity in the time Total phospholipid is determined as the of the first ALTE and the time at which phosphorus content of the chloroform extract bronchoscopy was performed is due to the fact by use of perchlorate to oxidise all elemental that a number of the infants and children were phosphorus in the residue to phosphate, referred for assessments long after their first which is then quantified colourimetrically as ALTE. The severity of the ALTE varied from phosphomolybdate employing a spectro- mild without cyanosis (n=2) to severe with photometer. metabolic acidosis (n= 6). Four ofthese infants survived and two died from SIDS. Fourteen index cases experienced recurrent bouts of STATISTICS cyanosis but were never documented as Descriptive statistics and the Fisher's exact test requiring hospital resuscitation for metabolic http://adc.bmj.com/ were used for analysis of the data. A p value of acidosis or respiratory failure. Four children 60Q05 was regarded as significant. had a clinical pattern consistent with prolonged expiratory apnoea. Four were classified as secondary ALTE and 12 as Results primary ALTE. Six controls also experienced There were 23 index cases and 26 controls apnoea and cyanosis as a result of severe airway whose diagnoses or presenting features are obstruction. on September 23, 2021 by guest. Protected copyright. shown in the table. The median age of onset of ALTE was 1 month (range 1-9 months). The SURFACTANT ASSAYS 60 The mean (SEM) amount of surfactant available for assay was 4-2 (1 6) mg in a the index cases and 4-7 (1.3) mg for the ) 50 controls. Four index cases and one control b had surfactant levels <0-1 mg. These were regarded as 'low' concentrations of surfactant. o 40 C SURFACE TENSION: SURFACE AREA LOOP 'O 30 ASSESSMENTS Of the 23 index cases, 17 had figure of eight cn type loop shapes, four anticlockwise loops, and I, . ,II ,II 1II ,II1,I,1I, ,z two normal 0 20 40 60 80 100 120 loops (figs 1-3). Loop shape classification Surface area of clearly separated index cases (% maximum) from controls: there being a highly significant Figure 3 A plot ofsurface tension (y) versus area (A) ofthe pool of a Langmuir trough for thefirst cycle of a difference (Fisher's exact test p<0 0001). On monolayer ofsurfactant extractedfrom a lung lavage repeat testing, the normal loops were regarded sample of an index case. Note how y.:A hysteresis is as abnormal but were not included as partially inverted (reversed) by comparison with a normal clockwise loop (fig 1) to give afigure ofeight. The degree abnormals in the statistical analysis. The three ofinversion is maximal at the initial (100%) area as infants who had ALTE and were siblings of quantified by (a -b). SIDS, all had abnormal loops. 504 Masters, Vance, Hills

Age ofonset ofALTE, age at bronchoscopy, and presentingfeatures ofindex and control hypoxic ischaemic events. Most with recurrent cases ALTE and PEA have continued to have some Arch Dis Child: first published as 10.1136/adc.71.6.501 on 1 December 1994. Downloaded from Case Age at onset of Age at bronchoscopyl form of ALTE beyond the time of assessment no ALTEs (months) lavage (months) Presentingfeature and into the second year of life. Four of six Index cases children appeared to be improved with oxygen 1 1 3 Recurrent apnoea/cyanosis treatment, however, there was not complete 2 1 4 Recurrent choking/cyanosis 3 1 120 Central alveolar hypoventilation. PEA resolution of ALTEs. 4 3 3 Severe apnoea, acidosis 5 2 11 Recurrent alarming. Apnoea of infancy 6 1 1 Recurrent choking. No cyanosis 7 9 9 Apnoea/no cyanosis Discussion 8 2 32 Apnoea/cyanosis 9 9 9 . Apnoea This is the first study to show a definable 10 1 1 Severe apnoea, acidosis abnormality in the physical properties of 11 2 5 Apnoea/cyanosis. Mechanical ventilation 12 1 9 Recurrent apnoea/cyanosis surfactant in children and infants with ALTE, 13 1 8 Recurrent apnoea/cyanosis. Reflux PEA, and SIDS. While other studies have 14 3 4 Apnoea/cyanosis, acidosis 15 4 20 Apnoea/cyanosis reported abnormalities in the chemical 16 1 76 Recurrent apnoea/cyanosis. PEA composition of surfactant, they did not 17 1 30 Recurrent apnoea/cyanosis 18 1 8 Apnoea/cyanosis. Reflux report in detail the physical properties of the 19 1 1 Recurrent apnoea/cyanosis surfactant despite the reporting ofvariations in 20 1 6 Recurrent apnoea/cyanosis. Acidosis. PEA 7 8 21 1 3 Recurrent apnoea/cyanosis the chemical composition oftheir samples.35 22 8 8 SIDS Indeed it is feasible that they were overlooked 23 1 1 SIDS Control cases or regarded as artefact as the degree of 1 5 Pulmonary hypoplasia. Tracheostomy hysteresis does change after the first loop even 2 2 Haemangioma. Tracheostomy 3 7 . Cerebral palsy in the normal situation.14 In our patients the 4 22 Subglottic stenosis reason for the abberation in loop shape and 5 22 Laryngotracheo bronchitis. Cricoid split 6 5 Tracheomalacia. Bronchitis cycle direction change is unclear. It may of 7 9 Dynamic tracheomalacia course be due to an aberration in the multi- 8 120 Scoliosis - ventilated 9 3 Laryngomalacia plicity of factors that govern adsorptive 10 6 Intractable seizure - ventilated properties of surfactant in contact with the 11 9 Laryngomalacia. Tracheomalacia 12 8 Tracheomalacia. Vascular ring platinum flag as manifest by a change in 13 23 Tracheomalacia contact angle,12 but it is unlikely to be related 14 8 Tracheomalacia. Laryngomalacia 15 3 Laryngomalacia to the quantity of surfactant as our results were 16 28 Laryngomalacia. Bronchitis independent of surfactant concentration. 17 7 Laryngomalacia. Tracheostomy 18 1 Laryngomalacia. Tracheomalacia Whether these findings are representative of 19 9 Laryngomalacia. Reflux the in vivo situation cannot be answered but it 20 2 Laryngomalacia. Bronchitis 21 2 Laryngomalacia would seem most likely that such changes in 22 76 Viral encephalopathy - ventilated physical properties are real, particularly as they 23 2 Laryngomalacia 24 72 Laryngomalacia. were totally reproducible over long periods of 25 28 Post foreign body atelectasis time. As surfactant is the major 'governor' of 26 7 Laryngomalacia surface and recoil forces within the lung at http://adc.bmj.com/ all but high volumes, it then would seem REPEAT LOOP ASSESSMENTS: REPRODUCIBILITY reasonable to assume that it might have a OF FINDINGS role in 'receptor triggering' and generally In order to gain some understanding of the influencing afferent neural feedback to the reproducibility of the initial findings, repeated brainstem. Given the nature of the loop testing of six index cases over a four to 12 abnormalities, it would then be possible for the month period was possible as the infants were receptors to 'read' the surface tension as being on September 23, 2021 by guest. Protected copyright. undergoing elective surgery requiring a general higher than is normal at low lung volumes and anaesthetic. There was 100% concordance; that thus risk the brain's interpretation that the is, the loop abnormality was sustained over lung is in an inspiratory mode when, in reality, time. it is in an expiratory mode. As respiratory events are most common in rapid eye movement sleep,15 the controller system is SLEEP STUDIES even more likely to be vulnerable as the Six infants had abnormal sleep studies in patient lung volumes, oxygen saturation state, that there were significantly more desatura- and hypoxic and hypercapnoeic thresholds tions and lower minimum saturations recorded fluctuate as do the frequency of apnoeas and in rapid eye movement sleep than normal dysrhythmic respirations that characterise this infants of the same age, as recorded in a sleep state. It is of some importance that six of previous study in our laboratory.13 The 12 infants who had polysomnographic studies desaturations in rapid eye movement sleep recorded significantly more desaturations and were attributed to combinations of dys- a greater degree of desaturation than recorded rhythmic respirations, apnoeas, paradoxical in normal infants of comparable age studied respirations, and hypopnoeas. within our laboratory. These findings would be consistent with a disorder of functional residual capacity regulation. OU1rCOME Indeed with these arguments, it is then not None of the infants with recurrent ALTE have necessary to postulate atelectasis in vivo or died despite the abnormalities in surfactant. even abnormalities in lung compliance16 as one One infant with PEA has developed cerebral would expect the reverse in this situation as the palsy as a result of recurrent and severe actual surface tension could be maintained Surfactant abnormalities in ALTE and SIDS 505

at values above that expected for the normal possibility of a disease continuum or a

expiratory cycle. This would provide the common unifying factor for ALTEs and SIDS. Arch Dis Child: first published as 10.1136/adc.71.6.501 on 1 December 1994. Downloaded from potential for the recurrent apnoea that We thank the University of New England department of physi- characterises these children and the rapid ology and Ms Abigail Morris, the scientist who performed the eye movement sleep state dominance of surfactant assays. apnoea without necessarily having sustained 1 Hoffman HJ, Damus K, Hillman L, Krongrad E. Risk abnormalities in compliance as implied by factors for SIDS. Results of the National Institutes of Fagan and Milner.16 The sustainability of our Health and Human Development SIDS cooperative epidemiological study. Ann NY Acad Sci 1988; 533: laboratory findings over time supports these 13-30. contentions as do the clinical findings of 2 Beckwith JB. Intrathoracic petechial hemorrhages: a clue to the mechanism of death in SIDS? Ann NYAcad Sci 1988; recurrent apnoea extending into the childhood 533: 37-47. years in some of the individuals as shown by 3 Morley CJ, Hill CM, Brown BD, Barson AJ, Davis JA. Surfactant abnormalities in babies dying from sudden the age distribution in the table. Also it is infant death syndrome. Lancet 1982; i: 1320-3. unlikely that these pathophysiological effects 4 Hill CM, Brown BD, Morley CJ, Davis JA, Barson AJ. Pulmonary surfactant. II. In sudden infant death syn- could be sustained in the presence of more drome. Early Hum Dev 1988; 16: 153-62. mature controller systems and more mature 5 Southall DP, Johnson P, Salmons S, et al. Prolonged expira- tory apnoea: a disorder resulting in episodes of severe respiratory mechanics where it could be arterial hypoxaemia in infants and young children. Lancet expected that the influence of chest wall 1985; ii: 571-7. 6 Southall DP, Samuels MP, Talbert DG. Recurrent cyanotic deformity during the respiratory cycle would episodes with severe arterial hypoxaemia and intrapul- be lost and expiratory reserve volumes are monary shunting: a mechanism for sudden death. Arch Dis Child 1990; 65: 953-61. increased thus negating the effects of a small 7 James D, Berry PJ, Fleming P, Hathaway M. Surfactant change in compliance. While there are no abnormality and the sudden infant death syndrome - a primary or secondary phenomenon? Arch Dis Child 1990; current data to support a potential role of 65: 744-8. surfactant in neural receptor function, there is 8 Gibson RA, McMurchie EJ. Changes in lung surfactant lipids associated with the sudden infant death syndrome. information supporting this contention in the Australian PaediatricJournal 1985; (suppl): 77-80. organs of other species. 17 9 Gibson RA, McMurchie EJ. Decreased lung surfactant disaturated phosphatidylcholine in sudden infant death While only four infants had non-idiopathic syndrome. Early Hum Dev 1988; 17: 45-55. ALTE, most reports on ALTE reveal a 10 Hills B, Masters IB, O'Duffy J. Abnormalities of surfactant in children with recurrent cyanotic episodes. Lancet 1992; significant number in whom other potential 339: 1323-4. causes can be found.18 This being so, our 11 Hills BA. A thermal surface phenomenon in the rabbit lung: possible basis for the conversion of heat into work. findings would allow for a more unifying JPhysiol (Lond) 1988: 402: 463-71. hypothesis and a way of differentiating those at 12 Hills BA. Contact-angle hysteresis induced by pulmonary surfactants.JApplPhysiol 1983; 54: 420-6. 'true risk'. 13 Masters IB, Goes AM, Healy L, O'Neil M, Stephens D, Harris MA. Age related changes in oxygen saturation over the first year of life: a longitudinal study. _J Paediatr Child Health 1994; 30: 423-8. Conclusion 14 Barrow RE, Hills BA. Surface tension induced by dipalmi- toly lecthin in vitro under physiological conditions. This study has shown consistent abnormalities JPhysiol (Lond) 1979; 297: 217-27. in 15 Thach T. Apnoea and the sudden infant death syndrome. in the physical properties of surfactant http://adc.bmj.com/ In: Saunders NA, Sullivan CE, eds. Lung biology in health children with recurrent ALTEs and, while it is and disease, sleep and breathing. New York: Marcel Dekker, unclear at this stage as to what factors are 1994: 649-72. 16 Fagan DG, Milner AD. Pressure volume characteristics of producing these abnormalities, these findings the in sudden infant death syndrome. Arch Dis Child may prove to be of great clinical importance in 1985;60:471-85. 17 Sbarbati A, Cesesi E, Accordini C. Surfactant-like material defining as well as differentiating ALTEs on the chemoreceptorial surface of the frog's taste organ: cases from other disorders that share similar an ultra structural and electon spectroscopic imaging presentations. Also these findings could prove study. J StructBiol 1991; 107: 128-35. 18 Kahn A, Rebuffat E, Franco P, et al. Apparent life threaten- on September 23, 2021 by guest. Protected copyright. to be an important way of identifying children ing events and apnoea of infancy. In: Backerman RC, Brouillette R, Hunt CE, eds. Respiratory control disorders in at risk of ALTEs or even SIDS, and combined infants and children. Baltimore: Wilkins and Wilkins, 1992: with previous publications3-10 support the 178-89.