- Email: [email protected]
Faecal calprotectin concentrations and diagnosis of necrotising enterocolitis
RESEARCH LETTERS
Contributors A C Moll, F E van Leeuwen, and J R M Cruysberg had the idea for the study. A C Moll and F E van Leeuwen designed the study. A C Moll, S M Imhof, J R M Cruysberg, A Y N Schouten-van Meeteren gathered data, A C Moll and F E van Leeuwen processed data and did statistical analysis. A C Moll, F E van Leeuwen, and M Boers drafted the manuscript. All investigators contributed to the interpretation of data and manuscript revisions.
Conflict of interest statement None declared.
Acknowledgments We thank W J Klokman from the Netherlands Cancer Institute for statistical advice. 1
2
3
4
5
Moll AC, Kuik DJ, Bouter LM, et al. Incidence and survival of retinoblastoma in the Netherlands: a register-based study 1862–1995. Br J Ophthalmol 1997; 81: 559–62. Klip H, Burger CW, de Kraker J, van Leeuwen FE. Risk of cancer in the offspring of women who underwent ovarian stimulation for IVF. Hum Reprod 2001; 16: 2451–58. Koudstaal J, van Dop PA, Hogerzeil HV, et al. Pregnancy course and outcome in 2965 pregnancies after in-vitro fertilisation in the Netherlands (in Dutch). Ned Tijdschr Geneeskd 1999; 143: 2375–80. Anteby I, Cohen E, Anteby, BenEzra D. Ocular manifestations in children born after in vitro fertilization. Arch Ophthalmol 2001; 119: 1525–29. Bruinsma F, Venn A, Lancaster P, Speirs A, Healy D. Incidence of cancer born after in-vitro fertilization. Hum Reprod 2000; 15: 604–07.
Departments of Ophthalmology (A C Moll MD, S M Imhof MD), Paediatric Oncology (A Y N Schouten-van Meeteren MD), and Clinical Epidemiology and Biostatistics (Prof M Boers MD, Prof F E van Leeuwen PhD), Vrije Universiteit Medical Centre, Amsterdam 1081 HV, Netherlands; Department of Ophthalmology, University Medical Centre, Nijmegen, Netherlands (Prof J R M Cruysberg MD); and Department of Epidemiology, Netherlands Cancer Institute, Amsterdam (Prof F E van Leeuwen) Correspondence to: Annette C Moll (e-mail: [email protected])
310
Faecal calprotectin concentrations and diagnosis of necrotising enterocolitis Daniel Carroll, Anthony Corfield, Richard Spicer, Pamela Cairns Calprotectin has been proposed as a useful marker of inflammatory bowel disease in children. We did a pilot study to establish whether it can be used to aid diagnosis of necrotising enterocolitis in preterm infants. Patients with clinical features of necrotising enterocolitis had raised faecal calprotectin concentrations at the time of diagnosis compared with matched controls (288·4 mg/L [SD 49·1] and 98·0 mg/L [60·6], respectively; p=0·0006). Faecal calprotectin might be a useful marker of gastrointestinal mucosal inflammation in neonates.
Lancet 2003; 361: 310–11
Necrotising enterocolitis is a serious disorder of preterm infants, with high mortality and morbidity. Diagnosis is made on the basis of clinical criteria since there are no specific diagnostic tests. Calprotectin is a 36-kDa calcium-binding and zinc-binding protein constituting 60% of soluble cytosol proteins in human neutrophil granulocytes.1 Faecal calprotectin concentrations are raised in various organic bowel diseases in adults.1 Calprotectin is resistant to degradation, and has been proposed as a useful laboratory marker of gastrointestinal inflammation in children and adults with active inflammatory bowel disease.2 Defects in or increased permeability of the mucosal barrier cause migration of large numbers of granulocytes into the intestinal lumen. If mucosal lesions are extensive, as in active inflammatory bowel disease, calprotectin concentrations are generally ten to twenty times the upper reference limit of 30 mg/L in adults.3 We identified seven consecutive patients with clinical features consistent with necrotising enterocolitis and who had required at least 7 days’ parenteral nutrition, intravenous antibiotics, and enteral starvation; and seven controls matched for age, sex, and gestational age. We obtained written informed consent from the parents of the infants, and obtained ethics approval from United Bristol Healthcare Trust ethics committee. We obtained stool samples, which were stored at –20ºC before analysis. Stool samples were obtained as soon as possible after the diagnosis of necrotising enterocolitis was made. All patients produced a stool within 24 h of diagnosis. We recorded the date and time at which each stool sample was obtained, patients’ gestational age, postnatal age at diagnosis, sex, feeding history, and clinical signs. Samples were thawed and analysed twice for faecal calprotectin concentrations with CALPREST (Eurospital, Trieste, Italy) and the mean concentration calculated. Data analysis was by Mann-Whitney U test with SPSS for Windows (version 10). 400 Faecal calprotectin concentration (mg/L)
A large retrospective cohort study from Sweden reported that 5·4% of children born after IVF had developmental problems, but that there was no increased risk of cancer in this group. This finding about cancer risk is lent support by results of a study by Bruinsma and colleagues5 that did not show a significantly increased incidence of cancer in children born after assisted conception in Australia. In a cohort of 9484 offspring of Dutch women who had ovarian stimulation before IVF, 16 cases of cancer were reported,2 which suggests that the risk of childhood cancer was not increased compared with the general population and with an internal reference group of subfertile women who were treated without IVF. Although IVF was done during 1980–95, no retinoblastoma cases were reported in children born during this time. Our five patients were all born in the Netherlands after 1995 between 1997 and 2001. That five cases arose during this time, but that none were seen during the earlier period is important, and represents a striking excess in disease frequency. Did something change in the IVF procedure itself (possibly the culture medium), or is our observation only a chance finding? Perhaps the same genetic factors are involved in infertility and retinoblastoma. However, none of the parents of our patients had had retinoblastoma, and we have not identified any published data about infertility problems of unaffected parents of retinoblastoma patients. Whether treatment with ovulation-inducing drugs increases the risk of childhood cancer is an important matter, especially with the rising numbers of women undergoing treatment for subfertility. Future investigators should consider the number of IVF treatments, other fertility drugs given before IVF, and the possibility that serious disorders in children conceived by IVF are diagnosed earlier than those in other children who do not have such close medical surveillance. Our finding requires further research to confirm the association and to explore a possible causal mechanism.
300
200
100
0 Controls
Necrotising enterocolitis patients
Faecal calprotectin concentrations
THE LANCET • Vol 361 • January 25, 2003 • www.thelancet.com
For personal use. Only reproduce with permission from The Lancet Publishing Group.
RESEARCH LETTERS
Patients with necrotising enterocolitis had a mean gestational age of 30 weeks (SD 4), a mean age at diagnosis of 12 days (5), four were boys, and three were girls. Three patients had mild necrotising enterocolitis (Bell’s stage Ib), one had definite necrotising enterocolitis (Bell’s stage IIb), and three had clinical signs consistent with advanced necrotising enterocolitis (Bell’s stage IIIa or IIIb). The four patients with stage IIb or above had pneumatosis intestinalis shown by plain abdominal radiographs. Controls also had a mean gestational age of 30 weeks (4), a mean age of 12 days (5) at the time of stool sample, four were boys, and three were girls. No patients in the control group developed symptoms consistent with necrotising enterocolitis. We detected a marked difference between the two groups: mean faecal calprotectin concentration was 288·4 mg/L (49·1) in patients with necrotising enterocolitis (range 210·0–340·0) compared with 98·0 mg/L (60·6) in controls (range 22·0–171·0); p=0·0006 (figure). Our results show a substantial difference between faecal calprotectin concentrations in children with clinical features consistent with necrotising enterocolitis and matched controls. However, concentrations varied widely in controls; many values were outside the normal reference range for adults.3 This variation might indicate fundamental differences in faecal calprotectin concentrations in neonates compared with adults. Our findings are supported by those of Olafsdottir and colleagues4 who showed raised faecal calprotecin concentrations in fullterm neonates. Rugtveit and Fagerhol5 showed age-dependent variation in faecal calprotectin concentrations in children, with much higher values in normal healthy infants at age 6 weeks than in adults. Gestational age could have some effect on normal faecal calprotectin concentration. Further research should assess the relation between faecal calprotectin concentration, type of infant food (early breast milk vs formula), and gestational age. Faecal calprotectin might be a useful marker of gastrointestinal mucosal inflammation in neonates. Contributors D Carroll obtained specimens, recruited patients, and analysed specimens. A Corfield provided technical assistance with the calprotectin assay. R Spicer identified patients, helped with data analysis, and obtained funding. P Cairns identified patients, provided the original idea for the study, and helped with data analysis.
Conflict of interest statement None declared.
Acknowledgments This work was supported by the Charitable Trustees of United Bristol Healthcare Trust. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. 1
2 3
4
5
Syringes with rubber-coated plungers and inactivation of surfactant Egbert Herting, Guido Stichtenoth, Bengt Robertson Injectable drugs are generally administered to newborns with 1 mL syringes. During in-vitro measurements of surface tension with a pulsating bubble surfactometer, we noticed that surfactant was inactivated after repeated aspiration into a 1 mL syringe with a rubber-coated plunger. Inactivation did not take place, however, when we used rubber-free two-part syringes with polyethylene pistons. Results of further studies showed that the silicone fluid used to lubricate the rubber surface of the syringe was inactivating the surfactant. Our findings suggest that contact with rubber surfaces should be avoided in the handling of surfactant material for biophysical assessment or clinical use.
Lancet 2003; 361: 311–13
Rubber, produced from natural or synthetic latex, is contained in many medical devices,1 and is in constant use in the daily care of patients. However, the material can contain more than ten different substances—eg, resins—that are not chemically bound but that help to guarantee its resilience, elasticity, colour, and impact resistance.1 Plasticisers—eg, those contained in polyvinylchloride containers and infusion bags or lines—can leach into blood products or solutions used in parenteral nutrition and dialysis.2 Deficiency of pulmonary surfactant is the underlying cause of respiratory distress syndrome in neonates, and the intratracheal administration of surfactant greatly reduces morbidity and mortality.3 To assess the biophysical activity of surfactant preparations in vitro, surface tension can be measured in a pulsating bubble surfactometer. In short, a bubble communicating with ambient air is generated in a test chamber filled with surfactant and pulsated by means of a piston system. The bubble undergoes cyclic changes in radius between 0·55 mm and 0·40 mm, corresponding to 50% area compression. The pressure changes are recorded and a computer calculates surface tension, using the law of Laplace. For many years, we have been using the pulsating bubble surfactometer system to try to optimise the protein and lipid composition of new surfactant preparations.4 While assessing a porcine modified natural surfactant at a low phospholipid concentration of 2·5 mg/mL, we were puzzled to note that identical preparations showed excellent biophysical activity—ie, rapid adsorption with minimum surface tension close to zero after a few seconds—when the measurements
Fagerhol MK, Andersson KB, Naess-Andresen CF, Brandtzaeg P, Dale I. Calprotectin (the L1 leukocyte protein). In: Smith VL, Dedman JR, eds. Stimulus response coupling: the role of intracellular calcium-binding proteins. Boca Raton: CRC Press, 1990: 187–210. Fagerhol MK. Calprotectin, a faecal marker of organic gastrointestinal abnormality. Lancet 2000; 356: 1783–84. Tibble J, Teahon K, Thjodleifsson B, et al. A simple method for assessing intestinal inflammation in Crohn’s disease. Gut 2000; 47: 506–13. Olafsdottir E, Aksnes L, Fluge G, Berstad A. Faecal calprotectin levels in infants with infantile colic, healthy infants, children with inflammatory bowel disease, children with recurrent abdominal pain and healthy children. Acta Paediatr 2002; 91: 45–50. Rugtveit J, Fagerhol MK. Age-dependent variations in fecal calprotectin concentrations in children. J Pediatr Gastroenterol Nutr 2002; 34: 323–24.
Department of Paediatric Surgery, Bristol Royal Hospital for Sick Children, Bristol BS2 8BJ, UK (D Carroll MRCS, A Corfield PhD, R Spicer FRCS, P Cairns MRCPCH) Correspondence to: Mr Daniel Carroll (e-mail: [email protected])
THE LANCET • Vol 361 • January 25, 2003 • www.thelancet.com
Figure 1: Three-part syringe with a rubber tip on the plunger (upper) and rubber-free two-part syringe (lower)
311
For personal use. Only reproduce with permission from The Lancet Publishing Group.
Contributors A C Moll, F E van Leeuwen, and J R M Cruysberg had the idea for the study. A C Moll and F E van Leeuwen designed the study. A C Moll, S M Imhof, J R M Cruysberg, A Y N Schouten-van Meeteren gathered data, A C Moll and F E van Leeuwen processed data and did statistical analysis. A C Moll, F E van Leeuwen, and M Boers drafted the manuscript. All investigators contributed to the interpretation of data and manuscript revisions.
Conflict of interest statement None declared.
Acknowledgments We thank W J Klokman from the Netherlands Cancer Institute for statistical advice. 1
2
3
4
5
Moll AC, Kuik DJ, Bouter LM, et al. Incidence and survival of retinoblastoma in the Netherlands: a register-based study 1862–1995. Br J Ophthalmol 1997; 81: 559–62. Klip H, Burger CW, de Kraker J, van Leeuwen FE. Risk of cancer in the offspring of women who underwent ovarian stimulation for IVF. Hum Reprod 2001; 16: 2451–58. Koudstaal J, van Dop PA, Hogerzeil HV, et al. Pregnancy course and outcome in 2965 pregnancies after in-vitro fertilisation in the Netherlands (in Dutch). Ned Tijdschr Geneeskd 1999; 143: 2375–80. Anteby I, Cohen E, Anteby, BenEzra D. Ocular manifestations in children born after in vitro fertilization. Arch Ophthalmol 2001; 119: 1525–29. Bruinsma F, Venn A, Lancaster P, Speirs A, Healy D. Incidence of cancer born after in-vitro fertilization. Hum Reprod 2000; 15: 604–07.
Departments of Ophthalmology (A C Moll MD, S M Imhof MD), Paediatric Oncology (A Y N Schouten-van Meeteren MD), and Clinical Epidemiology and Biostatistics (Prof M Boers MD, Prof F E van Leeuwen PhD), Vrije Universiteit Medical Centre, Amsterdam 1081 HV, Netherlands; Department of Ophthalmology, University Medical Centre, Nijmegen, Netherlands (Prof J R M Cruysberg MD); and Department of Epidemiology, Netherlands Cancer Institute, Amsterdam (Prof F E van Leeuwen) Correspondence to: Annette C Moll (e-mail: [email protected])
310
Faecal calprotectin concentrations and diagnosis of necrotising enterocolitis Daniel Carroll, Anthony Corfield, Richard Spicer, Pamela Cairns Calprotectin has been proposed as a useful marker of inflammatory bowel disease in children. We did a pilot study to establish whether it can be used to aid diagnosis of necrotising enterocolitis in preterm infants. Patients with clinical features of necrotising enterocolitis had raised faecal calprotectin concentrations at the time of diagnosis compared with matched controls (288·4 mg/L [SD 49·1] and 98·0 mg/L [60·6], respectively; p=0·0006). Faecal calprotectin might be a useful marker of gastrointestinal mucosal inflammation in neonates.
Lancet 2003; 361: 310–11
Necrotising enterocolitis is a serious disorder of preterm infants, with high mortality and morbidity. Diagnosis is made on the basis of clinical criteria since there are no specific diagnostic tests. Calprotectin is a 36-kDa calcium-binding and zinc-binding protein constituting 60% of soluble cytosol proteins in human neutrophil granulocytes.1 Faecal calprotectin concentrations are raised in various organic bowel diseases in adults.1 Calprotectin is resistant to degradation, and has been proposed as a useful laboratory marker of gastrointestinal inflammation in children and adults with active inflammatory bowel disease.2 Defects in or increased permeability of the mucosal barrier cause migration of large numbers of granulocytes into the intestinal lumen. If mucosal lesions are extensive, as in active inflammatory bowel disease, calprotectin concentrations are generally ten to twenty times the upper reference limit of 30 mg/L in adults.3 We identified seven consecutive patients with clinical features consistent with necrotising enterocolitis and who had required at least 7 days’ parenteral nutrition, intravenous antibiotics, and enteral starvation; and seven controls matched for age, sex, and gestational age. We obtained written informed consent from the parents of the infants, and obtained ethics approval from United Bristol Healthcare Trust ethics committee. We obtained stool samples, which were stored at –20ºC before analysis. Stool samples were obtained as soon as possible after the diagnosis of necrotising enterocolitis was made. All patients produced a stool within 24 h of diagnosis. We recorded the date and time at which each stool sample was obtained, patients’ gestational age, postnatal age at diagnosis, sex, feeding history, and clinical signs. Samples were thawed and analysed twice for faecal calprotectin concentrations with CALPREST (Eurospital, Trieste, Italy) and the mean concentration calculated. Data analysis was by Mann-Whitney U test with SPSS for Windows (version 10). 400 Faecal calprotectin concentration (mg/L)
A large retrospective cohort study from Sweden reported that 5·4% of children born after IVF had developmental problems, but that there was no increased risk of cancer in this group. This finding about cancer risk is lent support by results of a study by Bruinsma and colleagues5 that did not show a significantly increased incidence of cancer in children born after assisted conception in Australia. In a cohort of 9484 offspring of Dutch women who had ovarian stimulation before IVF, 16 cases of cancer were reported,2 which suggests that the risk of childhood cancer was not increased compared with the general population and with an internal reference group of subfertile women who were treated without IVF. Although IVF was done during 1980–95, no retinoblastoma cases were reported in children born during this time. Our five patients were all born in the Netherlands after 1995 between 1997 and 2001. That five cases arose during this time, but that none were seen during the earlier period is important, and represents a striking excess in disease frequency. Did something change in the IVF procedure itself (possibly the culture medium), or is our observation only a chance finding? Perhaps the same genetic factors are involved in infertility and retinoblastoma. However, none of the parents of our patients had had retinoblastoma, and we have not identified any published data about infertility problems of unaffected parents of retinoblastoma patients. Whether treatment with ovulation-inducing drugs increases the risk of childhood cancer is an important matter, especially with the rising numbers of women undergoing treatment for subfertility. Future investigators should consider the number of IVF treatments, other fertility drugs given before IVF, and the possibility that serious disorders in children conceived by IVF are diagnosed earlier than those in other children who do not have such close medical surveillance. Our finding requires further research to confirm the association and to explore a possible causal mechanism.
300
200
100
0 Controls
Necrotising enterocolitis patients
Faecal calprotectin concentrations
THE LANCET • Vol 361 • January 25, 2003 • www.thelancet.com
For personal use. Only reproduce with permission from The Lancet Publishing Group.
RESEARCH LETTERS
Patients with necrotising enterocolitis had a mean gestational age of 30 weeks (SD 4), a mean age at diagnosis of 12 days (5), four were boys, and three were girls. Three patients had mild necrotising enterocolitis (Bell’s stage Ib), one had definite necrotising enterocolitis (Bell’s stage IIb), and three had clinical signs consistent with advanced necrotising enterocolitis (Bell’s stage IIIa or IIIb). The four patients with stage IIb or above had pneumatosis intestinalis shown by plain abdominal radiographs. Controls also had a mean gestational age of 30 weeks (4), a mean age of 12 days (5) at the time of stool sample, four were boys, and three were girls. No patients in the control group developed symptoms consistent with necrotising enterocolitis. We detected a marked difference between the two groups: mean faecal calprotectin concentration was 288·4 mg/L (49·1) in patients with necrotising enterocolitis (range 210·0–340·0) compared with 98·0 mg/L (60·6) in controls (range 22·0–171·0); p=0·0006 (figure). Our results show a substantial difference between faecal calprotectin concentrations in children with clinical features consistent with necrotising enterocolitis and matched controls. However, concentrations varied widely in controls; many values were outside the normal reference range for adults.3 This variation might indicate fundamental differences in faecal calprotectin concentrations in neonates compared with adults. Our findings are supported by those of Olafsdottir and colleagues4 who showed raised faecal calprotecin concentrations in fullterm neonates. Rugtveit and Fagerhol5 showed age-dependent variation in faecal calprotectin concentrations in children, with much higher values in normal healthy infants at age 6 weeks than in adults. Gestational age could have some effect on normal faecal calprotectin concentration. Further research should assess the relation between faecal calprotectin concentration, type of infant food (early breast milk vs formula), and gestational age. Faecal calprotectin might be a useful marker of gastrointestinal mucosal inflammation in neonates. Contributors D Carroll obtained specimens, recruited patients, and analysed specimens. A Corfield provided technical assistance with the calprotectin assay. R Spicer identified patients, helped with data analysis, and obtained funding. P Cairns identified patients, provided the original idea for the study, and helped with data analysis.
Conflict of interest statement None declared.
Acknowledgments This work was supported by the Charitable Trustees of United Bristol Healthcare Trust. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. 1
2 3
4
5
Syringes with rubber-coated plungers and inactivation of surfactant Egbert Herting, Guido Stichtenoth, Bengt Robertson Injectable drugs are generally administered to newborns with 1 mL syringes. During in-vitro measurements of surface tension with a pulsating bubble surfactometer, we noticed that surfactant was inactivated after repeated aspiration into a 1 mL syringe with a rubber-coated plunger. Inactivation did not take place, however, when we used rubber-free two-part syringes with polyethylene pistons. Results of further studies showed that the silicone fluid used to lubricate the rubber surface of the syringe was inactivating the surfactant. Our findings suggest that contact with rubber surfaces should be avoided in the handling of surfactant material for biophysical assessment or clinical use.
Lancet 2003; 361: 311–13
Rubber, produced from natural or synthetic latex, is contained in many medical devices,1 and is in constant use in the daily care of patients. However, the material can contain more than ten different substances—eg, resins—that are not chemically bound but that help to guarantee its resilience, elasticity, colour, and impact resistance.1 Plasticisers—eg, those contained in polyvinylchloride containers and infusion bags or lines—can leach into blood products or solutions used in parenteral nutrition and dialysis.2 Deficiency of pulmonary surfactant is the underlying cause of respiratory distress syndrome in neonates, and the intratracheal administration of surfactant greatly reduces morbidity and mortality.3 To assess the biophysical activity of surfactant preparations in vitro, surface tension can be measured in a pulsating bubble surfactometer. In short, a bubble communicating with ambient air is generated in a test chamber filled with surfactant and pulsated by means of a piston system. The bubble undergoes cyclic changes in radius between 0·55 mm and 0·40 mm, corresponding to 50% area compression. The pressure changes are recorded and a computer calculates surface tension, using the law of Laplace. For many years, we have been using the pulsating bubble surfactometer system to try to optimise the protein and lipid composition of new surfactant preparations.4 While assessing a porcine modified natural surfactant at a low phospholipid concentration of 2·5 mg/mL, we were puzzled to note that identical preparations showed excellent biophysical activity—ie, rapid adsorption with minimum surface tension close to zero after a few seconds—when the measurements
Fagerhol MK, Andersson KB, Naess-Andresen CF, Brandtzaeg P, Dale I. Calprotectin (the L1 leukocyte protein). In: Smith VL, Dedman JR, eds. Stimulus response coupling: the role of intracellular calcium-binding proteins. Boca Raton: CRC Press, 1990: 187–210. Fagerhol MK. Calprotectin, a faecal marker of organic gastrointestinal abnormality. Lancet 2000; 356: 1783–84. Tibble J, Teahon K, Thjodleifsson B, et al. A simple method for assessing intestinal inflammation in Crohn’s disease. Gut 2000; 47: 506–13. Olafsdottir E, Aksnes L, Fluge G, Berstad A. Faecal calprotectin levels in infants with infantile colic, healthy infants, children with inflammatory bowel disease, children with recurrent abdominal pain and healthy children. Acta Paediatr 2002; 91: 45–50. Rugtveit J, Fagerhol MK. Age-dependent variations in fecal calprotectin concentrations in children. J Pediatr Gastroenterol Nutr 2002; 34: 323–24.
Department of Paediatric Surgery, Bristol Royal Hospital for Sick Children, Bristol BS2 8BJ, UK (D Carroll MRCS, A Corfield PhD, R Spicer FRCS, P Cairns MRCPCH) Correspondence to: Mr Daniel Carroll (e-mail: [email protected])
THE LANCET • Vol 361 • January 25, 2003 • www.thelancet.com
Figure 1: Three-part syringe with a rubber tip on the plunger (upper) and rubber-free two-part syringe (lower)
311
For personal use. Only reproduce with permission from The Lancet Publishing Group.