CLB-08746; No. of pages: 3; 4C: Clinical Biochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry
Short Communication
2
Hormone profiles in extremely preterm infants
3Q1
Ronda F. Greaves
4
Clinical Biochemistry, Discipline of Laboratory Medicine, School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia
O
1
30
Methods
31 32
Subjects were recruited from neonatal wards across three hospitals in Melbourne Australia. The studies were approved by the Human Research Ethics Committee of each hospital and neonatologists were consulted prior to obtaining written informed consent from the parents for sample collection. Phlebotomy sampling coincided with routine clinical sampling, with a maximum of 0.7 mL of whole blood collected for each sample. All samples were/are stored at −70 °C until analysis. The sample repositories are:
24 25 26 27
33 34 35 36 37 38 39 40 41 42
C
22 23
E
20 21
R
18 19
R
16 17
N C O
14 15
U
12 13
R-1: Serial serum samples were collected from 118 participants at birth (cord blood) and then at 1, 4, 7, 14, 21, 28 and 42 days after birth from babies born between 23 and 29 weeks gestation. All samples were tagged for hormone analysis by routine immunoassay
E-mail address: [email protected].
R-2: Serial serum samples were collected from 300 participants at two to three week intervals up to the equivalent of term from babies born between 24 and 32 weeks gestation. A total of 732 samples were collected; with a medium of three samples per participant. These samples are tagged for pituitary hormone analysis by immunoassay and steroid analysis by LC–MSMS. To date some samples have been utilised to study pituitary hormones. R-3: Paired blood (serum) and random urine samples were collected from 21 full term neonates and 21 preterm infants born b 32 weeks gestation. All samples are tagged for steroid metabolomic studies and are currently stored awaiting analysis.
P
28 29
Preterm birth, as defined by a gestational age of b 37 weeks at delivery, currently accounts for 5 to 18% of all births worldwide [1,2]. In recent decades neonatologists have invested significant effort to improve outcomes for preterm infants. Now neonatologists are good at keeping these young babies alive, with survival rates associated with preterm deliveries increasing from 85% in the mid-1990s to 93% reported in 2010; with a decrease in foetal deaths with increasing gestational age at birth [2,3]. Consequently, pathologies previously only considered for full term neonates are now being investigated in preterm infants. Preterm infants, particularly those who are very preterm (28–b 32 weeks) or extremely preterm (b 28 weeks), frequently have endocrine testing, due to multiple health issues including resistant hypotension, need for prolonged ventilatory support or unclear genital appearance. Immaturity of the endocrine system and its potential impact on morbidity is the subject of numerous research studies. Clinical reports suggest significant differences in serum hormone levels between extremely preterm infants compared to older preterm and full term infants. With the significant problem of little normative hormone data available for extremely preterm infants, our aim was to establish a biobank of samples to determine “normal” hormone patterns in very and extremely preterm infants.
D
8 9
methodology. All samples have now been utilised for various studies 43 [4,5]. 44
E
Introduction
T
7
R O
6 5
10 11
F
journal homepage: www.elsevier.com/locate/clinbiochem
45 46 47 48 49 50 51 52 53 54 55
Results
56
All studies planned for immunoassay analysis have now been conducted. A summary of these studies is provided in Table 1. As part of the data analysis to determine reference intervals all data generated (with the exception of free thyroxine) demonstrated a non-Gaussian distribution and required log transformation. Infants not surviving beyond the equivalent of term were excluded from statistical analysis.
57 58
Discussion
63
Interpretation of clinical measurements and laboratory results in preterm infants is a significant issue due to scarce age appropriate data [6,7]. The steroid hormone reference intervals generated in these immunoassay based studies provide clear guidance for the clinician to enable the correct interpretation of hormone results in neonates born less than 32 weeks gestation; reducing the risk of incorrect diagnosis due to misinterpretation of data. It is essential to recognise that the results of these immunoassay based studies cannot be routinely applied to mass spectrometry methods. Hence additional studies are planned to generate mass spectrometry based steroid hormone profile data.
64 65
Acknowledgements
74
This work has been conducted in conjunction with colleagues from the Murdoch Children's Research Institute: Professor Margaret Zacharin, Associate Professor Rodney Hunt, Mrs Janne Pitkin and others. Various funding has allowed this project to progress: NHMRC Grant ID
75
http://dx.doi.org/10.1016/j.clinbiochem.2014.05.040 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Greaves RF, Hormone profiles in extremely preterm infants, Clin Biochem (2014), http://dx.doi.org/10.1016/ j.clinbiochem.2014.05.040
59 60 61 62
66 67 68 69 70 71 72 73
76 77 78
2
Table 1 Summary of studies conducted and findings following immunoassay analysis of samples from the repositories.
Q2
t1:3
Hormone
t1:4 t1:5
Pituitary hormones GH R1
t1:6 t1:7
FSH
R1
15
t1:8 t1:9
LH
R2 R1
112 25
t1:10 t1:11 t1:12
R2
112
Prolactin FSH
R1
15
24–29 weeks gestation (median = 27 weeks) M with samples collected between 3 and 43 days of age
t1:13 t1:14
LH
R2 R1
113 21
24–35 weeks corrected age 24–29 weeks gestation (median = 27 weeks) with samples collected between 3 and 43 days of age
R2
113
24–35 weeks corrected age
t1:15 t1:16 t1:17 t1:18 t1:19 t1:20
Gender Reference Interval
U
112 participants with 676 results for this 23–29 weeks gestation with sampling at 1, 4, 7, 14, group overall 21, 28 and 42 days post-birth
N
C
DHEAS
E2
Progesterone
t1:23
Free thyroxine
M&F
24–29 weeks gestation (median = 28 weeks) with samples collected between 3 and 43 days of age (median corrected gestational age = 31 weeks at time of sample collection) 24–35 weeks corrected age 24–29 weeks gestation (median = 28 weeks) with samples collected between 3 and 43 days of age (median corrected gestational age = 31 weeks at time of sample collection) 24–35 weeks corrected age
F
O
Steroid and thyroid hormones Cortisol R1 112 participants with 637 to 679 results (analyte dependent) across the group
t1:22
t1:25
Preterm group
R
R
E
C
T
Prolactin
t1:21
t1:24
Repository n
23–29 weeks gestation with sampling at 1, 4, 7, 14, 21, 28 and 42 days post-birth
E
Analyser
Levels highest during the first week of life. Reference intervals at day 7 of life are: 7–121 mIU/L for babies born between 23 and 26 weeks gestation; and 8–188 mIU/L for babies born between 27 and 29 weeks gestation. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 1.6–167.0 IU/L (median 49.6 IU/L).
Siemens — Centaur
Based on first sample collected from each participant: 3.7–N200 IU/L. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 0.3–54.4 IU/L (median 7.5 IU/L).
Roche — Cobas 8000 e602 Siemens — Centaur
Based on first sample collected from each participant: 0.3–134.0 IU/L. Based on first sample collected from each participant: 374–5694 mIU/L. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 0.3–2.6 IU/L (median 0.9 IU/L). Based on first sample collected from each participant: 0.2–4.2 IU/L. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 0.1–8.5 IU/L (median 1.2 IU/L). Based on first sample collected from each participant: 0.1–10.9 IU/L. Based on first sample collected from each participant Based on first sample collected from each participant: 463–6037 mIU/L.
Roche — Cobas 8000 e602
D
M&F
Siemens — Immulite 2000
Siemens — Centaur
Roche — Cobas 8000 e602 Siemens — Centaur
Roche — Cobas 8000 e602
R.F. Greaves / Clinical Biochemistry xxx (2014) xxx–xxx
Please cite this article as: Greaves RF, Hormone profiles in extremely preterm infants, Clin Biochem (2014), http://dx.doi.org/10.1016/ j.clinbiochem.2014.05.040
t1:1 t1:2
Q3
Siemens — Immulite 2000 Levels highest during the first week of life. Reference intervals at day 7 of life are: 63–801 nmol/L for babies born between 23 and 26 weeks gestation; and 70–594 nmol/L for babies born between 27 and 29 weeks gestation. Levels highest during the first week of life. Reference intervals at day 7 of life are: 3.3–19.4 μmol/L for babies born between 23 and 26 weeks Q4 gestation; and 2.4–11.5 μmol/L for babies born between 27 and 29 weeks Q5 gestation. Levels ranged from b73 pmol/L to 1626 pmol/L over the six weeks. Reference intervals at day 7 of life are: 87–359 pmol/L for babies born between 23 and 26 weeks gestation; and b73–347 pmol/L for babies born between 27 and 29 weeks gestation. Levels highest during the first week of life. Reference intervals at day 7 of life are: 5.1–38.2 nmol/L for babies born between 23 and 26 weeks gestation; and 4.2–25.2 nmol/L for babies born between 27 and 29 weeks gestation. Levels as low as 2.6 pmol/L for the first 28 days with the nadir at 7 days for the overall group. Reference intervals at day 7 of life are: b2.6–14.0 pmol/L for babies born between 23 and 26 weeks gestation; and 5.2–18.6 pmol/L for babies born between 27 and 29 weeks gestation.
P
DHEAS = dihydroepiandrostane sulphate; E2 = estradiol; FSH = follicle stimulating hormone; GH = growth hormone; LH = luteinizing hormone.
R O
O
F
Q6
R.F. Greaves / Clinical Biochemistry xxx (2014) xxx–xxx
216757; education grants from Serono and Pfizer; and supply of the immunoassay kits courtesy of Roche Diagnostics and Siemens.
81
References
82 83 84 85 86 87 88 89 90 103
[1] World Health Organization Media Centre Fact sheet No 363: Preterm Birth — updated 2013. Available at www.who.int/mediacentre/factsheets/fs363/en/ [accessed 1st April 2014]. [2] Blencowe H, Cousens S, Oestergaard MZ, Chou D, Moller A-B, Narwal R, et al. National, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic analysis and implications. Lancet 2012;379:2162–72. [3] National Health, Medical Research Council. Care around preterm birth. Clinical practice guidelines. Australian Government Publishing Service; 1997.
[4] Greaves R, Hunt R, Chiriano A, Zacharin M. LH and FSH levels in extreme prematurity: development of reference intervals. Pediatrics 2008;121:e574–80. [5] Greaves RF, Donath S, Hunt RW. Serum hormone profiles over the first six weeks of life for preterm infants born less than 30 weeks gestation. XIIth International Congress of Paediatric Laboratory Medicine Berlin 2011. Clin Biochem 2011;44:531. [6] Greaves R, Hunt R, Zacharin M. Transient anomalies in genital appearance in some extremely preterm female infants may be the result of foetal programming causing a surge in LH and the over activation of the pituitary–gonadal axis. Clin Endocrinol 2008;69:763–8. [7] Greaves R, Kanumakala S, Read A, Zacharin M. Genital abnormalities mimicking congenital adrenal hyperplasia in premature infants. J Paediatr Child Health 2004;40:233–6.
U
N C O
R
R
E
C
T
E
D
P
R O
O
F
79 Q7 80
3
Please cite this article as: Greaves RF, Hormone profiles in extremely preterm infants, Clin Biochem (2014), http://dx.doi.org/10.1016/ j.clinbiochem.2014.05.040
91 92 93 94 95 96 97 98 99 100 101 102
Contents lists available at ScienceDirect
Clinical Biochemistry
Short Communication
2
Hormone profiles in extremely preterm infants
3Q1
Ronda F. Greaves
4
Clinical Biochemistry, Discipline of Laboratory Medicine, School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia
O
1
30
Methods
31 32
Subjects were recruited from neonatal wards across three hospitals in Melbourne Australia. The studies were approved by the Human Research Ethics Committee of each hospital and neonatologists were consulted prior to obtaining written informed consent from the parents for sample collection. Phlebotomy sampling coincided with routine clinical sampling, with a maximum of 0.7 mL of whole blood collected for each sample. All samples were/are stored at −70 °C until analysis. The sample repositories are:
24 25 26 27
33 34 35 36 37 38 39 40 41 42
C
22 23
E
20 21
R
18 19
R
16 17
N C O
14 15
U
12 13
R-1: Serial serum samples were collected from 118 participants at birth (cord blood) and then at 1, 4, 7, 14, 21, 28 and 42 days after birth from babies born between 23 and 29 weeks gestation. All samples were tagged for hormone analysis by routine immunoassay
E-mail address: [email protected].
R-2: Serial serum samples were collected from 300 participants at two to three week intervals up to the equivalent of term from babies born between 24 and 32 weeks gestation. A total of 732 samples were collected; with a medium of three samples per participant. These samples are tagged for pituitary hormone analysis by immunoassay and steroid analysis by LC–MSMS. To date some samples have been utilised to study pituitary hormones. R-3: Paired blood (serum) and random urine samples were collected from 21 full term neonates and 21 preterm infants born b 32 weeks gestation. All samples are tagged for steroid metabolomic studies and are currently stored awaiting analysis.
P
28 29
Preterm birth, as defined by a gestational age of b 37 weeks at delivery, currently accounts for 5 to 18% of all births worldwide [1,2]. In recent decades neonatologists have invested significant effort to improve outcomes for preterm infants. Now neonatologists are good at keeping these young babies alive, with survival rates associated with preterm deliveries increasing from 85% in the mid-1990s to 93% reported in 2010; with a decrease in foetal deaths with increasing gestational age at birth [2,3]. Consequently, pathologies previously only considered for full term neonates are now being investigated in preterm infants. Preterm infants, particularly those who are very preterm (28–b 32 weeks) or extremely preterm (b 28 weeks), frequently have endocrine testing, due to multiple health issues including resistant hypotension, need for prolonged ventilatory support or unclear genital appearance. Immaturity of the endocrine system and its potential impact on morbidity is the subject of numerous research studies. Clinical reports suggest significant differences in serum hormone levels between extremely preterm infants compared to older preterm and full term infants. With the significant problem of little normative hormone data available for extremely preterm infants, our aim was to establish a biobank of samples to determine “normal” hormone patterns in very and extremely preterm infants.
D
8 9
methodology. All samples have now been utilised for various studies 43 [4,5]. 44
E
Introduction
T
7
R O
6 5
10 11
F
journal homepage: www.elsevier.com/locate/clinbiochem
45 46 47 48 49 50 51 52 53 54 55
Results
56
All studies planned for immunoassay analysis have now been conducted. A summary of these studies is provided in Table 1. As part of the data analysis to determine reference intervals all data generated (with the exception of free thyroxine) demonstrated a non-Gaussian distribution and required log transformation. Infants not surviving beyond the equivalent of term were excluded from statistical analysis.
57 58
Discussion
63
Interpretation of clinical measurements and laboratory results in preterm infants is a significant issue due to scarce age appropriate data [6,7]. The steroid hormone reference intervals generated in these immunoassay based studies provide clear guidance for the clinician to enable the correct interpretation of hormone results in neonates born less than 32 weeks gestation; reducing the risk of incorrect diagnosis due to misinterpretation of data. It is essential to recognise that the results of these immunoassay based studies cannot be routinely applied to mass spectrometry methods. Hence additional studies are planned to generate mass spectrometry based steroid hormone profile data.
64 65
Acknowledgements
74
This work has been conducted in conjunction with colleagues from the Murdoch Children's Research Institute: Professor Margaret Zacharin, Associate Professor Rodney Hunt, Mrs Janne Pitkin and others. Various funding has allowed this project to progress: NHMRC Grant ID
75
http://dx.doi.org/10.1016/j.clinbiochem.2014.05.040 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Greaves RF, Hormone profiles in extremely preterm infants, Clin Biochem (2014), http://dx.doi.org/10.1016/ j.clinbiochem.2014.05.040
59 60 61 62
66 67 68 69 70 71 72 73
76 77 78
2
Table 1 Summary of studies conducted and findings following immunoassay analysis of samples from the repositories.
Q2
t1:3
Hormone
t1:4 t1:5
Pituitary hormones GH R1
t1:6 t1:7
FSH
R1
15
t1:8 t1:9
LH
R2 R1
112 25
t1:10 t1:11 t1:12
R2
112
Prolactin FSH
R1
15
24–29 weeks gestation (median = 27 weeks) M with samples collected between 3 and 43 days of age
t1:13 t1:14
LH
R2 R1
113 21
24–35 weeks corrected age 24–29 weeks gestation (median = 27 weeks) with samples collected between 3 and 43 days of age
R2
113
24–35 weeks corrected age
t1:15 t1:16 t1:17 t1:18 t1:19 t1:20
Gender Reference Interval
U
112 participants with 676 results for this 23–29 weeks gestation with sampling at 1, 4, 7, 14, group overall 21, 28 and 42 days post-birth
N
C
DHEAS
E2
Progesterone
t1:23
Free thyroxine
M&F
24–29 weeks gestation (median = 28 weeks) with samples collected between 3 and 43 days of age (median corrected gestational age = 31 weeks at time of sample collection) 24–35 weeks corrected age 24–29 weeks gestation (median = 28 weeks) with samples collected between 3 and 43 days of age (median corrected gestational age = 31 weeks at time of sample collection) 24–35 weeks corrected age
F
O
Steroid and thyroid hormones Cortisol R1 112 participants with 637 to 679 results (analyte dependent) across the group
t1:22
t1:25
Preterm group
R
R
E
C
T
Prolactin
t1:21
t1:24
Repository n
23–29 weeks gestation with sampling at 1, 4, 7, 14, 21, 28 and 42 days post-birth
E
Analyser
Levels highest during the first week of life. Reference intervals at day 7 of life are: 7–121 mIU/L for babies born between 23 and 26 weeks gestation; and 8–188 mIU/L for babies born between 27 and 29 weeks gestation. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 1.6–167.0 IU/L (median 49.6 IU/L).
Siemens — Centaur
Based on first sample collected from each participant: 3.7–N200 IU/L. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 0.3–54.4 IU/L (median 7.5 IU/L).
Roche — Cobas 8000 e602 Siemens — Centaur
Based on first sample collected from each participant: 0.3–134.0 IU/L. Based on first sample collected from each participant: 374–5694 mIU/L. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 0.3–2.6 IU/L (median 0.9 IU/L). Based on first sample collected from each participant: 0.2–4.2 IU/L. Data not log transformed due to sample size. Interim intervals developed by determination of the 2.5 and 97.5 percentiles which showed a range of: 0.1–8.5 IU/L (median 1.2 IU/L). Based on first sample collected from each participant: 0.1–10.9 IU/L. Based on first sample collected from each participant Based on first sample collected from each participant: 463–6037 mIU/L.
Roche — Cobas 8000 e602
D
M&F
Siemens — Immulite 2000
Siemens — Centaur
Roche — Cobas 8000 e602 Siemens — Centaur
Roche — Cobas 8000 e602
R.F. Greaves / Clinical Biochemistry xxx (2014) xxx–xxx
Please cite this article as: Greaves RF, Hormone profiles in extremely preterm infants, Clin Biochem (2014), http://dx.doi.org/10.1016/ j.clinbiochem.2014.05.040
t1:1 t1:2
Q3
Siemens — Immulite 2000 Levels highest during the first week of life. Reference intervals at day 7 of life are: 63–801 nmol/L for babies born between 23 and 26 weeks gestation; and 70–594 nmol/L for babies born between 27 and 29 weeks gestation. Levels highest during the first week of life. Reference intervals at day 7 of life are: 3.3–19.4 μmol/L for babies born between 23 and 26 weeks Q4 gestation; and 2.4–11.5 μmol/L for babies born between 27 and 29 weeks Q5 gestation. Levels ranged from b73 pmol/L to 1626 pmol/L over the six weeks. Reference intervals at day 7 of life are: 87–359 pmol/L for babies born between 23 and 26 weeks gestation; and b73–347 pmol/L for babies born between 27 and 29 weeks gestation. Levels highest during the first week of life. Reference intervals at day 7 of life are: 5.1–38.2 nmol/L for babies born between 23 and 26 weeks gestation; and 4.2–25.2 nmol/L for babies born between 27 and 29 weeks gestation. Levels as low as 2.6 pmol/L for the first 28 days with the nadir at 7 days for the overall group. Reference intervals at day 7 of life are: b2.6–14.0 pmol/L for babies born between 23 and 26 weeks gestation; and 5.2–18.6 pmol/L for babies born between 27 and 29 weeks gestation.
P
DHEAS = dihydroepiandrostane sulphate; E2 = estradiol; FSH = follicle stimulating hormone; GH = growth hormone; LH = luteinizing hormone.
R O
O
F
Q6
R.F. Greaves / Clinical Biochemistry xxx (2014) xxx–xxx
216757; education grants from Serono and Pfizer; and supply of the immunoassay kits courtesy of Roche Diagnostics and Siemens.
81
References
82 83 84 85 86 87 88 89 90 103
[1] World Health Organization Media Centre Fact sheet No 363: Preterm Birth — updated 2013. Available at www.who.int/mediacentre/factsheets/fs363/en/ [accessed 1st April 2014]. [2] Blencowe H, Cousens S, Oestergaard MZ, Chou D, Moller A-B, Narwal R, et al. National, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic analysis and implications. Lancet 2012;379:2162–72. [3] National Health, Medical Research Council. Care around preterm birth. Clinical practice guidelines. Australian Government Publishing Service; 1997.
[4] Greaves R, Hunt R, Chiriano A, Zacharin M. LH and FSH levels in extreme prematurity: development of reference intervals. Pediatrics 2008;121:e574–80. [5] Greaves RF, Donath S, Hunt RW. Serum hormone profiles over the first six weeks of life for preterm infants born less than 30 weeks gestation. XIIth International Congress of Paediatric Laboratory Medicine Berlin 2011. Clin Biochem 2011;44:531. [6] Greaves R, Hunt R, Zacharin M. Transient anomalies in genital appearance in some extremely preterm female infants may be the result of foetal programming causing a surge in LH and the over activation of the pituitary–gonadal axis. Clin Endocrinol 2008;69:763–8. [7] Greaves R, Kanumakala S, Read A, Zacharin M. Genital abnormalities mimicking congenital adrenal hyperplasia in premature infants. J Paediatr Child Health 2004;40:233–6.
U
N C O
R
R
E
C
T
E
D
P
R O
O
F
79 Q7 80
3
Please cite this article as: Greaves RF, Hormone profiles in extremely preterm infants, Clin Biochem (2014), http://dx.doi.org/10.1016/ j.clinbiochem.2014.05.040
91 92 93 94 95 96 97 98 99 100 101 102
