- Email: [email protected]
Evaluation of a multiplex liquid chromatography-tandem mass spectrometry method for congenital adrenal hyperplasia in pediatric patients
Accepted Manuscript Evaluation of a Multiplex Liquid Chromatography-Tandem Mass Spectrometry Method for Congenital Adrenal Hyperplasia in Pediatric Patients Jing Cao, Marilyn Sonilal, Stephen M. Roper, Mahesheema Ali, Sridevi Devaraj PII: DOI: Reference:
S2376-9998(18)30013-8 https://doi.org/10.1016/j.clinms.2018.07.001 CLINMS 38
To appear in:
Clinical Mass Spectrometry
Received Date: Revised Date: Accepted Date:
21 March 2018 22 June 2018 23 July 2018
Please cite this article as: J. Cao, M. Sonilal, S.M. Roper, M. Ali, S. Devaraj, Evaluation of a Multiplex Liquid Chromatography-Tandem Mass Spectrometry Method for Congenital Adrenal Hyperplasia in Pediatric Patients, Clinical Mass Spectrometry (2018), doi: https://doi.org/10.1016/j.clinms.2018.07.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of a Multiplex Liquid Chromatography-Tandem Mass Spectrometry Method for Congenital Adrenal Hyperplasia in Pediatric Patients Jing Cao a, b, Marilyn Sonilal b, Stephen M. Roper c, d, Mahesheema Ali a, b,, Sridevi Devaraj a, b * a
Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX
b
Department of Pathology, Texas Children’s Hospital, Houston, TX
c
Washington University School of Medicine, St Louis, MO
d
St. Louis Children’s Hospital, St Louis, MO
*Corresponding author Sridevi Devaraj, PhD, DABCC Professor of Pathology & Immunology Baylor College of Medicine Medical Director, Clinical Chemistry, Texas Children’s Hospital 6621 Fannin St, Houston, TX 77030 [email protected] Running title: Multiplexed adrenal steroid LC-MS/MS assay in CAH diagnosis Key words: congenital adrenal hyperplasia, adrenal steroid hormone, liquid chromatography, tandem mass spectrometry, interference
1 2 3
Abstract
4
patients with congenital adrenal hyperplasia (CAH) as confirmation of newborn screening (NBS)
5
or as initial diagnosis. This study reports the implementation of an adrenal steroid profiling
6
method with a turnaround time (TAT) of less than 24 hours using liquid chromatography and
7
tandem-mass spectrometry (LC-MS/MS). A lab-developed multiplex LC-MS/MS assay was used
8
to quantify levels of 11-deoxycortisol, cortisol, 17-hydroxy-progesterone (17-OHP),
9
androstenedione, and testosterone. Intra and interassay imprecision were found to be <10%.
10
Comparison with a reference laboratory revealed <20% bias for all 5 analytes and Deming
11
correlation coefficients >0.990. Linearity ranges were established from the lowest to upper limit
12
calibrator concentrations with 100- to 800-fold maximum dilution. Run to run carryover was
13
<0.1%, and acceptable matrix effect was observed (i.e., ion suppression enhancement <15%).
14
Compared to serum samples, ethylenediaminetetraacetic acid (EDTA) and heparin plasma had
15
large positive bias in the measurement of 11-deoxycortisol (62.2% and 60.2%, respectively) and
16
androstenedione (43.8% and 33.2%, respectively), while cortisol, 17-OHP and testosterone
17
showed less than 20% bias between sample types. Hemoglobin, bilirubin, or triglyceride
18
interference decreased 11-deoxycortisol measurement in EDTA plasma (-19.3%, -25.6%, and -
19
25.0%, respectively). Lipemia increased the measurement of testosterone by 28.9%. In summary,
20
our multiplexed LC-MS/MS method provided highly sensitive and specific measurement of
21
adrenal steroids. EDTA, heparin, hemolysis, icterus and/or lipemia may significantly impact
22
assay results and should be avoided. This method provides an effective strategy for improving
23
TAT in CAH testing and confirmation of NBS results.
24
List of Abbreviations: congenital adrenal hyperplasia (CAH), newborn screening (NBS),
25
turnaround time (TAT), liquid chromatography and tandem-mass spectrometry (LC-MS/MS),
26
17-hydroxy-progesterone (17-OHP), ethylenediaminetetraacetic acid (EDTA), 21-hydroxylase
27
deficiency (21OHyD), Limit of detection (LoD) and limit of quantification (LoQ), Clinical &
28
Laboratory Standards Institute (CLSI), ion suppression enhancement (ISE), coefficient of
29
variation (CV), 11-deoxycorticosterone (DOC).
Multiplexed adrenal steroid measurement provides critical diagnostic information for
30 31
Conflict of Interest Statement: The authors have no conflict of interest to disclose.
32
Introduction
33 34
The adrenal steroid hormone biosynthesis pathway gives rise to a number of structurally-
35
related glucocorticoids, mineralocorticoids, and sex steroids. Genetic defects in enzymes of this
36
pathway result in a group of autosomal recessive disorders named congenital adrenal hyperplasia
37
(CAH). Approximately 70% of infants affected by CAH have mineralocorticoid deficiency that
38
leads to salt-wasting syndrome. Severe forms may cause death in infancy due to shock,
39
hyponatremia and hyperkalemia. The most common form of CAH, 21-hydroxylase deficiency
40
(21OHyD), is included in the Newborn Screening (NBS) program in the United States. NBS
41
detects elevation in the preferred substrate of 21-hydroxylase, 17-hydroxy progesterone (17-
42
OHP), in dried heel stick blood spots using dissociation-enhanced lanthanide fluorescence
43
immunoassay. In order to confirm the diagnosis, measurement of multiple adrenal steroids, or
44
genetic testing of enzymes in the adrenal steroid pathway, is required [1].
45
The reasons for establishing an in-house multiplexed adrenal steroid panel in a pediatric and
46
women’s hospital are several fold. First, the turnaround time (TAT) of NBS usually exceeds 48-
47
72 hours [2] and, therefore, an in-house screening test is critical for prompt diagnosis of severe
48
salt-wasting CAH. Second, NBS testing has a low positive predictive value (about 1%) [3],
49
requiring a second-tier confirmatory test to assist in identifying patients with false positive
50
21OHyD screening results. Finally, mild forms of 21OHyD or CAH, other than 21OHyD, may
51
not show positive results during NBS, and patients may go on to develop adrenal steroid
52
disorders at an older age. A multiplexed adrenal steroid assay provides comprehensive testing of
53
CAH, including cosyntropin stimulation [4].
54
In this study, we report on the performance of a multiplexed adrenal steroid test panel
55
performed on a liquid chromatography tandem mass spectrometry (LC-MS/MS) platform, which
56
offers several advantages over immunoassays: 1) adrenal steroid hormones are structurally
57
similar to one another, and LC-MS/MS methods allow greater specificity [5], 2) hormones in the
58
cortisol synthesis pathways may be present at low concentrations, particularly in infants, and LC-
59
MS/MS methods are more sensitive [6], and 3) LC-MS/MS allows multiplexed analysis using
60
low sample volumes, which is especially useful for stimulation/suppression tests.
61
Preanalytical variables known to contribute to erroneous test results include: anticoagulants
62
and surfactants in blood collection tubes, lubricants from rubber stoppers, clot activators, and
63
separator gels [7-9]. We evaluated the impact of collection tube type on test results in effort to
64
demonstrate the practicability of implementing a second-tier CAH testing panel in a pediatric
65
hospital. The impact of sample integrity and interferences was also considered.
66 67
Materials and Methods
68 69 70
Experimental procedures Five steroids including 11-deoxycortisol, cortisol, androstenedione, 17-OHP, and
71
testosterone were measured using a laboratory-developed multiplex assay on a Nexera XR LC
72
system (Shimadzu, Kyoto, Japan) coupled with ABSciex Qtrap 5500 mass spectrometer (Sciex,
73
Framingham, MA). 50 uL of internal standard (IS, Chromsystems Mass Chrom steroids IS mix)
74
was added to 300 uL of sample, calibrator and control. 50uL of methyl-tert-butyl-ether (MTBE)
75
was added to the blank tube and 1.5 mL of MTBE was added to sample tubes. After thorough
76
mixing, samples were centrifuged at 4000 rpm for 10 minutes. 1 mL of the upper organic layer
77
was transferred and evaporated to dryness under nitrogen at 45°C, and reconstituted with 100 uL
78
of loading reagent (90:10 water: methanol). 25uL of each sample was injected on to a reversed-
79
phase column (Kinetex 2.6 µm C18 100 A 100 x 3 mm; Phenomenex, Torrance, CA) with an
80
approximate run time of 8 minutes. The gradient elution was composed of water (solvent A) and
81
methanol (solvent B), starting with a stepwise gradient in the order of 10% B for first 3 minutes,
82
followed by 40% B for 4 minutes, then 70% B for 3 minutes followed by 80% B for 50 seconds,
83
then 95% B for 3 minutes and finally 10% B for 3 min re-equilibration step. The flow rate was
84
550 µL/min and steroids were eluted between 4-6 minutes.
85
The mass spectrometer spray ionization source was operated with the following settings:
86
curtain gas: 20 psi, nebulizer gas (GS1): 25 psi, heater gas (GS2): 50 psi, ion source: turbo spray,
87
ion spray voltage: 5500 V, collision gas: medium. Unit mass resolution was set as 0.7 atomic
88
mass units full-width at half height in Q1 and Q3. Transitions of the steroids
89
(precursor/quantifier ion/qualifier ion) were: 11-deoxycortisol (347/97/109 m/z), cortisol
90
(363/97/327 m/z), androstenedione (287/97/109 m/z), 17-OHP (331/97/109 m/z), and
91
testosterone (289/97/109 m/z). Analyst version 1.6.2 was used for the data acquisition, and
92
MultiQuantTM version 3.0.1 was used for peak area integration, regression analysis, and
93
quantitation. Acceptance criterion of the calibration curve was defined as R2 > 0.990. All analyte
94
ion ratios needed to be within + 20% of the mean ion ratios of the calibration curve. Percent
95
accuracy needed to be within + 20% of theoretical values for standard 1, and + 15% of
96
theoretical values for Standards 2 to 6. Percent accuracy for quality controls needed to be + 20%
97
of theoretical values. Retention time differences between quantifier and qualifier ion needed to
98
be <0.04 minutes. It was required that the deuterated IS and the analyte of interest of the same
99
molecule have the same retention times. IS heights needed to be within + 35% of the mean IS
100
heights of the calibration curve.
101 102
Validation studies
103
For within run precision analysis, 20 replicates for each of the three levels of quality control
104
samples were analyzed in a single run. For between run analysis, this same sample group was
105
analyzed three times per day for 10 days. In-house test results were compared with a reference
106
LC-MS/MS method for accuracy. Samples from the College of American Pathology proficiency
107
testing were also used to verify accuracy of the assay. Limit of detection (LoD) and limit of
108
quantification (LoQ) were assessed following the Clinical & Laboratory Standards Institute
109
(CLSI) guidelines [reference] CLSI EP17-A2: Evaluation of Detection Capability for Clinical
110
Laboratory Measurement Procedures, 2nd Edition [10] using blank loading reagent and
111
calibrators, respectively. The LoD was defined as the lowest concentration at which the signal-
112
to-noise ratio remained above 3, and the LoQ was defined as the lowest calibrator concentration
113
with a coefficient of variation (CV) below 20%. The linearity range was assessed using assay
114
calibrators. The highest calibrator was diluted with blank calibrator to establish the maximum
115
dilution factor and the clinical reportable ranges for each analyte. A blank sample was included
116
after the highest calibrator concentration to assess carryover in each run. Three solvent blank
117
injections at the beginning and the end of every run were included to assess the occurrence of
118
within and between run carryover. The ion suppression enhancement (ISE) of matrix effect was
119
assessed by comparing analyte or IS spiked in dilution buffer, or in post-extraction buffer at the
120
highest calibrator concentration. Signals of steroids other than the analyte, or IS spiked in the
121
ISE study, were examined to evaluate endogenous interference.
122 123 124
Tube type and interference studies CLSI GP34-A: 2010 Validation and Verification of Tubes for Venous and Capillary Blood
125
Specimen Collection; The CLSI approved guidelines [11] were followed to evaluate the
126
influence of collection tubes on test results. With institutional review board approval and
127
informed consent, blood samples from 4 apparently healthy adults were collected in red-top gel
128
tubes, purple top tubes with EDTA, and green top tubes with heparin. After centrifugation,
129
serum, EDTA plasma, and heparin plasma samples were obtained and used in the collection tube
130
type study. Pooled serum was used in the interference study. Interference from hemoglobin,
131
bilirubin, and triglycerides were obtained from the Assurance interference test kit (Sun
132
Diagnostics, New Gloucester, ME). Interferences were examined at the following
133
concentrations: 1000 mg/dL hemoglobin, 70 mg/dL bilirubin, and 3000 mg/dL triglycerides.
134 135 136
Data analysis Analytical performance parameters were analyzed using Excel (Microsoft, Seattle, WA) and
137
EP Evaluator (Data Innovations, Burlington, VT). Linear regression was used in accuracy and
138
linearity range analyses. For interference and collection tube studies, difference plots were used
139
to evaluate bias between groups. ANOVA and paired t-tests were used to compare group
140
differences.
141 142
Results
143 144
Table 1 shows the analytical parameters of each analyte in the multiplexed CAH test panel.
145
Within- and between-run precision of all 5 analytes were below 10%. LoQs were established at
146
the lowest calibrator concentration and were above the LoDs. Linearity ranges were established
147
from the lowest to upper limit calibrator concentrations. Dilution folds from 100 to 800 were
148
validated. Run to run carryover was <0.1%, and ISE was in the acceptable range (i.e., <15%).
149
Measured concentration of steroids other than the analyte, or IS spiked in the ISE study, were
150
below the LoQ and, thus, indicate minimal endogenous interferences. A Deming regression
151
versus the reference method yielded a correlation coefficient of 0.990, which meets the
152
acceptability criteria, using 22 to 25 samples that spanned the linearity range for each analyte
153
(Figure 1).
154
Figure 2 shows the impact of each collection tube on test results, using the red-top serum
155
sample as reference. A positive bias of >30% was observed for both EDTA and heparin plasma
156
in the measurement of 11-deoxycortisol (62.2% and 60.2%, respectively) and androstenedione
157
(43.8% and 33.2%) relative to serum. Compared to 11-deoxycortisol and androstenedione, less
158
bias was noted for both EDTA and heparin in cortisol (<5%), 17-OHP (<15%) and testosterone
159
(<10%) measurements.
160
The impact of interferences, including hemolysis, icterus and lipemia on multiplexed adrenal
161
steroid hormone measurement in EDTA plasma is presented in Figure 3. Our study revealed a
162
negative biases in analysis of 11-deoxycortisol from hemoglobin, unconjugated bilirubin, and
163
triglycerides (-19.3%, -25.6%, and -25.0%, respectively). In addition, lipemia increased the
164
measurement of testosterone by 28.9%.
165 166
Discussion
167 168
The classic form of CAH, also known as the severe form, occurs in about 0.0067% of births
169
worldwide, while the prevalence of the non-classic, or mild form, ranges from 0.1% to up to 5%,
170
depending on ethnic group [12]. In severe CAH with mineralocorticoid deficiency, salt wasting
171
crises in infants may be life-threatening, and, therefore, prompt diagnosis and treatment is
172
needed. State-funded NBS programs screen for the enzyme defect found in >90% of CAH,
173
21OHyD, but the low positive predictive value of this assay leads to many false positive results
174
[3]. The mild forms, however, are unlikely to be identified by NBS and patients are likely to
175
develop sex steroid-related disorders at an older age.
176
Besides 21OHyD, other forms of CAH include 11β-hydroxylase deficiency, 17α-
177
hydroxylase/17,20-lyase deficiency, and 3β-hydroxysteroid dehydrogenase deficiency[13].
178
Depending on the specific enzyme deficiency, increases or decreases in adrenal steroids are
179
expected. For example, diagnosis of 21OHyD is based on elevated levels of 17-OHP. In 17OHD,
180
11-deoxycorticosterone (DOC) and corticosterone are elevated with low cortisol, androgens, and
181
estrogens; progesterone is also elevated, while aldosterone and renin are suppressed [14]. The
182
diagnostic characteristic of isolated 17,20-lyase deficiency is an elevated ratio (>50) of
183
17OHP:androstenedione [15], with all downstream (19-carbon) steroids reduced. Confirmation
184
of adrenocortical insufficiency usually requires a cosyntropin stimulation test, utilizing an assay
185
of cortisol, 17-OHP and androstenedione that consumes minimal sample would be preferred. It
186
is desirable to implement a multiplexed adrenal steroid assay in a pediatric and women’s hospital
187
due to the need to rapidly diagnose severe CAH (particularly in boys who do not present with
188
ambiguous genitalia), to confirm or rule out CAH diagnoses from positive NBS findings, to
189
diagnose CAH other than 21OHyD, and to diagnose mild CAH in older children [16]. Key
190
steroids for CAH diagnosis include 11-deoxycortisol, cortisol, DOC, corticosterone, 17-OHP,
191
androstenedione, and testosterone. Automated immunoassays are available for these steroids, and
192
LC-MS/MS based method using either heel stick dried blood spot or serum samples have been
193
reported [17, 18].
194
The multiplexed LC-MS/MS adrenal steroid assay reported in this study uses 300uL of
195
blood and has a simplified sample preparation procedure. Most importantly, the TAT of CAH
196
profiling has improved from 72 hours or more (send-out) to an average of 23 hours since the
197
introduction of this assay in-house, which has had a dramatically positive impact on clinical care.
198
The assay demonstrated good precision, accuracy, and broad linearity ranges. We additionally
199
validated dilutions ranging from 100 to 800 fold to allow for even wider clinically reportable
200
ranges. Carry over was <0.1% and matrix effect was <15%. This assay has the potential to
201
include additional steroids, such as dehydroepiandrosterone and corticosterone, for which
202
development is currently underway.
203
Pediatric labs are often faced with limited blood sample volumes and/or comprised sample
204
quality due to issues that can arise during blood draw. While the volume of sample required for a
205
send out is typically 1 mL, our laboratory has developed a multiplex assay that requires only 300
206
uL, which further allowed additional tests to be performed on the same blood drawIn this study,
207
we demonstrated that 11-deoxycortisol and androstenedione were strongly affected versus the
208
other three analytes, when substituting serum with EDTA or heparin plasma (Figure 2). It is
209
suspected that the bias from EDTA and heparin plasma could be due to ion suppression. Our
210
results corroborate several other reports in the literature that addressed interference from blood
211
collection tube additives on steroid measurements by LC-MS/MS [7-9]. Additionally, lipemia
212
interference resulted in >10% bias for three of the five analytes, (i.e., 11-deoxycortisol,
213
androstenedione and testosterone), while hemolysis and icterus interferences resulted in a >10%
214
bias for 11-deoxycortisol (Figure 3). We are planning to include additional exogenous and
215
endogenous interference candidates in future studies in order to more fully understand the
216
potential components of result bias.
217
In conclusion, our study addressed practical concerns for implementing an in-house CAH
218
steroid assay. We found that a sample volume of 300 µL can be sufficient to run multiplexed
219
adrenal steroid tests; TAT within a day can be achieved, which is a significant reduction over
220
send-out testing; serum is the preferred sample matrix and interferences from hemolysis, icterus
221
and lipemia should be avoided. We encourage other pediatric hospitals to consider adopting an
222
in-house multiplex adrenal steroid assay for its capacity to serve both as a second-tier CAH
223
diagnosis tool to behind NBS, and a to timely provider of initial CAH diagnoses.
224 225
Acknowledgement
226
Ching Nan Ou Fellowship in Clinical Chemistry at Texas Children’s Hospital References
227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
1. 2. 3. 4. 5.
6. 7. 8.
9. 10.
Speiser, P.W., et al., Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 2010. 95(9): p. 4133-60. TDSHS. https://www.dshs.texas.gov/newborn/default.shtm. [cited 2018; Available from: https://www.dshs.texas.gov/newborn/default.shtm. Kwon, C. and P.M. Farrell, The magnitude and challenge of false-positive newborn screening test results. Arch Pediatr Adolesc Med, 2000. 154(7): p. 714-8. Kushnir, M.M., et al., Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories. Clin Biochem, 2011. 44(1): p. 77-88. Minutti, C.Z., et al., Steroid profiling by tandem mass spectrometry improves the positive predictive value of newborn screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab, 2004. 89(8): p. 3687-93. Stanczyk, F.Z. and N.J. Clarke, Advantages and challenges of mass spectrometry assays for steroid hormones. J Steroid Biochem Mol Biol, 2010. 121(3-5): p. 491-5. Bowen, R.A. and A.T. Remaley, Interferences from blood collection tube components on clinical chemistry assays. Biochem Med (Zagreb), 2014. 24(1): p. 31-44. Shi, R.Z., H.H. van Rossum, and R.A. Bowen, Serum testosterone quantitation by liquid chromatography-tandem mass spectrometry: interference from blood collection tubes. Clin Biochem, 2012. 45(18): p. 1706-9. Fang, Y., et al., GJB2 as Well as SLC26A4 Gene Mutations are Prominent Causes for Congenital Deafness. Cell Biochem Biophys, 2015. 73(1): p. 41-4. CLSI, Evaluation of Detection Capability for Clinical Laboratory Measurement
Procedures., in Clinical & Laboratory Standards Institute EP17-A2. 2012. 11. CLSI, Validation and Verification of Tubes for Venous and Capillary Blood Specimen Collection., in CLSI GP34-A. 2010. 12. Merke, D. and M. Kabbani, Congenital adrenal hyperplasia: epidemiology, management and practical drug treatment. Paediatr Drugs, 2001. 3(8): p. 599-611. 13. White, P.C. and P.W. Speiser, Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev, 2000. 21(3): p. 245-91. 14. Kater, C.E. and E.G. Biglieri, Disorders of steroid 17 alpha-hydroxylase deficiency. Endocrinol Metab Clin North Am, 1994. 23(2): p. 341-57. 15. Geller, D.H., et al., The genetic and functional basis of isolated 17,20-lyase deficiency. Nat Genet, 1997. 17(2): p. 201-5.
259 260 261 262 263 264 265
16. 17.
18.
Soldin, S.J. and O.P. Soldin, Steroid hormone analysis by tandem mass spectrometry. Clin Chem, 2009. 55(6): p. 1061-6. Kulle, A.E., et al., LC-MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers. Eur J Endocrinol, 2015. 173(4): p. 517-24. Boelen, A., et al., Determination of a steroid profile in heel prick blood using LC-MS/MS. Bioanalysis, 2016. 8(5): p. 375-84.
Table 1. Analytical parameters of multiplexed adrenal steroid assay. LoD: limit of detection, LoQ: limit of quantification. CRR: clinical reportable range. ISE: ion suppression enhancement. 11-deoxycortisol (ng/dL)
Cortisol (µg/dL)
17-OHP (ng/dL)
<5.1%
<3.5%
<6.1%
<3.5%
<3.9%
<6.2%
<6.2%
<6.2%
<8.6%
<6.2%
0.9970
0.9963
0.9973
0.9970
0.9967
4.4
0.3
2.5
4.5
1.3
LoQ
9.0
1.0
10.0
18.0
5.0
Linearity Range
9.0- 1390.0
1.0-28.8
10.0-1510
18.0- 1400
5.0- 1180
Maximum Dilution
200
800
200
100
200
Carryover
<0.1%
<0.1%
<0.1%
<0.1%
<0.1%
14.2%
2.4%
3.0%
8.8%
12.4%
Within-run Precision 1 (N=20) Between-run Precision 1 (N=30) Accuracy (R value) 2 (N=25) LoD
ISE
3
1
Data are shown as < the highest CV among three levels.
2
Deming regression correlation coefficients are shown.
3
Positive percent values indicate enhancement.
Androstenedione Testosterone (ng/dL) (ng/dL)
Figure 1. Scatter plots of adrenal steroids between the reference laboratory and Texas Children’s Hospital (TCH) laboratory. The red dotted line indicates fitted Deming regression, and the equations are displayed. The black dotted line indicates line of identity. Figure 2. The impact of collection tube type on multiplexed adrenal steroid hormone measurements. Reference sample tube type: red-top serum tube. Figure 3. The impact of hemolysis, icterus and lipemia interferences on multiplexed adrenal steroid hormone measurements. 1000 mg/dL hemoglobin, 70 mg/dL bilirubin, and 3000 mg/dL triglycerides were spiked in pooled serum sample and % difference from non-spiked pooled sample was calculated.
Evaluation of a Multiplex Liquid Chromatography-Tandem Mass Spectrometry Method for Congenital Adrenal Hyperplasia in Pediatric Patients
Highlights •
Multiplexed adrenal steroids measurements are critical for proper diagnosis of CAH.
•
This study evaluates measurement of 11-deoxycortisol, cortisol, 17-OHP, androstenedione, and testosterone.
•
The assay has advantages of small sample volume, simple sample preparation, and short turnaround time, which are in high demand in emergency settings and for cosyntropin stimulation tests.
•
Interference from hemolysis, icterus and lipemia, EDTA or heparin plasma as compared to serum samples, was found to significantly impact some assay results and should be avoided.
S2376-9998(18)30013-8 https://doi.org/10.1016/j.clinms.2018.07.001 CLINMS 38
To appear in:
Clinical Mass Spectrometry
Received Date: Revised Date: Accepted Date:
21 March 2018 22 June 2018 23 July 2018
Please cite this article as: J. Cao, M. Sonilal, S.M. Roper, M. Ali, S. Devaraj, Evaluation of a Multiplex Liquid Chromatography-Tandem Mass Spectrometry Method for Congenital Adrenal Hyperplasia in Pediatric Patients, Clinical Mass Spectrometry (2018), doi: https://doi.org/10.1016/j.clinms.2018.07.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of a Multiplex Liquid Chromatography-Tandem Mass Spectrometry Method for Congenital Adrenal Hyperplasia in Pediatric Patients Jing Cao a, b, Marilyn Sonilal b, Stephen M. Roper c, d, Mahesheema Ali a, b,, Sridevi Devaraj a, b * a
Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX
b
Department of Pathology, Texas Children’s Hospital, Houston, TX
c
Washington University School of Medicine, St Louis, MO
d
St. Louis Children’s Hospital, St Louis, MO
*Corresponding author Sridevi Devaraj, PhD, DABCC Professor of Pathology & Immunology Baylor College of Medicine Medical Director, Clinical Chemistry, Texas Children’s Hospital 6621 Fannin St, Houston, TX 77030 [email protected] Running title: Multiplexed adrenal steroid LC-MS/MS assay in CAH diagnosis Key words: congenital adrenal hyperplasia, adrenal steroid hormone, liquid chromatography, tandem mass spectrometry, interference
1 2 3
Abstract
4
patients with congenital adrenal hyperplasia (CAH) as confirmation of newborn screening (NBS)
5
or as initial diagnosis. This study reports the implementation of an adrenal steroid profiling
6
method with a turnaround time (TAT) of less than 24 hours using liquid chromatography and
7
tandem-mass spectrometry (LC-MS/MS). A lab-developed multiplex LC-MS/MS assay was used
8
to quantify levels of 11-deoxycortisol, cortisol, 17-hydroxy-progesterone (17-OHP),
9
androstenedione, and testosterone. Intra and interassay imprecision were found to be <10%.
10
Comparison with a reference laboratory revealed <20% bias for all 5 analytes and Deming
11
correlation coefficients >0.990. Linearity ranges were established from the lowest to upper limit
12
calibrator concentrations with 100- to 800-fold maximum dilution. Run to run carryover was
13
<0.1%, and acceptable matrix effect was observed (i.e., ion suppression enhancement <15%).
14
Compared to serum samples, ethylenediaminetetraacetic acid (EDTA) and heparin plasma had
15
large positive bias in the measurement of 11-deoxycortisol (62.2% and 60.2%, respectively) and
16
androstenedione (43.8% and 33.2%, respectively), while cortisol, 17-OHP and testosterone
17
showed less than 20% bias between sample types. Hemoglobin, bilirubin, or triglyceride
18
interference decreased 11-deoxycortisol measurement in EDTA plasma (-19.3%, -25.6%, and -
19
25.0%, respectively). Lipemia increased the measurement of testosterone by 28.9%. In summary,
20
our multiplexed LC-MS/MS method provided highly sensitive and specific measurement of
21
adrenal steroids. EDTA, heparin, hemolysis, icterus and/or lipemia may significantly impact
22
assay results and should be avoided. This method provides an effective strategy for improving
23
TAT in CAH testing and confirmation of NBS results.
24
List of Abbreviations: congenital adrenal hyperplasia (CAH), newborn screening (NBS),
25
turnaround time (TAT), liquid chromatography and tandem-mass spectrometry (LC-MS/MS),
26
17-hydroxy-progesterone (17-OHP), ethylenediaminetetraacetic acid (EDTA), 21-hydroxylase
27
deficiency (21OHyD), Limit of detection (LoD) and limit of quantification (LoQ), Clinical &
28
Laboratory Standards Institute (CLSI), ion suppression enhancement (ISE), coefficient of
29
variation (CV), 11-deoxycorticosterone (DOC).
Multiplexed adrenal steroid measurement provides critical diagnostic information for
30 31
Conflict of Interest Statement: The authors have no conflict of interest to disclose.
32
Introduction
33 34
The adrenal steroid hormone biosynthesis pathway gives rise to a number of structurally-
35
related glucocorticoids, mineralocorticoids, and sex steroids. Genetic defects in enzymes of this
36
pathway result in a group of autosomal recessive disorders named congenital adrenal hyperplasia
37
(CAH). Approximately 70% of infants affected by CAH have mineralocorticoid deficiency that
38
leads to salt-wasting syndrome. Severe forms may cause death in infancy due to shock,
39
hyponatremia and hyperkalemia. The most common form of CAH, 21-hydroxylase deficiency
40
(21OHyD), is included in the Newborn Screening (NBS) program in the United States. NBS
41
detects elevation in the preferred substrate of 21-hydroxylase, 17-hydroxy progesterone (17-
42
OHP), in dried heel stick blood spots using dissociation-enhanced lanthanide fluorescence
43
immunoassay. In order to confirm the diagnosis, measurement of multiple adrenal steroids, or
44
genetic testing of enzymes in the adrenal steroid pathway, is required [1].
45
The reasons for establishing an in-house multiplexed adrenal steroid panel in a pediatric and
46
women’s hospital are several fold. First, the turnaround time (TAT) of NBS usually exceeds 48-
47
72 hours [2] and, therefore, an in-house screening test is critical for prompt diagnosis of severe
48
salt-wasting CAH. Second, NBS testing has a low positive predictive value (about 1%) [3],
49
requiring a second-tier confirmatory test to assist in identifying patients with false positive
50
21OHyD screening results. Finally, mild forms of 21OHyD or CAH, other than 21OHyD, may
51
not show positive results during NBS, and patients may go on to develop adrenal steroid
52
disorders at an older age. A multiplexed adrenal steroid assay provides comprehensive testing of
53
CAH, including cosyntropin stimulation [4].
54
In this study, we report on the performance of a multiplexed adrenal steroid test panel
55
performed on a liquid chromatography tandem mass spectrometry (LC-MS/MS) platform, which
56
offers several advantages over immunoassays: 1) adrenal steroid hormones are structurally
57
similar to one another, and LC-MS/MS methods allow greater specificity [5], 2) hormones in the
58
cortisol synthesis pathways may be present at low concentrations, particularly in infants, and LC-
59
MS/MS methods are more sensitive [6], and 3) LC-MS/MS allows multiplexed analysis using
60
low sample volumes, which is especially useful for stimulation/suppression tests.
61
Preanalytical variables known to contribute to erroneous test results include: anticoagulants
62
and surfactants in blood collection tubes, lubricants from rubber stoppers, clot activators, and
63
separator gels [7-9]. We evaluated the impact of collection tube type on test results in effort to
64
demonstrate the practicability of implementing a second-tier CAH testing panel in a pediatric
65
hospital. The impact of sample integrity and interferences was also considered.
66 67
Materials and Methods
68 69 70
Experimental procedures Five steroids including 11-deoxycortisol, cortisol, androstenedione, 17-OHP, and
71
testosterone were measured using a laboratory-developed multiplex assay on a Nexera XR LC
72
system (Shimadzu, Kyoto, Japan) coupled with ABSciex Qtrap 5500 mass spectrometer (Sciex,
73
Framingham, MA). 50 uL of internal standard (IS, Chromsystems Mass Chrom steroids IS mix)
74
was added to 300 uL of sample, calibrator and control. 50uL of methyl-tert-butyl-ether (MTBE)
75
was added to the blank tube and 1.5 mL of MTBE was added to sample tubes. After thorough
76
mixing, samples were centrifuged at 4000 rpm for 10 minutes. 1 mL of the upper organic layer
77
was transferred and evaporated to dryness under nitrogen at 45°C, and reconstituted with 100 uL
78
of loading reagent (90:10 water: methanol). 25uL of each sample was injected on to a reversed-
79
phase column (Kinetex 2.6 µm C18 100 A 100 x 3 mm; Phenomenex, Torrance, CA) with an
80
approximate run time of 8 minutes. The gradient elution was composed of water (solvent A) and
81
methanol (solvent B), starting with a stepwise gradient in the order of 10% B for first 3 minutes,
82
followed by 40% B for 4 minutes, then 70% B for 3 minutes followed by 80% B for 50 seconds,
83
then 95% B for 3 minutes and finally 10% B for 3 min re-equilibration step. The flow rate was
84
550 µL/min and steroids were eluted between 4-6 minutes.
85
The mass spectrometer spray ionization source was operated with the following settings:
86
curtain gas: 20 psi, nebulizer gas (GS1): 25 psi, heater gas (GS2): 50 psi, ion source: turbo spray,
87
ion spray voltage: 5500 V, collision gas: medium. Unit mass resolution was set as 0.7 atomic
88
mass units full-width at half height in Q1 and Q3. Transitions of the steroids
89
(precursor/quantifier ion/qualifier ion) were: 11-deoxycortisol (347/97/109 m/z), cortisol
90
(363/97/327 m/z), androstenedione (287/97/109 m/z), 17-OHP (331/97/109 m/z), and
91
testosterone (289/97/109 m/z). Analyst version 1.6.2 was used for the data acquisition, and
92
MultiQuantTM version 3.0.1 was used for peak area integration, regression analysis, and
93
quantitation. Acceptance criterion of the calibration curve was defined as R2 > 0.990. All analyte
94
ion ratios needed to be within + 20% of the mean ion ratios of the calibration curve. Percent
95
accuracy needed to be within + 20% of theoretical values for standard 1, and + 15% of
96
theoretical values for Standards 2 to 6. Percent accuracy for quality controls needed to be + 20%
97
of theoretical values. Retention time differences between quantifier and qualifier ion needed to
98
be <0.04 minutes. It was required that the deuterated IS and the analyte of interest of the same
99
molecule have the same retention times. IS heights needed to be within + 35% of the mean IS
100
heights of the calibration curve.
101 102
Validation studies
103
For within run precision analysis, 20 replicates for each of the three levels of quality control
104
samples were analyzed in a single run. For between run analysis, this same sample group was
105
analyzed three times per day for 10 days. In-house test results were compared with a reference
106
LC-MS/MS method for accuracy. Samples from the College of American Pathology proficiency
107
testing were also used to verify accuracy of the assay. Limit of detection (LoD) and limit of
108
quantification (LoQ) were assessed following the Clinical & Laboratory Standards Institute
109
(CLSI) guidelines [reference] CLSI EP17-A2: Evaluation of Detection Capability for Clinical
110
Laboratory Measurement Procedures, 2nd Edition [10] using blank loading reagent and
111
calibrators, respectively. The LoD was defined as the lowest concentration at which the signal-
112
to-noise ratio remained above 3, and the LoQ was defined as the lowest calibrator concentration
113
with a coefficient of variation (CV) below 20%. The linearity range was assessed using assay
114
calibrators. The highest calibrator was diluted with blank calibrator to establish the maximum
115
dilution factor and the clinical reportable ranges for each analyte. A blank sample was included
116
after the highest calibrator concentration to assess carryover in each run. Three solvent blank
117
injections at the beginning and the end of every run were included to assess the occurrence of
118
within and between run carryover. The ion suppression enhancement (ISE) of matrix effect was
119
assessed by comparing analyte or IS spiked in dilution buffer, or in post-extraction buffer at the
120
highest calibrator concentration. Signals of steroids other than the analyte, or IS spiked in the
121
ISE study, were examined to evaluate endogenous interference.
122 123 124
Tube type and interference studies CLSI GP34-A: 2010 Validation and Verification of Tubes for Venous and Capillary Blood
125
Specimen Collection; The CLSI approved guidelines [11] were followed to evaluate the
126
influence of collection tubes on test results. With institutional review board approval and
127
informed consent, blood samples from 4 apparently healthy adults were collected in red-top gel
128
tubes, purple top tubes with EDTA, and green top tubes with heparin. After centrifugation,
129
serum, EDTA plasma, and heparin plasma samples were obtained and used in the collection tube
130
type study. Pooled serum was used in the interference study. Interference from hemoglobin,
131
bilirubin, and triglycerides were obtained from the Assurance interference test kit (Sun
132
Diagnostics, New Gloucester, ME). Interferences were examined at the following
133
concentrations: 1000 mg/dL hemoglobin, 70 mg/dL bilirubin, and 3000 mg/dL triglycerides.
134 135 136
Data analysis Analytical performance parameters were analyzed using Excel (Microsoft, Seattle, WA) and
137
EP Evaluator (Data Innovations, Burlington, VT). Linear regression was used in accuracy and
138
linearity range analyses. For interference and collection tube studies, difference plots were used
139
to evaluate bias between groups. ANOVA and paired t-tests were used to compare group
140
differences.
141 142
Results
143 144
Table 1 shows the analytical parameters of each analyte in the multiplexed CAH test panel.
145
Within- and between-run precision of all 5 analytes were below 10%. LoQs were established at
146
the lowest calibrator concentration and were above the LoDs. Linearity ranges were established
147
from the lowest to upper limit calibrator concentrations. Dilution folds from 100 to 800 were
148
validated. Run to run carryover was <0.1%, and ISE was in the acceptable range (i.e., <15%).
149
Measured concentration of steroids other than the analyte, or IS spiked in the ISE study, were
150
below the LoQ and, thus, indicate minimal endogenous interferences. A Deming regression
151
versus the reference method yielded a correlation coefficient of 0.990, which meets the
152
acceptability criteria, using 22 to 25 samples that spanned the linearity range for each analyte
153
(Figure 1).
154
Figure 2 shows the impact of each collection tube on test results, using the red-top serum
155
sample as reference. A positive bias of >30% was observed for both EDTA and heparin plasma
156
in the measurement of 11-deoxycortisol (62.2% and 60.2%, respectively) and androstenedione
157
(43.8% and 33.2%) relative to serum. Compared to 11-deoxycortisol and androstenedione, less
158
bias was noted for both EDTA and heparin in cortisol (<5%), 17-OHP (<15%) and testosterone
159
(<10%) measurements.
160
The impact of interferences, including hemolysis, icterus and lipemia on multiplexed adrenal
161
steroid hormone measurement in EDTA plasma is presented in Figure 3. Our study revealed a
162
negative biases in analysis of 11-deoxycortisol from hemoglobin, unconjugated bilirubin, and
163
triglycerides (-19.3%, -25.6%, and -25.0%, respectively). In addition, lipemia increased the
164
measurement of testosterone by 28.9%.
165 166
Discussion
167 168
The classic form of CAH, also known as the severe form, occurs in about 0.0067% of births
169
worldwide, while the prevalence of the non-classic, or mild form, ranges from 0.1% to up to 5%,
170
depending on ethnic group [12]. In severe CAH with mineralocorticoid deficiency, salt wasting
171
crises in infants may be life-threatening, and, therefore, prompt diagnosis and treatment is
172
needed. State-funded NBS programs screen for the enzyme defect found in >90% of CAH,
173
21OHyD, but the low positive predictive value of this assay leads to many false positive results
174
[3]. The mild forms, however, are unlikely to be identified by NBS and patients are likely to
175
develop sex steroid-related disorders at an older age.
176
Besides 21OHyD, other forms of CAH include 11β-hydroxylase deficiency, 17α-
177
hydroxylase/17,20-lyase deficiency, and 3β-hydroxysteroid dehydrogenase deficiency[13].
178
Depending on the specific enzyme deficiency, increases or decreases in adrenal steroids are
179
expected. For example, diagnosis of 21OHyD is based on elevated levels of 17-OHP. In 17OHD,
180
11-deoxycorticosterone (DOC) and corticosterone are elevated with low cortisol, androgens, and
181
estrogens; progesterone is also elevated, while aldosterone and renin are suppressed [14]. The
182
diagnostic characteristic of isolated 17,20-lyase deficiency is an elevated ratio (>50) of
183
17OHP:androstenedione [15], with all downstream (19-carbon) steroids reduced. Confirmation
184
of adrenocortical insufficiency usually requires a cosyntropin stimulation test, utilizing an assay
185
of cortisol, 17-OHP and androstenedione that consumes minimal sample would be preferred. It
186
is desirable to implement a multiplexed adrenal steroid assay in a pediatric and women’s hospital
187
due to the need to rapidly diagnose severe CAH (particularly in boys who do not present with
188
ambiguous genitalia), to confirm or rule out CAH diagnoses from positive NBS findings, to
189
diagnose CAH other than 21OHyD, and to diagnose mild CAH in older children [16]. Key
190
steroids for CAH diagnosis include 11-deoxycortisol, cortisol, DOC, corticosterone, 17-OHP,
191
androstenedione, and testosterone. Automated immunoassays are available for these steroids, and
192
LC-MS/MS based method using either heel stick dried blood spot or serum samples have been
193
reported [17, 18].
194
The multiplexed LC-MS/MS adrenal steroid assay reported in this study uses 300uL of
195
blood and has a simplified sample preparation procedure. Most importantly, the TAT of CAH
196
profiling has improved from 72 hours or more (send-out) to an average of 23 hours since the
197
introduction of this assay in-house, which has had a dramatically positive impact on clinical care.
198
The assay demonstrated good precision, accuracy, and broad linearity ranges. We additionally
199
validated dilutions ranging from 100 to 800 fold to allow for even wider clinically reportable
200
ranges. Carry over was <0.1% and matrix effect was <15%. This assay has the potential to
201
include additional steroids, such as dehydroepiandrosterone and corticosterone, for which
202
development is currently underway.
203
Pediatric labs are often faced with limited blood sample volumes and/or comprised sample
204
quality due to issues that can arise during blood draw. While the volume of sample required for a
205
send out is typically 1 mL, our laboratory has developed a multiplex assay that requires only 300
206
uL, which further allowed additional tests to be performed on the same blood drawIn this study,
207
we demonstrated that 11-deoxycortisol and androstenedione were strongly affected versus the
208
other three analytes, when substituting serum with EDTA or heparin plasma (Figure 2). It is
209
suspected that the bias from EDTA and heparin plasma could be due to ion suppression. Our
210
results corroborate several other reports in the literature that addressed interference from blood
211
collection tube additives on steroid measurements by LC-MS/MS [7-9]. Additionally, lipemia
212
interference resulted in >10% bias for three of the five analytes, (i.e., 11-deoxycortisol,
213
androstenedione and testosterone), while hemolysis and icterus interferences resulted in a >10%
214
bias for 11-deoxycortisol (Figure 3). We are planning to include additional exogenous and
215
endogenous interference candidates in future studies in order to more fully understand the
216
potential components of result bias.
217
In conclusion, our study addressed practical concerns for implementing an in-house CAH
218
steroid assay. We found that a sample volume of 300 µL can be sufficient to run multiplexed
219
adrenal steroid tests; TAT within a day can be achieved, which is a significant reduction over
220
send-out testing; serum is the preferred sample matrix and interferences from hemolysis, icterus
221
and lipemia should be avoided. We encourage other pediatric hospitals to consider adopting an
222
in-house multiplex adrenal steroid assay for its capacity to serve both as a second-tier CAH
223
diagnosis tool to behind NBS, and a to timely provider of initial CAH diagnoses.
224 225
Acknowledgement
226
Ching Nan Ou Fellowship in Clinical Chemistry at Texas Children’s Hospital References
227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
1. 2. 3. 4. 5.
6. 7. 8.
9. 10.
Speiser, P.W., et al., Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 2010. 95(9): p. 4133-60. TDSHS. https://www.dshs.texas.gov/newborn/default.shtm. [cited 2018; Available from: https://www.dshs.texas.gov/newborn/default.shtm. Kwon, C. and P.M. Farrell, The magnitude and challenge of false-positive newborn screening test results. Arch Pediatr Adolesc Med, 2000. 154(7): p. 714-8. Kushnir, M.M., et al., Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories. Clin Biochem, 2011. 44(1): p. 77-88. Minutti, C.Z., et al., Steroid profiling by tandem mass spectrometry improves the positive predictive value of newborn screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab, 2004. 89(8): p. 3687-93. Stanczyk, F.Z. and N.J. Clarke, Advantages and challenges of mass spectrometry assays for steroid hormones. J Steroid Biochem Mol Biol, 2010. 121(3-5): p. 491-5. Bowen, R.A. and A.T. Remaley, Interferences from blood collection tube components on clinical chemistry assays. Biochem Med (Zagreb), 2014. 24(1): p. 31-44. Shi, R.Z., H.H. van Rossum, and R.A. Bowen, Serum testosterone quantitation by liquid chromatography-tandem mass spectrometry: interference from blood collection tubes. Clin Biochem, 2012. 45(18): p. 1706-9. Fang, Y., et al., GJB2 as Well as SLC26A4 Gene Mutations are Prominent Causes for Congenital Deafness. Cell Biochem Biophys, 2015. 73(1): p. 41-4. CLSI, Evaluation of Detection Capability for Clinical Laboratory Measurement
Procedures., in Clinical & Laboratory Standards Institute EP17-A2. 2012. 11. CLSI, Validation and Verification of Tubes for Venous and Capillary Blood Specimen Collection., in CLSI GP34-A. 2010. 12. Merke, D. and M. Kabbani, Congenital adrenal hyperplasia: epidemiology, management and practical drug treatment. Paediatr Drugs, 2001. 3(8): p. 599-611. 13. White, P.C. and P.W. Speiser, Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev, 2000. 21(3): p. 245-91. 14. Kater, C.E. and E.G. Biglieri, Disorders of steroid 17 alpha-hydroxylase deficiency. Endocrinol Metab Clin North Am, 1994. 23(2): p. 341-57. 15. Geller, D.H., et al., The genetic and functional basis of isolated 17,20-lyase deficiency. Nat Genet, 1997. 17(2): p. 201-5.
259 260 261 262 263 264 265
16. 17.
18.
Soldin, S.J. and O.P. Soldin, Steroid hormone analysis by tandem mass spectrometry. Clin Chem, 2009. 55(6): p. 1061-6. Kulle, A.E., et al., LC-MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers. Eur J Endocrinol, 2015. 173(4): p. 517-24. Boelen, A., et al., Determination of a steroid profile in heel prick blood using LC-MS/MS. Bioanalysis, 2016. 8(5): p. 375-84.
Table 1. Analytical parameters of multiplexed adrenal steroid assay. LoD: limit of detection, LoQ: limit of quantification. CRR: clinical reportable range. ISE: ion suppression enhancement. 11-deoxycortisol (ng/dL)
Cortisol (µg/dL)
17-OHP (ng/dL)
<5.1%
<3.5%
<6.1%
<3.5%
<3.9%
<6.2%
<6.2%
<6.2%
<8.6%
<6.2%
0.9970
0.9963
0.9973
0.9970
0.9967
4.4
0.3
2.5
4.5
1.3
LoQ
9.0
1.0
10.0
18.0
5.0
Linearity Range
9.0- 1390.0
1.0-28.8
10.0-1510
18.0- 1400
5.0- 1180
Maximum Dilution
200
800
200
100
200
Carryover
<0.1%
<0.1%
<0.1%
<0.1%
<0.1%
14.2%
2.4%
3.0%
8.8%
12.4%
Within-run Precision 1 (N=20) Between-run Precision 1 (N=30) Accuracy (R value) 2 (N=25) LoD
ISE
3
1
Data are shown as < the highest CV among three levels.
2
Deming regression correlation coefficients are shown.
3
Positive percent values indicate enhancement.
Androstenedione Testosterone (ng/dL) (ng/dL)
Figure 1. Scatter plots of adrenal steroids between the reference laboratory and Texas Children’s Hospital (TCH) laboratory. The red dotted line indicates fitted Deming regression, and the equations are displayed. The black dotted line indicates line of identity. Figure 2. The impact of collection tube type on multiplexed adrenal steroid hormone measurements. Reference sample tube type: red-top serum tube. Figure 3. The impact of hemolysis, icterus and lipemia interferences on multiplexed adrenal steroid hormone measurements. 1000 mg/dL hemoglobin, 70 mg/dL bilirubin, and 3000 mg/dL triglycerides were spiked in pooled serum sample and % difference from non-spiked pooled sample was calculated.
Evaluation of a Multiplex Liquid Chromatography-Tandem Mass Spectrometry Method for Congenital Adrenal Hyperplasia in Pediatric Patients
Highlights •
Multiplexed adrenal steroids measurements are critical for proper diagnosis of CAH.
•
This study evaluates measurement of 11-deoxycortisol, cortisol, 17-OHP, androstenedione, and testosterone.
•
The assay has advantages of small sample volume, simple sample preparation, and short turnaround time, which are in high demand in emergency settings and for cosyntropin stimulation tests.
•
Interference from hemolysis, icterus and lipemia, EDTA or heparin plasma as compared to serum samples, was found to significantly impact some assay results and should be avoided.