Pregnancy and the rise of the CDT levels, do we need to ...

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Pregnancy and the rise of the CDT levels, do we need to update

the cut-off values?

Versie: Eerste versie /eindversie (doorhalen wat niet van toepassing is)

Naam student: Laurens Haverkate

studentnummer: 11338636

Opdracht, versie: Scriptie

Inleverdatum: 23-10-2020

Aantal woorden: 5502

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Pregnancy and the rise of the CDT levels, do we need to update the cut-off values?

Abstract

Extensive consumption of alcohol during pregnancy can lead to severe complications

for the unborn child. Therefore an objective unbiased screening method is necessary

to detect excessive intake of alcohol during pregnancy. Carbohydrate-deficient

Transferrin (CDT) levels in serum have become a common biomarker for chronic

alcohol abuse. However, several studies showed that CDT levels might be elevated

during pregnancy, for reasons unrelated to alcohol intake. The aim of this study is to

investigate the changes in CDT values during pregnancy and to determine accurate,

trimester dependent reference values. To achieve this, 439 serum samples of 147

pregnant women that were followed during pregnancy and post-partum were

analysed by the high-performance liquid chromatography (HPLC) and N-Latex

immunophelometric assay. From these measurements, new reference values were

calculated. This study shows that there is a trimester-dependent increase of CDT levels

for both the HPLC and N-Latex immunoassay. The estimated reference values for CDT

analysis were 1.52%, 1.94%, 2.04% and 1.37% for trimester 1, 2 , 3 and four weeks post-

partum for the HPLC and 1.49%, 1.76%, 1.75% and 1.44% for the N-Latex

immunoassay. This study shows that CDT levels clearly increase during pregnancy

and that there is a need for higher cut-off values to prevent wrongful accusations of

chronic alcohol abuse.

Introduction

Extensive consumption of alcohol during pregnancy can lead to several health

complications for the unborn child. A daily disproportionate consumption of alcohol

has major implications on the development of the unborn child and could lead to

severe disorders, such as Fetal Alcohol Syndrome (FAS), miscarriage, intrauterine fetal

death and limited development of cognitive and behavioural characteristics (Gupta et

al.¸ 2016). As such it is important to refrain of alcohol during pregnancy. Considering

the stigma associated with drinking during pregnancy, pregnant women with

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drinking problems might not always share this information with healthcare providers.

Therefore, an objective unbiased screening method to detect excessive intake of alcohol

independent of the sincerity of the patient is needed.

Analysis methods for recently consumed alcohol comprise the detection of alcohol in

blood and/or breath, in which ethanol can be detected for +/- 12 hours (Helander et al.,

2002) and ethylglucoride (EtG), an alcohol breakdown product which can remain

elevated in urine for up to five days after drinking (Wurst et al., 2000). In order to detect

prolonged excessive ethanol intake, analysis of Carbohydrate-deficient Transferrin

(CDT) in serum has become the most common biomarker for detection of chronic

ethanol abuse (Howlett et al.¸ 2018). CDT is defined as the ratio of monosialo- and

disialo-transferrin (one or two sialic acids) to the total amount of transferrin and it is

known that a daily intake of 50-80 gram of ethanol for two weeks alters the

composition of the glycosylation-sites of Transferrin by elevating the CDT levels

(Stibler et al., 1991). CDT levels can remain elevated for 2-3 weeks, depending on the

amount of consumed alcohol and the period of time (Bortolotti et al., 2018) In more

extreme cases it lasts for approximately a month before the CDT levels are restored to

normal (Hock et al., 2005).

Transferrin, a glycoprotein for iron transport, contains two possible positions for

glycosylation. These sites contain two N-linked oligosaccharide chains that can be

bound by one or more sialic acid residues which results in six isoforms of transferrin.

The formation of Acetaldehyde out of alcohol leads to a higher hydrolysis of sialic acid

and less binding of carbohydrate chains, because sialyltransferase is inhibited (Stibler

et al.¸1991). Therefore, chronic alcohol intake is associated with an increase in

carbohydrate deficient transferrin isoforms, which makes CDT a key factor in

identification of alcoholics and in the subsequent procedures such as driving license

restrictions (NVKC, 2015).

Because of these severe consequences of CDT analysis, it is important that the results

of CDT analysis are clear and reliable to prevent incorrect accusations of chronic

ethanol abuse. The current cut-off values are determined for the general population,

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however previous reports suggest that these might not be suitable for pregnant women

(Bortolotti et al., 2020). Several studies described the physiological alterations in

pregnant women in whom increasing CDT values could lead to false-positive

identification of chronic alcohol abuse (Kenan et al., 2011; Bianchi et al., 2011). While

the intake of alcohol during pregnancy is highly discouraged, alcohol consumption by

pregnant women is still a problem in several countries, for instance in Europe almost

16% of the resident women consume alcohol during pregnancy (Mårdby et al., 2017)

and in the United States and in Canada 10%-15% of the pregnant women consumed

ethanol during pregnancy (Popova et al., 2017). Moreover, the study of Popova et al.

(2017) stated that 3% of the women were considered to partake in binge drinking

during pregnancy. These previous reports show that an objective parameter for

alcohol is necessary for pregnant women. However, this parameter should be

unbiased by physiological alterations during pregnancy to prevent incorrect

accusations of chronic alcohol consumption.

The aim of this study is to investigate the changes in CDT values during pregnancy

and to determine accurate, trimester dependent reference values. Therefore, plasma

samples of 147 pregnant women will be analyzed by two analytically distinct

experimental approaches, the High-performance liquid chromatography (HPLC) and

the N-Latex CDT immunonephelometric assay. The HPLC method presented by

Helander et al. (2003) is considered as the reference method (Helander et al., 2003;

Delanghe et al.¸ 2007; Schellenberg et al., 2016).

Methods

Subjects and samples

Blood serum was collected from 147 during pregnancy in the first, second and third

trimester and one month post-partum. The CDT levels of the samples taken one month

post-partum are considered to represent the normal non-pregnant condition. Samples

were collected by the Amphia Hopsital in Breda and analyzed at the Meander Medical

Centre in Amersfoort. The sera were left-overs from previous research. Samples were

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stored at -70 ◦C until analysis. The participating women were considered as healthy

and their gestation as normal.

Methods

In this study the High-performance liquid chromatography (HPLC) protocol of the

Meander Medical Centre was used and this is based on the HPLC as described by

Helander et al. (2003). Before performing an analysis with the HPLC, 100 µl of each

sample was incubated for at least 4 hours after saturation with 10 µl FeNTA and

addition of 10 µl of a lipid precipitation mix, consisting of dextran sulfate (20 g/L) and

sodium chloride (1 mol/L). Subsequently, the samples were centrifuged (10 minutes at

14000 rpm) and 100 µl of the supernatant was brought into 400 µl Aquadest, which

was centrifuged again for 10 minutes at 14000 rpm. Afterwards, 450 ul of this

supernatant was pipetted in a microvial for analysis. The liquid flow was 1 ml/min.

The HPLC separates the distinct transferrin isoforms by using anion exchange

chromatography. Subsequently, the absorbance was measured at 470 nm.

The used set-up comprised an HPLC device from Shimadzu outfitted with an LC-

20AT prominence pump including a low-pressure gradient control valve FCV-

10ALvp, a DGU-14A degasser and a SIL-20AC autosampler. The used UV detector

was a UV-vis detector SPD-20AV prominence. To separate the distinct transferrin

glycoforms an Source® 15Q PE 4.6/100 anion-exchange chromatography column (GE

Healthcare, Uppsala, Sweden) was used.

Each run included three controls: two control samples for laboratory precision

(Clinchek-control serum, Recipe; via AKSA Medical), so-called Clinchek 1 (%CDT

range 1.06 – 1.76) and Clinchek 2 (%CDT range 2.92 – 4.39) and a pool of sera leftovers

of CDT analysis of the laboratory of the Meander Medical Centre (expected %CDT

range 1.62%-1.82%).

For the HPLC assay CDT is defined as the fraction of disialo-transferrin (as percent of

the total amount of transferrin). The CDT cut-off value for defining elevated %CDT

values is 1,7%.

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The chromatographs (N = 431) were visually inspected by using Shimadzu Class-agent

software for aberrant HPLC chromatographs such as Transferrin variants, di-

tribridging, icteric and hemolytic samples.

The second way to determine the %CDT levels of the serum samples was by

performing a N-Latex immunophelometric assay on the Atellica Neph 630

nephelometer (Siemens Healthineers). The used protocol of the Meander Medical

Centre is based on the general Manufacture factional settings as provided by Siemens

Healthineers and as described by Delanghe et al. (2007). At least 250 ul serum was

necessary for analysis. Furthermore, transferrin levels were measured by the N-Latex

immunoassay. CDT for the N-latex immunophelomtric assay is defined as the sum of

asialo-, monosialo- and disialotransferrin (relative to the total amount of transferrin).

In this assay a monoclonal antibody is used that is designed to bind specifically the

transferrin glycoforms without 1 or 2 complete N-glycans and glycoforms with empty

glycosylation sites, in other words, the asialo-, monosialo- and disialotransferrin

glycoforms. To enable a better comparison with the results of the HPLC, which defines

the DST fraction as CDT, and by using harmonization procedures from the

International Federation of Clinical Chemistry (IFCC), the output of the N-latex

immunoassay is converted by using the following formula, %CDT/IFCC = (N-Latex

%CDT - 0,97)/0,7 as reported by Delanghe et al. (2007). The cut-off value for elevated

CDT levels for the N-Latex immunoassay is 1.7% The used reagents and controls are

taken from the standard N-Latex CDT kit (Siemens Healthineers) and in addition a

serum pool, consisting of leftover sera from regular CDT analyses from the laboratory

of the Meander Medical Centre, was used as extra control (expected %CDT range

1.80%-2.20%).

Statistical analysis

The outcome of both experimental approaches was analyzed using Statistical Package

for the Social Sciences (SPSS) version 24. The distribution of each trimester was

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examined using the Shapiro-Wilk test (significance at p< 0.05), because this test is more

powerful than the Kolmogorov-Smirnov test (Steinskog et al. 2007; Ghasemi &

Zahediasl, 2012). The homogenity of the Variances was tested with the Levene’s test.

Since the pregnant women were followed during pregnancy, the samples of each

trimester and post-partum were considered dependent and therefore to analyze the

differences between the trimesters for CDT a one-way ANOVA and a Tukey’s post-

hoc test were performed at a significance level of p < 0.05 (Lee & Lee, 2018). Extra

attention was given to a subgroup of patients with a complete collection of samples

(samples present for all periods under investigation). To analyze the differences

between the trimesters of this group, the results were analyzed by using an one-way

ANOVA and a Tukey’s post-hoc test for the HPLC output and a Sign test for the N-

Latex immunoassay output (significance at p < 0.05).

Finally, the cut-off values were determined. To do so, the upper limit of the 95%

confidence interval, in other words the 97.5th percentile, was calculated following the

formula: µ + 1.96 * SD, whereby µ is the mean and SD the standard deviation of the

corresponding period.

Results

From 147 patients 123, 120, 100 and 96 serum samples from trimester 1, 2, 3 and post-

partum respectively were available for CDT analysis. Visual control of the output of

the HPLC and the N-Latex immunoassay led to exclusion of 6 samples, because of

Transferrin variants (N = 5). An extraordinary high CDT level (%CDT = 3.13 as

measured by the HPLC; %CDT = 2.54 as measured by the N-Latex immunoassay) was

observed in one sample with both assays in trimester 2 and since this was the only

sample of this patient, this sample was excluded, because it raised suspicion of chronic

alcohol consumption. Moreover, after data analysis it was considered an outlier (the

data and figures can be found in the supplemental data). One sample of trimester 1

did not contain enough serum for both experiments and was only analyzed in the

HPLC. In the end, the data consisted of 122, 117, 99 and 95 samples from trimester 1,

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2, 3 and post-partum for the HPLC and 122, 117, 99 and 95 samples for the N-Latex

immunoassay.

A summary of the results of the statistical analysis can be found in table 1. The Shapiro-

Wilk test showed that the Trimester 1, 2, 3 and the Post-Partum samples were normally

distributed for both methods at a significance of p<0.05. In the supplemental data can

be found that the assumption of equal variances was not violated (significance at

p<0.05).

Table 1. Descriptive statistical analysis of CDT levels in pregnant women in trimester 1, 2,3 and 4

weeks post-partum, measured by 1a. a high-performance liquid chromatography (HPLC) and 1b. a N-

Latex immunoassay.

1a. High-Performance liquid chromatography (HPLC)

HPLC Trimester 1 Trimester 2 Trimester 3 Post-partum

Sample size 122 117 99 95

Mean 1.11 1.5421 1.6243 1.0376

95% Confidence Interval for Mean 1.0728 1.5952 1.5824 1.0035

1.1472 1.5790 1.6663 1.0716

Median 1.15 1.55 1.6400 1.0500

Variance

0.043 0.041 0.044 0.028

Std. Deviation 0.20755 0.20151 0.21015 0.16713

Minimum 0.50 0.96 1.05 0.53

Maximum 1.59 1.94 2.11 1.53

Range 1.09 0.98 1.06 1.00

Interquartile Range

0.27 0.29 0.28 0.24

Skewness -0.439 -0.439 -0.244 -0.094

Kurtosis 0.275 -0.152 0.069 0.797

Shapiro-Wilk test for normal distribution P = 0.072 P = 0.076 P = 0.788 P = 0.664

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1b. N-Latex immunoassay N-Latex immunoassay Trimester 1 Trimester 2 Trimester 3 Post-partum

Sample size 121 117 99 95

Mean 1.0429 1.2470 1.2165 0.9860

95% Confidence Interval for Mean 1.0015 1.1995 1.1620 0.9371

1.0843 1.2945 1.2709 1.0349

Median 1.0600 1.21 1.21 0.96

Variance 0.053 0.067 0.074 0.058

Std. Deviation

0.22982 0.25954 0.27291 0.24010

Minimum 0.51 0.43 0.54 0.44

Maximum 1.69 1.89 1.92 1.77

Range 1.18 1.46 1.38 1.33

Interquartile Range 0.31 0.39 0.37 0.30

Skewness 0.145 0.159 0.188 0.397

Kurtosis 0.014 0.121 -0.004 0.811

Shapiro-Wilk test for normal distribution P = 0.694 P = 0.231 P = 0.856 P = 0.238

Table 2 shows an overview of the results of the one-way ANOVA and Tukey’s post-

hoc test of the HPLC and the N-Latex immunoassay (significance at p < 0.05).

During pregnancy the CDT values increase strongly compared to the post-partum

value and the CDT values in trimester 2 and 3 are significantly higher than those post-

partum (see table 2 and figure 1) with both assays. In trimester 1 the increase was only

modest and significant only for the HPLC assay.

Several samples are approaching or even exceeding the cut-off values for both

experimental approaches. These results are visualized in figure 1 as histograms (1a;

1b) and box plots (1c; 1d). In these figures, the red line at the histograms represent the

normal distribution and in the boxplot the horizontal line inside each box represents

the median, the box frame represents the interquartile range (IQR), the vertical lines

represent the range of the dataset, except the outliers and the red dotted line represents

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the current CDT cut-off value of 1.7%. Outliers are defined as the function of 1.5 of the

IQR.

Table 2. Results of testing differences in CDT levels between trimesters of pregnancy and post-partum

for significance by performing an one-way ANOVA – Tukey Post-Hoc test. 2a. High-performance

liquid chromatography (HPLC) and 2b. N-Latex immunoassay. The N-latex immunoassay output is

corrected to IFCC values.

One-way ANOVA – Tukey Post-Hoc test HPLC N-Latex immunoassay

Trimester 1 Trimester 2 P < 0.001 P < 0.001

Trimester 3 P < 0.001 P < 0.001

Post-partum P = 0.039 P = 0.348


Trimester 3 P= 0.013 P = 0.809

Post-partum P < 0.001 P < 0.001


Trimester 2 P = 0.013 P = 0.809

Post-partum P < 0.001 P < 0.001

Post-partum Trimester 1 P = 0.039 P = 0.348

Trimester 2 P < 0.001 P < 0.001

Trimester 3 P < 0.001 P < 0.001

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1a.

1b.

1c. 1d.

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Figure 1. CDT levels of each trimester of pregnancy and four weeks post-partum measured by a high-

performance liquid chromatography (HPLC) and N-Latex immunoassay visualized in Histograms (1a.

HPLC; 1b. N-Latex immunoassay) and box-plots (1c. HPLC; 1d. N-Latex immunoassay). The red line

at the histograms represent the normal distribution and in the boxplot the horizontal line inside each

box represents the median, the box frame represents the interquartile range (IQR), the vertical lines

represent the range of the dataset, except the outliersOutliers are defined as the function of 1.5 of the

IQR. The red dotted lines represent the current CDT cut-off value of 1.7%. The p-values are stated in

table 2.

The results of analysis of the subpopulation of patients with four available samples

(N = 53 for HPLC; N = 52 for N-Latex immunoassay), analyzed with an one-way

ANOVA and Tukey’s post-hoc test for the HPLC results and a Sign test for the N-Latex

immunoassay, can be found in Table 3 and are visualized in figure 2, 3 and 4 and as

can be seen CDT levels are elevated in trimester 1, 2 and 3 compared to post-partum.

In trimester 2 and 3 for both the HPLC and the N-Latex immunoassay the rise of the

CDT levels was significant. Trimester 1 was not significantly elevated in both

experimental approaches.

Table 3. Results of testing differences in CDT levels between trimesters of pregnancy and post-partum

for significance by performing an one-way ANOVA – Tukey Post-Hoc test for the 3a. High-performance

liquid chromatography (HPLC; N = 53) and a Sign test for the 3b. N-Latex immunoassay output (N =

52). This group receives extra attention since it possible to analyze the results during all distinct periods.

One-way ANOVA – Tukey Post-Hoc test

Sign test

(HPLC)

(N-Latex)

HPLC N-Latex immmunoassay


Trimester 3 P < 0.001 P < 0.001

Post-partum P = 0.125 P = 0.263


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2d.

Trimester 3 P= 0.312 P = 0.038

Post-partum P < 0.001 P < 0.001


Trimester 2 P = 0.312 P = 0.038

Post-partum P < 0.001 P < 0.001

Post-partum Trimester 1 P = 0.125 P = 0.263

Trimester 2 P < 0.001 P < 0.001

Trimester 3 P < 0.001 P < 0.001

2a. 2b.

2c.

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performance liquid chromatography (HPLC) and N-Latex immunoassay visualized in Histograms (2a.

HPLC; 2b. N-Latex immunoassay) and box-plots (2c. HPLC; 2d. N-Latex immunoassay). The red line

at the histograms represent the normal distribution and in the boxplot the horizontal line inside each

box represents the median, the box frame represents the interquartile range (IQR), the vertical lines

represent the range of the dataset, except the outliers and the red dotted line represents the current CDT

cut-off value of 1.7%. Outliers are defined as the function of 1.5 of the IQR. This group receives extra

attention since it possible to analyze the results during all distinct periods. P-values are stated in table

3.


performance liquid chromatography (HPLC). 53 women provided serum samples for CDT analysis at

every trimester and four weeks post-partum. The numbers 1, 2, 3 and 4 on the X-axis, represent

trimester 1, 2 3 and post-partum.

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Figure 4. CDT levels of each trimester of pregnancy and four weeks post-partum measured by a N-Latex

immunoassay. 53 women provided serum samples for CDT analysis at every trimester and four weeks

post-partum. The numbers 1, 2, 3 and 4 on the X-axis, represent trimester 1, 2 3 and post-partum.

Finally, trimester-dependent cut-off values were determined. The results of these

calculations can be found in table 4.

In the Netherlands the cut-off values used to determine excessive alcohol abuse take

the inter-laboratory and biological variation into account. Specifically, for the Dutch

situation an extra section is added for the Dutch approach.

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Table 4. Descriptive statistical analysis to determine CDT cut-off values for trimester 1, 2,3 and four

weeks post-partum (5a. HPLC; 5b. N-Latex immunoassay). The upper limit represents the 97.5th

percentile and is calculated with the formula: µ + 1.96 * SD, whereby µ is the mean and SD the standard

deviation of the corresponding period.

4a. High-performance liquid chromatography (HPLC)

HPLC Trimester 1 Trimester 2 Trimester

3

Post-partum

97.5th percentile 1.52% 1.94% 2.04% 1.37%

HPLC reference values determined

by Bortolotti et al. (2020)

1.45% 2.01% 2.05% -

Current cut-off value 1.7% 1.7% 1.7% 1.7%

Estimated cut-off value 1.7% 1.94% 2.04% 1.7%

4b. N-Latex immunoassay


97.5th percentile 1.49% 1.76% 1.75% 1.44%


Estimated cut-off value 1.7% 1.76% 1.75% 1.7%

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The CDT reference values in the Netherlands

In the Netherlands, a different approach is used to determine the cut-off value of elevated CDT. The international cut-

off value for elevated CDT of 1.7% is used, however a critical value is added for compensation of interlaboratory and

biological variation. Therefore, the cut-off value for elevated CDT in the Netherlands is 2.0%.

The formula for the cut-off value in the Netherlands is 97,5th percentile + critical difference. (NVKC, 2015) The upper

limit of the 95% confidence interval, in other words the 97.5th percentile, was determined following the formula: µ + 1.96

* SD, whereby µ is the mean and SD the standard deviation of the corresponding period. The formula for the critical

difference is: µ1 – µ0 = µ1 – µ0 = z * √[2] * √ [CVa,b^2 + CVb,w^2] * GW/100, where z is the amount of standard deviations

for the chosen interval (1.65 for the 95% confidence interval) CVa,b the analytical variation between laboratories (for

HPLC, EQUALIS = 8.8%, for N-Latex, Equalis = 6.6%), CVb,w the biological variance (4.7%; Helander et al.; 2003) and

GW is the involved limit-value, in this case the output of the 97.5th percentile. (NVKC, 2015) The results of these

calculations can be found in table 6.?-----

Table 5. Descriptive statistical analysis to determine CDT cut-off values for trimester 1, 2,3 and four weeks post-partum (5a. HPLC;

5b. N-Latex immunoassay). The upper limit represents the 97.5th percentile and is calculated with the formula: µ + 1.96 * SD,

whereby µ is the mean and SD the standard deviation of the corresponding period. The critical value is calculated with the formula

µ1 – µ0 = µ1 – µ0 = z * √[2] * √ [CVa,b^2 + CVb,w^2] * GW/100 in which z represents the amount of standard deviations for the

chosen interval (1.65 for the 95% confidence interval) CVa,b the analytical variation between laboratories (for HPLC, EQUALIS

= 8.8%, for N-Latex, Equalis = 6.6%), CVb,w the biological variance (4.7%) and GW is the involved limit-value, in this case the

output of the 97.5th percentile.

5a. High-performance liquid chromatography (HPLC)


97.5th percentile 1.52% 1.94% 2.04% 1.37%

Critical difference 0.35% 0.45% 0.47% 0.32%


New cut-off value 2.0% (1.87%*) 2.39% 2.51% 2.0% (1.69%*)

5b. N-Latex immunoassay


97.5th percentile 1.49% 1.76% 2.75% 1.44%

Critical difference 0.28% 0.33% 0.33% 0.27%


New cut-off value 2.6% (1.78%*) 2.6% (2.09%*) 2.6% (2.08%*) 2.6% (1.71%*)

*The estimated cut-off in this study, which is not exceeding the current cut-off

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Discussion

Extensive consumption of alcohol during pregnancy can lead to severe complications

for the unborn child (Gupta et al.¸ 2016). Therefore an objective unbiased screening

method is necessary to detect excessive intake of alcohol during pregnancy. There are

several possible biomarkers to detect alcohol intake, however for prolonged alcohol

consumption CDT turns out to be a suited and accurate biomarker, since it remains

elevated for 2-3 weeks after excessive alcohol consumption, enabling long-term

detection of chronic alcohol abuse (Howlett et al.¸ 2018; Stibler et al., 1991; Bortolotti et

al., 2018). Yet, it is observed that pregnant women may already have elevated CDT

levels, unrelated to alcohol intake (Kenan et al., 2011; Bianchi et al., 2011; Bortolotti et

al., 2020).

The aim of this study was to examine the CDT levels during pregnancy and to

determine new cut-off values for this population. 439 serum samples of 147 pregnant

women collected during the three trimesters of pregnancy and four weeks post-

partum were analysed by high-performance liquid chromatography (HPLC) and the

N-latex Immunoassay. A significant increase in CDT-levels was found with the HPLC

for trimester 1, 2 and 3 compared to the post-partum samples. In trimester 2 and 3, the

CDT levels were approaching and exceeding the cut-off value of 1.7%. To be more

specific, 27.4% of the samples in trimester 2 and 40.4% in trimester 3 had a %CDT of

1.7% or higher. No elevated samples were observed in trimester 1 and post-partum.

For the N-latex immunoassay similar results were observed, although the rise of CDT

levels in trimester 1 was not significant. Moreover, 4.3% of the samples in trimester 2,

5.1% in trimester 3 and 1.1% in post-partum had a %CDT of 1.7% or higher.

Furthermore, transferrin levels increased significantly in trimester 2 and 3 as measured

by the N-Latex immunoassay (supplemental data). This study confirms that CDT

levels are rising over time during pregnancy and that the current cut-off value of 1.7%

is not justified for trimester 2 and 3 and could lead to a false-positive identification of

extensive alcohol consumption. The estimated reference values for CDT analysis were

1.52%, 1.94%, 2.04% and 1.37% for trimester 1, 2 , 3 and four weeks post-partum for the

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HPLC and 1.49%, 1.76%, 1.75% and 1.44% for the N-Latex immunoassay. Considering

the harmonisation of the CDT assays, these results could be combined towards a

general CDT cut-off values during pregnancy: 1.7% for trimester 1, 1.92% for trimester

2, 2.04% for trimester 3 and 1.7% for four weeks post-partum. Note that such an

approach may not be justified because of the differences between the N-latex and

HPLC assay, which remain present after harmonisation (Jacobs et al., 2020).

The elevation of CDT levels during pregnancy was expected, since in previous

research of Kenan et al. (2011), Bianchi et al. (2011) and Bortolotti et al. (2020) increased

CDT levels in pregnant women were observed over time. Noteworthy, the previously

mentioned studies all used the HPLC for determining the CDT levels. Only one study

was found that performed N-Latex immunoassay for serum samples of women during

pregnancy, but this study of Niemelä et al. (2016) showed only a significant increase of

the CDT levels for women that consumed alcohol during pregnancy. No elevated CDT

levels were found in women who refrained from drinking alcohol during pregnancy

or in abstainers of alcohol (Niemelä et al., 2016). Our study used two distinct CDT

analysis methods, has a large sample size and provides an overview of the CDT levels

in the same pregnant women over time. The study of Bortolotti et al. (2020) is

interesting, because this study has a comparable aim and has a similar sample size, in

contrast to the smaller sample sizes of Kenan et al. (2011) and Bianchi et al. (2011).

However, the trend of elevation of CDT levels during pregnancy and the mean CDT

ratio of each study is similar or close to the observed results of this study. Kenan et al.

(2011) found mean %CDT levels of 1.07 and 1.61 in trimester 1 and 3, Bianchi et al.

(2011) found %CDT levels of 1.01, 1.30 and 1.53 in trimester 1, 2 and 3 and Bortolotti et

al. (2020) found %CDT levels of 1.07, 1.43 and 1.59 for trimester 1, 2 and 3, all measured

by the HPLC. Importantly, in contrast to our approach of using the post-partum levels

as baseline CDT values, Bortolotti et al. (2020) used a reference group. In our approach

we assumed that CDT levels would have returned to baseline values in one month

post-partum. Indeed, Bortolotti et al. (2018) and Hock et al. (2005) have shown that CDT

values return to normal post-partum. The excellent agreement of our post-partum

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results with results of the control group of Bortolotti et al. (2020) supports this

conclusion. The reference values estimated with a HPLC by Bortolotti (1.45%, 2.01%

and 2.05% for trimester 1, 2 and 3) are also very similar to the reference values of the

HPLC determined in this study. The differences for each trimester is 0.07 %CDT or

less. Overall, our study, in combination with those described in literature

demonstrated that the current CDT cut-off value is not adequate for pregnant women

and that a revision of the reference values is needed in this group.

The mechanism leading to elevated CDT levels during pregnancy is not clear.

However, this study also observed rising transferrin levels during pregnancy

(supplemental data) and previous research showed that human serum sialic acids are

dropping during pregnancy (Rajan et al.; 1983). The rising transferrin levels combined

with the dropping serum sialic acids levels may indicate an enhanced production of

transferrin with short carbohydrate chains. In general, rising transferrin levels during

pregnancy could result in altered levels of isoforms. This is supported by a study by

Rajan et al. (1983) which also observed that the sialyltransferase levels do not increase

during pregnancy. These observations suggest that a possible disbalance of available

transferrin, sialyltransferase and sialic acids could lead to reduced glycosylation of

transferrin.

Noteworthy is the difference between the HPLC and the N-Latex immunoassay,

despite harmonisation and standardisation procedures by the IFCC. This was already

observed in a study of Jacobs et al. (2020), which observed more variation between the

experimental approaches at low and high CDT levels. The results of this study likely

support this observation, since the discrepancy between the two analysis methods is

larger in trimester 2 and 3 compared to trimester 1 and post-partum. However, an

analysis of these different outputs is not performed in this study because a direct

method comparison was not the aim of this study. It is also remarkable that a study of

Niemelä et al. (2016) also showed that CDT levels did not increase during pregnancy

when measured with the N-Latex immunoassay. Although it remains unclear where

this variation comes from, it is important to realize that these detecting methods are

21

fundamentally different. The HPLC measures all glycoforms, including CDT (as

disialotransferrin) and the N-Latex immunophelometric assay detects the lack of

binding of monoclonal antibodies without one or two N-glycans. The HPLC is a very

robust method and it is possible that an immunoassay is susceptible to interference,

especially during physiological alterations of the blood composition during

pregnancy. The observed discrepancy between these two analysis methods in this

study, the study of Jacobs et al. (2020) and in the study of Niemelä et al. (2016), suggest

that there might be need for a revision of the harmonisation procedures, for example

the formula for the correction of the N-Latex immunoassay output to IFCC-CDT

values.

In addition, the distinct trends of both analysis methods is noteworthy. As can be seen

in figure 3 and 4, the CDT levels of many samples analysed with the N-Latex

immunoassay are decreasing after trimester 2, while the CDT levels of samples

measured by the HPLC are dropping after trimester 3. In general, detecting a visual

trend in the results of the N-Latex immunoassay is difficult. Another remarkable

outcome of this study is the variation in the p-values of the Shapiro-Wilk test for

normal distribution between the trimesters: the p-values of, for example, the HPLC

trimester 3 and post-partum are higher than the p-values of HPLC trimester 1 and 2

and also variation is seen in the skewness and the kurtosis. A possible explanation for

this could be that the rate of physiological alterations in women is probably higher at

the beginning of gestation leading to more variation.

The number of studies investigating CDT levels during pregnancy is small, while CDT

analysis plays a major role in the detection of chronic alcohol abuse. However, the

results of previous studies are mostly consistent in showing elevation of the CDT levels

during pregnancy. Although in most countries alcohol consumption during

pregnancy is discouraged, it is important to have an objective and unbiased detecting

method, but also to prevent false-positive accusations. Moreover, early detection of

chronic ethanol abuse could be important because of the severe implications for the

foetus. This study provided a better insight in the CDT levels during pregnancy and

22

showed that the current cut-off value for elevated CDT is not sufficient for pregnant

women and that there is need for trimester dependent cut-off values. Further study

could assess other CDT analysis methods, like capillary electrophoresis. It is also

recommended to acquire an improved understanding of CDT levels during

pregnancy. Additional studies will be needed to examine the possible mechanism that

is causing the elevated CDT levels. Suggestions for these studies are examining serum

sialyltransferase and sialic acids levels during pregnancy. This study shows that the

CDT levels are clearly increased during pregnancy and that there is a need for higher

cut-off values to prevent false positive detections of chronic alcohol abuse. Moreover,

this study demonstrates the importance of critically assessing the application of

general cut-off values and guidelines for specific subgroups. Indeed, for pregnant

women, the use of higher, trimester dependent CDT cut off values contributes to

improvements in the CDT policy and the corresponding judicial and medical

processes to prevent injustice.

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1c. 1d.

Supplemental data

Figure 1. Normal Q-Q plots and boxplots of trimester 2 of the HPLC analysis of a sample with (1a; 1c)

and without (1b; 1d) a remarkable high %CDT level of 3.13 (sample id is 140). This sample was

excluded, because of suspicion of chronic alcohol consumption and because it turned out to be an outlier.

The red line at the histograms represent the normal distribution and in the boxplot the horizontal line

inside each box represents the median, the box frame represents the interquartile range (IQR), the

vertical lines represent the range of the dataset, except the outliers. Outliers are defined as the function

of 1.5 of the IQR.

1a. 1b.

26

Normality plots of each trimester of each detecting method

HPLC Trimester 1

27

HPLC Trimester 2

HPLC Trimester 3

28

HPLC post-partum

29

N-Latex immunoassay trimester 1


30


31

N-Latex immunoassay post-partum

Statistical Analysis:

Test of homogenity of Variances Levene

statistic df1 df2 significance

HPLC 2.581 3 429 0.053

N-Latex immunoassay 1.244 3 428 0.293

32

Descriptives

HPLC

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum Maximum Lower Bound Upper Bound

1 122 1,1100 ,20755 ,01879 1,0728 1,1472 ,50 1,59

2 117 1,5421 ,20151 ,01863 1,5052 1,5790 ,96 1,94

3 99 1,6243 ,21015 ,02112 1,5824 1,6663 1,05 2,11

4 95 1,0376 ,16713 ,01715 1,0035 1,0716 ,53 1,53

Total 433 1,3285 ,32182 ,01547 1,2981 1,3589 ,50 2,11

Multiple Comparisons

Dependent Variable: HPLC

Tukey HSD

(I) Period (J) Period

Mean Difference

(I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

1 2 -,43205* ,02566 ,000 -,4982 -,3659

3 -,51434* ,02683 ,000 -,5835 -,4451

4 ,07242* ,02714 ,039 ,0024 ,1424

2 1 ,43205* ,02566 ,000 ,3659 ,4982

3 -,08229* ,02708 ,013 -,1521 -,0124

4 ,50447* ,02739 ,000 ,4338 ,5751

3 1 ,51434* ,02683 ,000 ,4451 ,5835

2 ,08229* ,02708 ,013 ,0124 ,1521

4 ,58676* ,02849 ,000 ,5133 ,6602

4 1 -,07242* ,02714 ,039 -,1424 -,0024

2 -,50447* ,02739 ,000 -,5751 -,4338

3 -,58676* ,02849 ,000 -,6602 -,5133

*. The mean difference is significant at the 0.05 level.

ANOVA

HPLC

Sum of Squares df Mean Square F Sig.

Between Groups 27,865 3 9,288 236,115 ,000

Within Groups 16,876 429 ,039

Total 44,742 432

33

Descriptives

Latex

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum Maximum Lower Bound Upper Bound

1 121 1,0429 ,22982 ,02089 1,0015 1,0843 ,51 1,69

2 117 1,2470 ,25954 ,02399 1,1995 1,2945 ,43 1,89

3 99 1,2165 ,27291 ,02743 1,1620 1,2709 ,54 1,92

4 95 ,9860 ,24010 ,02463 ,9371 1,0349 ,44 1,77

Total 432 1,1254 ,27287 ,01313 1,0996 1,1512 ,43 1,92

ANOVA

Latex

Sum of Squares df Mean Square F Sig.

Between Groups 5,221 3 1,740 27,721 ,000

Within Groups 26,870 428 ,063

Total 32,091 431

Multiple Comparisons

Dependent Variable: Latex

Tukey HSD

(I) Period (J) Period

Mean Difference

(I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

1 2 -,20412* ,03249 ,000 -,2879 -,1203

3 -,17357* ,03396 ,000 -,2611 -,0860

4 ,05689 ,03435 ,348 -,0317 ,1455

2 1 ,20412* ,03249 ,000 ,1203 ,2879

3 ,03054 ,03422 ,809 -,0577 ,1188

4 ,26101* ,03460 ,000 ,1718 ,3503

3 1 ,17357* ,03396 ,000 ,0860 ,2611

2 -,03054 ,03422 ,809 -,1188 ,0577

4 ,23046* ,03599 ,000 ,1377 ,3233

4 1 -,05689 ,03435 ,348 -,1455 ,0317

2 -,26101* ,03460 ,000 -,3503 -,1718

3 -,23046* ,03599 ,000 -,3233 -,1377

*. The mean difference is significant at the 0.05 level.

34

Transferrin levels measured by the N-Latex immunoassay

Transferrin levels were examined and it turned out that only trimester 2 was accordant

a normal distribution (p = 0.655). In contrary, the Shapiro-Wilk test showed that

trimester 1, 3 and post-partum did not correspond to a normal distribution, because

p<0.05. Therefore, a non-parametric test was needed for examination of the increasing

transferrin levels. A Sign test was applied to analyze the increase of the transferrin

concentrations and as can be found in table 6, transferrin levels are. This is visually

supported by figure 6.

Table 7. Results of testing differences in transferrin levels between trimesters of pregnancy and post-

partum for significance by performing a Sign test.

Tri 1 – tri2 Tri 1 – tri 3 Tri 1 - PP Tri 2 – tri 3 Tri 2 - PP Tri 3 - PP

P-value P<0.001

P < 0.001 0.094 P<0.001 P < 0.001 P < 0.001

Figure 7. Transferrin levels of each trimester of pregnancy and four weeks post-partum measured by a

N-Latex immunoassay visualized in a Histogram (3a) and a box-plot (3b) The red line at the histograms

represent the normal distribution and in the boxplot the horizontal line inside each box represents the

median, the box frame represents the interquartile range (IQR), the vertical lines represent the range of

the dataset. Outliers are defined as the function of 1.5 of the IQR. Palatino Linotype

7a. 7b.

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