A C TA Obstetricia et Gynecologica

AOGS M A I N R E S E A R C H A R T I C L E

Pharmacokinetics of 250 μg anti-D IgG in the third trimester

of pregnancy: an observational study
ELEONOR TIBLAD1 , AGNETA WIKMAN2 , ANDERS RANE3 , YVONNE JANSSON1 & MAGNUS WESTGREN1
Departments of 1 Obstetrics and Gynecology, 2 Immunology and Transfusion Medicine, and 3 Clinical Pharmacology,
Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden

Key words Abstract

Antenatal anti-D prophylaxis,
pharmacokinetics, anti-D immunoglobulin,
antenatal Rh prophylaxis, plasma
concentration
Correspondence
Eleonor Tiblad, Department of Obstetrics and
Gynecology, Karolinska University Hospital,
Huddinge, K47, S-141 86 Stockholm, Sweden.
Email: eleonor.tiblad@karolinska.se
Conflict of interest
The authors have stated explicitly that there
are no conflicts of interest in connection with
this article.
Please cite this article as: Tiblad E, Wikman A,
Rane A, Jansson Y, Westgren M.
Pharmacokinetics of 250 μg anti-D IgG in the
third trimester of pregnancy: an observational
study. Acta Obstet Gynecol Scand
2012;91:587–592.
Received: 8 November 2011
Accepted: 10 February 2012
Objective. We present a pharmacokinetic study evaluating a single intramuscular
dose of 250 μg anti-D immunoglobulin in the third trimester of pregnancy. The aim
of the study was to determine the kinetic profile and duration of detectable levels
of anti-D. Design. Prospective observational study. Setting. Antenatal outpatient
clinic. Population. Healthy Rhesus D (RhD)-negative pregnant women with an
RHD-positive fetus. Methods. Serial plasma anti-D quantitations following antenatal
administration of anti-D immunoglobulin were performed using flow cytometry.
Kinetic profiles for anti-D levels were generated from the concentration values
at predetermined sampling time points. The half-lives were calculated by linear
regression analysis. Main outcome measures. Time vs. concentration profile, half-
life and anti-D concentration ≥1 ng/mL close to term. Results. The maximal plasma
concentration of anti-D was usually seen at 3–10 days postinjection, with a median
value of 25 ng/mL. The half-life varied between individuals, with a median of
23 days. We found detectable levels of anti-D IgG within two weeks of parturition
in 11 of 12 women. Conclusions. The preparation of anti-D immunoglobulin used
in the present study, if administrated in pregnancy week 28–30, is associated with
detectable levels of anti-D in most women at the time of delivery. Although the
half-time is 23 days, it is uncertain whether all mothers have adequate anti-D
concentrations at term. Alternative strategies may be evaluated in the future, with
repeated administration of antenatal prophylaxis at term rather than conventional
postpartum administration of anti-D.

DOI: 10.1111/j.1600-0412.2012.01377.x Abbreviations: IgG, immunoglobulin G; RBC, red blood cell; RhD, Rhesus D; Clast,
last concentration measured; Cmax, maximal plasma concentration.

individuals can therefore not easily be translated to preg-

Introduction
Anti-D postpartum prophylaxis has dramatically reduced the
number of Rhesus D (RhD)-sensitized pregnancies globally.
Nevertheless, a significant number of women become sen-
sitized during an ongoing pregnancy owing to spontaneous
fetomaternal hemorrhage. Detectable levels of fetomaternal
hemorrhage occur in about 45% of pregnant women dur-
ing the third trimester (1). It has been shown in several
studies that routine antenatal anti-D prophylaxis to RhD-
negative women reduces the risk of sensitization significantly
(2–4). Pregnant women differ from non-pregnant individu-
als regarding distribution volumes and metabolism of drugs
(5–7). Results from pharmacokinetic studies in non-pregnant
nant women. There are several different brands of polyclonal
Key Message
Routine antenatal Rh prophylaxis in the third trimester of
pregnancy can prevent sensitization in Rhesus D (RhD)-
negative pregnant women. Plasma concentrations of anti-
D immunoglobulin in pregnant women after a single
intramuscular injection of 250 μg anti-D at gestational
age 28–30 weeks show a comparable pharmacokinetic
profile to previously evaluated anti-D immunoglobulin
preparations.


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C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 587–592 587
Pharmacokinetics of antenatal anti-D
anti-D immunoglobulin (IgG) preparations, and their use
varies between countries. Studies on 300 μg anti-D with the
one- or two-dose regimen, as well as on single doses of 200
and 100 μg anti-D using the two-dose regimen, have been
published and show comparable results (4,8–10). One study
on a 250 μg anti-D preparation in pregnant women was pub-
lished in 1982. Since then, the manufacturing procedure for
human plasma-derived polyclonal anti-D IgG has changed,
resulting in improved purity and substantially increased viral
safety (11), which might have changed the pharmacokinetic
properties of the immunoglobulins. Analytical methods have
also improved over the years, allowing for reliable quantita-
tion of antibody levels at lower plasma concentrations.
We present a pharmacokinetic study evaluating a single
intramuscular dose of 250 μg (1250 i.u.) polyclonal anti-D
IgG preparation in the third trimester of pregnancy. The aim
of the study was to determine the kinetic profile and duration
of detectable levels of anti-D.
Material and methods
We performed a prospective study with open label admin-
istration of 250 μg Rhesonativ R
(batch C006A8708; Oc-
tapharma, Stockholm, Sweden) in gestational week 28–30.
Sixteen RhD-negative women with a negative first and
second trimester alloantibody screening test were recruited.
They were all enrolled in an ongoing prospective study in
the Stockholm region based on first trimester determina-
tion of cell-free fetal DNA RHD genotyping in maternal
plasma. In that study, all RhD-negative women carrying an
RHD-positive fetus are offered antenatal anti-D prophylaxis
in gestational week 28–30. The RHD type of the fetus was
confirmed by umbilical cord serology at birth. All women
received oral and written information and provided written
consent to participate in the study.
Eligible participants were healthy women without previous
history of pregnancy complications, with a singleton preg-
nancy and estimated due date confirmed by ultrasound ex-
amination in the second trimester. Exclusion criteria were de-
fined as follows: women who had received anti-D prophylaxis
earlier in the current pregnancy, who had multiple pregnancy
or presence of maternal erythrocyte alloantibodies at time of
inclusion, or medication other than vitamins, folic acid and
iron supplement. Before inclusion, a routine obstetric history
was checked, as well as body height, weight and blood pres-
sure. Kidney and liver function were assessed by urinalysis
(urinary albumin and creatinine), and a plasma sample was
analysed for creatinine, cystatin C, alanine aminotransferase,
aspartate aminotransferase, serum albumin and calculation
of glomerular filtration rate.
Rhesonativ R
is a polyclonal anti-D IgG for intramuscular
use, prepared from human plasma with a high anti-D titer.
588 Acta Obstetricia et Gynecologica Scandinavica 
C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 587–592
E. Tiblad et al.
The manufacturing process consists of cold ethanol fraction-
ation with additional purification procedures. The plasma
donors are tested for hepatitis A, B and C, human immunod-
eficiency virus and parvovirus B19. In order to increase viral
safety further, a virus inactivation step based on treatment
with organic solvent/detergent is included in the manufac-
turing process.
Serial plasma anti-D quantitations following intramuscu-
lar administration of a single dose of 250 μg (1250 i.u.) anti-
D immunoglobulin were performed. Blood sampling time
points were set before the start of the study to days 0, 3,
10±2, 14±2, 28±2, 42±2, 56±2, 63±2, 70±2, 77±2 and
84±2 postinjection. The concentration of anti-D was anal-
ysed by flow cytometry. Briefly, 50 μL plasma was incu-
bated with 50 μL enzyme-treated R0r (cDe/cde) red blood
cell suspension for 30 min. After three washes, the anti-
D-coated red blood cells were incubated with fluorescein
isothiocyanate-conjugated F(ab-)2 antihuman IgG (Jackson
Immuno Research, West Grove, PA, USA) for 20 min, and
after two washes the mean fluorescence intensity was mea-
sured by flow cytometry (FACSCalibur, Becton, Dickinson
and Company, Franklin Lakes, USA). The samples were run
in duplicate and reported as the mean value of two. All the
samples from each single woman were analysed in the same
batch. The mean fluorescence intensity was compared against
a standard curve based on an international reference anti-D
(NIBSC 73/515). As an internal control, a sample with known
anti-D concentration was used. The concentration was re-
ported in nanograms per milliliter. The enzyme treatment
of the red blood cells was performed in order to increase
the sensitivity of the method. The lowest detectable value
was 1 ng/mL.
Time vs. concentration profiles for anti-D levels were gen-
erated from the concentration values at the predetermined
sampling time points. The half-life was estimated from all
available data points, except when the number of data points
was too low or when a second dose was given during the
study period. Plasma samples taken after administration of
extra Rh prophylaxis for medical indications were excluded
from the pharmacokinetic analysis. The half-lives and elimi-
nation constant (k) values were calculated by linear regression
analysis. Pharmacokinetic parameters from the set of evalu-
able data were calculated by medians and range or means
and standard deviations. The last concentration measured
(C last ) was included in the statistics if it was collected no
more than two weeks prior to parturition in a delivery at
term.
The study was approved by the Regional Ethics Com-
mittee (no. 2010/862-31/1). The Medical Products Agency
decided a permission not to be required for this study
because Rhesonativ R
is already registered and in use in
Sweden.
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E. Tiblad et al. Pharmacokinetics of antenatal anti-D

Table 1. Obstetric data and gestational age at anti-D administration.

Woman Body weight Age Anti-D Gestational age

number on day 0 (kg) (years) Parity administration (weeks) at delivery (weeks) Comments
+6 +5
1 79 30 1 29 39
2 98 38 2 30+0 39+3
3 73 23 1 29+3 39+2
4 97 30 1 29+1 40+1
5 64 32 1 29+0 39+5
6 90 23 2 30+0 39+3
7 68 21 0 29+1 37+3
8 72 33 0 29+0 39+6
9 60 34 3 28+2 38+1 Intrauterine growth restriction −38%. Missed all
appointments after day 28 sample.
10 80 37 1 29+3 39+1
11 79 22 0 29+3 38+0 Extra Rh prophylaxis due to external cephalic
version on days 22 and 36 postinjection.
12 82 30 0 30+4 41+6
13 104 25 2 29+4 41+1
14 62 29 0 29+2 34+3 Intrauterine growth restriction −38%.
Premature cesarean section.
15 87 32 1 28+4 40+3 Extra Rh prophylaxis due to external cephalic
version on day 58 postinjection.
16 86 30 0 29+4 38+6 Mild pre-eclampsia at gestational age 38 weeks.

Results
Obstetric characteristics, pregnancy complications and ges-
tational age at administration of anti-D of all women are
listed in Table 1. Pregnancy was uneventful in a majority of
the women.
A total of 124 out of 134 (92.5%) possible samples were
collected and analysed. Of these 124 samples, 116 (93.6%)
were collected at the predefined time points, seven samples
within three days and one within four days. No samples were
collected on day 84, because all women had delivered by
that time. There were nine missing samples owing to missed
appointments. Numbers of samples collected at each time
point are presented in Table 2.
Pharmacokinetic parameters are presented in Table 3.
Maximal plasma concentration of anti-D (C max ) was usu-
ally seen at 3–10 days postinjection, with a median value of
25 ng/mL, but with a wide range of 9–58 ng/mL. Four women
had the same plasma anti-D concentrations on days 3 and
10, indicating that the true C max lay somewhere within that
time period. In general, lower values of C max were found in
women with high bodyweight. The half-life (t 1/2 ) varied in an
expected way between individuals, with a median of 23 days.
We found detectable levels of anti-D IgG within two weeks of
parturition in 11 of 12 women (C last ). Figure 1 illustrates the
mean concentrations of anti-D IgG with standard deviations
for all samples analysed at each time point. Only four women
delivered after 40 weeks of gestation. The plasma analyses in
a woman who previously had undergone bariatric surgery
(patient number 13) resulted in a highly divergent pattern,
and her values were not included in the pharmacokinetic
analysis. During the entire study, no adverse drug reactions
were reported.

Table 2. Number of blood samples collected at each time point.

Gestational age at
anti-D Gestational age
administration at last sample
(weeks)∗ Day 0 Day 3 Day 10 Day 14 Day 28 Day 42 Day 56 Day 63 Day 70 Day 77 Day 84 (weeks)∗
+3 +2 +4
29 (28 –30 ) 16 16 15 16 15 13 13 11† 6‡ 3‡ 0 38+3 (32+1 –41+1 )

∗
Median (range).
† Three samples excluded from analysis because of extra antenatal anti-D and bariatric surgery.
‡ Two samples excluded from analysis because of extra antenatal anti-D and bariatric surgery.


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Pharmacokinetics of antenatal anti-D E. Tiblad et al.

Table 3. Pharmacokinetic parameters of anti-D in plasma of RhD- a rapid increase in concentrations over the first 3–10 days.
negative women. This is in accordance with previous studies using 250 μg and
Gestational
other concentrations of anti-D (8–11). We observed a normal
Woman C max t max C last age at C last variability in half-life between individuals, with a median of
number t 1/2 k (ng/mL) (day) (ng/mL) (weeks) 23 days. Our sampling interval was not set to define the ex-
act day of maximal plasma concentrations. Rather, we were
1 12.5 −0.055 28 3 1 37+5
interested in the duration of measurable prophylactic levels
2 24.1 −0.029 15 10 4 38+6
3 13.7 −0.051 27 14 1 38+3
of anti-D close to term. In our study, most women delivered
4 30.1 −0.023 9 10 2 39+0 before gestational week 40. We were able to collect blood
5 20.9 −0.033 40 3 4 38+5 samples at 10 weeks postinjection in four uncomplicated
6 23.0 −0.030 18 10 4 38+2 pregnancies. All had detectable levels of anti-D, ranging from
7 15.7 −0.044 19 10 2 36+6 1–4 ng/mL. One of those did not deliver by 11 weeks after
8 27.5 −0.011 25 3 4 38+6 antenatal anti-D, and she still had detectable levels at that
9 15.2 −0.045 58 3 n.a. 32+1
time. At 12 weeks after injection, all women had delivered.
10 27.0 −0.026 16 3 3 38+1
11 – – 46 3 n.a. 37+5
If extrapolating the individual pharmacokinetic linear curves
12 30.3 −0.023 16 3 4 41+1 in the 12 women with measurable C last , nine would have had
13 – – – – 0 40+3 anti-D IgG concentrations ≥1 ng/mL at the time of delivery
14 19.4 −0.036 37 3 n.a. 33+2 (data available on request). It is known from previous studies
15 24.7 −0.028 19 10 n.a. 39+2 that detectable levels of anti-D are not found in all women af-
16 20.9 −0.033 47 3 8 38+3 ter 10 weeks postinjection (12). Bichler et al. found antibody
Note: The value of C last was not calculated if extra anti-D prophylaxis levels of 1 ng/mL or more in only 60% of women 11 weeks
had been administrated during pregnancy or if delivery was preterm. Ab- after administration of 300 μg anti-D, while MacKenzie et al.
breviations: t 1/2 , plasma half-life in days; k, elimination constant; C max , reported that only 30% of women in their study with a two-
maximal concentration in nanograms per milliliter; t max , postinjection dose regimen of 100 μg anti-D IgG had antibody levels of
day of C max ; C last , last concentration before delivery; and n.a., not ap- 2 ng/mL at 12 weeks postinjection (8, 9). In our study, a
plicable.
single dose of 250 μg of anti-D IgG resulted in comparable
anti-D concentrations at term. Our finding of lower maxi-
mal concentrations of anti-D IgG in obese women compared
Discussion with those of normal weight has previously been reported
The pharmacokinetic profile of 250 μg anti-D IgG admin- by other authors (9). Bodyweight does not seem to influence
istered intramuscularly at gestational week 28–30 showed the duration of detectable levels, which means that protection

Figure 1. Mean plasma concentrations of anti-D IgG days after intramuscular injection (in nanograms per milliliter ±SD). The data points represent
different numbers of women owing to missing values towards the end of the study period: day 3, n=15; day 10, n=14; day 14, n=14; day 28, n=12;
day 42, n=10; day 56, n=11; day 63, n=8; and day 70, n=4.

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E. Tiblad et al.
against sensitization is expected to be the same independently
of bodyweight.
Variability in residual anti-D levels and in half-life depends
on individual IgG clearance from plasma and consumption
of anti-D. Uptake from muscular compartments may vary
as well. Differences in results regarding concentrations and
frequency of detectable levels of anti-D between studies are
influenced by sensitivity and coefficient of variation in the
assays used.
There is a lack of studies on pharmacokinetics of anti-D
IgG in pregnant women using modern analytical techniques,
such as flow cytometry. Earlier studies were performed us-
ing the AutoAnalyzer, which provides results with a higher
coefficient of variation than flow cytometry. The AutoAn-
alyzer measures the capacity of antibodies to form aggluti-
nates. Immunoglobulin M has a high capacity of agglutina-
tion; hence, analysis of alloantibodies by hemagglutionation
involves a risk of too high values due to inclusion of im-
munoglobulin M antibodies. With flow cytometry, only la-
beled IgG antibodies are measured, and the results are more
accurate.
The frequency of fetomaternal hemorrhage with detectable
levels of fetal RBCs in the maternal circulation is about
40–70% in the third trimester of pregnancy, depending on
the study and method used (1,13,14). It is not exactly clear
how much anti-D is needed to prevent sensitization in a
pregnant woman. According to the World Health Organi-
zation, 25 μg would neutralize 2 mL of whole fetal blood
(15). Only 0.90% of pregnant women will have a fetoma-
ternal hemorrhage during pregnancy week 30–39 that will
exceed 1 mL of fetal blood (12). Of these, about 30% will be
non-responders to a possible sensitizing event (15). MacKen-
zie et al. calculated that 25 μg of anti-D immunoglobulin
would be equal to a plasma concentration of 2.4 ng/mL in
a pregnant woman (9). In summary, half of this, 1.2 ng/mL,
would be enough to neutralize a fetomaternal hemorrhage
of 1 mL fetal blood during the third trimester. The original
dose studies of anti-D prophylaxis that form the basis for rec-
ommendations were carried out in male prison volunteers,
who differ from pregnant women in many aspects, as follows:
(i) distribution volume; (ii) metabolism of drugs; and, per-
haps most importantly, (iii) the immune system in a pregnant
women is “reprogrammed” towards active tolerance to pater-
nal antigens (16). Translation of what would be a protective
dose of anti-D in a pregnant woman cannot easily be done.
Antibody-mediated clearance of antibody-coated fetal RBCs
may not be the only mechanism of protection. Polyclonal
anti-D IgG may also have immunomodulatory functions,
inhibiting an inflammatory immune response (17). Despite
low or undetectable levels of anti-D prophylaxis at term, there
is convincing clinical evidence that routine antenatal anti-D
prophylaxis is efficient in reducing the incidence of RhD sen-
sitization (2–4). As the exact mechanism of protection by

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Acta Obstetricia et Gynecologica Scandinavica 
C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 587–592
Pharmacokinetics of antenatal anti-D
anti-D prophylaxis remains unknown, this is the most im-
portant end-point.
In most maternity units, postnatal anti-D prophylaxis is
provided within 72 h of delivery if cord blood serology re-
veals an RhD-positive child. With first trimester genotyping
of cell-free fetal DNA, the fetal RHD status will be known
before birth. It could be discussed if the conventional post-
partum anti-D prophylaxis should be administered to the
mother at term to ensure an adequate anti-D IgG plasma
concentration in the 40th to 42nd week of gestation, as well
as after delivery. This question needs to be addressed in a
prospective randomized trial.
In summary, the frequency of plasma concentrations of
anti-D ≥1 ng/mL at term with the preparation of 250 μg
polyclonal anti-D IgG studied is similar to what has been
reported with other brands; hence, we conclude it safe to
use a single dose of 250 μg as routine antenatal prophylaxis.
However, a considerable number of women will not have
measurable levels of anti-D at term or post-term, and it can
be considered to offer the conventional postpartum dose at
term to women not yet delivered to provide further protection
against sensitization.
Funding
The study was funded by Octapharma pharmaceutical com-
pany. The funding included the provision of anti-D pro-
phylaxis used in the study, as well as covering the costs for
recruiting women and the collection and analysis of plasma
samples. None of the authors has financial interests in the
company. Analysis of data and writing of the manuscript was
done by the authors independently of the company.
Acknowledgements
Margaretha Ström, Maria Fursäter and Maria Karlsson as-
sisted in collection and preparation of all the blood samples
in the study, as well as in administration of anti-D prophy-
laxis. We also thank Doris Hugoson for running the serology
titrations and flow cytometry quantitations.
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