(9770 x g, 10 miii) and an aliquot of the supernate was
tested for drug metabolites with “Abuscreen” (Roche Diagnostic Systems, Nutley, NJ) radioimmunoassay
kits, according to the manufacturer’s
instructions.
To determine
the efficiency of the method, we added
known amounts of morphine glucuronide and benzoylecgonine to 0.5-1.0 g of mecomum
from non-drug-dependent
infants and assayed, recovering
84%-97% of the added
morphine and 70%-105% of the cocaine metabolite.
Cocaine and morphine metabolites were detected in the
meconium of the 10 drug-dependent infants as late as the
third postnatal day:
gLg/gof stool
Cocaine metaboflte
Day 1
Day 2
6.34
2.33
3.23
1.93
1.77
10.84
5.38
4.54
(-)
2.38
(-)
Mean
SD
2.17
1.07
9.67
11.28
Morphine metabolite
Day
3
(-)
1.17
0.30
3.67
Day 1
Day 2
Day 3
3.27
1.71
0.56
1.18
(-)
1.17
(-)
1.21
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
7.23
6.05
1.03
(-)
(-)
(-)
(-)
0.69
0.97
0.54
2.15
1.07
3.72
2.42
(-)
(-)
5.38
12.10
2.44
2.30
(-)
12.11
17.78
3.55
5.95
0.72
3.31
6.24
1.15
2.15
2.62
3.86
4.30
(-)
0.89
assays (4-6) and, to our knowledge, three immunoassays
are commercially available today.’
Planning to measure TNF in serum and plasma of patients with various ailments, we evaluated the influence of
blood-collecting systems.
Venous blood from 10 healthy volunteers was collected
into the following tubes: sterile polystyrene tubes [Falcon
Plastics, Becton Dickinson (B-D), Belgium] without (a) and
with (b) heparin (Novo Industri,
Bagsvaerd, Denmark)
added to give a final colicentration of 10 kilo-USP unitsfL;
(c) sterile glass tubes containing 0.1 mL of 150 g/L K3-EDTA
(Vacutainer Systems, A 3200 QS from B-D); (d) sterile glass
tubes containing 143 USP units of sodium heparin (Vacutamer Systems, A 3200 KA, B-D); and (e) sterile glass tubes
containing 143 USP units of sodium heparin (Venoject;
Terumo-Europe,
Leuven, Belgium).
The specimens were kept at 4#{176}C
and processed as follows.
The blood in tube a was allowed to clot and the serum was
recovered. Shortly after venipuncture
(time t0) 2 mL of
anticoagulated
blood was taken from tubes b, c, d, and e, and
plasma was harvested. Subsequently
the remaining contents of tubes b, d, and e were incubated at 37#{176}C
and plasma
was sampled 2 h (t2) and 4 h (t4) later. TNF was measured in
all specimens with a commercially available TNFa-radioirnmunoassay (IRE-Medgenix).
The following tabulation summarizes our results.
1.41
below the Abuscreen detection limitin the supemate (<14 ng/mL for
cocaine, <24 ng/mL for morphine).
TNF, ng/L, detected
In
(-),
No drug metabolites were found in the stools of control
infants. Mean concentrations
of drug metabolites in the
stools were highest during the first two days, starting to
decrease on the third. Mecomum from postnatal days 1 and
2 were presumably
formed in utero and therefore reflected
exposure to drugs abused by the mother, whereas stools
from the third day were probably an admixture of meconiurn and milk stools, the latter being essentially drug free.
We hope that this method can also be applied to detect
other drug metabolites in meconiurn. In a preliminary study
to detect cannabinoids in meconium, we found that added
methanol was essential in the preliminary extraction.
References
1. Halatead AC, Godolphin W, Lockitth
G, Segal S. Timing of
specimen collection is crucial in urine screening of drug dependent
mothers and newborns. Clin Biochem 1988;21:59-61.
2. Ostrea Jr EM, Ascensio D, Naluz A, Simkowski K, Subramanian
MG, Abel E. The detection of heroin, cocaine and cannabunoid
metabolites in the stools of infants of drug dependent mothers:
clinical significance. Pediatr Res 1987;21:240.
influence of Blood-Collecting Systems on
Concentrations of Tumor Necrosis Factor in Serum and
Plasma, Geert Leroux-Roels,
Fritz Offner, Jan Philippe,
and Alex Verrneulen (Dept. of Intern. Med., University
Hospital, De Puntelaan 185, B-9000 Gent, Belgium)
Tumor necrosis factor (TNF), a monocyte/macrophagederived protein, displays many biological activities. It may
play a role in the pathogenesis of several infectious and
inflammatory
processes, but its precise role in vivo has not
yet been defined (1,2). TNF concentrations in human serum
were first measured with a biological assay based on the
cytocidal effect of TNF (3), but more recently with immune-
Sublect
Tube Tub
b,
Tube
Tubed
Tubee
0
no.
137
101
106
120
160
4764 11460
2110 7370
2030 4870
71
2490 5660
6230 10040
3400 6900
2940 8170
2260 4930
1
78
59
2
3
4
5
61
120
50
85
6
7
138
76
153
67
112
90
95
42
73
113
85
116
8
93
58
109
85
9
10
95
84
131
96
107
119
64
93
182
53
65
75
(4
to
to
68 1624 3174
100 1003 1764
138 294
411
762
1072
115 1905
195 737
87
2790
127
811
923
1139
Mean
89.9 88.4105.9
97.53431
134 589
805
93 1603 2785
6200
99 649 1185
7638115.6 997.71604.8
SD
25.6 35.2 37.4
28.8 1572
2407 35.6
5730 10780
2360
529.5 971.4
Prolonged incubation of blood in tube b did not alter the
TNF content (data not shown). However, incubation of tube
e for 2 hat 37 #{176}C
increased the mean value for TNF to 997.7
(SD 529.5) ng/L and 2 h later the mean value was 1604.8
(SD 971.4) ngfL. This phenomenon was even more pronounced in tube d, for which these respective TNF concentrations were 3431 (SD 1572) and 7638 (SD 2407) ng/L. To
see if this increase was induced by endotoxin contaminants
in the tubes, we measured the endotoxin
content
of these
containers. For that purpose, we rinsed five tubes of each
type with 1 mL of pyrogen-free
water and measured the
endotoxun concentration
of the washing fluids with the
Coatest (Kabi Vitrum, Stockholm,
Sweden). The mean
endotoxin
content of heparinized
B-D tubes (tube d) was
24.68 (SD 3.57) ng, whereas Terumo tubes (tube e) contained
1.28 (SD 1.13) ng of endotoxin. No endotoxin was found in
tubes a, b, or c.
Heparinized
evacuated tubes from B-D and Terumo are
widely used in clinical practice and blood specimens are
‘Biokine’” TNF Test Kit (T Cell Sciences, Cambridge, MA),
TNF-a-RIA (IRE-Medgenix, Fleurus, Belgium), and TNFa Test
(Genzyme Corp., Boston, MA).
CLINICALCHEMISTRY, Vol. 34, No. 11, 1988 2373
often kept in such tubes for prolonged periods, so those
studying TNF or endotoxin should be aware of these observations.
References
1. Beutler B, Cerami A. Cachectin and tumor necrosis factor as two
sides of the same biological coin. Nature (London) 1986;320:584-8.
2. Old L. Tumor necrosis factor. Sci Am 1988;258:41-9.
3. Waage A, Halstensen A, Espevik T. Association between tumour
necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet 1987;i:355-7.
4. Franchimont P, Reuter A, Gsen Ph, Bernier J, Vrindts-Gevaert
Y. Tumor necrosing facthr an mediateur de la fonction macrophagique. Actions biologiques et dosage radio-iminunologique. Med
Hyg 1987;45:2160-8.
5. Balkwill F, Osborne R, Burke F, et al. Evidence for tumour
necrosis factor/cachectin production in cancer. Lancet 1987;n:122932.
6. Teppo A-M, Maury C. Radioimmunoassay of tumor necrosis
factor in serum. Clin Chem 1987;33:2024-7.
Measurement
of Cortisol with EMIT and Amerlite
immunoassays,
L. W. J. J. M. Westerhuis (Dept. of Clin.
Chem., Gregorius Hospital, Brunssuin,
The Netherlands)
We examined the performance
of a new, commercially
available competitive immunoassay
of cortisol, based on
enhanced chemiluminescence
(Amerlite, Amersham International). A polyclonal sheep antibody to cortisol, the cortisol in the patient’s sample, and a cortisol derivative labeled
with horseradish peroxidase (EC 1.11.1.7) are incubated in
microtiter wells coated with a donkey anti-sheep antibody.
After unbound material
is aspirated
and the wells are
washed, the light-generating
reaction is started by adding
luminol, hydrogen peroxide, and a substituted
phenol as
enhancer. The light emitted is measured in an automated
analyzer 2 to 20 mm later.
We compared the performance of the Amerlite cortisol
assay with that of the zssrr homogeneous immunoassay
(Syva Co., Palo Alto, CA) of cortisol, adapted,
as described
(1), to a Cobas Bio centrifugal
analyzer equipped with a
DENS software program (Hoffmann-La
Roche, Inc.). CVs for
cortisol values in patients’ sera and control sera were as
follows:
Cortlsol concn,
pWoI/L
Amerlite
Mean
SD
2. (36)
Between-run: control sera’
1. (11)
2. (9)
3. (11)
0.11
0.89
0.01
0.05
-
References
1. !zquierdo JM, Quiro A, Alvarez J. Homogeneous enzyme unmunoasaay for cortisol with a centrifugal analyzer. Clin Chem
1984;30:1824-6.
2. Cornbleet PJ, Gochman
coefficients in method
197925:432-8.
N. Incorrect
comparison
least-square regression
analysis. Clin Chem
of the Abbott TOx#{174}
Assay for Amphetamine
in Post-Mortem Urine Samples, P. Kintz, A. Tracqui, P.
Specificity
Mangin, A. Lugnier, and A. Chaumont
(Inst. de Med.
Legale, 11, rue Humann, 67085 Strasbourg Cedex,
France)
CV,%
Within-run: Patients’ sera
1. (36)#{176}
From these CVs we conclude that the Amerlite assay is
precise over the range examined here, with good run-to-run
reproducibility,
whereas the EMIT assay shows a poorer
performance, especially at low concentration of ana.lyte.
To assess the accuracy of both methods, we analyzed
control sera 2 and 3 and compared the measured cortisol
values with the assigned target values. We found acceptable
accuracy for the Amerlite method and a negative bias for
the EMIT method. The reason for this negative bias is not
clear. Results obtained by both methods for cortisol in 101
patients’ sera (range, 0.05 to 1.40 pmo]/L) were evaluated by
orthogonal regression analysis (2), the regression equation
being: Amerlite = (0.91 EMiT)
0.01 pmol/L (r = 0.928).
Overall, results obtained with the EMIT method were about
10% greater than those obtained with the Amerlite method.
Calibration stability of the Amerlite system was assessed
by measuring the six kit standards in duplicate with each
run during five weeks. The standard curve remained stable
and changed by <5% during this period, which means that
the economy of standardization
protocol (single measurement of four standards) is justified.
Evidently the Amerlite enhanced luminescence
immunoassay of cortisol provides analytically
reliable results. The
instrumentation
system is easy to handle in a routine
hospital laboratory. Low cortisol values determined with the
EMIT method should be considered
with caution, because of
the poorer analytical performance
of the EMIT procedure.
CVs for the EMIT method are probably high because the
difference in absorbance for the highest and lowest calibrators is usually very small. Moreover, only tiny absorbance
values are measured at low concentrations of cortisol.
5.8
6.2
0.39
0.99
0.40
0.02
0.07
0.02
23.8
0.97
0.07
7.4
0.41
0.05
0.08
0.04
0.05
0.02
Systematic post-mortem toxicological screenings at our
Forensic Institute, with an Abbott TDx fluorescence polarization detector, revealed in numerous cases the apparent
presence of considerable amounts of amphetamine derivafives in the urine tested; moreover, the concentrations
detected appeared to increase with the time after death.
Because substances produced by putrefaction can interfere
EMIT
Within-run: Patients’ sera
1. (20)
2. (20)
Between-run: control sera
1. (20)
2. (8)
3. (8)
0.53
0.32
11.0
:
#{149}
No. of determinations listed in parentheses.
bControl serum 1: Seronorm, Nyegaard,Norway.Controlsera 2 and 3: Sera
from the Dutch ExternalQualityAssuranceScheme (LWBA) with assigned
values (mean ± SD) of 0.90 ± 0.10 and 0.42 ± 0.05 rnoI/L, respectively.
2374 CLINICALCHEMISTRY, Vol. 34, No. 11, 1988
with toxicological analyses-especially
putrefactive bases
(most frequently f3-phenethylanune,
tyramine, and tryptamine) interfere with amphetamine
(1)-we
wanted to
determine whether the Abbott TDx could be used to screen
for amphetamine derivatives in post-mortem urine.
According to the manufacturer,
the TDx Amphetamine/
Methamphetamine
procedure
demonstrates
1.63-1.80%
cross-reactivity between amphetamine
and 3-phenethylamine, and 1.14-2.20% cross-reactivity
between amphetamine and tyramine. We checked these cross-reactivity
data