British Journal of Anaesthesia 1995; 74: 430-437
LABORATORY INVESTIGATIONS
Radioimmunoassay for plasma histamine: a study of false positive
and false negative values
D. LAROCHE, F. DUBOIS, J.-L. GERARD, C. LEFRANCOIS, B. ANDRE, M.-C. VERGNAUD,
L. DUBUS AND H. BRICARD
Summary
In order to achieve a correct diagnosis of drug
anaphylaxis using a radioimmunoassay devoid of
interferences, we have studied factors leading to
false positive or false negative values of plasma
histamine. Different steps in sample collection were
studied systematically in 30 normal volunteers.
False positive values were found in haemolysed
samples, with histamine concentrations being correlated with haemoglobin concentrations, and
where plasma was aspirated from the white-cell
layer. There was no significant increase when a
tourniquet or vacuum tubes were used, or when
blood tubes were left at 4 °C overnight. In 12
patients who experienced an anaphylactic reaction,
histamine disappeared from blood 10 times more
slowly than expected. False negative values were
found in two pregnant women and one heparinized
patient. Histamine was remarkably stable in vitro in
blood or plasma samples, whereas it disappeared
rapidly when plasma from a pregnant woman or a
heparinized patient was added to the sample. We
conclude that false positive and false negative
values are rare when using this radioimmunoassay.
(Br. J. Anaesth. 1995; 74: 430-437)
Key w o r d s
Histamine. Measurement techniques, radioimmunoassay.
There are many reasons for adverse reactions to
drugs. Anaphylactic reactions are of particular
interest as their severity increases with repeated
administration of the drug responsible. Biochemical
diagnosis may be obtained by measuring plasma
histamine concentrations soon after the reaction
[1,2]. However, concentrations were rarely measured in routine practice, for two reasons: sampling
recommendations [3] were difficult to ascertain in
emergency conditions and false positive results may
be obtained when basophils are disrupted during or
after sample withdrawal; and the half-life of histamine in plasma is assumed to be very short
(minutes) [4] because of metabolization by two
pathways, methylation by methyltransferase and
oxidation by diamine oxidase [5]. Thus falsenegative values may be obtained if blood is obtained
late after the reaction or if histamine is metabolized
in the sample itself. The assays for histamine
determination during the 1980s often lacked sensitivity or specificity [6], and controversial results
were obtained in a quality control study [7]. A
radioimmunoassay, using a succinyl-glycinamide
derivatization of histamine, has been available for a
few years [8]. This assay is easy to use, sensitive and
specific [9], and appears to be reliable in clinical
conditions [10, 11]. The sampling recommendations,
assessed for the classic fluorimetric assay, may not
apply when using the radioimmunoassay. Furthermore, in vivo metabolism of histamine under clinical
conditions, that is unexpected anaphylactic reactions, has only been evaluated in a few patients
[12,13] and the 1-min half-life found in volunteers
perfused with histamine [4] may not be valid under
such conditions.
The aim of this study was to determine which
conditions for sample collection or handling could
lead to false positive or false negative values for
plasma histamine concentrations when using the
radioimmunoassay.
Materials and methods
CONTROL SUBJECTS AND SAMPLING PROCEDURE
The study was approved by the local Committee for
the Protection of Volunteers for Biomedical Research
and informed consent was obtained from all subjects.
Blood was obtained consecutively in seven tubes,
within 1 or 2 h after lunch, from each of 30 healthy
volunteers (12 male) aged 27.3 (range 18-45) yr. On
the left forearm, a tight tourniquet was placed 5 min
before introducing a 22-gauge Vacutainer needle
into an antecubital vein, and connecting it to 2-ml
glass vacuum tubes (Vacutainer, Becton Dickinson).
These tubes contained, in order: tube 1, ethylenediaminetetraacetic acid (EDTA); tube 2, EDTA;
tube 3, heparin; last tube, called No. 7, EDTA. At
the same time a fluoroethylene-propylene catheter
(Abbocath T 20G) was placed, without a tourniquet,
DOMINIQUE LAROCHE*, MD, PHD (Service of Nuclear Medicine);
FREDERIC DUBOIS, MD, JEAN-LOUIS GERARD, MD, CLAUDE
LEFRANCOIS, MD, BERNARD ANDRE, MD, LAURENT DUBUS, MD,
HENRI BRICARD, MD (Department of Anaesthesiology); MARIECLAUDE VERGNAUD, MD (Department of Pneumonology); Centre
Hospitalier Regional Universitaire, Caen, France. Accepted for
publication: November 5, 1994.
*Address for correspondence: Service de Medecine Nucleaire,
CHRU de Caen, F-14033 Caen Cedex, France.
Histamine: false positive and false negative values
into a vein of the right forearm and blood was
obtained by gravity through the catheter into three
EDTA-containing open tubes, numbered consecutively 4 to 6 (inclusive). Tube Nos 4 and 6 were
constructed from polypropylene and tube 5 from
sterile glass (Vacutainer, Becton Dickinson). The
whole sampling procedure took less than 5 min for
each subject. Tube Nos 1-6 were placed immediately
in ice-cold water and centrifuged at 4 °C and 800 g
(1500 rpm) for 10 min. Thereafter, plasma was
aspirated gently without approaching the white cell
layer and frozen at — 20 °C until assayed. Subsequently, from tube No. 2, another plasma sample
was aspirated near or inside the white cell layer (tube
No. 8) and frozen at —20 °C. During the course of
the study, spontaneous haemolysis occurred in three
samples from two subjects and catheterization failed
in two others. Tube Nos 1-6, but not tube No. 7
obtained from these four subjects were rejected.
Whole blood in tube No. 7 was left either at room
temperature (20 °C) for 1 (n = 8) or 18 h (« = 10), or
at 4°C for 18 (« = 9) or 40 h (n = 7) before
centrifugation. Furthermore, blood samples obtained from seven other volunteers were divided into
three aliquots which were centrifuged at either 4 °C
or 15 °C or without refrigerating the centrifuge.
SAMPLES WITH INCREASED HISTAMINE
CONCENTRATIONS
Two types of samples with increased concentrations
of histamine were used: plasma samples obtained in
EDTA-containing tubes from patients who had
anaphylactoid reactions, which were stored frozen at
— 20 °C, and blood samples from normal volunteers
challenged in vitro with anti-IgE immunoglobulin
(approximately 8 ml of blood was obtained in
heparinized tubes (Vacutainer, Becton Dickinson)
and incubated at 37 °C. Subsequently, 50 nl of
monoclonal
anti-human-IgE
immunoglobulin
(Immunotech) was added and incubation was prolonged for 30 min). Histamine concentrations in the
plasma samples were 10-1100 nmol litre"1.
Stability of histamine in whole blood
After in vitro degranulation, blood samples were
separated into two aliquots. One was centrifuged
immediately and the other was left for 2 h at room
temperature (« = 9) or overnight (18 h) at 4 °C (n =
7). In another experiment, five blood samples were
divided into three aliquots which were centrifuged at
4 °C, 15 °C or without refrigeration.
Stability of histamine in plasma
Three aliquots of plasma, obtained after in vitro
degranulation of blood from five volunteers, were
placed in polypropylene, polyethylene or sterile glass
tubes. The tubes were left for 30 min at room
temperature, then frozen at — 20 °C until assayed.
Plasma from three patients who experienced anaphylactoid reactions and plasma obtained after in
vitro degranulation were left in polypropylene tubes
431
at room temperature for up to 48 h or at 4 °C for up
to 72 h, before measurement of histamine. Four
plasma samples obtained during anaphylactoid reactions were subjected to five freeze-thaw cycles,
with measurement of histamine after each cycle.
Seven samples obtained during anaphylactoid reactions were diluted 1/10 or 1/50 in kit buffer before
assay. The remaining volume of diluted plasma was
frozen at — 20 °C and re-assayed 1 week later.
IN VIVO HISTAMINE METABOLISM
Patients with increased histamine concentrations
Two consecutive blood samples were obtained in
EDTA-containing tubes from 12 patients (six male),
aged 21-82 yr, during anaphylactoid reactions for
measurement of plasma histamine and tryptase. All
had immediately reacted after i.v. administration of
a drug (neuromuscular blocker, n — 8; beta-lactam,
n = 11 gelatine, n = 1; protamine, n = 1; aprotinin, n
= 1). Ten patients recovered well but two who had
experienced severe and prolonged anoxia died some
weeks later.
IN VITRO HISTAMINE METABOLISM
In order to evaluate the rate of histamine metabolism
in the sample tube during specific clinical conditions,
a plasma sample containing histamine 300 nmol
litre"1, obtained through in vitro degranulation, was
mixed (vol/vol) with plasma from a pregnant woman
(34 gestational weeks), from a heparinized patient
(after 40 min of extracorporeal circulation) or from
a normal control. The mix was incubated at 37 °C for
80 min or at 4 °C for 22 h.
ASSAYS
Histamine was measured after alkylation by radioimmunoassay [8] (Immunotech, Luminy, France).
In this study, the lower limit of detection of the assay
was 0.5 nmol litre"1 (1 nmol litre"1 = 0.11 ng ml"1).
The usual limit for pathological values is
9 nmol litre"1 [3]. The within-assay coefficient of
variation is 10% for a plasma sample containing
histamine 10 nmol litre"1. For each control subject,
the eight tubes were processed within the same assay
series. In haemolysed samples, haemoglobin was
measured by differential spectroscopy (normal range
< 5 umol litre"1) [14]. Tryptase was measured by an
immunoradiometric assay [15] and urinary methylhistamine by a competitive radioimmunoassay [16]
(Kabi Pharmacia Diagnostics, Uppsala, Sweden).
The normal range is < 2 ng litre"1 for tryptase and
< 380 (imol per mole of creatinine for urinary
methylhistamine [17]. All measurements were made
in duplicate. When necessary, samples were diluted
in the buffer provided with the kit.
STATISTICAL ANALYSIS
Data are expressed as mean (SD). Means were
compared by analysis of variance. Values were
compared by Wilcoxon or Kruskal-Wallis tests when
British Journal of Anaesthesia
432
the number of compared samples was small. Linear
correlation was performed using the method of least
squares. P < 0.05 was considered statistically significant.
10
Results
SEARCHING FOR FALSE POSITIVE RESULTS IN NORMAL
CONTROLS
Blood sampling
Sampling conditions are summarized in table 1.
Individual histamine concentrations measured in the
30 volunteers are shown in figure 1, according to the
different sampling conditions.
Mean histamine concentrations in tubes centrifuged immediately were, respectively (n = 30): tube
1, 2.04(1.02) (range 0.7-5.2) nmol litre"1; tube 2,
1.80 (0.80) (0.8-3.7) nmol litre"1; tube 3, 2.04 (0.72)
(1.1-3.7) nmol litre"1; tube 4, 2.06(1.11) (0.7-6.2)
Table 1 Summary of the different withdrawal conditions used
for obtaining blood samples in 30 volunteers for measurement
of plasma concentrations of histamine. X = condition fulfilled,
G = glass, P = polypropylene, H = heparin; E = EDTA.
*Tube left at room temperature or 4 °C for various times
c
E
2
.2 2
X
4°C
15 °C
WR
Figure 2 Histamine concentrations measured after
centrifugation at 4 °C, 15 °C or without refrigeration (WR), of
blood aliquots from seven different subjects.
nmol litre"1; tube 5, 1.90 (0.94) (0.4-5) nmol litre"1;
and tube 6, 2.19(0.91) (0.5-3.9) nmol litre"1. The
overall mean was 2.01 (0.92) nmol litre"1. Analysis of
variance showed no significant difference between
means.
One of the patients (represented by filled triangles
in fig. 1) had increased values in tubes 4 and 5 (6.2
and 5 nmol litre"1, respectively). The values in the
other tubes were 1.4—3.3 nmol litre"1.
Tube number
Sampling
condition
1
First ml discarded
Tourniquet
Catheter
Vacuum
Tube material
Anticoagulant
2
3
X
4°C
X
—
X
G
E
X
X
—
X
G
E
X
X
X
—
X
G
H
X
Immediate
centrifugation
X
X
X
5
6
7
Stability of histamine in whole blood
X
—
P
E
X
X
—
X
—
G
E
X
X
—
X
—
X
X
—
X
G
E
*
X
X
X
Blood tube No. 7 was left at room temperature for
1 or 18 h, or at 4 °C for 18 or 40 h. Histamine
concentrations were compared with concentrations
in tube No. 2 centrifuged immediately from the same
patient. There was no significant difference between
histamine concentrations in tube Nos 7 and 2 except
for a slight increase after 18 h at room temperature
(2.67(1.07) vs 1.81 (1.02) nmol litre"1; Wilcoxon
test, P < 0.01).
4
—
p
E
X
10 -
Centrifuge temperature
9
Blood aliquots from seven subjects were centrifuged
at different temperatures: mean histamine concentrations were 3.66 (1.64) nmol litre"1 (range 2.07.4 nmol litre"1) at 4 °C; 1.81(0.48) (1.1-2.5)
nmol litre"1 at 15 °C; and 2.41 (0.57) (1.6-3.4) nmol
litre"1 without refrigeration (fig. 2). The difference
was significant (P < 0.01, Kruskal-Wallis test). In
one case, a large increase in plasma histamine concentration was observed at 4 °C. When this patient
was excluded, the difference was still significant
(P<0.02); values found after centrifugation at
15 °C were consistently smaller than in the other
conditions.
8-
= 6o
|
A
•
c
E 4-
•
2-
I
0
1
2
I !
3
4
Tube number
Plasma aspiration
5
6
7
Figure 1 Individual histamine concentrations in 30 volunteers,
according to the sampling conditions 1-6 (see table 1).
=
pathological range (9 nmol litre"1 = 1 ng ml"1).
Histamine concentrations exceeded 9 nmol litre"1
in six of 30 samples obtained near the buffy coat
(tube No. 8). The concentration range was 0.6> 150 nmol litre"1. The mean concentration was
12.3 (28.3) nmol litre"1, whereas in plasma aspirated
from the same tube far from the buffy coat (tube No.
Histamine: false positive and false negative values
433
40 -,
_ 1000 -
|30H
O
"o
E
_c
<o 1 0 0 c
£200)
c
E
<S 10-
10 J
0
10
20
30
40
50
Haemoglobin (nmol litre" 1 )
Figure 3 Linear correlation between histamine and
haemoglobin concentrations in eight spontaneously haemolysed
plasma samples (r2 = 0.992).
1
2), it was 1.8 (0.8) nmol litre" (Wilcoxon test,
P < 0.05). The measured value was related to the
level of plasma reached by the pipette. The value
remained unchanged when approaching the cell
layer until the tip of the pipette entered the white cell
layer, then the value increased dramatically.
Spontaneous haemolysis
Two control subjects were rejected from the study
because haemolysis occurred, respectively, in 1 or 2
samples: histamine concentrations were 5.5, 12.4
and 11.4 nmol litre"1, whereas the mean values in the
other tubes from the same subjects were, respectively, 1.74(0.48) and 3.18 (0.70) nmol litre"1. In
order to evaluate the effect of haemolysis, we
measured histamine and haemoglobin concentrations
in 10 spontaneously haemolysed plasma samples
obtained for other purposes. Histamine concentrations ranged from 2.1 to 32 nmol litre"1 and haemoglobin from 3.2 to 49 umol litre"1. Two samples
had
increased
haemoglobin
(13.5
and
33.6 umol litre"1, respectively) but normal histamine
concentrations (2.8 and 5.5 nmol litre"1). A linear
correlation (r2 = 0.992) was found for the eight other
pairs of values (fig. 3).
SEARCHING FOR FALSE NEGATIVE RESULTS
In vitro stability studies
Stability of histamine in whole blood. We compared
histamine concentrations in blood aliquots centrifuged immediately after degranulation with concentrations in aliquots left for 2 h at room temperature
or 18 h at 4 °C before centrifugation. No significant
difference was found between the values in tubes
centrifuged immediately and in the other tubes
(Wilcoxon test). Similarly, the effect of temperature
during centrifugation was studied at 4 °C, 15 °C or
without refrigeration. The Kruskal—Wallis test
showed no significant difference between histamine
concentrations in the three aliquots.
Stability of histamine in plasma. The effect of storage
of plasma in polypropylene, polyethylene or sterile
Figure 4 Effect of freezing diluted plasma samples at —20 °C
on histamine concentrations (values corrected for dilution). 1 =
Immediate assay, 2 = assay after freezing.
glass tubes was studied. No significant difference
was found for histamine concentrations in the
different tubes (Kruskal-Wallis test).
No significant difference was found between
histamine concentrations measured immediately
after centrifugation or after incubation of plasma at
room temperature for up to 48 h, or at 4 °C for up to
72 h (Kruskal-Wallis test).
Five successive freeze—thaw cycles were performed
for four plasma samples. There was no significant
difference between the values after 1, 2, 3, 4 or 5
cycles (Kruskal-Wallis test). Moreover, a plasma
sample has been stored frozen at — 20 °C for 3 years,
with histamine concentrations of 1116 nmol litre"1
after the reaction, 1060 nmol litre"1 4 months later
and 1047 nmol litre"1 3 years later.
Diluted plasma samples were assayed immediately
after the dilution process or frozen at — 20 °C before
assay. The measured histamine concentrations were
smaller in frozen diluted samples (Wilcoxon test, P
< 0.05); the difference was greater when the initial
concentration was smaller (fig. 4).
In vivo histamine metabolism
Blood was obtained twice from 12 patients who
experienced anaphylactic reactions. The selection
criteria were an increase in histamine and tryptase
concentrations during the reaction. All had increased
plasma histamine concentrations in the first sample
(853(1343) (38-5100) nmol litre"1), obtained 560 min (30 (21) min) after the onset of the reaction,
and plasma tryptase concentrations were 7—
500 ug litre"1. The mean histamine concentration in
the second sample was 65 (95) (5-300) nmol litre"1,
30-200 min (72 (43) min) after the first. As shown in
figure 5, all but one patient had persistently increased
histamine concentrations 60 min after the onset of
the reaction.
Clinical cases with false negative results
Case 1. A pregnant woman, aged 35 yr, was administered suxamethonium and thiopentone for Caesarean section. Immediately, erythema, hypotension
and mild bronchospasm occurred. Blood was obtained 30 min after the onset of the reaction. Plasma
histamine concentration was < 0.5 nmol litre"1,
whereas plasma tryptase was 35 ug litre"1 (normal
British Journal of Anaesthesia
434
value < 2 ug litre"1)- A second blood sample, obtained 1 h later, contained histamine < 0.5 nmol
litre"1 and tryptase 47 ug litre"1. Urine samples
contained methylhistamine 1180 nmol per mole of
creatinine (normal range < 380 nmol mol"1). Eight
weeks later, skin tests and specific IgE measurements
showed evidence of anaphylaxis to suxamethonium.
Case 2. A pregnant woman, aged 32 yr, had a
Caesarean section for a twin pregnancy. She received
suxamethonium and thiopentone and developed
acute bronchospasm, followed by diffuse urticaria
and severe hypotension 10 min later. A blood sample
was withdrawn immediately, but left at 4 °C overnight. Plasma histamine concentration was
0.6 nmol litre"1 and tryptase 63 ug litre"1. In a second
sample, 1 h later, histamine concentration was
0.6 nmol litre"1 and tryptase 86 ug litre"1. Urinary
methylhistamine concentration was 2160 umol per
mole of creatinine. Neuromuscular blocker-specific
IgE was present in high concentrations and skin tests
were positive for suxamethonium.
Case 3. A 56-yr-old man was administered fentanyl,
fiunitrazepam, thiopentone, pancuronium, Haemaccel and heparin before extracorporeal circulation
(ECC) for coronary bypass operation. When ECC
was started, acute hypotension was observed, which
was treated by acceleration of ECC, ephedrine and
fluid perfusion. A blood sample was obtained within
10 min of the reaction. Histamine concentration was
1.5 nmol litre"1 and tryptase 4.3 ug litre"1. One hour
later, histamine concentration was < 0.5 nmol litre"1
and tryptase 9.4 ug litre"1. Urinary methylhistamine
concentration was 1665 umol per mole of creatinine.
The molecule responsible was not identified.
In vitro histamine metabolism
We studied the rate of disappearance of histamine
from a plasma sample containing histamine,
300 nmol litre"1, mixed with plasma from a pregnant
woman (mix No. 1), from a heparinized patient (mix
No. 2) or from a normal subject (mix No. 3). When
10000i
£ 1000o
S
c
100-
50
100
150
Time (min)
200
Figure 5 Histamine concentrations (log) in two successive
samples from 12 patients who experienced anaphylactoid
reactions.
250
1000-1
~ 100-
~
10-
20
40 60 80
Time (min)
100
Figure 6 In vitro disappearance of histamine at 37 °C from
plasma containing histamine 300 nmol litre"1, mixed (vol/vol)
with normal plasma ( • ) , plasma from a pregnant woman (D)
or plasma obtained after 40 min of extracorporeal circulation
(O).
incubation was performed at 37 °C, histamine disappeared from mix Nos 1 and 2 with an apparent
half-life of 20 min (fig. 6). At 4 °C, the half-life was
130 min for mix No. 1 and 110 min for mix No. 2.
After 22 h at 4 °C, histamine concentrations were
4.2 nmol litre"1 in mix No. 1 and 1.8 nmol litre"1 in
mix No. 2. Histamine concentrations did not change
in mix No. 3 during the whole experiment.
Discussion
The aim of this study was to determine which
conditions could lead to false positive or false
negative values for plasma concentrations of histamine when using the radioimmunoassay, in order
to achieve an accurate diagnosis of anaphylaxis. The
monoclonal antibody used here is highly specific for
histamine; the cross-reactivity ratio for N-methylhistamine is 1/14500 and for histidine 1/250000 [8].
Numerous compounds known to be potentially
capable of interfering with measurements of histamine failed to interfere in this assay [9]. This assay
compares favourably with other techniques, according to a quality control study [18]. It was shown to
have equal sensitivity and accuracy as the fluorimetric assay, and to be convenient to use for plasma
samples at the Munich Consensus Development
Conference on Histamine Determination [19]. According to the manufacturer, the sensitivity is
0.2 nmol litre"1. In this study it was 0.5 nmol litre"1
(78 determinations over a 2-yr period). The variation
coefficient was 10% for a concentration of
10 nmol litre"1 (21 determinations) and in the normal
concentration range [9]. False positive results were
investigated in normal volunteers after lunch, as
food may increase plasma histamine concentrations.
The mean histamine concentration was 2.01 (0.92)
nmol litre"1, whereas it was 0.8 (0.4) nmol litre"1 in
13 fasting controls [10] (P < 0.01). Other laboratories reported values of 3.33 (1.62) nmol litre"1 [8]
in 14 non-allergic subjects or 1.74 (0.72) nmol litre"1
[9] in 40 normals using the same reagents.
From our results we suggest that a simplified
sampling procedure may be used comprising direct
venous puncture and sampling into glass vacuum
tubes containing either EDTA or heparin. The
Histamine: false positive and false negative values
threshold usually reported for pathological levels,
obtained with the fluorimetric method, is 1 ng ml"1,
that is 9 nmol litre"1 [3]. This value appears to be
appropriate for the radioimmunoassay also. The
stability of basophils in blood has been studied at
room temperature and at 4 °C, and during centrifugation at various temperatures. Although there
was a tendency for histamine to increase when blood
was left overnight at room temperature or for 2 days
at 4 °C, the samples could be left for 1 h at room
temperature or for 1 night at 4 °C without significant
changes in histamine concentrations, indicating that
histamine does not leak from basophils readily
enough to give false positive values. The values
obtained after centrifugation at different temperatures were very close, except when the centrifugation
temperature was 4 °C. This could be explained by
poor temperature regulation, with variations of
+ 2 °C or 3 °C, and potential freezing leading to cell
lysis. Thus we suggest die use of an intermediate
temperature of 15 °C. Although use of a nonrefrigerated centrifuge is possible, it induces a slight
increase in histamine concentrations. As centrifugation was performed at 4 °C for tubes 1-6, it is
possible that the higher values obtained apparently
at random in some samples could result from an
excessively low temperature.
During this study, false positive values were
observed in two different situations: (i) when
spontaneous haemolysis occurred (three of 224
samples, i.e. 1.2 %), histamine concentrations ranged
from normal to moderately increased. Histamine
increased concomitantly with an increase in haemoglobin in most cases. Spontaneous haemolysis
during sampling affects a few cells: haemoglobin
concentrations are in the micromolar range in
haemolysed plasma and millimolar in whole blood,
and histamine concentrations in haemolysed plasma
are 20-50 times smaller than in whole blood.
However, the red colour is readily noticed. Measurements of haemoglobin may be useful in interpreting
an increased histamine concentration in a reddish
sample obtained after an anaphylactoid event; (ii)
when plasma was aspirated from the upper cell layer,
a dramatic increase in histamine concentration
occurred. In such cases, no apparent change was
noticed in the tested plasma, contrary to the
haemolysed samples.
We were also interested in detecting possible false
negative results caused by the disappearance of
histamine either in vivo or in vitro. The in vivo halflife of plasma histamine has been calculated as
1-2 min in volunteers receiving histamine infusions
[4] and was shown to be in the minute range during
challenges [20] or protocols [21]. All of these values
were obtained in subjects with moderately increased
peak histamine concentrations. We selected for this
study 12 patients with increased concentrations of
plasma histamine during anaphylactic reactions to
i.v. administered drugs. All had increased concentrations of tryptase, which is a specific marker of mast
cell activation virtually absent from normal serum
[22]. The initial histamine concentrations were
increased markedly in the majority of patients
(38-5100 nmol litre"1). As no intermediate values
435
were obtained, the exact half-life could not be
calculated; however its value was probably closer to
20 than to 2 min. The longer half-life could be
explained by saturation of enzymatic metabolism, by
continued release or by reduced clearance because of
the disease process. The combination of a high initial
histamine concentration and a longer half-life than
expected allowed histamine to remain at pathological
levels 1 h after the onset of reaction in 11 of 12
patients.
The stability of histamine was studied further in
vitro. As large quantities of different plasma obtained
during anaphylactoid reactions were not easily
available, in some experiments we used normal
blood challenged in vitro with anti-IgE. On such
occasions the anticoagulant was heparin instead of
EDTA. However, heparin has no effect in vitro on
histamine metabolism and we have verified in one
patient that histamine concentrations were identical
in EDTA and heparin-containing tubes obtained at
the same time after an anaphylactoid event. Whole
blood could be left for 2 h at room temperature or
overnight at 4 °C without significant change in
histamine concentration. Blood could be centrifuged
at 4 °C, 15 °C or without refrigeration, without
changing histamine values. It had been suggested
that histamine could be adsorbed on tube walls,
especially where sterile glass is used, and that some
contaminants of the tube material could interfere
with the assay [23], but we could not confirm this. It
is likely that contaminants interfered with light
emission during the fluorimetric assays, whereas
radioimmunoassays are not affected by such conditions, as they are highly specific for the measured
molecule. Dyer and colleagues reported that histamine added to normal plasma was stable for 30 min
at room temperature or at 4 °C, but did not
experiment further [24]. In this study, endogenous
histamine was stable in separated plasma for 48 h at
room temperature or 72 h at 4 °C.
It is usual to suggest that one should not re-freeze
plasma samples that have previously been frozen.
However, five freeze-thaw cycles did not change
histamine concentrations in this study. When diluted
plasma samples were frozen, a dramatic decrease in
histamine concentrations was observed. The radioimmunoassay used in this report proceeds through
an alkylation step leading to stable alkylated histamine [8]. When diluted histamine samples were
alkylated before freezing, there was no decrease in
histamine concentration (results not shown). Thus,
when in vitro histamine-release tests are performed
from diluted blood samples, supernatants should be
either assayed immediately or alkylated before
freezing if the assay cannot be performed immediately.
False negative values have been identified in two
different clinical conditions: pregnancy and extracorporeal circulation. Tryptase concentrations are
increased in patients undergoing anaphylactoid reactions [10,25]. Thus the finding of increased
concentrations of tryptase in each of our three
patients indicated mast cell degranulation, and
obviously in vivo histamine release, which was
confirmed by increased methylhistamine concen-
British Journal of Anaesthesia
436
trations in urine. There are two reasons for the low
value of histamine: a shorter half-life in vivo and in
vitro degradation. Several reports have shown that
diamine oxidase activity increases during pregnancy,
with maximum activation during the third gestational trimester [26, 27]. It increases to similar levels
30 min after injection of large doses of heparin [28].
In patients administered low doses of heparin, the
effect appears to be dose-dependent [29]. We tried to
appreciate the in vivo catabolism of histamine under
similar clinical conditions: mixing plasma containing
large quantities of endogenous histamine with plasma from a pregnant woman or a heparinized patient
led to the disappearance of histamine from the
sample within 80 min at 37 °C, or 1 night at 4 °C,
whereas histamine was remarkably stable in plasma
from subjects with normal metabolism. Our results
confirm those of Morel and Delaage who showed
that exogenous histamine added to plasma from a
pregnant woman disappeared from the sample within
10 min whereas alkylated histamine did not [8].
These findings indicate that precautions should be
taken in order to avoid false negative values for
histamine in pregnant women and heparinized
patients: blood should be refrigerated and plasma
frozen as soon as possible, preferably after alkylation
of histamine. In contrast, patients with normal
metabolism of histamine do not require such precautions, as histamine is remarkably stable in their
plasma samples.
This study has shown that a great number of
precautions used with the fluorimetric assay are not
necessary with the radioimmunoassay, and that false
positive results are rare. Moderately increased
concentrations (< 50 nmol litre"1) may be found in
haemolysed samples. High values are found where
plasma is aspirated from the white cell layer. Thus
laboratory staff must be well informed of the
dramatic consequences in subsequent diagnosis.
During anaphylactic events, histamine disappeared
from plasma at a slower rate than expected, and high
values were found 1 h, and in some patients 2 h, after
the onset of the reaction. This allows emergency
treatment to be administered before sampling is
performed. Clinical conditions where histamine
metabolism is activated are rare. They include
pregnancy and high-dose heparinization. In such
cases false negative values are likely to occur. Plasma
tryptase should be measured in order to ensure an
accurate diagnosis, although the clinical sensitivity
of this marker is lower than that of histamine [17].
Histamine measurements should be undertaken
every time a clinical reaction suggesting anaphylaxis
occurs after drug administration. When resuscitation
is unsuccessful or after-effects are anticipated, such
measurements may give forensic evidence [11].
Where the reaction mechanism is unclear, they may
be helpful. Furthermore, during drug or food
challenges they can be of great interest for linking
together mechanisms and symptoms. As routine,
reliable techniques are available, and as false negative, and also false positive, results are rare and well
documented, there is no justification, apart from
cost, for not performing histamine measurements
every time drug anaphylaxis is suspected.
Acknowledgements
This work was supported by a grant from the National Health
Ministry. The anaesthetists who participated in the study are
kindly acknowledged. We thank the Laboratory of Nuclear
Medicine for excellent technical assistance, the Laboratory of
Haemostasis for Cardiovascular Diseases (M.-C. Khayat, MD)
for performing the haemoglobin measurements and Immunotech
International for providing the histamine kits.
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