To the Editor,
Immediate hypersensitivity reactions are related to mast cell and/or
basophil activation. The mediators released such as tryptase and
histamine are involved in clinical symptoms and are key parameters that
contribute to diagnosis. Serum tryptase concentrations peak between 30
minutes and 4 hours following the reaction. Tryptase release is
considered a robust marker of mast cell degranulation but is not
informative in mild reactions.1 Histamine is released
at the early beginning of the reaction but has a short half-life. The
concentration value can be altered by pre-analytic
conditions.2 Anaphylactic reactions can occur during
night and week-end when laboratories can’t take charge of samples. To
our knowledge, no thorough study of the stability in whole blood of
these markers in anaphylaxis has been published. The aim of our study
was to evaluate the impact of whole blood sample storage conditions
(temperature and delay before centrifugation and plasma collection) on
the reliability of tryptase and histamine measurements.
Blood samples from 14 patients suspected of anaphylactic reactions
(grade 2 to 4 of the Ring and Messmer scale)3 and from
10 volunteers were collected on EDTA after signed informed consent
(CCPPRB Caen Basse-Normandie protocol 2004-32). The description of the
patient anaphylactic episodes appears in Table 1.
When received in the lab, an aliquot of whole blood was processed for
diagnostic (reference measurement) and the remaining was divided in
aliquots stored at room temperature (RT) or at +4°C for 24, 72 hours or
7 days (patients) or 2, 6, 24 or 72 hours for controls before
centrifugation and plasma collection.
Total tryptase concentrations were measured by an automated
fluoroimmunoassay (ThermoFisher, Phadia SAS, ). Increased tryptase is
defined as ≥ 1.2 x basal value + 2
µg.L-1.4 In our hands, tryptase
uncertainties of measurement for low and high concentrations (9
µg.L-1 and 38.2 µg.L-1) are 17% and
16% respectively, in accordance with published
results.5 Plasma histamine concentrations were
measured by a radioimmunoassay (Beckman Coulter, Immunotech, France).
Increased values defined by the manufacturer are >10
nmol.L-1, in accordance with published
data.6 In our hands, histamine uncertainties of
measurement for low and moderate concentrations (4.7
nmol.L-1 and 12.9 nmol.L-1) were
22% and 25%, respectively.
The differences between the concentrations measured before and after
storage were compared by paired two-tailed t-tests using SAS software.
Results were considered significant for p < 0.05.
As shown in Figure 1A, storage conditions did not modify tryptase
concentrations (linear regression: slope=1.079, R²=0.9675). Tryptase
concentrations appeared stable in whole blood left at +4°C for 7 days or
72 hours at RT.
Histamine concentrations in patient samples were not modified during 72h
at +4°C (Figure 1B) or at RT (Figure 1C). In the control group at RT,
histamine concentrations were significantly increased at 6 hours
(p=0.005) although moderately increased and staying within the limits of
uncertainty measurement and never reaching the positivity threshold
(Figure 1E). After 24 hours at RT a false positivity was observed for 8
of 10 samples (p<0.0001) (Figure 1E). At +4°C, histamine
concentrations were significantly increased after 24 hours
(p<0.0001) but remained in the limits of uncertainty
measurement and under the threshold of positivity (Figure 1D). After 72h
at +4°C, histamine concentrations exceeded the limits of uncertainty
measurement and the positivity threshold for 4 samples of 10 (Figure
1D).
Tryptase and histamine measurements are recommended to prove
degranulation in anaphylaxis.7 Anaphylactic reactions
occur unexpectedly. It is thus important to master sample shipment and
processing before mediator measurement.
The knowledge of possible artifacts modifying the measured values is
necessary for the biochemist to address accreditation criteria of
pre-analytic requirements (ISO 15189 standard) and for the physician to
rely on trustable diagnostic data.
Tryptase stability in whole blood had not been described. Our data has
shown no impact on results after 72h at RT or 7 days at +4°C. Tryptase
stability in plasma or serum has been evaluated by the manufacturer who
ensured stability for 48h at RT or 5 days at +2°C to +8°C
(Thermofisher).8 Thus, measured values of tryptase
appear highly reliable.
Histamine stability in whole blood had only been evaluated in controls
and false positive results may be attributed to passive release from
basophil during prolonged storage.2 We observed no
impact for patient blood samples after 72h at +4°C or at RT. In
contrast, false positive results were observed in controls after storage
at RT during 24h or at +4°C during 72h. Histamine is known to be stable
in the plasma obtained after centrifugation up to 4 days at RT for
patients and controls.9
According to these results, we suggest that whole blood samples can be
stored at +4°C up to 72h for histamine and 7 days for tryptase when the
laboratory is not available immediately. In any case, the biochemist
must accept all these unrenewable samples. It is his role to take into
account the pre-analytical conditions to interpret the results and
provide helpful information to the physician.
Keywords : pre-analytic; tryptase; histamine; whole
blood.