Tryptases are proteases located in mast cell granules and in small quantities in basophils. The determination of tryptase values in serum has proven to be useful as a marker of mast cell activation in both anaphylaxis and mastocytosis.
To date, the role tryptase play in vivo has still not been well defined . In vitro studies postulate that they participate in the inactivation of fibrinogen as well as in the inhibition of fibrinogenesis, the activation of collagenase in synovial cells, the inactivation of some neuropeptides that have a bronchodilating function, such as VIP, the stimulation of fibroblast proliferation and of the synthesis of mRNA by procollagen in culture and chemotactic activity carried out by eosinophils.
Since tryptase is expressed almost exclusively in mast cells and has a longer half-life than does histamine, these proteases are sensitive and specific markers of mast cell degranulation.
DETERMINATION OF SERUM TRYPTASE
Mast cells play a key role in allergic reactions and increase under inflammatory conditions. Once activated, they release a variety of mediators that trigger the signs and symptoms involved in allergic reactions, such as anaphylaxis. These mediators include histamine and tryptase. Tryptase is a protein found in mast cells. There are two proformes (monomeric), α-tryptase and ß-tryptase and the concentration reflects the number of mast cells, while the mature β-tryptase (tetrameric) indicates the activation of mast cells. The total serum tryptase i.e. the two proformes α-tryptase and ß-tryptase plus mature tryptase, may be measured.
Normal tryptase values are 1-16 g / l.
Human mast cells play a central role in inflammatory processes and are activated, in particular, during allergic reactions. That is why an increase in tryptase serum levels is considered a marker of allergic reaction and allows for the diagnosis of "anaphylactic shock". 1
Mast cells are activated by allergens through an IgE mediated mechanism, resulting in a release of tryptase that shows increased serum levels. The peak is reached 15-120 minutes after the onset of the reaction, the tryptase level then slowly decreases within 3-6 hours later. There is usually a return to baseline levels about 24 hours after the onset of the reaction. Therefore the "tryptase curve” is performed in the presence of a suspected "allergic reaction", with assays every three hours for the first 24 hours.
However, it should be born in mind that systemic anaphylactic reactions without tryptase increase may be provoked by biological mechanisms which are unrelated to mast cells. Although elevated levels of tryptase in post-mortem samples may indicate a fatal anaphylactic reaction as the cause of death, moderately high levels have been observed in post mortem samples not attributable to anaphylactic reactions.
Other pathologies also show an increase in mast cells 2 and, therefore, in baseline tryptase:
- Acute myelogenous leukemia
- Indolent systemic mastocytosis
- Aggressive systemic mastocytosis
Basal tryptase dosage is considered important in atopic individuals who are to be given immunotherapy (especially for hymenoptera venom) or surgical intervention, as high basal tryptase levels are considered to be expression of an increased risk of adverse reactions in the course of immunotherapy, or surgery.
THE METHOD USED FOR THE DETERMINATION OF TRYPTASE
Anti-tryptase, covalently coupled to the system which is used for the assay, reacts with the tryptase in the patient's serum sample. Anti-tryptase antibody conjugated with the enzyme to form a complex is added after the washing step. The unbound anti-tryptase enzyme is eliminated after the incubation step by subsequent washing and the immunocomplex is brought into contact with the development substrate. The released fluorescence is measured once the reaction has been stopped. The results are then evaluated by converting the responses of the samples into concentrations using the calibration curve. Samples of venous plasma and serum (EDTA or heparin) can be used. The samples may be kept at room temperature only for the purpose of shipping, otherwise from 2 ° -8 ° C if the determination is performed within one week of sampling. If the serum concentrations are determined later than one post sampling week they are to be stored at -20 ° C.
It is advisable that sampling be carried out no earlier than 15 minutes from the onset of the event supposed to have been responsible for the activation of the mast cells, up to a maximum 3 post-onset hours. The time lapse between the reaction and sample collection must be noted. A further blood sample after 24 to 48 hours (depending on the amplitude of activation) is to be taken so as to confirm the return to baseline levels.
Should there be the suspicion of elevated baseline tryptase levels, or pre-existing mastocytosis, then one or more samples, 1 or 2 weeks later, are to be taken. Any post-mortem samples must be taken within 48 hours from the time of death.
Normally, there is no need to dilute the sample, with the exception of values over and above that of 200 g / l.
Dose range: undiluted sample: 1-200 mg / l.
The minimum detection limit is <1.0 g / l.
The tryptase dose technology is based on an extremely high total binding capacity, achieved through a high binding capacity per milligram of cellulose in combination with an optimal amount of cellulose in each solid phase. This ensures the binding of all the relevant antibodies, regardless of the antibody affinity, providing a reduced non-specific binding.
The ImmunoCAP solid phase (method commonly used for the determination of the dose of serum tryptase) is constituted by a derivative of cellulose enclosed in a capsule. The highly branched hydrophilic polymer provides an ideal microenvironment for allergens, binding irreversibly and while maintaining their native structure.
• Hogan AD, Schwartz LB. Markers of mast cell degranulation. Methods 1997; 13: 43-52.
• Schwartz LB, Bradford TR, Rouse C, Irani A- M, Van der Zwan JK, Van der
Linden P-W G. Development of a new, more sensitive immunoassay for human tryptase: use in systematic anaphylaxis.J Allergy Clin Immunol 1994; 14(3):190-204.
•1Schwartz LB. Diagnostic value of tryptase in anaphylaxis and mastocytosis. Immunol Allergy Clin North Am. 2006 Aug; 26(3):451-63.
•2Van Doormaal JJ, van der Veer E, van Voorst Vader PC, Kluin PM, Mulder AB, van der Heide S, Arends S, Kluin-Nelemans JC, Oude Elberink JN, de Monchy JG. Tryptase and histamine metabolites as diagnostic indicators of indolent systemic mastocytosis without skin lesions. Allergy. 2012 May; 67(5):683-90.