WO2001009617A1

WO2001009617A1 - Anaphylaxis screening

Info

Publication number: WO2001009617A1
Application number: PCT/AU2000/000908
Authority: WO
Inventors: Brian Baldo
Original assignee: Nsl Health Limited
Priority date: 1999-08-03
Filing date: 2000-08-01
Publication date: 2001-02-08
Also published as: AUPQ199799A0

Abstract

A single-test assay to determine the likelihood of anaphylaxis to neuromuscular blocking drugs in a subject, the assay comprising (a) reacting a sample of the subject's serum with a morphine-like compound; and (b) testing for an interaction betweem the morphine-like compound and immunogloblulin E antibodies in the serum, wherein an interaction is indicative of the likelihood of anaphylaxis to neuromuscular blocking drugs in the subject. Use of a morphine-like compound in an assay to determine the likelihood of anaphylaxis to neuromuscular blocking drugs in the subject.

Description

ANAPHYLAXIS SCREENING

Technical Field

The present invention relates generally to medical tests and assays, particularly to a screening test for allergy to drugs used in anaesthesia.

Background Art

Although anaphylaxis during anaesthesia is a rare condition, its diagnosis has important implications particularly in providing safe future anaesthesia. At present, diagnosis requires the determination of both the nature of the reaction and the drug or other agents responsible for the reaction. Elevated levels of mast cell tryptase (MCT) approximately 1 to 4 hours after a reaction occurs is the single most useful test to confirm an anaphylactic event (Baldo BA, Fisher MM. Diagnosis of IgE-dependent anaphylaxis to neuromuscular blocking drugs, thiopentone and opioids.

Ann Fr Anesth Reanim 1993: 12:173-181; Fisher MM, Baldo BA. The diagnosis of fatal anaphylactic reactions during anaesthesia; employment of immunoassays for mast cell tryptase and drug-reactive IgE antibodies. Anaesth Intens Care 1993: 21:353-357) but this test does not identify the causative agent. The most valuable test to identify the drug responsible is skin testing which is usually carried out 4 to 6 weeks after the reaction (Fisher MM. Intradermal testing after anaphylactoid reaction to anaesthetic drugs; practical aspects of performance and interpretation. Anaesth Intens Care 1984; 12:115-120). Radioimmunoassays (RIAs) for the detection of drug-reactive IgE antibodies (Baldo BA, Harle DG. Drug allergenic determinants. In: Baldo BA, ed. Molecular Approaches to the Study of Allergens. Basel: Karger 1990; Monogr Allergy, 28: 11-51 ;Baldo BA, Pham NH. Structure-activity studies on drug-induced anaphylactic reaction. Chem Res Toxicol 1994; 7:703-721) provide important supplementary tests. The RIAs can also sometimes confirm the diagnosis by identifying the drug responsible in patients whose skin tests are negative and the in vitro tests offer the advantage that they can be carried out with blood taken at the time of the reaction. This increases the tests^' value in patients following cancellation of surgery due to known or suspected sensitivity and when surgery must be performed before skin testing can be reliably undertaken.

The major limitations to the use of the RIAs for neuromuscular blocking drugs (NMBDs) is that the preparation of the materials used in the tests is usually only possible in dedicated laboratories and. with the exception of the assay to detect IgE antibodies to succinylcholine. the correlation of results obtained with other RIAs for NMBDs with skin test results is not always good (Fisher MM. Baldo BA. Anaphylaxis during anaesthesia: current aspects of diagnosis and prevention. Europ J Anaesth 1994; 11:263-284).

In 1983, the present inventor showed that morphine was a potent inhibitor of the binding in vitro of some IgE antibodies to NMBDs in sera from a few subjects who had experienced an anaphylactic reaction following exposure to these drugs during anaesthesia (Baldo BA, Fisher MM.

Anaphylaxis to muscle relaxant drugs: Cross-reactivity and molecular basis of binding of IgE antibodies detected by radioimmunoassay. Molec Immunol 1983: 12:1393-1400). The mechanism of this inhibition was suspected to be cross-reactive recognition of substituted ammonium groups which are shared by the NMBDs. morphine and a number of other drugs and chemicals (Baldo

BA. Fisher MM. Substituted ammonium ions as allergenic determinants in drug allergy. Nature 1983: 306:262-264). Since that time, a range of available NMBDs have been used in routine RIA screening of sera from subjects with suspected anaphylaxis to a NMBD following drug administration during anaesthesia.

From extensive studies, the present inventor has found that one test can be used to determine the likelihood of anaphylaxis to a wide range of NMBDs thus replacing the need to carry out costly multiple tests to determine the possibility of anaphylaxis during anaesthesia.

Disclosure of Invention

In an attempt by the present inventor to determine the value of morphine-like compounds in solid-phase form as a potential diagnostic aid in suspected cases of NMBD-induced anaphylaxis. results of RIAs for the detection of drug-reactive IgE antibodies were compared with the clinical picture and skin test findings in a large group of patients defined as experiencing anaphylaxis on the basis of a positive serum MCT result and a positive skin test to a NMBD(s).

In a first aspect, the present invention provides a single-test assay to determine the likelihood of anaphylaxis to neuromuscular blocking drugs

(NMBDs) in a subject, the assay comprising: (a) reacting a sample of the subject's serum with a morphine-like compound: and

(b) testing for an interaction between the morphine-like compound and IgE antibodies in the serum, wherein an interaction is indicative of the likelihood of anaphylaxis to NMBDs in the subject.

Preferably, the morphine-like compound is selected from morphine, heroin, codeine, meperidine (pethidine), methadone, and fentanyl. More preferably, the compound is morphine.

The assay is preferably in the form of a solid-phase radioimmunoassay where the morphine-like compound is bound to a solid support. During the reacting of the serum with the morphine-like compound, any IgE antibodies in the serum which are reactive to NMBDs. bind to the morphine-like compound. After the binding reaction, the sample is then treated with a radiolabelled anti-human IgE antibody which binds to the bound IgE. This second binding is detected by measuring radioactivity associated with the test sample. The presence of radioactivity is indicative that IgE antibodies reactive to NMBDs were present in the serum and therefore indicating an interaction with the morphine-like compound.

It will be appreciated, however, that other means of detecting bound IgE including non-radioactive immunoassys would also be suitable.

The clear advantage of the present invention is that there is no need to test subjects' sera for specific reactions to many NMBDs in separate specific tests for each NMBD of interest as is currently required to determine whether a subject may suffer an adverse reaction at or during anaesthesia. In order to ensure that any positive reaction in the test is relevant, confirmation that the reaction is a true anaphylactic response is desirable. The present inventors have found that a second test can be employed to further enhance the diagnostic potential of the present invention.

In a second aspect, the present invention provides a double-test assay to determine the likelihood that a reaction to a neuromuscular blocking drug

(NMBD) in a subject is a true anaphylactic response, the assay comprising:

(a) carrying out the assay according to the first aspect of the present invention: and

(b) carrying out a second assay on the serum of a subject having an interaction between a morphine-like compound and IgE antibodies in the serum in assay (a) to determine whether the subject experienced a true anaphylactic response, wherein a positive reaction in (b) is indicative of the likelihood of experiencing a true anaphylactic response to NMBDs in the subject.

Preferably, assay (b) is a test for the presence of elevated mast cell tryptase (MCT) serum levels after obtaining serum from the subject within a desired sampling period from the reaction with the morphine-like compound. Preferably, the desired sampling period is 0.5 to 4 hours (optimal), but may be up to about 10 hours after the reaction, depending on the subject. The MCT assay is preferably carried out by employing an immunoassay, preferably radioimmunoassay, for the presence of human mast cell tryptase

(MCT).

It will be appreciated that other tests to determine whether a subject is capable of having a true anaphylactic response would also be suitable for assay (b). Such tests include skin (prick or intradermal) with suspected NMBDS. morphine or other alkaloids.

In a third aspect, the present invention provides use of an assay utilising a morphine-like compound to measure the presence of IgE antibodies in serum of a subject as a one-test assay to determine the likelihood of anaphylaxis to neuromuscular blocking drugs (NMBDs) in the subject.

Throughout this specification, unless the context requires otherwise. the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any description of prior art documents herein is not an admission that the documents form part of the common general knowledge of the relevant art in Australia.

In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples. Modes for Carrying Out the Invention

METHODS AND PATIENTS

Intradermal testing was carried out 4-6 weeks after the reaction with drugs diluted to concentrations determined as unlikely to produce reactions as a result of direct histamine release (Fisher MM. Intradermal testing after anaphylactoid reaction to anaesthetic drugs: practical aspects of performance and interpretation. Anaesth Intens Care 1984: 12:115-120).

RIAs for drugs (Baldo BA. Fisher MM. Anaphylaxis to muscle relaxant drugs:

Cross-reactivity and molecular basis of binding of IgE antibodies detected by radioimmunoassay. Molec Immunol 1983: 12:1393-1400) and the immunoassay for tryptase determination have been described (Enander I,

Matsson P. Nystrand }. Andersson A. Eklund E. Bradford TR, Schwartz LB.

A new radioimmunoassay for human mast cell tryptase using monoclonal antibodies. J Immunol Methods 1991; 138:39-46: Fisher MM, Baldo BA. The diagnosis of fatal anaphylactic reactions during anaesthesia: employment of immunoassays for mast cell tryptase and drug-reactive IgE antibodies.

Anaesth Intens Care 1993; 21:353-357). All serum samples for MCT determination were taken 30 min to 4 hours after the adverse reaction

(Schwartz LB. Yunginger JW. Miller J. Bokhari R, Dull D. Time course of appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis. J Clin Invest 1989: 83:1551-1555). Six patients who died but survived for 4 hours after the reaction and had a positive serum tryptase level in the postmortem blood were included in the study.

Results of the immunoassays were considered in the following groups:

Group 1. Patients who had an elevated serum MCT level and showed a positive skin test to at least one NMBD.

Group 2. Patients who had an elevated serum MCT level and showed a positive skin test to a drug other than a NMBD. Group 3. Patients who had suspected anaphylaxis but a serum MCT level that was not elevated and skin tests to NMBDs were negative.

Group 4. Patients who had suspected anaphylaxis, serum MCT levels were not elevated and no skin testing was performed. Control sera were obtained from normal, healthy laboratory workers and cord sera, used as an IgE-'free^' control, from the Department of Obstetrics. Royal North Shore Hospital

Specificity, sensitivity, efficacy and positive predictive values for the morphine and NMBD RIAs were calculated using the methods of Galen and

Gambino (Galen RS, Gambino SR. Beyond Normality: The predictive value and efficiency of medical diagnoses. J Wiley and Sons. New York 1st Ed. 1975).

RESULTS

With the aim of subdividing the 347 subjects suspected of experiencing an anaphylactic reaction following anaesthesia into homogeneous groups for meaningful comparison, MCT levels were determined in the serum of each subject. Subjects with elevated MCT levels were then further divided into those who were skin test positive to a NMBD given at the time of the reaction

(Group 1, 118 subjects) and those who were skin test-negative to these drugs but positive to a different drug given at the time of the reaction (Group 2. 45 subjects). Of the 184 subjects whose serum MCT levels were not elevated, 56 were skin test negative to the drugs administered (Group 3) while in 128 cases, skin tests were not performed (Group 4).

Results of the RIAs for the NMBDs and morphine carried out on the sera from each of the 4 different groups are shown in Table 1. Whereas in Group 1. IgE antibodies to the NMBD given at the time of the reaction were detected in only 56 of 108 subjects. 100 of 118, or 85% of subjects, had IgE antibodies that reacted with morphine. Breaking down these numbers into the subgroups formed from the 8 different NMBDs revealed that the RIA to detect suxamethonium sensitivity was the drug test that correlated best with skin test findings (—70%). IgE was detected in only 2 of 6 and 2 of 8 subjects who reacted following administration of atracurium and alcuronium respectively while the frequencies of antibodies detected to the steroid-like NMBDs vecuronium, pancuronium and rocuronium were also low. By contrast, the morphine RIA picked up 67 of 69 (-97%). 9 of 10 and 15 of 15 reactors to suxamethonium. vecuronium and rocuronium respectively. Only 4 of 8 and. interestingly, only 3 of 12 reactors to alcuronium and atracurium respectively reacted positively to the morphine solid phase (Table 1). Table 1. Incidence of positive immunoassays for the detection of IgE antibodies to morphine and NMBDs in 347 patients who experienced an adverse reaction during anaesthesia

Number positive for IgE antibodies

Group Number Morphine NMBDs

1. MCT elevated

Positive skin test for

NMBD(s) 118 100/118 56/108

Breakdown for individual NMBDs:

Suxamethonium 69 67/69 47/69 Vercuronium 10 9/10 2/10 Rocuronium 15 15/15 3/13 Pancuronium 2 0/2 0/2 Alcuronium 8 4/8 2/8 Atracurium 12 3/12 2/6 Gallamine 1 1/1 0/0 Mivacurium 1 1/1 0/0

2. MCT elevated

Positive skin test for druj; other than NMBD 45 2/45 ^J 0/45

3. MCT not elevated

Skin test negative 56 1/56³ 0/56

4. MCT not elevated

No skin tests performed 128 2/128⁴ 0/128

One patient reacted to flucloxacillin.

One patient reacted to morphine: Result probably reflects presence of true IgE antibodies to morphine.

Subject had a convincing clinical reaction to alcuronium.

Two tests marginally elevated with uptakes of 3.1% and 3.4%. Upper limit of normal = 3%. TABLE 2. Performance comparisons of morphine and NMBD RIAs in tests for the detection of IgE antibodies in 347 patients suspected of anaphylaxis to a NMBD(s)

Test Specificity¹'² Sensitivity^1,3 Positive predictive Efficiency^1,5 value^1,4

Morphine RIA 98% 85% 96% 94% NMBD RIAs 100% 52% 100% 82%

1

Values calculated according to Galen and Gambino¹⁸

Defined as negativity in health

Defined as positivity in disease

Defined as predictive value of a positive result

Defined as the percentage of all results that are true results

In Groups 2, 3 and 4. IgE antibodies to NMBDs were not detected in any of the sera but antibodies to morphine were detected in 2, 1 and 2 subjects respectively. Two patients had elevated MCT levels, positive skin tests to a drug (not a NMBD) given prior to the reaction, and morphine antibodies. One reacted to flucloxacillin and one to morphine. The one patient with a positive morphine RIA. negative skin tests and a normal MCT level had a convincing clinical reaction and may be allergic to alcuronium. Two patients in whom MCT was not elevated and no skin testing was performed had low levels of IgE antibodies reactive with morphine ( uptakes of 3.1% and 3.4%: upper limit of uptake with normal sera. 3%).

The test has been used under clinical situations where 235 patients have been screened. Of the total tested. 159 were found to be both positive for NMBDs and morphine. Importantly, all positive morphine subjects assayed correlated to NMBD indicating a likelihood of anaphylaxis to at least one neuromuscular blocking drug.

DISCUSSION

One of the major uncertainties in evaluating diagnostic test results in suspected cases of anaphylaxis to anaesthetic drugs is the possibility that a negative result to the suspected drug(s) may occur in a subject who experienced some or all of the signs associated with an anaphylactic or anaphylactoid reaction due to another, non-allergic, mechanism. In such cases it is difficult to determine whether the result is due to a weakness inherent in the test, such as lack of sensitivity, or whether the negative finding truly reflects the real situation. Use of the MCT assay enables the investigator to determine whether or not a suspected reaction was truly anaphylactic that is, it identifies an homogeneous study group since, when serum tryptase is elevated, an anaphylactic (or less commonly an anaphylactoid) reaction is the most likely mechanism (Fisher MM, Baldo BA.

Mast cell tryptase in anaesthetic anaphylactoid reactions. Brit J Anaesth 1998: 80: 26-29). When this information is used with results of skin tests responses to drugs, homogeneous groups of anaphylactic and non- anaphylactic reactors can be further studied in vitro for the presence or absence of drug-reactive serum IgE antibodies. Results presented here demonstrate how closely the drug RIAs for IgE antibodies correlate with the group (Group 1) defined by elevated MCT levels and positive skin test responses to NMBDs compared to the other three groups. Further, the absence or low frequency of positive reactions in Groups 2. 3 and 4 (Table 1) suggests that positive results to both the morphine and NMBD RIAs are clinically significant, that is likely to be associated with reactions (Table 2).

The early studies showed that IgE antibodies in the sera of subjects who had anaphylaxis to a NMBD were complementary to substituted ammonium groups on the NMBDs and other drugs and chemicals including morphine. This led to the hypothesis that cross-linking of mast cell-bound

IgE antibodies, the prelude to allergic mediator release, may occur via the ammonium ion antigenic determinants on protein-bound or even free NMBDs since all NMBDs are at least divalent with respect to substituted ammonium groups. Experimental verification of release of mediators from human leucocytes by free NMBDs appears to have been provided (Vervloet D.

Arnaud A. Senft M, Dor P. Didier A, Bongrand P, Charpin J. Anaphylactic- reactions to suxamethonium. Prevention of mediator release by choline. J Allergy Clin Immunol 1985; 76:222-225: Didier A, Cador D. Bongrand P. Furstoss R. Fourneron P. Senft M. Philip-Toet F. Charpin D, Charpin J. Vervloet D. Role of the quaternary ammonium ion determinant in allergy to muscle relaxants. } Allergy Clin Immunol 1987: 79:578-584.). Compounds such as morphine that cross-react with NMBD-reactive antibodies via a single substituted ammonium group would not. in the free state, be expected to mediate allergic release by cross-linking IgE bound to mast cells and basophils. Although anaphylaxis to morphine has been reported together with morphine-reactive IgE antibodies, allergic reactions to the narcotic are rare .

What is now apparent from the present results is that the morphine RIA proved to be the best single in vitro test for the detection of IgE antibodies that react with substituted ammonium ions and hence for the in vitro diagnosis of NMBD-induced anaphylaxis. Detection of IgE antibodies in the morphine RIA showed an 86% correlation with the 118 subjects with an elevated MCT level and identified as skin test-positive to an NMBD given immediately prior to the reaction. This compares very favourably with the NMBD RIAs where results with the compounds with steroid-like structures

(vecuronium, pancuronium. rocuronium) are poor, chiefly because of technical difficulties arising from the chemical structures of these drugs. For the morphine assay, correlations between the detection of IgE antibodies on the one hand and the subjects with MCT elevation and a positive skin test (Group 1. Table 1) on the other hand, were excellent (90-97%) for suxamethonium. vecuronium and rocuronium. Interestingly, only 3 of 12 patients who reacted to atracurium were picked up as positive in the morphine RIA. A possible explanation for this is that the reactions were not anaphylactic. Lorenz^'s group has identified a group of patients who have extreme reactions to the direct histamine releasing effects of atracurium

(Lorenz W. Sitter H. Stinner B. Duda D. Kapp B, Gstrein B. Dietz W. Doenicke A. Dick W. Controlled clinical trials and cross-sectional studies with plasma histamine measurements and histamine receptor antagonists: solving the problem of preoperative Hi- + H2-prophylaxis by asking new questions? Agents and Actions. Suppl. 1991; 33:197-230) and. in a situation similar to that described with Haemaccel^®, such reactions could be associated with positive skin tests and elevated MCT levels.

The relevance of these findings is that the morphine RIA offers an improvement over the battery of RIAs for individual NMBDs (Table 2) and provides a valuable adjunct to skin test and tryptase determination in the diagnosis of allergic reactions to NMBDs. Its usefulness is particularly apparent when a diagnosis is needed in skin test-negative patients or prior to skin tests which, optimally, are usually carried out about one month after a reaction. In addition to its superior performance, the use of the morphine RIA instead of the battery of tests for individual NMBDs greatly reduces the time, labour and cost of in vitro testing in cases of suspected anaphylaxis to NMBDs and we have altered our laboratory practice to perform only the morphine RIA.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are. therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A single-test assay to determine the likelihood of anaphylaxis to neuromuscular blocking drugs in a subject, the assay comprising:

(a) reacting a sample of the subject's serum with a morphine-like compound: and

(b) testing for an interaction between the morphine-like compound and immunogloblulin E antibodies in the serum, wherein an interaction is indicative of the likelihood of anaphylaxis to neuromuscular blocking drugs in the subject.

2. The assay according to claim 1 wherein the morphine-like compound is selected from the group consisting of morphine, heroin, codeine, meperidine (pethidine). methadone, and fentanyl.

3. The assay according to claim 2 wherein the morphine-like compound is morphine.

4. The assay according to any one of claims 1 to 3 in the form of a solid- phase radioimmunoassay where the morphine-like compound is bound to a solid support.

5. A double-test assay to determine the likelihood that a reaction to a neuromuscular blocking drug in a subject is a true anaphylactic response, the assay comprising:

(a) carrying out the assay according to any one of claims 1 to 4; and

(b) carrying out a second assay on the serum of a subject having an interaction between a morphine-like compound and immunogloblulin E antibodies in the serum in assay (a) to determine whether the subject experienced a true anaphylactic response, wherein a positive reaction in (b) is indicative of the likelihood of experiencing a true anaphylactic response to neuromuscular blocking drugs in the subject.

6. The assay according to claim 5 wherein the second assay (b) is a test for the presence of elevated mast cell tryptase serum levels after obtaining serum from the subject within a desired sampling period from the reaction with the morphine-like compound.

7. The assay according to claim 6 wherein the desired sampling period is up to about 10 hours after the reaction.

8. The assay according to claim 7 wherein the desired sampling period is about 0.5 to 4 hours after the reaction.

9. The assay according to any one of claims 6 to 8 wherein the test for the presence of elevated mast cell tryptase serum levels is carried out using an immunoassay for the presence of human mast cell tryptase.

10. The assay according to claim 9 wherein the immunoassay is a radioimmunoassay.

11. The assay according to claim 5 wherein the second assay (b) is a skin (prick or intradermal) test with suspected one or more neuromuscular blocking drugs, morphine or other alkaloids.

12. Use of an assay utilising a morphine-like compound to measure the presence of IgE antibodies in serum of a subject as a one-test assay to determine the likelihood of anaphylaxis to neuromuscular blocking drugs in the subject.