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Abstract
Introduction Perioperative anaphylaxis (POA) can lead to significant complications. Therefore, accurate identification of allergens for POA patients is critical to ensure the safety of future surgical and anaesthetic procedures. Existing perioperative allergen detection methods face challenges in sensitivity and specificity. The passive mast cell activation test (pMAT) has recently emerged as a potential diagnostic tool. Our study aims to evaluate the diagnostic efficacy of pMAT for identifying perioperative allergens, with a focus on non-depolarising neuromuscular blocking agents, the most common culprits of POA.
Methods and analysis This prospective diagnostic accuracy study will measure the diagnostic accuracy of pMAT in POA patients. Participants will undergo skin testing (ST), basophil activation testing (BAT) and pMAT. The diagnostic validity of pMAT will be assessed based on the results of ST and BAT. The assessment of diagnostic accuracy will include sensitivity, specificity, likelihood ratios, and false-positive and false-negative rates while measurement of the consistency rate will assess reliability.
Ethics and dissemination This study has been approved by the Institutional Review Board of China-Japan Friendship Hospital (2023-KY-247). Results will be disseminated through academic presentations and peer-reviewed journal publications and will provide valuable scientific data and some new insights into the diagnostic accuracy of pMAT.
- anaesthetics
- allergy
- medicine
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STRENGTHS AND LIMITATIONS OF THIS STUDY
Passive mast cell activation test (pMAT) diagnoses allergens by quantifying the percentage of mast cell activation postexposure.
pMAT can be performed on stored serum samples, which may potentially improve diagnostic accessibility.
We will evaluate the diagnostic accuracy of pMAT in identifying perioperative allergens.
The use of laboratory of allergic diseases 2 (LAD2) cells in our study may not fully recapitulate primary human mast cell responses.
Introduction
Perioperative anaphylaxis (POA) represents a significant but rare crisis encountered during anaesthesia and surgical procedures. The reported incidence of POA exhibits substantial geographic variation, with rates ranging from 1:18 600 to 1:353 in suspected cases.1 2 In China, the prevalence of suspected severe POA is currently 1 in 11 360 cases.3 The severity of POA is underscored by mortality rates ranging from 1.4% to 4.8%.4–6 Additionally, POA is associated with prolonged hospitalisation and escalated healthcare costs.7 The rapid exposure to numerous potential allergens during the perioperative period exacerbates the challenge of identifying the precise allergen responsible for an event. Conventional diagnostic methods, such as skin testing (ST) and basophil activation testing (BAT), often fall short in their diagnostic capacity, leaving many POA cases unresolved.8 This diagnostic gap can significantly hamper the anaesthesiologist’s ability to select appropriate drugs for anaesthesia, which may affect surgical management.
The passive mast cell activation test (pMAT) has recently emerged as an attractive supplement to ST and BAT to diagnose allergens by sensitising donor/humanised mast cells with patient sera. This method has been recognised for its increased sensitivity and specificity in the context of peanut allergy.9 The potential complementary role of this technology in the diagnosis of chlorhexidine and rocuronium allergy has also been preliminarily validated.10 11 This study aims to investigate the diagnostic accuracy of the pMAT for perioperative allergens, particularly non-depolarising neuromuscular blocking agents (NMBAs).
Methods and analysis
Study overview and design
This investigation will be conducted as a prospective diagnostic accuracy study within a network of hospitals that participated in our previous epidemiologic research. We intend to enrol 27 hospitals from this network, each of which will be tasked with reporting cases of suspected POA. A designated physician from the anaesthesiology department of each participating hospital will be responsible for case reporting. The reported cases will be screened again by two experienced anaesthesiologists according to predefined inclusion and exclusion criteria. Eligible patients would be included in this study and invited to undergo allergen test in the China-Japan Friendship Hospital. The study has been rigistered in Chinese Clinical Trial Registry (ChiCTR2400084268) and the patient recruitment is scheduled to take place from May 2024 to May 2025.
The primary objective of this study is to evaluate the diagnostic accuracy of pMAT in identifying specific perioperative allergens, especially NMBAs. ST and BAT will be used as reference standards for evaluating the diagnostic accuracy of pMAT. A positive result in either test will permit the diagnosis of allergens. Besides, subject to sufficient enrollment, the study aims to expand its diagnostic scope to include other perioperative agents, such as sedatives and opioids. Figure 1 provides a schematic flow chart delineating the study protocol.
A flow chart of the study design. BAT, basophil activation testing; NMBAs, neuromuscular blocking agents; pMAT, passive mast cell activation test; ST, skin testing.
Sample size considerations
As a diagnostic accuracy study, the number of patients in the case group required for the diagnostic test is estimated by sensitivity, and the number of patients in the control group required for the diagnostic test is estimated by specificity. The specific number is calculated according to the following formula:
As shown in previous researches, the sensitivity and specificity of pMAT in detecting potential allergens are approximately 97% and 92%, respectively.9 Considering a two-sided significance level of 0.05 (α) and an allowable error of 0.1 (δ), our calculations suggest that a minimum of 12 patients should be enrolled in the case group and 29 patients in the control group. The primary objective of the study is to evaluate the diagnostic accuracy of pMAT for NMBAs as perioperative allergens. Furthermore, this study aims to explore the diagnostic accuracy of pMAT for other potential perioperative allergens, including opioids and sedativs. The number of cases required for each drug will be the same as calculated above.
Case report
The case will be reported using a standardised online data entry system specifically developed for this study. Each reporter will receive a unique identifier and password to access a secure website for submitting case details. Once submitted, the data will be stored in an online repository that can only be accessed by investigators involved in our study team.
The report for this study will be divided into two parts. The first will focus on anaphylaxis clinical manifestations, including cardiovascular system, respiratory system, as well as dermatological and mucosal reactions, alongside patient baseline information and anaesthesia records for verification. Designated physicians in each hospital will be kindly asked to report these data promptly after the occurrence of POA.
The second part will offer a comprehensive overview, covering details on anaesthesia and surgery, medical history, POA, perioperative medication and rescue treatment. Investigators will complete this section of the report by referring to the patient’s medical records or by telephone follow-ups.
Inclusion criteria
Adults aged 18 and older who have been diagnosed with POA are eligible for this study. Patients with POA may exhibit various clinical manifestations within 15 min after the administration of anaesthetic drugs during the perioperative period. Criteria for inclusion include:
Circulatory system: hypotension (systolic blood pressure ≤70 mm Hg), tachycardia (heart rate ≥110 beats/min) or rapid progression to severe arrhythmia, circulatory failure or cardiac arrest.
Respiratory system: increased secretion of saliva and sputum, laryngospasm, bronchospasm, increased airway pressure (peak ≥40 mm Hg), wheezing, moist rales in the lungs and hypoxaemia (oxygen saturation ≤90%).
Digestive system: nausea, vomiting or diarrhoea.
Dermatological and mucosal reactions: skin flushing, various rashes (especially wind-blown papules), subcutaneous angioedema, systemic skin oedema and oedema of nasal, ocular and pharyngeal mucosa.
To ensure accuracy, two investigators will independently assess the allergic cases, with a third investigator making the final decision in case of disagreement.
Exclusion criteria
Patients with systemic manifestations meeting the inclusion criteria but failing to make differential diagnosis from non-allergic diseases such as asthma, haemorrhagic shock, deep anaesthesia or pulmonary embolism.
Patients who are unable to discontinue antihistamines or glucocorticoids prior to BAT or pMAT, which may affect the results.
Patients with severe immune system diseases, haematological system diseases or skin diseases that may impact the allergen detection results.
Anaphylaxis occurring within 4–6 weeks prior to study entry.
Patients who decline to participate in the study.
Skin tests
ST will be performed by two experienced anaesthesiologists in the postanaesthesia care unit (PACU). We present a list of potential ST drug concentrations according to guideline recommendations (table 1).12 13 Morphine serves as a positive control while normal saline (0.9%) is used as a negative control for all tests. The brief steps for the ST are as follows:
Maximum concentration of drugs in ST and BAT
The patient assumes an upright position with the entire arm exposed. Either the medial forearm or lateral upper arm is selected as the test site. The skin is disinfected with 75% alcohol, and the test is performed after the alcohol has evaporated.
In the skin prick test (SPT), the skin is pricked with a disposable prick needle through a drop of the test drugs and the needle is then discarded. The resulting weal diameter is recorded after 15–20 min.
The intradermal test (IDT) entails injecting 0.02–0.05 mL of the preprepared test substance into the dermis using a 1 mL syringe, resulting in a weal diameter of approximately 2–3 mm. After 15–20 min, the maximum diameter of the weal and erythema is recorded.
Positive results would be interpreted as follows: SPT results are read after 15–20 min, and a weal with a diameter of ≥3 mm is considered positive. If SPT is negative or inconclusive, an IDT would be performed on the volar side of the forearm. The results of IDT are measured after 20 min, and a weal with an increase in diameter ≥3 mm compared with the original bleb would be considered positive.12
Specimen collection and storage
When the patients come to China-Japan Friendship Hospital for identifying perioperative allergens, we will collect blood samples for BAT and pMAT. The BAT will be performed with whole blood within 4–6 hours after the collection, and the whole blood sample will be stored on ice. We will separate the serum by centrifugation with the other part of blood sample for pMAT. As pMAT primarily involves IgE in the serum, unlike BAT which requires living cells in the sample, the serum used for pMAT can be frozen if it will not be detected immediately after collection.
Basophil activation tests
The experimental procedures will adhere to the recommendations from the Flow CAST kit (Bühlmann Laboratories AG, Switzerland), and the results of BAT will be analysed by flow cytometry. After venipuncture, a 5 mL of blood will be collected into an EDTA tube and sensitised with the suspected allergen within 4 hours. Table 1 provides a list of drugs concentrations used in BAT.8 14 15 For each tube, basophil activation is induced by adding 50 µL of the patient’s whole blood sample, 50 µL of the corresponding stimulator (with the stimulation buffer serving as the patient’s background control and FcεRI antibody as the stimulus quality control) and 100 µL of the stimulation buffer.
After that, FITC anti-human CD63 and PE anti-human CCR3 would be added to stain cells. The remaining red blood cells are removed by a lysis reaction, and the cells are resuspended in a wash buffer and examined by flow cytometry. For a correct evaluation and standardisation of the results, the activation gate should be set with 2%–2.5% basophil of background exhibiting spontaneous activation according to the requirements of the kit. Under this circumstance, BAT will be considered positive if the percentage of activated basophils ≥5% and the stimulation index (SI=activated percentage of basophils divided by the percentage of basophils in the negative control group) ≥2%.
Culture of human mast cells laboratory of allergic diseases 2 (LAD2)
The human mast cells (MCs) laboratory of allergic diseases 2 (LAD2) were kindly provided by A.Kirshenbaum and D. Metcalfe (National Institutes of Health, NIH, Bethesda, Maryland, USA). Cells will be maintained in StemPro-34 medium supplemented with StemPro nutrient supplement, 1% penicillin—streptomycin solution, 2 mM L-glutamine, and 100 ng/mL human stem cell factor in an incubator containing 5% CO2 at 37℃. The culture medium will be replaced by half every 7 days, and the cells will be maintained at a density of 2–5×105 cells/mL.
Passive mast cell activation tests
For pMAT, LAD2 (5×105 cells/mL) will be passively sensitised with patient sera overnight in a humidified CO2 incubator at 37℃. Subsequently, 100 µL of sensitised LAD2 is preincubated with interleukin-33 (IL-33) (100 ng/mL) (PeproTech) for 20 min at 37℃. IL-33 could prime sIgE/FcεRI-mediated activation yet it does not induce degranulation. The preincubated LAD2 are stimulated with Tyrode’s buffer 100 µL (Solarbio, Beijing) as negative control, anti-IgE 100 µL as positive control (final concentration 10 µg/mL), and 100 µL suspected allergens at final concentrations 1000, 100 and 10 fold dilutions of the original drug solution for 20 min at 37℃. Reactions are stopped by placing the cells on ice. Subsequently, supernatants will be removed by centrifugation (500 g for 5 min at 4℃). Cells are stained with monoclonal anti-human CD117-APC (clone 104D2; BioLegend, California, USA), anti-human CD203c-PeCy7 (clone NP4D6; BioLegend) and anti-human CD63-FITC (clone H5C6; BioLegend) for 40 min at 4℃. Additionally, DAPI will be used for staining during flow cytometry to distinguish between live and dead cells. Finally, the cells will be washed and resuspended in phosphate-buffered saline. Degranulation of LAD2 will be measured as surface upregulation of the lysosomal degranulation marker CD63. The activation of mast cells by different drug concentrations will be repeated twice to avoid discrepancies. If the results exhibit contrasting trends, a third test will be performed for confirmation.
Flow cytometry
Flow cytometry will be performed on a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, California, USA). Correct compensation settings for antibodies conjugated with fluorochromes are performed using CytExpert (Beckman Coulter). Flow cytometric data are analysed using FlowJo V.10.8.1 software (BD Bioscience, Franklin Lakes, New Jersey, USA). In this study, mast cells will be further gated as CD117-and CD203c-positive in flow cytometry. In case of a system error, it is recommended that more than 1500 mast cells be counted per sample, in accordance with the relative study.10 The gate is set based on the single stained and fluorescence minus one sample.
Statistical analysis
The statistical analysis of this study will be conducted using by SPSS V.27.0 (SPSS), with a critical value of 0.05 for statistical significance. The data obtained in this study will be analysed as follows:
Variables will be presented using standard summary statistics, such as mean±SD or median and IQR (IQR: 25th–75th percentile) for continuous variables, depending on the normality of the distribution. Frequencies and proportions will be used for categorical variables. The χ2 test or Fisher’s exact test will be used for categorical variables, and Student’s t-test or Mann-Whitney U test for continuous variables, depending on normality and homogeneity of variance.
The receiver operating characteristic curve (ROC) curve will be used to determine the optimal cut-off value for a positive diagnosis of potential allergens. Similar to BAT, the cut-off will also satisfy the criterion of SI≥2.16 Validity and reliability will be used for further evaluation of pMAT in each group with different drug concentrations. Validity will be assessed using sensitivity, specificity, false positive rate, false negative rate, likelihood ratio, area under curve of ROC curve and Youden index. The reliability of each test result will be estimated using consistency rate and consistency test.
Ethics and dissemination
The Institutional Review Board of China-Japan Friendship Hospital (2023-KY-247) has provided ethical approval for this prospective diagnostic accuracy study. All potential participants will be fully informed of the study objectives, methods, potential risks and measures to mitigate such risks before providing written informed consent. The research team members will explain the purpose of the study, testing procedures, sample collection, potential risks and our ability to manage and mitigate these risks. Subsequent sample collection and allergen testing will only be conducted with the patient’s informed consent. Throughout the testing process, patients will have the right to withdraw from the study at any time. It is also important to note that although there are some risks associated with this study, they have been assessed and are considered to be manageable. ST can be painful and may cause severe allergic reactions, but the incidence of this risk is very low. The ST will be performed in PACU, fully equipped to deal with such risks, with rescue drugs and equipment on hand and continuous monitoring of vital signs. A senior anaesthesiologist will be required to supervise the ST of the two investigators. If patients experience any discomfort or unexpected situations during the study, whether related to the study or not, they would inform doctors at any time, and appropriate medical treatment would be provided.
The benefits of participation are significant. Patients suspected of POA will receive complimentary diagnostic evaluations for anaesthetic allergens. The test will identify the patient’s perioperative allergens and guide the anaesthetic medication for future anaesthetic planning. The data from this study will assist public health departments, healthcare providers, and scientific groups in China to develop better guidelines and expert opinions related to POA, providing a solution for more patients with a more accurate identification of allergens.
In conclusion, the minimal risks associated with physical discomfort during blood collection and mild allergy are outweighed by the significant benefits of informing rapid diagnosis of POA and identification of perioperative allergens. The results of this study will be disseminated through academic presentations and publications in peer-reviewed journals.
Discussion
POA is a critical issue with potentially serious consequences. Although its incidence is relatively low, the overall impact is considerable, especially in high-throughput surgical environments. In mainland China, where the annual surgical volume will exceed 81.03 million by 2022,17 the projected POA incidence rate of 1 in 11 360 means that approximately 7100 patients could be affected each year.3 Therefore, accurate identification of the specific agents responsible for POA is a clinical imperative to prevent re-exposure during subsequent surgical and anaesthetic procedures.
Diagnosing the specific allergen for POA can be challenging as patients are exposed to multiple potential allergens within a short time when POA occurs. Current allergen identification methods have limitations that can lead to diagnostic failure. Drug provocation, the gold standard for allergen detection, is rarely used due to the potentially serious consequences and ethical concerns. ST, which is the most recommended and commonly used method, sometimes has insufficient sensitivity. Serum-specific IgE tests, which are limited in the variety of allergens they can detect, still require further validation of their sensitivity and specificity. Over the last two decades, BAT has emerged as a reliable complementary diagnostic tool to identify allergens, study cross-reactivity and monitor therapy.18 19 It provides the ability to measure in vivo basophil activation of individual basophils using flow cytometry. However, performing BAT is technically challenging as it requires specialised personnel and equipment, fresh samples and expertise. Moreover, the technique would be lost as a diagnostic tool in patients whose cells have a non-responder status.
In vitro pMAT is emerging as a promising alternative. Unlike BAT, pMAT uses serum samples that can be frozen, stored and transported to a recognised reference centre experienced in mast cell lines and/or cultures. These centres are capable of conducting batch testing with the necessary quality controls. Although still in its early stages, the utility of pMAT, which employs passively sensitised human cultured mast cells to investigate FcεRI/sIgE-mediated mast cell activation, is promising. In a study by Bahri et al, mast cells were derived from CD117+CD34+ human progenitor cells and sensitised with sera from patients allergic to peanut, exhibited allergen-specific degranulation.9 In this study, mast cell degranulation will be assessed by the quantification of CD63 and CD107a.
Recent researches on pMAT have also focused on identifying perioperative allergens. Two studies conducted by Jessy Elst in 2020 and 2023, respectively, evaluated the effectiveness of pMAT in diagnosing allergens to chlorhexidine and rocuronium.10 11 The study concluded that pMAT is a valuable complement to ST and BAT for drug-induced FcεRI/sIgE-mediated anaphylaxis. Furthermore, pMAT exhibits higher accuracy in identifying potential allergens. However, it is important to note that further studies are still required before pMAT can be widely implemented. The technique requires drug-specific validation and other factors or technical details might play a role.
This study aims to improve the diagnosis of potential perioperative allergens by enrolling patients for pMAT evaluation. The main objective of this study is to assess the diagnostic accuracy of NMBAs. It is important to note that the study will not be restricted to one single drug. If enough patients with POA are enrolled, pMAT will be used to identify other potential drug allergens with the same sample size mentioned previously, thereby drawing a more comprehensive conclusion on pMAT.
In this study, we choose not to differentiate blood precursors to produce mast cells, a process that requires substantial amounts of peripheral blood and specific expertise, potentially limiting the broader applicability of the pMAT in allergen diagnostics. Instead, we will use a more readily available cell line. Among the commonly used human mast cell (hMC) lines, HMC-1 cells are not suitable for our purposes because they lack surface expression of high-affinity IgE receptor FcεRI, which is critical for our research protocol. Conversely, LAD2 cells express this necessary receptor and will be chosen to facilitate the evaluation of pMAT. However, it should be acknowledged that there are differences between LAD2 cells and primary hMCs, such as a lower content of proteases in LAD2 cell.20 Nevertheless, the comparative ease of use of LAD2 cells supports our commitment to demonstrating their utility and diagnostic accuracy in pMAT, potentially facilitating the adoption of the test and encouraging broader research engagement. It is also important to note that prolonged culture of LAD2 cells may reduce their degranulation capacity.21 Despite initiating our study with a new batch of LAD2 cells, we will vigilantly monitor the degranulation response of the mast cells, particularly the degranulation ratio in each patient’s positive control, to preclude any impact on our results.21
Currently, drug provocation tests may still be considered the gold standard for allergen diagnosis.22 However, it should be noted that drug provocation carries potential risks and ethical concerns, which may pose difficulties during ethical review. Therefore, in this study, we will choose ST and BA as our preferred for allergen diagnosis. This limitation is inherent in our study and may be difficult to rectify, as it is a common issue in the majority of allergen diagnosis research. It is acknowledged that both ST and BAT have unsatisfactory sensitivity, which may result in false negatives in the gold standard. We aim to explain the complementary role of pMAT through further stratified analysis.
A total of at least 12 patients with a confirmed drug allergy and 29 patients without will be enrolled in this study, which meets the requirements of the study design. However, it is our intention to enrol a larger number of patients if possible in order to obtain more reliable conclusions.
Currently, pMAT is a promising technique for allergen identification. This study aims to investigate the feasibility and value of using the LAD2 cell line within a standardised pMAT framework for allergen detection. The anticipated results have the potential to expand the current understanding of the applicability of pMAT in allergen diagnostics and may contribute to improved clinical outcomes during the perioperative period.
Ethics statements
Patient consent for publication
Footnotes
XL and JZ contributed equally.
Contributors ZheL and HC designed and wrote the manuscript; ZhenL supervised the manuscript and created the data analysis plan; XL and JZ designed, supervised and edited the manuscript as guarantor.
Funding This work will be supported by National High Level Hospital Clinical Research Funding (No.2023-NHLHCRF-YYPPLC-ZR-10).
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.