The association between neutrophil-to-lymphocyte ratio and anaphylaxis refractory to epinephrine treatment

2.1 Study design and population

This was a retrospective, observational study that enrolled anaphylaxis patients arriving at a regional emergency department (ED) of a teaching hospital in South Korea between January 2015 and December 2019. Adults (> 18 years) who were diagnosed with moderate to severe anaphylaxis, according to the criteria of the National Institute of Allergy and Infectious Disease (NIAID) and the Food Allergy and Anaphylaxis Network (FAAN), and the grading system suggested by Brown SG were included in the study [10, 11]. The diagnostic criteria and the grading systems for anaphylaxis are described in Table 1 and Table 2. Patients who were transferred in from other hospitals, who suffered prehospital cardiac arrest, or who had no laboratory data available were excluded.

The term ‘refractory anaphylaxis’ is not clearly defined. We used the definition from a previous study [6], which described refractory anaphylaxis as a condition that required more than two doses of 0.3 mg intramuscular epinephrine for complete symptom resolution. According to the definition, the enrolled patients were divided into two groups: refractory and non-refractory anaphylaxis. Our study was approved by the Institutional Review Board of Uijeongbu St. Mary’s Hospital on March 25, 2020 (number UC20RASI0037).

2.2 Anaphylaxis management

Patients with anaphylaxis were treated by following previously established guidelines [1]. Epinephrine 0.3 mg (0.3 mL of a 1 mg/mL (1 : 1,000) solution) was injected via an intramuscular route as soon as anaphylaxis was recognized. A repeat injection of intramuscular epinephrine was ordered if symptoms did not resolve after five or more minutes after the first injection. If symptoms persisted, intravenous infusion of epinephrine or other vasopressors such as dopamine or norepinephrine were considered. Intubation or other resuscitative measures were taken as needed.

We recommended that all patients be admitted for prolonged observation. Patients with persisting symptoms, or who were being treated by an intravenous infusion of epinephrine, dopamine, or norepinephrine, or who were intubated were admitted to the intensive care unit for proper management.

2.3 Data collection

We collected data on patient demographics and characteristics via electronic medical records. Data collected were as follows: age, sex, use of emergency medical services, past history of diseases (hypertension, diabetes mellitus (DM), cardiovascular disease, chronic pulmonary disease, and malignancy), known previous allergies to elicitors, elicitors that caused anaphylaxis (categorized as food, drugs, contrast media, insect venom, others, and idiopathic), symptoms related to skin and mucous tissue (skin rash, pruritis, swelling of lips, tongue and uvula), cardiovascular symptoms (hypotension, dizziness, collapse, loss of consciousness, and incontinence), respiratory symptoms (dyspnea, wheezing, stridor, and hypoxemia), and gastrointestinal symptoms (cramping abdominal pain and intractable vomiting), initial mean arterial pressure (MAP), heart rate, body temperature, number of intramuscular epinephrine injections, dose of administered intravenous epinephrine (mg), use of vasopressors (dopamine or norepinephrine), H₁ blocker, H₂ blocker, and corticosteroids, rate of hospital admission, and hospital length of stay in days.

Initial laboratory findings were also collected at the time of ED arrival as follows: hemoglobin (g/dL), platelet count (10⁹/L), white blood cell (WBC) count (10⁹/L) with differentials, which included percentages (%) and absolute count (10⁹/L) of segmented neutrophils, lymphocytes, monocytes, eosinophils, and basophils, glucose (mg/dL), blood urea nitrogen (mg/dL), creatinine (mg/dL), sodium (mmol/L), potassium (mmol/L), chloride (mmol/L), and C-reactive protein (mg/L). CBC was measured using an automated blood cell counter (Sysmex XE-2100). NLR was calculated as the ratio of segmented neutrophil counts to lymphocyte counts in the same blood sample.

2.4 Statistical analysis

Categorical variables are displayed as numbers (percentages). All continuous variables were tested for normal distribution using the Shapiro-Wilk test and are presented as the means ± standard deviation (SD) for normally distributed data and as medians (interquartile range (IQR)) for data not normally distributed. For comparison of patient characteristics and laboratory findings between groups, the chi-square test and Fisher’s exact test for categorical variables, the Student’s t test for normally distributed continuous variables, and the Mann-Whitney test for non-normally distributed continuous variables were used. The independent association between baseline characteristics, including NLRs, and the occurrence of refractory anaphylaxis were analyzed using multivariate logistic regression. Variables with a P value < 0.1 on univariate logistic regression analysis were manually entered into the model. Furthermore, the receiver operating characteristic (ROC) curve was drawn and the area under the curve (AUC) was calculated to determine whether NLR was a good predictor of refractory anaphylaxis. The optimal cut-off value was obtained by calculating the Youden index.

All statistical analyses were performed using MedCalc Statistical Software version 19.6 (MedCalc Software, Ostend, Belgium) and GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, California, USA). A P value < 0.05 was considered statistically significant.

4.1 WBC and differential counts in anaphylaxis

Studies on WBC and differential counts of anaphylaxis patients are still scarce today. A previous study by Tang et al. revealed that both WBC counts and neutrophil percentages were elevated compared with their baseline values in patients with anaphylaxis attacks [15]. However, because all patients included in our study had anaphylaxis attacks, the results may not be directly comparable. A more recent study by Kim et al. compared laboratory results of patients with mild or moderate anaphylaxis reactions with patients with severe anaphylaxis [16]. They were unable to find statistically significant differences in WBC counts between groups. However, their study did not focus on the differences in neutrophil counts, lymphocyte counts, or NLRs between groups. Furthermore, their categorization of the study patients was different from ours. Therefore, the findings in our study are relatively new.

Low NLRs indicate that either neutrophil counts are decreased or lymphocyte counts are increased in the blood sample. In our study, patients with refractory anaphylaxis showed lower neutrophil percentages, lower absolute segmented neutrophil counts, and higher lymphocyte percentages than those with non-refractory anaphylaxis, while there were no statistical differences in absolute lymphocyte counts. This means that there was a decrease in absolute segmented neutrophil counts in patients with refractory anaphylaxis. Similar findings were described previously by Yanagawa et al.[17]. Their study had shown that patients with anaphylactic shock, compared with patients with hemorrhagic, cardiogenic, or septic shock, had relatively decreased neutrophil and increased lymphocyte percentages. Although the study population was different from ours, it partly corroborates our findings in that more severe anaphylaxis patients show decreased neutrophil and increased lymphocyte percentages in WBC and differential tests.

4.2 Neutropenia in refractory anaphylaxis

The mechanism behind neutropenia in refractory anaphylaxis is difficult to speculate. Traditionally, it is believed that anaphylactic reactions are induced by mast cells or basophils [18]. Both IgE antibodies and circulating immune complexes (IC) are known to trigger mast cells and basophils to release mediators, which cause smooth muscle spasm, vasodilation, myocardial depression, and laryngeal edema [2, 19]. However, previous animal studies have shown that neutrophils also play a significant role in anaphylaxis [20]. Neutrophils are considered to be immediate effectors of anaphylactic reactions by directly encountering IC, particularly those formed of IgGs [2, 21]. Neutrophils activated by ICs are known to release platelet-activating factors, leukotrienes, and histamines, which are all potent mediators of severe allergic reactions [22]. This may explain the elevation of neutrophil counts in patients with anaphylaxis attacks because neutrophil activation will mobilize neutrophils to enter the bloodstream [23]. Also, it is surmised that patients with more severe anaphylactic reactions may have a greater number of neutrophils, which is contrary to the results of our study [24].

One possible explanation is that refractory anaphylaxis may utilize a different immune pathway from a typical anaphylaxis. It is presumed that in contrast to non-refractory anaphylaxis, nitric oxide (NO) may play a critical role in refractory anaphylaxis [19]. Platelet-activating factors, leukotrienes, and histamines may enhance NO production by activating endothelial cell NO synthases, which activate guanylate cyclase to synthesize cyclic guanylate monophosphate (cGMP) from guanosine triphosphate [25]. Overproduction of NO and cGMP contributes to vasodilation and distributive shock [13]. Anaphylactic shock induced by this NO-cGMP pathway may not rapidly respond to standard intramuscular epinephrine treatment [26]. A previous in vitro study had shown that NO mediates the apoptosis of neutrophils by enhanced reactive oxygen species production and caspase-8 activation [27]. It is possible that overproduced NO may accelerate neutrophil apoptosis, resulting in neutropenia. However, the relationship between the NO-cGMP pathway and lower absolute segmented neutrophil counts is still not fully understood, and future studies are warranted.

4.3 Incidence of refractory anaphylaxis

The incidence of refractory anaphylaxis in our study (25.4%) was much more frequent than described in the literature. A previous study by Korenblat et al. reported that 17 out of 105 patients (16.2%) required three or more intramuscular epinephrine injections [28]. A more recent analysis of the European Anaphylaxis Registry by Francuzik et al. reported that only less than 1% of severe anaphylaxis cases were refractory to epinephrine treatment [6]. The variation in disease prevalence may be because of epidemiologic factors. During the study period in South Korea, epinephrine autoinjectors were only available at the Korea Orphan and Essential Drug Center with a prescription, and intramuscular epinephrine injection by emergency medical technicians was prohibited by law. Thus, a vast majority of anaphylaxis patients received intramuscular epinephrine only after arriving at the hospital. Delays in epinephrine treatment is associated with more severe, even fatal anaphylactic reactions [29]. This may have caused more frequent refractory anaphylaxis events than observed in previous studies. Also, the treatment protocol of our emergency center may be more aggressive than the standard care given at other centers; 110 out of 126 patients (87.3%) received intramuscular epinephrine injections, which is more frequent than previously described, and it may have affected the ratio [30].

4.4 Use of point-of-care testing devices

Since anaphylaxis is a medical emergency requiring clinical diagnosis and immediate management, laboratory tests such as CBCs may not be performed rapidly enough to influence emergency physicians’ treatment decisions. One potential solution to the problem may be the utilization of point-of-care testing (POCT) devices. There are several commercial POCT devices to accurately measure WBC and differential counts within minutes [31, 32]. This approach has been previously studied in severe asthma, and it has shown promising results [33]. These POCT devices to obtain NLRs may be used as an alternative to traditional CBC teststest, but its use in anaphylaxis may need to be validated.

4.5 Limitations

Our study had several limitations. This was a single-center, retrospective study with a small sample size, so it was exposed to potential confounders and selection bias. A multicenter prospective study is required to confirm our findings. Refractory anaphylaxis is still not clearly defined, and our definition is not universally accepted. A consensus for a definition of refractory anaphylaxis may be needed. Most patients did not undergo skin or provocation tests to confirm the elicitors of their anaphylactic reactions. The causes of anaphylaxis may not be accurate because we relied solely on patient history. The blood samples used for analysis were usually taken as soon as the patient arrived at our hospital, but the exact timing of blood sampling and intramuscular epinephrine injection were not clarified, and epinephrine injections may have influenced CBC and differential test results. Additionally, some patients may have taken medications before arriving at our emergency department that may have affected results, including allergy medications and chemotherapeutic agents, and that were not fully addressed in the medical records.