A nomogram prediction of citrate reaction during mononuclear cell collection in solid tumor patients

Citrate reaction is one of the main adverse events in peripheral blood mononuclear cell (MNC) collection. The aim of this study was to elucidate the risk factors for citrate reaction in patients with advanced solid tumor collection and to construct a nomogram to predict the risk.

identify the risk factors of citrate reaction. According to the results of the multivariate logistic model, nomogram was established and receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value of the model. Conclusions: The MNC collection process is safe. The incidence of citrate reaction in the collection of peripheral blood MNCs from patients with advanced solid tumor is related to the age, gender, and processed circulating blood volume of patients. The nomogram can be used to assess a patient's risk of citrate reaction. Yong

| INTRODUCTION
In recent years, chimeric antigen receptor T-cell (CAR T) immunotherapy has emerged as a novel cancer therapy, in which CAR T cells targeting CD19 have shown potent efficacy in patients with relapsed and refractory hematologic tumors of B cell origin. 1 CAR T cells can selectively and directly recognize and kill tumor cells without the requirement of antigen presentation, which is not restricted by the major histocompatibility complex. 2 CAR T-cell therapy has also shown great potential in the treatment of advanced solid tumors. A number of phase I clinical trials of CAR T-cells in solid tumors are underway worldwide, with peripheral mononuclear cell (MNC) collection from solid tumor patients as a critical first step in CAR T-cell trials. 3,4 A large number of literature reports and clinical practices have found that adverse reactions, especially the citrate reaction in the collection of peripheral MNCs cause discomfort in patients.
Citrate, a calcium chelator, is an anticoagulant that prevents blood clotting during extracorporeal circulation during apheresis. Citrate in the blood is rapidly metabolized by the liver and eliminated by the kidneys. Symptoms of hypocalcemia associated with high citrate concentrations often occur during therapeutic plasma exchange and MNC collection. 5 These symptoms range from mild (eg, numbness around the mouth and extremities, dizziness) to moderate (eg, nausea, muscle cramps) and severe (rarely, eg, tetany and arrhythmias). 6 About 1.5% to 5% of patients experienced moderate to severe citrate-induced hypocalcemia response during collection, requiring medical intervention. 7 Therefore, predicting the probability of hypocalcemia in solid tumor patients, in advance, can better prevent the occurrence of moderate and severe reactions, to ensure the smooth acquisition. Therefore, 148 MNC collection patients from Shanghai Mengchao Cancer Hospital were retrospectively studied. In this study, the risk factors of citrate-induced hypocalcemia reaction during collection were identified, and other adverse reactions during collection were observed and recorded.

| Inclusion of patients
From January 1, 2021 to December 30, 2021, peripheral blood MNCs were collected from 148 patients with advanced solid tumor in Shanghai University Affiliated Mengchao Cancer Hospital. All patients met the blood collection criteria: neutrophil count ≥3 Â 10 9 /L, platelet ≥80 Â 10 9 /L, hemoglobin ≥90 g/L, absolute value of monocyte lymphocyte ≥0.9 Â 10 9 /L, hematocrit ≥30%, and normal liver and kidney function. Patients with active viral or bacterial infections, severe organ dysfunction medical histories including immunodeficient and systemic autoimmune diseases were excluded from the study. Patients that had prescribed treatment with glucocorticoids or immunosuppressive agents 1 month before the study were also excluded. All patients signed the informed consent for MNC collection before treatment.

| MNC collection
The Fresenius COM. TEC blood cell separator (Fresenius Kabi, Bad Homburg, Germany) collects peripheral blood MNCs mainly by specialized apheresis nurses. The lymphocyte collection program in auto MNC was selected. Specialized nurses in the department carried out blood cell separator operation. The information of patients included gender, height, weight, hematocrit (Hct), white blood cell (WBC) counts, and the initial ratio of anticoagulant to whole blood 1:10. The ratio of anticoagulant to whole blood was reset to 1:12 after a cycle of systemic blood. For patients with platelets greater than 200 * 10 9 / L, the mode of manual collection can be adopted in the second cycle. The default value of white membrane menu parameter was used at the beginning of each cycle, and it could be adjusted according to the thickness of white cell layer after one to two cycles. If the white cell layer could not be observed or was less than 2 mm, circulation volume was then increased 20 to 50 mL from the original single circulation volume. The total circulation volume was set according to the requirements of the final product quantity. The whole blood flow rate was 35 to 65 mL/ min. Apheresis collection parameters are summarized in Table 1.
T A B L E 1 Apheresis collection parameters.

| Statistical analysis
Statistical analysis was carried out utilizing SPSS Statistical computer program 25.0 (IBM, New York, New York, USA). Measurement data were presented as the means ± SD, and classification variable data were presented in terms of percentage. Multivariate and univariate analyses were performed by logistic regression model to determine the risk factors for citrate reaction. The RMS package of R software (version 3.5.1) was applied to construct a nomogram based on the independent predictors. The nomogram's performance was tested using calibration curves and the concordance index (C-index), which were derived and presented for this purpose. By plotting Y (sensitivity) and X (specificity), the receiver operating characteristic (ROC) curves of the nomogram model were constructed. 8 3 | RESULTS

| Safety of MNC collection
In the process of MNC collection, 35(23.6%) of the 148 patients developed citrate reaction, including 2 patients only with nausea and vomiting, 25 patients only with numbness in extremities and perioral area, 3 patients with chest tightness and numbness in extremities and perioral area, and 4 patients who developed not only nausea and vomiting but also numbness in extremities and perioral area, and 1 patient with transient hypotension and numbness in extremities and perioral area. These are mild reactions that lasted about 30 minutes. According to the common Adverse Event Evaluation criteria version 5.0, all of them were graded 1 to 2 and were alleviated by oral or intravenous administration of calcium gluconate.

| The nomogram and its predictive performance
The nomogram model for estimating the risk of citrate reaction was composed of three independent risk factors. Each individual risk factor had a specific score, and the individualized grade of each included patient was defined by the sum of points from the three predictors. The total score of the scale (0-100) predicted the risk probability of citrate reaction (Figure 1). The performance of the nomogram was measured by ROC curve, and the area under the curve (AUC) of the model was 0.799 ( Figure 2).

| DISCUSSION
With the development of immunotherapy, peripheral blood MNCs (PBMC) including lymphocytes and monocytes, are more and more widely used to produce CAR T cells, DC vaccines, and natural killer (NK) cells, and so on. The collection of peripheral blood MNCs from tumor patients is the key first step of adoptive cellular immunotherapy, and is different from the collection of PBMC from healthy donors. This group of people tend to be older and have hardening of blood vessels and some have different degrees of influence on vascular function caused by lack of exercise for a long time. In some cases, the PBMC content in peripheral blood is low due to repeated chemoradiotherapy. Some of the liver detoxification function and the ability to metabolize citrate are decreased due to various reasons leading to prolonged collection time. However, only a few studies have focused on the influencing factors of adverse reactions in the acquisition process. In this study, three factors were identified as risk factors for the occurrence of citrate reactions in the collection. In addition, we developed an easy-to-use prognostic nomogram that integrated three predictors to predict the risk of citrate occurrence in these patients. After verification, the constructed line chart shows good prediction accuracy. Jill Adamski et al. 6 showed that patients with multiple myeloma were more prone to citrate reaction during MNC collection compared with patients with other hematologic diseases and solid tumors. Our study group considered that the occurrence of citrate reaction in solid tumor patients may be related to multiple factors. To date, no such large-scale study has explored risk factors of citrate reaction in solid tumor patients during MNC collection. Our study suggests that gender, age, and circulating blood volume are independent predictors of numbness in solid tumor patients during collection. According to our model, the risk of numbness in female patients (sex = 2) was 5.718 times higher than that in male patients (sex = 1), and female patients were more prone to citrate reaction. The risk of citrate reaction decreased with age. Irani et al. 9 showed a significantly increased risk of citrate reaction in young women during therapeutic plasma exchange, similar to the results of this study. It is also not clear why older males have a decreased risk of citrate reaction. The decreased rate of citrate response may be due to a reduction in symptom perception as men age. In this study, serum calcium concentration before blood collection was found to not be a risk factor for the occurrence of citrate response. We will further collect serum calcium concentration during and after the operation to analyze whether they are risk factors for the occurrence of citrate reaction.
In this study, we observed 35 patients with citrate reaction, all were mild/moderate reactions. No arrhythmia, hand and foot convulsions, or other severe reactions were observed. The patients we considered for inclusion in the clinical trial of CAR T-cell therapy had normal liver and kidney function, rapid calcium chelate metabolism, resulting in a mild citrate reaction, and oral calcium gluconate treatment could relieve symptoms.
In conclusion, the risk factors for citrate reaction in solid tumor patients during MNC collection include age, gender and circulating blood volume. During MNC collection, more attention should be paid to young female solid tumor patients, and the symptoms of citrate reaction and post-treatment measures should be fully informed before the operation. Furthermore, we will further investigate whether precollection prophylactic oral calcium gluconate can reduce the probability of citrate reaction in young female solid tumor patients. We developed a novel and practical nomogram model which can accurately predict the probability of citrate reaction.