A Novel Blood Test to Predict Safe (Non-trigger) Foods for Infants and Toddlers With Food Protein-induced Enterocolitis Syndrome (FPIES)
Phase: NA
Conditions: Food Protein-Induced Enterocolitis Syndrome, Allergies, Pediatric Disorder
Sponsor: University of Michigan

Title: Novel Blood Test to Predict Safe Foods for Infants and Toddlers With Food 
Protein-induced Enterocolitis Syndrome (FPIES)
PI: Mohamad El Zaatari, PhD
NCT: NCT04644783
IRB Protocol Version Approval Date: 9/3/2020

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A NOVEL BLOOD TEST TO PREDICT SAFE (NON-TRIGGER) FOODS FOR INFANTS AND TODDLERS 
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WITH FOOD PROTEIN-INDUCED ENTEROCOLITIS SYNDROME (FPIES). 
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PRINCIPAL INVESTIGATOR: Mohamad El-Zaatari PhD. Research Assistant Professor, Division of 
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Gastroenterology, Department of Internal Medicine, University of Michigan. 
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CO-INVESTIGATORS: 
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i) Georgiana Sanders MD. Professor of Internal Medicine, Associate Professor of Pediatrics and Research
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Associate Professor of Mary H Weiser Food Allergy Center, Medical School, University of Michigan. 
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ii) Charles Schuler MD. Assistant Professor of Internal Medicine, Division of Allergy and Immunology.
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iii) John Y. Kao MD. Associate Professor, Division of Gastroenterology, Department of Internal Medicine,
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University of Michigan. 
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VERSION 2; ***. 
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I. PROTECTION OF HUMAN SUBJECTS
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1. Risks to Human Subjects
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A. Human Subjects Involvement, Characteristics, and Design
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Describe and justify proposed human involvement 
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The objective of this project is to validate a blood test that can distinguish safe from trigger foods for 
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protein-induced enterocolitis syndrome (FPIES). 
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Background: FPIES is an allergic disease that leads to repetitive profuse vomiting in response to solid food. 
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In severe cases, when patients fail to thrive, FPIES requires nasogastric tube installment or total parenteral 
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nutrition (TPN). The major challenge in managing FPIES is the identification of safe (non-trigger) foods in a 
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timely manner, while avoiding repetitive allergic reactions and recovery periods. This study proposes a 
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solution to the problem by developing a new blood assay that screens a large number of foods (more than 
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20) in a culture plate. Its purpose is to rapidly predict what the safe foods are. The outcome will overcome
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the time-lapse required to identify such safe foods, which could normally extend past a year or longer, by 
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which time the child may have failed to thrive or developed food aversions due to association with negative 
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experiences. 
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FPIES leads to repetitive hospital and ER visits with 2,064 encounters occurring at the University of 
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Michigan Health System (UMHS) in the past year (from 11/01/17 until 11/01/18; data obtained using 
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Datadirect for cohort discovery results; FPIES ICD10 K52.2). The latter number of encounters only 
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comprises newly diagnosed patients. 
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In this study, FPIES patients will be recruited from the UMHS patient cohort. From 11/01/17 – 11/01/18, this 
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cohort comprised 223 newly diagnosed patients at UMHS. Two sub-groups will be identified as follows: i) 
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children exhibiting FPIES reactions to 2-3 foods, ii) children exhibiting FPIES reactions to 4 or more foods. 
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The reason for separating patients into these two groups is due the existence of two separate groups [one 
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with 2-3 food triggers, and another with more than 4 food triggers] as previously described by the UMHS 
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Allergists who care for FPIES patients (Volertas S., Hudson E., McMorris G., and G. Sanders. Annals of 
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Allergy, Asthma & Immunology 121:S119 2018). 
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The plan is to enroll up to 20 patients with FPIES: up to10 exhibiting reactions to 2-3 foods, and up to 10 
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exhibiting FPIES reactions to 4 or more foods. Recruitment will take place at the University of Michigan 
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Allergy Clinics, and University of Michigan Pediatric outpatient clinics. 
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Hypotheses:  
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1. Our blood test that we developed will distinguish safe from trigger foods for FPIES patients.  
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2. The mechanism of FPIES involves innate immunity and the identified heatmap pattern for trigger foods 
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will remain after foods are tolerated. 
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Specific aims: 
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1,  Assess the assay’s precision by its predictive accuracy in distinguishing safe foods from trigger foods.  
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2. Evaluate the reliability of the test when using a panel of standardized, frozen foods vs freshly prepared 
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foods 
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3. Demonstrate persistence of the immune reaction to trigger foods by comparing results of the test after 
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trigger foods are tolerated to the initial results 
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Exploratory aim: 
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1. Gain further insight into the pathophysiology of FPIES through single cell RNASeq 
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Statistical Design: A random-effects logit model will be used to model the binary outcome (safe or trigger 
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food) as a function of the 9 biomarker measurements in the assay. The random effect in the logit model will 
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take into consideration of the correlated data measured within the same subject. A cluster Receiver 
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operating characteristic (ROC) curve analysis will be used to assess the precision of the assay. Specifically, 
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we will compute the area under the cluster ROC curve (AUC), along with a 95% confidence interval (CI). 
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The assay is predictive if the lower limit of the 95% CI is above 0.5, which is the null value indicating no 
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predictive ability. 
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Brief description of the proposed study  
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FPIES is a severe food allergy to a wide variety of foods in babies and toddlers. Usually, the symptoms 
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resolve between the ages of 3 to 5 years old. However, the children are at risk of failing to thrive, and 
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requiring a nasogastric tube or TPN. The objective of the study is to validate a newly developed blood assay 
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that will rapidly predict safe (non-trigger) foods for the affected children. In our preliminary data (Case Study 
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HUM00155216), the blood assay safely predicted more than 10 consecutive safe foods without triggering 
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an allergic response. Prior to the assay, allergic reactions and ER visits were repeatedly occurring.  
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Subject Recruitment: Subjects will be recruited through the University of Michigan Allergy Clinics, and 
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University of Michigan Pediatric outpatient clinics:  
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1. Patients may be referred by the clinician caring for the patient. 
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2. Pre-screening by reviewing FPIES visits in the past 2 years will be performed. Data Direct, EMERSE 
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and billing codes may be used to identify potential subjects. The patient's primary allergist or 
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pediatrician will be informed about study team’s contact with the identified patients. 
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o Patients will then be contacted by email and/or telephone by the clinical members of the 
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study team to explain the study and determine whether they are interested in participating. 
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Opt out information will be provided in the email and by phone contact. 
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3. Patients may self-refer: 
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o The study will be listed in UMHealthResearch.org 
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o Flyers describing the study, with study team contact information, will be posted in the 
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participating clinics. 
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Inclusion Criteria: 
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1. Patients aged 1 month – 7 years old with a physician diagnosis of FPIES: 
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a. Group 1: Documented reactions to 2-3 trigger foods with recurrent delayed vomiting. 
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b. Group 2: Documented reactions to 4 or more trigger foods with recurrent delayed vomiting. 
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Exclusion Criteria: 
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1. Patients without a physician diagnosis of FPIES. 
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2. Patients who are currently on medications that suppress the immune system. 
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3. Patients who do not have at least 2 trigger foods identified. 
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4. Patients who have a history of an organic GI disease (e.g., inflammatory bowel disease, celiac 
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disease, biliary disorders, bowel resection), cardiac, pulmonary, neurologic, renal, endocrine, or 
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gynecological pathology 
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5. Lack of parental or guardian informed consent. 
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Study Design 
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1. Potential participants will be contacted by email and / or telephone after pre-screening of physician-
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diagnosed FPIES patients who have visited the UM allergy clinics within the past three years, or who 
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are currently in contact with the UM allergy clinic nutritionist. An option for the patients to opt out will 
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be provided. The patient's primary allergist or pediatrician will be informed about study team’s 
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contact with the identified patients.  Patients may be referred by their primary allergist during a clinic 
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visit. 
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2. If a parent/guardian expresses interest in the study, they will be sent/given a copy of the informed 
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consent to review. 
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3. An appointment will be scheduled with a study team member at the UM allergy clinics. 
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4. The protocol will be discussed face-to-face with parent/guardian with an opportunity to ask questions 
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and review the informed consent document. After written informed consent is signed by one 
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parent/guardian, study procedures can begin.  Parent/guardian will be given a copy of the signed 
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informed consent and it will be imaged into the chart.  
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5. Parent/Guardian will be asked to fill out Questionnaire One at the time of entry into the study. 
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6. Parent/Guardian will be instructed regarding scheduling the blood draw for analysis:   
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a. Only known “safe foods” (foods that do not induce repetitive vomiting) are to be consumed 
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for one week before blood draw. 
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b. Blood draw will be performed at the MLabs Blood Draw Stations at Michigan Medicine 
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outpatient clinics.  
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7. Patient will have 6 ml of blood drawn for analysis: 
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a. 5.5 ml of blood for testing of potential food reactions 
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b. 0.5 ml of blood for RNA/DNA analysis, which will be stored. 
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8. After blood draw, diet will be managed per the primary clinician and nutritionist. 
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a. Parents will be given Questionnaire Two to fill out detailed information about all foods 
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introduced until the results of the assay are received. 
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b. During that time, food introduction will continue as directed by primary allergist and/or 
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nutritionist 
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9. Blood will be analyzed by the PI in a research lab at the University of Michigan to determine 
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differences in the PI’s assay between potentially tolerated and not tolerated foods (see description of 
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laboratory test lines 216-232)   
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10. After identification of potentially “safe foods” to be introduced in the future, the information will be 
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given to the clinical study team and child’s clinician, who will contact parents by phone or email 
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(parental preference).  
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a. Parents will be asked to detail current nutritional status, including foods tolerated or not 
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tolerated since the initial blood draw.  Results will be recorded by the study team and the 
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parents will be asked to return Questionnaire Two. 
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b. After discussion within the clinical team (including clinical research team and child’s 
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clinicians), parents will be instructed to introduce new foods identified as potentially safe into 
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the diet, one at a time for seven days. 
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c. Results of these trials will be recorded on Questionnaire Three and returned to the study 
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team 3 month intervals. 
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11. A second blood draw will be performed, at least 2 weeks after the initial blood draw. 6 cc will be 
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drawn: 
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a. To explore the consistency of a standard panel of foods vs individually prepared foods 
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b. To perform additional analyses including single cell RNASeq (scRNA-seq) on WBCs 
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following treatment with foods. 
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12. Subjects will be followed at 3 month intervals with review of tolerated foods, number and types of 
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known triggers assessed.  When a subject is tolerating two or more triggers foods for 3 or more 
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months: 
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a. Questionnaire Four will be given to fill out and return 
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b. Subject will have 6 mL drawn for analysis 
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i. 5.5 mL to rerun the initial assays 
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ii. 0.5 mL for RNASeq on WBCs following treatment with foods. 
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Study Grid 
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B. Sources of Materials  
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The research material obtained from the human subjects  
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1. 6ml of blood in a K2 EDTA (lavender top) blood tube withdrawn by specialized phlebotomists in 
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the blood draw unit (0.5 ml of this will be used to isolate DNA for potential future sequencing under a 
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separate IRB). White blood cells (WBCs) will be isolated from 5.5 ml of blood using ACK buffer lysis of red 
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blood cells, within 1 hour of blood collection, as performed previously for the Case Study (HUM00155216). 
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Blood will be kept on ice in the K2 EDTA tubes during the waiting period (< 1 hour). WBCs will be plated in 
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24 well plates, and exposed to different food homogenates for 3 hours. Total RNA will be extracted, and RT-
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qPCR for our gene panel will be used to generate the heatmap.  
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A second blood draw may be requested from a limited number of patients, at least 2-4 weeks after 
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the initial blood draw, for scRNA-seq. In this case, the blood will be prepared in the same manner as for the 
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initial draw above, but instead of extracting RNA, samples will be submitted to the UofM Advanced 
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Genomics Core for them to perform scRNA-seq on the samples. The data will be analyzed using the cloupe 
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software by 10x Genomics.  
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A third blood draw may occur (or second blood draw if scRNA were not performed for that patient) 
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after the patient recovers from several triggers. The patient will be screened at 3 month intervals, so the 
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third (or second if no scRNA-seq) will be performed for this purpose at least 3 months after the initial blood 
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draw. In this case, the cells will be prepared in the same manner as the initial blood draw, but treated with 
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only a limited number of safe, trigger foods, and outgrown trigger foods.  
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2. At the termination of the protocol samples and data will cease to be accessed, or otherwise 
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accessed again after IRB approval transferred to another existing protocol. There are no plans to destroy 
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records or samples. 
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Protection of Information: 
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All data will be kept on REDCap, a password-protected, HIPAA compliant, web-based application 
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developed by Vanderbilt University to capture data for clinical research and create databases and projects. 
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The databases use instruments such as surveys and forms as research capture tools. Projects are self-
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sufficient and secure databases that can be used for normal data entry or for surveys across multiple 
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distinct time points. Only study team members will have access to this data.   
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1. A master list that includes identifiable data will be kept in a REDCap “Study Housekeeping” 
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database that includes subject number, patient name, parent names, MRN, contact information 
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including address, email, phone number, documentation of blood draw, samples saved and filling 
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out of questionnaires. 
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2. All data variables will be kept in a separate “Research Data” REDCap database.  Subjects will be 
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identified only by subject number in this database.  Data will include: 
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a. Results of blood tests 
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b. Questionnaire answers 
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c. Age 
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d. Number of food reactions 
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e. Identified trigger foods 
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f. Identified tolerated foods. 
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3. Data downloaded for analysis will have only subject number included. 
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4. Stored RNA, DNA or cDNA samples will contain only the subject number without personal 
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identifiers. 
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5. Parents/guardians may request that stored blood samples be destroyed at the end of the study. 
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C. Potential Risks  
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Describe potential risks  
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The risk in this study arises due to incorrect predictions by the test. The current test being validated is not 
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an FDA-approved or cleared test. However, current standard of care recommends which foods to trial in a 
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random fashion, and incorrect predictions frequently occur leading to reactions. Hence this study will not 
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pose an added risk to the current standard of care. The indication of what to trial will be communicated to 
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the patients by the allergist and nutritionist as currently occurs in the standard of care. However, the only 
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difference is that the allergist and nutritionist will in this study use this assay for indication regarding what 
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foods to trial. If a food is falsely identified as safe by the assay but triggers a reaction, then these reactions 
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will be managed by the nutritionist and allergist as per current standard of care for failed trials. 
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Blood draw from infants and toddlers is a safe procedure with minimal risks. Trained and experienced 
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phlebotomists at UMHS perform blood draws on infants and toddlers routinely. The procedure may cause 
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anxiety for the infant/toddler. The test can result in a small bruise or mild soreness for the child at the site of 
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blood draw. The bruise can last for a few days.  
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A risk of breach of confidentiality always exists in all studies.  
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Describe alternate treatments and procedures  
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The parent/child can proceed with introduction of new foods per guidance of the nutritionist.  This is based 
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on historically better tolerated foods in the general FPIES population and may or may not apply to the 
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patient. 
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2. Adequacy of protection against risks 
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A. Recruitment and Informed Consent
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Plan for recruitment 
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All FPIES patients will be recruited from the University of Michigan Allergy Clinics, and University of 
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Michigan Pediatric outpatient clinics. The patients will be identified by pre-screening of the UM FPIES 
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patient cohort. Pre-screening will be performed by members of the Division of Allergy and Immunology at 
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Michigan Medicine. Patients will not be contacted by the study team until pre-approval is obtained from the 
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primary allergist or pediatric clinician. Suitable FPIES patients who fulfill the inclusion and exclusion criteria 
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will be contacted by email and / or phone. An option to opt out will be provided. If patients are interested in 
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participating, a meeting with the study team will be scheduled at the UM allergy clinics. The patients will be 
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presented with the consent form by the study team member to consider consenting for the study and the 
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blood draw procedure. Patients will be told that their decision whether to participate in the study will not 
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affect their clinical care. 
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Describe the circumstances of consent 
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A study coordinator or clinical study team member familiar with the study protocol will obtain informed 
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consent for all participants. Consent/assent will be obtained prior to any study measures/questionnaires. 
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Each participant will receive a verbal and written explanation of the purposes, procedures, risks, and 
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potential benefits of the study in language appropriate for the individual. 
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B. Protections Against Risk
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Planned procedures for protecting against risk 
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1. The risk of a false result will be managed by the allergist and nutritionist as per current standard of care in
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managing reactions to failed trials. 
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2. The risk from blood draw is minimal and routinely performed by the phlebotomists at UMHS. 
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3. All data variables will be kept in a separate “Research Data” REDCap database.  Subjects will be
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identified only by subject number in this database. 
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4. A separate “Housekeeping” REDCap database will be used to link the patient identifiable information and
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the study number. 
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3. Potential benefit of the proposed research to the research subjects and others
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Potential benefits 
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There may be direct benefit to the patient by the potential of identifying tolerated foods, thus avoiding the 
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adverse reactions of frequent emesis and potential dehydration, as well as the trauma parents encounter 
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when giving their infant a food that causes such reactions. There may be benefit to the larger community of 
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FPIES patients if this protocol indicates the new assay can identify tolerated vs non-tolerated foods. 
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Discuss why risks to subjects are reasonable in relation to benefits 
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Patients already encounter false predictions and failed trials with allergic reactions regularly. The indication 
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from the assay is expected to predict safe foods at a higher rate than the guesswork utilized in the current 
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standard of care. 
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4. Importance of the knowledge to be gained
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Discuss importance of the knowledge to be gained 
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Validation of this test has the potential to modify patient care strategy for patients with FPIES. As such, 
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random guessing of safe foods would be replaced by a scientific “guide” to what might be hypoallergenic to 
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the body’s immune cells using this test. 
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Discuss why the risks are reasonable in relation to the importance of the knowledge to be gained  
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Currently, trialing food is random. Obtaining a guide for what might be a safe food, by utilizing our proposed 
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assay, would be invaluable. Incorrect predictions cannot pose more risk than random selection of foods to 
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trial. Hence, there is only benefit to be gained if the test is predictive or partially predictive. 
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II. INCLUSION OF WOMEN AND MINORITIES  
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Inclusion of Women  
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This study will include children of both genders.  
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Inclusion of Minorities  
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The disease is overwhelmingly predominant in White Caucasian children (Based on personal communication with 
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allergists caring for FPIES patients at UofM clinics, and the following published abstract: R. Tarrant and A. and Byrne. Clinical 
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Presentation and Food Allergens Associated with Food Protein-Induced Enterocolitis Syndrome – a frequently misdiagnosed rare 
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form of gastrointestinal food hypersensitivity. European Society of Pediatric Gastroenterology, Hepatology and Nutrition 62(1); DOI: 
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10.13140/RG.2.1.1621.2087). Therefore, for the purpose of feasibility of meeting the required proposed patient 
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numbers for statistical analyses, we have chosen a majority of White Caucasian subjects for this initial 
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study. If patients from other ethnicities – who do not usually have FPIES enroll – they will not be excluded 
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from the study. 
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Inclusion of Children  
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Children with FPIES will be included in this study. 
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III. PLANNED ENROLLMENT TABLE 
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Total Planned 
Enrollment: 
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TARGETED/PLANNED ENROLLMENT: Number of Subjects 
Ethnic Category 
Females 
Males 
Total 
Hispanic or Latino 
1 
1 
2 
Not Hispanic or Latino 
9 
9 
18 
Ethnic Category: Total of All Subjects * 
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10 
20 
Racial Categories 
 
American Indian/Alaska Native 
      
      
      
Asian 
1 
1 
2 
Native Hawaiian or Other Pacific Islander  
      
      
      
Black or African American  
1 
1 
2 
White 
8 
     8 
16 
Racial Categories: Total of All Subjects * 
10 
10 
20 
 
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