| Background and purposeLaryngopharyngeal reflux disease occurs in about 10%of otolaryngological patients,and hoarseness is associated with laryngeal reflux in 50%of patients.Since the symptoms and signs of LPRD have no specificity,the current LPRD diagnosis dilemma is triggered.24 hours laryngopharyngeal pH monitoring technology was considered in the diagnosis LPRD gold standard,but it is still used diagnostic standard of gastroesophageal reflux disease,the pH<4 times and time percentage as a diagnostic LPRD parameters,and LPRD is given priority to with greater than 4 of weak acid reflux,rarely appear below 4 reflux,makes the diagnosis LPRD sensitivity is low,the missed diagnosis is high.Compared with laryngeal and pharyngeal pH monitoring,the sensitivity of pepsin in the diagnosis of LPRD can reach 100%,which is considered as the most promising method for the diagnosis of LPRD.Pepsin is regarded as a good reflux marker and an important pathogenic factor of LPRD.This article focused on Pepsin and LPRD to explore the diagnostic significance of Pepsin in oropharyngeal pH environment and saliva on LPRD,pepsin expression of tissue level in LPRD,so as to improve the clinical theoretical basis for the pepsin-related diagnosis of LPRDMaterials and methodsResearch objectsFrom October 2018 to November 2019,Petient suffering from laryngopharygeal reflux related disease was visited Otolaryngology department of Nanfang hospital of Southern Medical University,after finishing speciality check-up,who needs hospitalization care.There are 79 patients was finished those examination,such as Fiber electronic laryngoscope,RSI,RFS,24h Dx-pH detection and saliva from 5 times of different period;Using upright Ryan index>9.41 or supine Ryan index>6.8 as the diagnostic criteria of LPRD,LPRD has 21 patients,among them 17 patients have collected false vocal cord tissue.LPRD patients had no airway or digestive tract anatomical variation,no history of surgery or underlying diseases,no recent history of smoking or alcohol abuse,and no recent history of PPI drug use.From June 2016 to December 2017,healthy volunteers were recruited by the department of Otolaryngology department of Nanfang hospital of Southern Medical University There were no symptoms related to laryngopharyngeal reflux,no acute inflammation,no history of underlying diseases,and no history of PPI medication.The 24h Dx-ph examination,RSI and RFS were completed,and saliva was collected from 5 times of different period and a tiny mucosal tissue from False vocal cord,were collected.This study was approved by the ethics committee of Nanfang hospital of Southern Medical University,and all enrolled subjects were informed and agreed to participate in the study.Research methods1.Scale collectionThe included subjects were filled in the reflux symptom scale(RSI)and reflux signs scale(RFS)respectively.2.24-hour oropharyngeal pH monitoringThe Dx-pH monitoring system of American Retech company was used to connect the equipment.After confirming the normal operation of the equipment,the electrode probe was inserted from one side of the anterior nostril,the probe was placed about 0.5cm above and below the uvula,and the external electrode was fixed on one side of the cheek.The patient was asked to record the time of eating,lying down and symptom occurrence according to the actual situation.After the 24-hour monitoring,the machine was removed.Date View V3 software was used to analyze data and output dynamic oropharyngeal pH waveform,related pH parameters and Ryan index.3.Pepsin detection of salivaThe collected saliva was removed from the liquid nitrogen,reheated to 4℃ by steps,and centrifuged for 15min(1000rpm)in a 4℃ centrifuge.Pepsin concentration of saliva samples was detected by human pepsin detection kit(CEA632Hu)from wuhan cloud-clone company4.Pepsin immunohistochemical staining of ventricular band tissuesTake room in areas of mucosal tissue fixation,dehydration and embedding,made from wax block,will organize wax block slice,organizations will be cut off after removing wax,paraffin antigen repair,endogenous peroxidase blocking,closed and serum antibody incubation,DAB chromogenic,such as redyeing,transparent,cover will be room for interpretation with pepsin immunohistochemical results.The chromogenic degree was scored according to the chromogenic depth in the cytoplasm of squamous epithelial cells.0 points were scored as negative for no chromogenic color,1 point was scored as weak positive for light brown,2 points was scored as positive for tan,and 3 points were scored as strong positive for granular dark tan.According to the proportion of color rendering in color rendering tissues,1 point is scored for color rendering less than 25%,2 points is scored for color rendering 25%～50%,3 points is scored for color rendering 51%～75%,and 4 points is scored for color rendering greater than 75%.According to the product of chromogenic degree and chromogenic proportion,the expression degree is divided into four grades,0 is denoted as negative,1-4 is denoted as weak positive,1 is denoted as weak positive,5-8 is denoted as positive,2 is denoted as positive,and 9-12 is denoted as strong positive5.Statistical analysisSPSS 20.0 version was used for data processing,and the measurement data were subject to t test for normal distribution,and non-parametric test for non-normal distribution.The enumeration data were described by percentage or rate,and chi-square test or Fisher’s exact probability test were used.The test level P<0.05 was statistically significant.Results1.Oropharyngeal pH changes in the LPRD patientsThe minimum pH value of oropharyngeal in the LPRD was significantly lower than the control.The mean minimum pH value of the LPRD was 4.04±1.18,while the mean minimum pH value of the control was 5.85±0.164.When the oropharyngeal pH baseline was set at 6.5,6,5.5 or 5,the total time of reflux exposure and the percentage of reflux exposure time in the LPRD group were significantly higher than the control.The oropharyngeal pH≤5,(5,5.5],(5.5,6],the total exposure time of reflux in the LPRD was significantly longer than the control.2.Changes in pepsin concentration of saliva at different period in the LPRD patientsThe pepsin concentration in the saliva of the same individuals fluctuated greatly at different period in the same day,Individuals was the highest when they were awake in the morning.The pepsin concentration of saliva in the LPRD was significantly different from the control at 1 hour after dinner and 30 minutes before bedtime(P<0.05).Saliva sampling of 1 hour after dinner was significantly more effective in diagnosing LPRD than 30 minutes before bedtime.The cutoff value for optimal pepsin concentration of saliva 1 hour after dinner was 2.11ng/ml,and the sensitivity and specificity of LPRD diagnosis at this threshold was 76.2%and 100%3.Pepsin expression in False vocal cord tissues in the LPRDThe expression level of Pepsin in LPRD patients’ False vocal cord tissues was statistically different from the control(P<0.05).The positive rate of Pepsin expression in LPRD false vocal cord mucosal tissues was significantly higher than the control.The positive rate of pepsin expression in the LPRD with was 100%,among which 23.5%were strongly positive,41.1%were positive,35.2%were weakly positive,and no negative expression was found.The positive rate of Pepsin expression in the control was 18.2%,of which 18.2%were weak positive,81.8%were negative,and there was no strong positive or positive expressionConclusion1.The LPRD had prolonged exposure to weak acid with pH lower than 6 in the oropharynx compared with the control.2.The pepsin concentration was greatly fluctuated at 5 different period and the pepsin concentration in saliva was the highest in the morning awake.The cutoff value for optimal pepsin concentration of saliva 1 hour after dinner was 2.11ng/ml3.Pepsin expression was found in normal human false vocal cord.The expression level of Pepsin in false vocal cord of LPRD patients is higher.It is suggested that the moderate positivity pepsin expression in false vocal cord may be used as a cutpoint for the diagnosis of LPRD;this threshold could obtain good specificity to diagnose LPRD. |
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