Establishment Of Reference Ranges For Levels Of Coagulation Factors In Cord Blood And Study On Its Influence

BackgroundNeonatal bleed more easily than elderly children, about50%neonatal have bleed atbirth. The causes of bleeding were multiple anomalies, including platelet count (orquality), vascular wall, and coagulation system. The latter also includes hereditary andincludes hereditary and secondary coagulation factor deficiency. It is important reasonof newborn hemorrhage.Sever cases will have the central nervous system sequelae, evenbe death due to intracranial hemorrhage. Due to the complex etiology and diverse andnonspecific clinical manifestations of hemorrhagic disease in neonatal period, earlydiagnosis is not easy. About1/3children with hereditary coagulation factor deficiencyare lack of clear family history. Therefore，detection of the levels of coagulation factorsis an important basis on diagnosis and differential diagnosis.Compared with elder children and adults, there are certain characteristics of the levels ofcoagulation factors in neonatal period. Firstly, newborn is at a relatively low coagulationstate. The levels of coagulation factors are still lower than the normal adults until atbirth. At10-11weeks’ gestation, coagulation factors synthesized. At19-27weeks’gestation, fetal blood can be detected all coagulation factors, and their levels increasewith gestational age. Secondly, the coagulation system develops rapidly in the postnatal.The levels of coagulation factors are in dynamic process, and their development is notconsistent.Based on above reasons, it is need to establish reference ranges for levels of coagulationfactors in different period, that should meet the statistical requirements, investigateinfluence the levels of coagulation factors. It provides a reliable basis on the diagnosisand differential diagnosis of hemorrhagic disease.Cord blood can be drawn convenient and available at birth. In this study, we expect toanswer the questions as follow: How the levels of coagulation factors in cord blood? Isthere relationship between levels in cord blood and dynamic changes of coagulation factors levels in postnatal? Whether to predict the hemorrhagic disease caused bycoagulation factors deficiency?Reviewed related literatures, there are few researches on the levels of coagulationfactors in cord blood. And the results are not consistent, due to different sample size,different inclusion criteria, different detection methods, different statistical methods,and even different race of subjects. Researches on influence factors of coagulationfactors levels are fewer, that is fewer influence factors. It is still not able to reflect thematernal perinatal situation and neonatal birth physiological and pathological factors onlevels of coagulation factor in cord blood comprehensively and systematically.ObjectiveThrough detecting the levels of coagulation factors in cord blood，establish a referenceranges for levels of coagulation factors in cord blood from normal newborn, and analyzeinfluence on levels of coagulation factors in cord blood, included the physiological andpathological status of mater and the fetus/newborn.Contents1. Detect the levels of FII、FV、FVII、FVIII、FIX、FX、FXI and FXII in cord blood.2. Establish the reference range for levels of coagulation factors in cord blood fromnormal newborn.3. Analyze influence on levels of coagulation factors in cord blood, included thephysiological and pathological status of mater and the fetus/newborn.Methods1. Design and use questionnaire: the questionnaire mainly based on the principle,content, problem proposing way and matters need attention of questionnaire design inmedical statistics, combined with the aim and the background of this study. There arefour parts with51question，included neonatal basic information, situation at birth,maternal situation in pregnancy and health condition of family members. Carried out asmall scale pre-experiment before using questionnaire, and modified some question thatinterviewers can not easily answer. Complete the information by field investigations and clinical records review.2. Subject: newborns and their mothers who born in Guangzhou Women and ChildrenHospital from2011November to2012January.3. The collection, disposal and detection of specimen: prepare the Vacuum Vascular(1:9)Folic acid sodium anticoagulation tube). After ligationing the newborn umbilical cord,we would be collect3.6ml umbilical venous blood immediately, inject1.8ml blood intotwo tubes. Upside down and mix the blood10times, centrifuge with within30minutes.Drain the upper plasma and save it at Negative80degrees. Use CA-1500Automaticblood coagulation analyzer and related reagents, we have established the standardcurves of levels each of coagulation factors, had quality control and detected levels ofeight kinds of coagulation factors including FII、FV、FVII、FVIII、FIX、FX、FXIand FXII in umbilical cord blood plasma which we had collected.4. Each cord blood specimen has its identification markings with the specimen numbersand the hospitalization ID of pregnant mother. In the questionnaire, the informationobtained by open-ended question and filling will record in the excel document with thetext forms. The information obtained by close questions and selecting will record in theexcel document with the coded number. The detection results of each coagulationfactors will be record in the excel documents. The information of questionnaire and thedetection data should be verification by2men for2times, to ensure the data inputaccurate, complete and true.5. Establishment of reference ranges for levels of coagulation factors in cord blood:according to the C28-A3document, it needs at least120cases for establishing referenceranges of each item. In this study, we determine the inclusion criteria of normalnewborn firstly. Then screen from all subjects to obtain the normal newborn. Detect thenormal newborn’s levels of coagulation factors in cord blood，to establish referenceranges. The normal newborns conditions:①Pregnancy age is less than35old age;②Pregnant mother without hematologic system related diseases history and its familyhistory;③Pregnant mother without Pregnancy Complications, and not use any drugsbefore delivery（including vitamin K product, etc）;④Natural labor newborn；⑤Gestational age≥37weeks~<42weeks;⑥birth weight≥2500g~≤4000g, All is suitablefor gestational age;⑦Newborn babies without asphyxia damage in history,1minuteApgar score after birth>7points,5minute Apgar score after birth>7points；⑧newborn babies before discharge eat sleep well and no bleeding tendency. Establishment ofReference range for levels of coagulation factors in the normal umbilical cord blood:Using statistical software to normality test results on all kinds of coagulation factorslevels in normal newborn cord blood. Data which comply with the normal distribution,is determined its95%reference by mean±1.96s in bilateral, by mean+1.645s inunilateral upper limit and mean-1.645s in unilateral lower limit; it is determined its99%reference by mean±2.58s in bilateral, by mean+2.326s in Unilateral upper limit andmean-2.326s in unilateral lower limit. Data not meet the normal distribution can beexpressed in percentile, determine its95%reference by P2.5~P97.5in bilateral; by P95inunilateral upper limit and P5unilateral lower limit; determine its99%reference by inP0.5~P99.5in bilateral; by P99in unilateral upper limit and P1unilateral lower limit.6. Analysis of influence on levels of coagulation factors in cord blood: The maternalinfluence during pregnancy on cord blood levels of coagulation factors:①maternalage;②prenatal infections;③etiological diagnosis;④gestational diabetes mellitus(GDM);⑤gynecological diseases in pregnancy;⑥Pregnancy induced hypertensionsyndrome. The influence of physiological and pathological conditions on cord bloodcoagulation factors activity level:①gender;②the gestational age;③birth weight;④the mode of delivery;⑤multiple pregnancies;⑥the intrauterine hypoxia;⑦otherinternal and surgical diseases;⑧family history of hemorrhagic disease. p<0.05wasconsidered statistically significant. t-test use for the data complying with normaldistribution and homogeneity of variance; Mann-Whitney U test use for the data notcomplying with normal distribution and heterogeneity of variance.Results1. The use of the questionnaire: In this study, a total collection of cord blood is291, itcorresponds to the pregnant women all received a questionnaire survey. Response ratewas100%. Get all information in the questionnaire by field investigations and clinicalrecords review.2. Collection results of cord blood: Total291cord blood samples were collected anddivided into several groups, including136in group of natural newborn,31in group of cesarean delivery,30in group of infant with intrauterine hypoxia,25in group of twinpregnancy,13in group of mother with GDM,12in group of premature infants,10ingroup of pregnancy with gynecological diseases,10in group of over-weight infant,8ingroup of≥35-year-old mothers with first delivery,8in group of infection in perinatal,7samples with2or more abnormal factors in mother or newborn,1case died due to theinfant died within1hour after birth.3. Results of standard curve establishment and experimental quality control: standardcurves of coagulation factors have been establish (see Figure2). All samples weredeparted5batches to detect, conducted5times experiments quality control. Eachquality control result is within the reference range provided by reagent introduction.4. Reference ranges for levels of coagulation factors in cord blood: the95%referenceranges of FII、FV、FVII、FVIII、FIX、FX、FXI and FXII in cord blood are27.04~49.02,53.30~116.40,27.80~56.70,19.16~113.06,19.85~35.65,24.20~48.00,24.40~42.20and9.20~54.60respectively. The95%reference ranges of eight coagulation factors incord blood are23.56~52.50,53.30~116.40,27.80~56.70,4.31~127.91,17.35~38.15,24.20~48.00,24.40~42.20and9.20~54.60respectively.5. Influential factors in cord blood coagulation factors: FII and FIX in the group of≥35-year-old mothers with first delivery are more active than the normal (p=0.009,0.028);every coagulation factor between the GDM group and the not GDM group is withoutstatistical difference(p>0.05); FVIII in the group of Pregnancy associated withgynecologic diseases is less active than the normal (p=0.043), FII, VII and FX are moreactive than the normal (p=0.032,0.024,0.022); every coagulation factor between themale group and the female group is without statistical difference(p>0.05); FII, FV, FIXand FXI in the group of premature infant are less active than the normal (p=0.031,0.037,0.000,0.002); FII and FVII in the group of birth weight>4.0kg are more active than thenormal (p=0.043,0.043); FVIII in the group of cesarean section is less active than thenormal (p=0.004); FVIII, FIX and FXI in the group of twin pregnancy are less activethan the normal (p=0.002,0.000,0.028); FII and FVIII in the group of intrauterinehypoxia are less active than the normal (p=0.032,0.012) Conclusions1. Reference ranges for levels of coagulation factors in cord blood: the95%referenceranges of FII、FV、FVII、FVIII、FIX、FX、FXI and FXII in cord blood are27.04~49.02,53.30~116.40,27.80~56.70,19.16~113.06,19.85~35.65,24.20~48.00,24.40~42.20and9.20~54.60respectively. The95%reference ranges of eight coagulation factors incord blood are23.56~52.50,53.30~116.40,27.80~56.70,4.31~127.91,17.35~38.15,24.20~48.00,24.40~42.20and9.20~54.60respectively. Levels of FVIII and FXII inthis study are lower than the result of foreign literature, but consistent with domesticreports.2. Our results show that, in addition to the pregnant mother suffering from GDM andneonatal sex, the remaining influence factors have certain effects on the levels ofcoagulation factors in cord blood. Levels of FII and FIX in the group of≥35-year-oldmothers with first delivery are higher; Level of FXI in the group of perinatal infection islower; Level of FVIII in the group of pregnancy complicated with uterine myoma islower, Levels of FII, VII and FX are higher; Levels of FII, FV, FIX and FXI in thegroup of premature infant are lower; Levels of FII and FVII in the group of birthweight>4.0kg are higher; Levels of FII and FVIII intrauterine hypoxia are lower;Levels of FVIII in the group of cesarean section is lower; Levels of FVIII, FIX and FXIin the group of twin pregnancy are lower.3. Our results are inconsistent with domestic and foreign reports. The reason may beassociated with inclusion criteria, the number of cases, detection methods, thecharacteristics of the coagulation factor and race.4. Our study use136cases to establish the reference ranges for levels of coagulationfactors in cord blood. Our sample cases are significantly more than other researches atpresent, and meet the requirement of C28-A3document. Our influence factors are morecomprehensive than other researches. We hope that this study is able to objectivelyreflect the levels of coagulation factors in cord blood in domestic.