The Detection Of Amyloid-beta In Alzheimer’s Disease Peripheral Blood And The Study Of It In Clinical Application

Objective: Amyloid cascade hypothesis believes that amyloid beta(Amyloid beta, Aβ) is the main reason of Alzheimer’s Disease(Alzheimer’s diseases, AD). The deposition of Aβ in the extracellular of brain neurons has become AD diagnostic markers. Currently, the testing means targeting Aβ mainly include neuroimaging methods and cerebrospinal fluid biomarkers detection. However, neuroimaging diagnostic methods, can provide evidence for the diagnosis of AD, but whether the tracer used can be clean by the metabolic system has not yet been determined, and the side effects to human body is difficult to assess. The non- labelling technique lacks of specificity, easily leading to misdiagnosis or false positive results, thus brings difficulties to clinical diagnosis. The purpose of this paper is to study the role of Aβ in the diagnosis and pathogenesis of AD through establishing a simple and easy way to detect the Aβ in peripheral circulation. Compared with neuroimaging markers and CSF detection, peripheral specimen acquires simple and easy, and can reflect the brain disease outcomes, has significant advantages in disease diagnosis, treatment, and pathogenic mechanism study.Methods: 1. According to the strict into and discharge standards, selecting 50 AD patients(AD group) and 50 gender, age matched healthy persons(HC group) in general hospital of Tianjin medical university neurology department in March 2012 to October 2014 AD. Extracting vein, EDTA- 2 na anticoagulation whole blood. Set aside.2. Confirmed the existing of Aβ fibrils/aggregates in plasma and red cell membrane by protein immunoblot(Western blot, WB) and immunofluorescence experiments.3. Analyzed the plasma amyloidosis fluorescence intensity in AD group and HC group by Thioflavin T(Thioflavin T, Th T) specific experiment.4. The detection of blood physics index and lipid level in AD group and HC group.5. The count of elongated cells in AD group and HC group and analyze the corelation between elongated cells quantity and Simple intelligent state examination(Simple getting state examination, MMSE).6. Discuss the effect of plasma to Th T binding Aβ fibrils/aggregates on the red cell membranein Th T specificity experiment.7. Studied the amyloid binding-positive subjects(% of total) and amyloid binding-positive RBCs(% of total) in AD and HC group.8. By Leica Application Suit 2.0.1 software, analyze the Th T positive red cell morphology.Results: 1. Plasma amyloid fluorescence intensity AD group is(16.63±5.67) U/ml, HC is(12.83±0.27) U/ml.AD group plasma amyloid fluorescence intensity was significantly higher than that of control group(P < 0.05).2. AD and HC group whole blood viscosity(low cut 1.001/s) are(18.16±3.6) and( 17.92±0.4)m Pa.s, respectively(P >0.05); Whole blood viscosity(low cut 5.00 1/s) are(8.64±1.45) and(8.40±0.37)m Pa.s, respectively(P >0.05); Whole blood viscosity(low cut 30.00 1/s) are(5.32±0.76) and(5.09±0.16)m Pa.s, respectively(P > 0.05);Whole blood viscosity(high cut200.001/s)are(4.16±0.54) and(3.74±0.15)m Pa.s, respectively(P<0.05); Plasma concentrations are(1.40±0.10) and(1.33±0.07)m Pa.s, respectively(P>0.05); Blood sedimentation are(11.8±6.18) and(8.30±6.09)mm/h, respectively(P>0.05); Hematocrit are(0.43±0.61) and(0.45±0.31) respectively(P>0.05); Whole blood high shear relative index are(2.94±0.22) and(2.74±0.26), respectively(P>0.05); Whole blood low shear relative index are(12.79±1.78) and(14.24±1.32), respectively(P>0.05); Blood sedimentation equation k value are(39.61±13.08) and(32.26±15.27), respectively(P<0.05); Red blood cell aggregation index are(4.34±0.36) and(4.13±0.14), respectively(P>0.05);Whole blood low shear viscosity reduction are(38.36±2.77) and(38.81±2.50)m Pa.S, respectively(P < 0.05); Whole blood high shear viscosity reduction are(6.33±0.40) and(6.19±0.49 m Pa.S, respectively(P<0.05); Erythrocyte rigidity index are(4.50±0.34) and(4.70±0.50) m Pa. S, respectively(P>0.05); TK erythrocyte deformation index are(0.82±0.08) and(0.81±0.07), respectively(P>0.05).3. AD and HC group total cholesterol are(4.71±1.24)and(3.95±0.22),respectively(P<0.05);HDL are(1.21 ± 0.35)and(1.40±0.17),respectively P>0.05; Low density lipoprotein are(2.70 ±0.84)and(2.78±0.36),respectively P>0.05.4. There is relation among whole blood viscosity(high shear part), plasma amyloid levels and serum total cholesterol content in AD.The multivariate linear regression analysis gained the regression equation =2.798+0.070X1+0.027X2(, whole blood high shear viscosity; X1,plasma amyloid levels; X2, total cholesterol).5. The AD group increase red blood cell volume(16.8%) is significantly higher than the HC group(6.7%)(P<0.01). There is a negative correlation between increase red blood cell volume quantity and MMSE in AD(r =-0.773).6. ThT can recognize Aβ amyloid fibrils and/or aggregation associated with RBC specifically and the dyeing results is not disrupted by plasma7. Our study showed that 98%of AD peripheral RBCs were amyloid binding-positive(ranging from 2-30 %),while only 39% that of RBCs(ranging from 2-3.4 %) in healthy controls.8. Aβ fiber/aggregates binding to red blood cells leads to increase red blood cell volume, morphological changes, mainly for four types.Conclusion 1. Analysis of plasma amyloidosis,plasma total cholesterol levels and blood physical index of patients with AD, the results of three biomarkers were significantly higher than that of HC group, and plasma amyloidosis and total cholesterol levels can lead to blood viscosity plasma physics index(high part) abnormal, which may serve as auxiliary examination indexs.2. To explore a method for detection of blood cell amyloid, realize the detection of amyloidosis of red blood cells, granulocyte, monocyte, this method can be used for the detection of blood cell amyloidosis.3. We reported for the first time the range of amyloidosis red blood cell in patients with AD, and identified the range of it is(2%-30%), which may be used as an indicator for examination of AD.4. Th T can specific identification of peripheral circulation in Aβ fiber/aggregates on the red cell membrane,and we found Th T positive red blood cell morphological changes, suggesting that Aβ fiber/aggregates can result in red blood cell morphology change, indicate cell amyloid may participate in the pathological mechanism of AD.