医学课件血红蛋白电泳的意义和判断.ppt

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pour modifier les styles du texte du masque,Deuxime niveau,Troisime niveau,Quatrime niveau,Cinquime niveau,*,目的,血红蛋白结构和功能的回顾,Sebia CAPILLARYS 2/Minicap,血红蛋白检测说明,血红蛋白临床结果说明,1,血红蛋白的结构,血红蛋白分子是由,4,条多肽链,(2,对,),和,4,个可以结合铁和氧气的血红素,.,Normal hemoglobins,contain,alpha,beta,delta,and gamma,globin chains,.,2,三维结构,4,亚单位,2 alpha,链,2 alpha genes,3,非,alpha,链,:beta,delta,gamma,1,b,gene,1,d,gene,2,g,genes,正常结果,Hb A:,a2b2,Hb F:,a2g2,Hb A2:,a2d2,+,-,琼脂糖凝胶,Hydrasys,(Sebia),碱性缓冲液,成人,新生儿,血红蛋白遗传病类型,(HEMOGLOBINOPATHIES)?,血红蛋白疾病,血红蛋白性质变化,:,血红蛋白分子病,结构异常,一个或数个氨基酸变异或缺失,都将会导致血红蛋白分子的电性发生改变,常见血红蛋白变异体,b,-globin:,S,C,D,Punjab,E,血红蛋白含量变化,:,地中海贫血,合成或合成调控异常,:,b,globin,b,Thalassemias,a,globin,a,Thalassemias,合并型,:,血红蛋白分子病,Hemoglobinopathies/,地贫,Thalassemias,血红蛋白遗传病,遗传性疾病基因学图谱,正常基因,异常基因,异常血红蛋白类型,目前,已经证实的有超过,1000,种以上的变异体,(,a,b,g,and,d),大部分变异体出现频率较低,并且没有明显临床症状,4,种主要变异体,(Hb S,Hb C,Hb E and Hb D,Punjab,),出现频率较高,(,特定人群,),携带者会出现健康问题,儿童可能在出生时即伴随有严重症状,血红蛋白疾病的区域分布图,Thalassemias,常见与地中海区域,:,意大利,西班牙,希腊,近东地区,非洲北部,.,同样常见于东南亚,:,泰国,印度,越南,中国,印尼,.,临床症状,*,Beta,地贫重症型,-,严重贫血,(,需要长期输血治疗,),*Beta,地贫中间型,*Beta,地贫轻微型,-,无临床症状,Beta,地中海贫血,常见区域,bo,没有,Beta,链合成,b+,合成能力下降,Alpha,地中海贫血,临床症状,:,根据,Alpha,基因的表达状态,有,4,个不同程度的,Alpha,地贫症状,*1,个基因表达沉默,:,静止型,(,非常难检测到,在出生会测到,Hb Barts 1-2%),*2,个基因表达沉默,:,轻型,alpha,地贫,在出生会测到,Barts 2-10%,*3,个基因表达沉默,:,血红蛋白,Hb H,病,出生时出现,2-30%Barts,儿童,-,成人出现,10-15%Hb H,*4,个基因表达沉默,:Hb Barts,胎儿水肿综合征,(,死胎,),常见区域,常见与远东地区,非洲和地中海区国家,其他特征,Hb H,Hb A2,Hb F,贫血,小红细胞,靶细胞,异常,出生时少量的,(2%)Hb Barts,0,正常,正常,0,0,0,一条链缺失,出身时,Hb Barts(5%),0,正常或减少,正常,0,0,0,2,条链缺失,不稳定血红蛋白溶血性贫血,(Hb H),10-15%,减少,也许增加,轻微的到严重的,+,+,3,条链缺失,胎儿水肿综合症,(hydrops fetalis),0,0,100%Hb Barts,严重的,-,-,4,条链缺失,基因型,诊断,血常规,临床症状,检测结果,aa/aa,无,Hb A:N,HbA2:N(2-3.5%),-a/aa,a,-Thalassaemia,2,杂合体,(,静止型,),轻微小红细胞症,MCV:75-80fL,MCH:20-24,无,Hb A and HbA2:N,Hb Barts 0-3%at birth,Confirmation by molecular biology,-a/-a,a,-Thalassaemia,2,纯合体,小红细胞症,血红蛋白减少,Hb:11-12 g/dL,无明显临床症状,Hb A and HbA2:N,Hb Barts 2-8%at birth,-/aa,a,-Thalassaemia,1,杂合体,MCV:65-75,MCH:20-24,需要分析序列分析或,DNA,序列分析,-/-a,HbH,小红细胞症,血红蛋白减少,Hb:8-11 g/dL,MCV:55-65,MCH:20-24,出现溶血脾脏肿大,HbA2 N or,Hb Barts 5%,Hb H 10-20%,-/-,a,-Thalassaemia,1,纯合体,(,死胎,),严重贫血,水肿胎,Hb:8-11 g/dL,MCV 100-110,MCH,无法存活,死胎,Absence of HbA and HbF,Hb Barts 80-100%,Hb Portland 5%,镰刀,型,红细胞血症,变异体出现在,6 GAG(,谷氨酸,),GTG(,缬氨酸,),N,SS,AS,abnormal gene,normal gene,N,HbS,N,N,HbS,AS,N,HbA,HbS,N,N,SS,HbA,HbS,Chromosomes 16,a2,a1,a2,a1,Homozygote,Chromosomes 11,Htrozygote,d,b,d,b,A,g,A,g,G,g,G,g,d,b,d,b,A,g,A,g,G,g,G,g,Chromosomes 16,a2,a1,a2,a1,Homozygote,Chromosomes 11,Heterozygote,d,b,d,b,A,g,A,g,G,g,G,g,d,b,d,b,A,g,A,g,G,g,G,g,C,型血红蛋白分子病,变异体位置,:AA 6:GAG(,谷氨酸,),AAG(Lys),HbA,Heterozygote AC,HbA,HbC,N,HbA,SC Compound,heterozygoty,HbA,HbC,N,HbS,Htrozygote composite,AC,SC,Chromosomes 16,a2,a1,a2,a1,Homozygote,Chromosomes 11,Htrozygote,d,b,d,b,A,g,A,g,G,g,G,g,d,b,d,b,A,g,A,g,G,g,G,g,Chromosomes 16,a2,a1,a2,a1,SC heterozygote compound,Chromosomes 11,Heterozygote,d,b,d,b,A,g,A,g,G,g,G,g,d,b,d,b,A,g,A,g,G,g,G,g,D,型血红蛋白分子病,常见区域,:,常见于印度,.,特点,:,变异体位置,b,121 Glu,(,谷氨酸,负电荷,),Gln,(,谷氨酰胺,不带电荷,),(D-Los Angeles=D-Punjab),(6 different Hb D according to the position of the mutation),碱性缓冲液中,:,电荷下降,M,电泳速度变度变慢,靠近,Hb S,临床症状,:,基因杂合体,:,无症状,(,仅在电泳中发现,D,区带,),Hb A fraction:65 70%,Hb D fraction:30 35%,基因纯合体,:,轻微贫血,Hb A fraction:absence,Hb D fraction:100%,E,型血红蛋白分子病,常见区域,:,常见与东南亚地区,.,特点,:,变异位点,b,26 Glu,(,谷氨酸,负电荷,),Lys,(,赖氨酸,正电荷,),碱性缓冲液,:,总体带电荷下降,凝胶电泳中,出现在,HbC,附近,临床症状,:,杂合体,:,无症状,Hb A fraction:65 70%,Hb E fraction:30 35%,基因纯合体,:,轻微贫血,Hb A fraction:absence,Hb E fraction:100%,下列情况下,需要进行血红蛋白检测,临床和遗传问题,(,产前诊断,),贫血,小红细胞症,新生儿筛查,对来自地贫高发区人口,进行筛查,对诊断有帮助的其他信息,患者临床症状,年龄,籍贯和其他相关的背景,(,输血记录,),血液全套检测,网状细胞级别和进行细胞形态镜检,Hb,电泳,(EDTA,抗凝管,),离子平衡和溶血情况,血红蛋白检测的几种方法,目前,血红蛋白检测步骤如下,:,-,电泳法对,Hb,进行检测,或者使用等电聚焦电泳法,-,使用,HPLC,法进行确认,.,凝胶电泳结果识别表,19,在,pH,梯度的凝胶片上,根据蛋白不同的等电点,对蛋白进行分离,Hb,电泳位置取决于缓冲液,PH,值,蛋白电泳至其等电点位置后,停止泳动,At pI=pH,Hb does not have a net charge and stops migrating,电泳分离效果好,但时间长,.,需要经验丰富才能获得良好结果及进行结果判断,等电聚焦电泳法,20,介于普通电泳和等电聚焦电泳之间的新方法,:,-,通过毛细管电泳法,即可以获得与,IEF,方法一样的分辨率,同时检测结果量值,Hb A2/Hb F,同样精确,.,-,出现异常变异体后,:,使用下列方法确认,:,*ITANO test(solubility test),*HPLC,液相色谱,*Genotyping,基因筛查,Capillarys 2,Minicap,全自动毛细管电泳法进行血红蛋白检测,Capillarys 2/Minicap,毛细管电泳法原理,Peltier,温控桥,Anode,+,Cathode,-,检测器,氘光灯,电泳,高电压,此时,电渗力,(EOF),远远强于电场力,.,因此,蛋白被动从正极向负极端移动,.,正电极,+,EOF,负电极,-,Electro,migration,蛋白电泳,DETECTION,INJECTION,带正电荷的缓冲液,毛细管壁负离子鞘,Capillarys/Minicap,血红蛋白电泳,血红蛋白条带,在电渗力的作用下,8,分钟即完成电泳过程,然后,在,415nm,紫外光下被检测并显示出来,.,正常质控品,必须在每天检测前使用,Capillarys:,-,检测速度,:40,样品,/,小时,试管架,1,至,7,号位放置样品,8,号位放,4ml,溶血素,Minicap:,-,检测速度,:10,样品,/,小时,试管架,1,至,26,号位放置样品,27,号位放溶血素,(8,个样品消耗,5ml,溶血素,),Capillarys/Minicap,血红蛋白检测标本要求,抗凝管采血,(EDTA,heparin,citrate).,如果需要对输血患者采血,需要在输血前采集血液,.,使用去除血浆后的红细胞,(,隔夜或,5000 rpm,5,分钟离心后样品,)+4C,以上,标本最多存放,7,天,.,需要长期存放,可以洗涤红细胞后,放置于,-80C(,可存放至少,3,个月,),Capillarys Minicap,血红蛋白条带识别表,Capillarys/Minicap,区带识别表,Z 15:,Hb H,Z 14:/,Z 13:Hb-J Rovigo,Hb N-Baltimore,J-Kaoshiung,Z 12:,Hb Barts,Hb Providence,Hb J-Mexico,Hb J-Baltimore.,Z 11:Hb Kaoshiung(New York),denaturated Hb A,Z 10:Hb Hope,Hb M-Iwate,Z 9:,Hb A,Hb Camperdown,Hb Phnom Penh,Z 8:Acetylated Hb F,Hb Altanta,Hb Athens-GA,Z 7,:,Hb F,denaturated Hb S,Hb Porto-Allegre.,Z 6:,Hb D-Punjab,Hb Philadelphia,Hb Korle-Bu,Hb Lepore,Hb K,ln,denaturated Hb E.,Z 5:,Hb S,Hb Hasharon,Hb Handsworth,denaturated Hb O-Arab.,Z 4:,Hb E,denaturated Hb C,Hb K,ln,Hb A2 variants,M-Iwate,Hb A2 variants,Z 3:,Hb A2,Hb O-Arab,Z 2:,Hb C,Hb Constant Spring,Setif HbA2 variant,Z 1:Hb,d,A2 Hb,a,A2,Hasharon Hb A2 variant,Winnipeg Hb A2 variant,正常,Hb A,2,质控品,条带结果可录为,QC,29,Hb AFSC,质控品,可用来作为参照曲线,便于异常结果的判断,30,Hbs A,F,A,2,检测结果可通过颜色进行区分,异常区带显示为灰色,31,主要异常血红蛋白变异体,Z15 Hb H,Z12 Hb Bart,Z10 Hb Hope,Z9 ,Hb A,Z7 ,Hb F,Z6 Hb D Punjab,Z5 ,Hb S,Z4 Hb E,Z3 ,Hb A2,Z2 ,Hb C,HbE,HbS,HbH,HbS,变异体,Hb A2,正常值,6 Glu Val,Hb A=,22,Hb S=,2,S,2,Hb A2=,22,33,Capillarys/Minicap,血红蛋白试剂优势,:,无需前处理,(,不需要洗血和溶血,),，直接检测,血红蛋白,C,和,E,能从,A2,分离开,根据,S,型和,D,型的细微电荷区别,进行区分,.,对于处于,A,和,S,之间的,F,给予个性化和聚焦的精确量化,.,对,A2,型血红蛋白有很好的分辨率和分离效果,Hb Barts,和,Hb H,可以很好的检测和量化,糖化型血红蛋白电泳出现在主体峰内,不影响检测结果,异常结果分析,35,Case 2:31 Y Female,血红蛋白,12.8,红细胞压积,37.4 (I),A=84.1%,F=1.5%,A2=,5.5%,36,31 Y Female,Hgb 12.8,Hematocrit 37.4 (II),Alk,Acid,F,S,C,A,2,C,S,F,-thalassemia-,轻型,A,2,A,A,A,A,C,P,C,P,A=93.4%,F=1.1%,A2=5.5%,37,Hb A 92.5%,Hb F 2.6,Hb A2 4.9%,Hb F,Hb A,Hb A2,67 Y female from Tunisia,Hgb 9.1,hematocrit 28,MCV 62,小红细胞症,;beta thalassemia,轻型,Hb H 4%,Hb A 94.7%,Hb A2 1.3%,Hb A,Hb A2,Hb H,Child 9 Y,from Laos,Hgb 8,hematocrit 27.9,MCV 54.1,严重小红细胞症,;,血红蛋白,H,病，需要基因筛查,Hb H and Barts-Alpha-thalassemia,H Barts,40,Case 1:,13 Y Male,Hgb 8.1,Hct 24.0 (I),A2=,6.1%,S=82.8%,41,Case 1:13 Y Male,Hgb 8.1,Hematocrit 24.0 (II),Alk,Acid,F,S,C,S,A,2,+F,C,S,F,S,Hgb F/S,A2=4.6%,+beta thalassemia minor,A,2,A,F,A,C,P,C,P,42,Case 2:65 Y Male,Hgb 9.4,Hematocrit 27.3 (I,),Co-migration,with A2,A=62.1%,A2=31.7%*,43,Case 2:Hb E Migrates Separately From Hb A2,26 GluLys,44,21Y,pregnant woman,Hgb 13.1,hematocrit 38.6,Hb A 95%,Hb F 2.1%,Hb A2 2.9%,Normal pattern,Hb F slightly increased(pregnancy),Baby 2 months old,father from Martinique,mother from Central Africa,Hgb 11,hematocrit 29.8,Hb F 49.5%,Hb S 25.9%,Hb A2 1.8%,Hb C 22.8%,Hb F,Hb S,Hb A2,Hb C,SC compound heterozygoty,Diabetic patient,54 Y,Hgb 14.5,hematocrit 43.5,MCV 88,HbA1c measurement(HPLC Tosoh),Hb A 54.7%,Hb X 41.7%,Hb A2 3.6%,Hb A,Hb A2,Hb X,Normal hemogram;compatible with heterozygote A/D;,To be identified by genotyping,Hb A 51.8%,Hb A2 2.9%,Hb C 35.9%,Hb A,Hb A2,Hb C,Diabetic patient,61 Y,Hgb 11.6,hematocrit 34.8,MCV 85,Slight anemia;compatible with heterozygote A/C,To be identified by genotyping,Adopted child,4 Y,originated from Haiti,Hgb 10.9,Hematocrit 31.3,MCV 76.9,Hb A 97.5%,Hb A2 1.4%,Hb X 1.1%,Hb A,Hb A2,Hb X,Slight anemia;heterozygous Delta mutation,asymptomatic,Diabetic patient,73 Y,Hgb 10.2,hematocrit 30.8,MCV 87.3,Alpha chain mutation;genotyping confirmation:,Hb Winnipeg,a,75 Asp Tyr,Hb A-Wi,Hb A2-Wi,Hb A,Hb A2,Hb A 90.3%,Hb F 3.8%,Hb A2 5.9%,Hb A,Hb F,Hb A2,Hb A 64%,Hb S 33.1%,Hb A2 2.9%,Hb A,Hb S,Hb A2,Hb A 2.2%,Hb F 67.3%,Hb S 29.1%,Hb A2 1.4%,Hb A,Hb F,Hb S,Hb A2,Father:Beta thalassemia minor Mother:Heterozygote AS,Baby 2 months old:Association S-beta thalassemia,Hb Lepore on Minicap/Capillarys,A,N,Alc.,A,2,Lepore,N,Ac.,A,0,+,Lepore,A,1,Hb A2,Hb A,Hb F,Hb Lepore,AFSC control overlaying,Hb Lepore,b,和,d,链重新组合形成,.,在碱性缓冲液下电泳,:,总体带电荷下降,电泳位置出现,在,S,型血红蛋白附近,基因杂合体,:,Hb Lepore fraction:5 15%,Clinical signs of minor,b,thalassemia,基因纯合体,:,Hb Lepore fraction:about 30%,Clinical signs of homozygous,b,thalassemia,常见区域,:,Lepore,(-Boston Washington):,常见于意大利人群,;,同样常见于在罗马尼亚,澳大利亚和墨西哥人群,.,特点,:,a,47 Asp,His,Hb Hasharon,Hb A,Hb A2,Hb F,Hb Hasharon,Hb Ha-A2,Hb O-Arab,b,121 Glu,Lys,Hb A2+O-Arab,Hb F,Hb A,DIAGNOSTIC FLOW-CHART,A NEW FAST HEMOGLOBIN ELECTROPHORESIS METHOD FOR DRIED BLOOD SPOTS ON SEBIA CAPILLARYS AUTOMATED SYSTEM,现有的新生儿血红蛋白筛查方法,(SCD),新生儿血红蛋白检测,:,*,等电聚焦,(IEF),*,高效离子交换液相色谱法,(CE-HPLC).,毛细管电泳方法可取代常规,操作复杂的等电聚焦方法,新生儿血红蛋白快速检测法,取血板,BSD600,全自动打孔器,全自动溶血,样品稀释和检测,溶血剂,样品,条码识别：,取血板条码样品稀释杯条码,使用纯净水洗脱样品,与,IEF,方法比较,毛细管方法优势,对于常见异常血红蛋白变异体有,100,区分能力,(Hb S,C,D,E),对其他非常见变异体有良好检测能力,对不同类型样品拥有相同检测能力：新生儿,早产儿或者进行过输血患儿样品,全程检测溯源性,:,取血板和样品稀释杯使用条码,高效性,(48,个样品,/,小时,),576,样品,/,天,4,小时人机分离操作,最多可进行,3X192,个样品检测,可选择使用与毛细管匹配的全自动打孔机,完全自动化处理样品,无需人工进行样品转移,避免交叉污染,该项目检测完全自动化,:,从前处理到检测,直到结果分析,全程无需人工干预,.,使用标准取血板,在室温下进行干燥,.,干燥的取血板可在,2-8C,下,保存,1,个月,.,新生儿血红蛋白检测的样品要求,通过颜色区分正常及异常样品,正常血样,:,with Hb F and Hb A,(+acetylated Hb F),异常血样,提供样品检测完整溯源性,通过样品,ID,试剂杯和全自动打孔器的数据记录完成数据交换,异常样品自动显示,电泳图谱说明,:,*,检测到条带的位置,图谱会自动定位,*,鼠标经过的区带位置,会显示出该区带的所有可能出现的异常血红蛋白名称,.(N1 to N13).,新生儿血红蛋白检测条带名称识别表,N 13,:Hb Barts,N 12,:/,N 11,:Hb Hope,Gamma chain variants,denatured Hb A,N 10,:Hb A,N 9,:Denatured Hb F,N 8,:/,N 7,:Hb F,N 6,:/,N 5,:Hb D-Punjab,Hb G-Philadelphia,Hb Korle-Bu,N 4,:Hb S,acetylated gamma chain variants,N 3,:Hb E,Hb F-Ouled Rabah,gamma chain variants,Methemoglobin,N 2,:Hb A2,Hb O-Arab,Hb F-Ouled Rabah,gamma chain variants,N 1,:Hb C,正常血样,Hb F,Hb A,Birth:19/01/06,Sample collection:24/01/06,Term:39 w.,Weight:3445 g,输血后检测结果,Birth:29/01/06,Sample collection:01/02/06,Term:38 w.,Weight:3100 g,F/S homozygote,Birth:05/01/06,Sample collection:08/01/06,Term:37 w.,Weight:3490 g,Hb S,Hb F,Denatured,Hb F,早产儿体内的,F/S,Denatured,Hb F,Hb F,Hb S,Birth:20/02/06,Sample collection:23/02/06,Term:34 w.,Weight:1300 g,F/SC compound heterozygote,Denatured,Hb F,Hb F,Hb S,Hb C,Birth:30/01/06,Sample collection:02/02/06,Term:40 w.,Weight:3900 g,F/AS heterozygote,Hb A,Hb F,Hb S,Birth:16/12/06,Sample collection:19/12/06,Term:36 w.,Weight:2780g,F/C homozygote,Hb F,Hb C,Denatured,Hb F,Birth:20/12/05,Sample collection:24/12/05,Term:39 w.,Weight:3160 g,F/AC heterozygote,Birth:18/12/06,Sample collection:21/12/06,Term:40 w.,Weight:3035 g,F/AC in a premature newborn,Birth:17/10/05,Sample collection:20/10/05,Term:30 w.,Weight:2330 g,F/AD heterozygote,Birth:29/01/06,Sample collection:31/01/06,Term:37 w.,Weight:3280 g,F/E homozygote,Hb E,Hb F,Denatured,Hb F,Birth:10/11/06,Sample collection:13/11/06,Term:40 w.,Weight:?,F/AE heterozygote,Birth:10/03/06,Sample collection:13/03/06,Term:39 w.,Weight:3420 g,F/AE in a premature newborn,Birth:21/07/05,Sample collection:24/07/05,Term:35 w.,Weight:1840 g,F/A0-Arab heterozygote,Birth:14/02/06,Sample collection:17/02/06,Term:40 w.,Weight:3120 g,F/AKorle-Bu heterozygote:,Birth:31/10/06,Sample collection:03/11/06,Term:40 w.,Weight:3790 g,Barts in a term newborn,Birth:05/04/06,Sample collection:08/04/06,Term:38 w.,Weight:2790 g,对新生儿血红蛋白的定量检测,AF control,Hb A,Hb F,Hb A2,Hb quantification,Normal blood,Hb A,Hb F,Hb quantification,Ratio A/F,F/AS heterozygote,Hb A,Hb F,Hb quantification,Hb S,Ratio A/S,F/AC heterozygote,Hb A,Hb F,Ratio A/C,Hb C,Hb quantification,Hb Barts in a newborn,Hb A,Hb F,Hb quantification,Hb Barts,Cord blood analysis,特定程序下,使用普通血红蛋白试剂盒,CORD BLOOD:normal sample,CORD BLOOD:,a,-Thalassemia,CORD BLOOD:,Heterozygote A/D-Punjab,CORD BLOOD:,Heterozygote A/S,总结,血红蛋白疾病,:,CAPILLARYS,新生儿和脐带血血红蛋白检测可作为等电聚焦方法和液相色谱法的替代技术,如需对特殊检测结果进行确认,可选择其他方法,(,液相色谱,基因筛查,),Thank you for your attention,