血红蛋白电泳的意义和判断ppt课件.ppt
ADULT AND NEWBORN Hemoglobin screening with capillarys,2,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,3,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.,三维结构,4 亚单位2 alpha链2 alpha genes3 非 alpha链 : beta, delta, gamma1 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 DPunjab 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 Thalassemiasa 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 DPunjab)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,镰刀型红细胞血症,变异体出现在 6 GAG (谷氨酸) GTG (缬氨酸),C型血红蛋白分子病,变异体位置:AA 6: GAG (谷氨酸) AAG (Lys),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 b121 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: absenceHb 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 b26 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: absenceHb 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.,20,凝胶电泳结果识别表,21,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,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,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,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 HZ 14 : /Z 13 : Hb-J Rovigo, Hb N-Baltimore, J-KaoshiungZ 12 : Hb Barts, Hb Providence, Hb J-Mexico, Hb J-Baltimore.Z 11 : Hb Kaoshiung(New York),denaturated Hb AZ 10 : Hb Hope, Hb M-IwateZ 9 : Hb A, Hb Camperdown, Hb Phnom PenhZ 8 : Acetylated Hb F, Hb Altanta, Hb Athens-GAZ 7 : Hb F, denaturated Hb S, Hb Porto-Allegre. Z 6 : Hb D-Punjab, Hb Philadelphia, Hb Korle-Bu, Hb Lepore, Hb Kln,denaturated Hb E.Z 5 : Hb S, Hb Hasharon, Hb Handsworth, denaturated Hb O-Arab.Z 4 : Hb E, denaturated Hb C, Hb Kln, Hb A2 variants, M-Iwate Hb A2 variantsZ 3 : Hb A2, Hb O-ArabZ 2 : Hb C, Hb Constant Spring, Setif HbA2 variantZ 1 : Hb dA2 Hb aA2, Hasharon Hb A2 variant, Winnipeg Hb A2 variant,30,正常Hb A2 质控品,条带结果可录为QC,31,Hb AFSC质控品, 可用来作为参照曲线,便于异常结果的判断,32,Hbs A, F,A2 检测结果可通过颜色进行区分,异常区带显示为灰色,主要异常血红蛋白变异体,Z15 Hb HZ12 Hb BartZ10 Hb HopeZ9 Hb A Z7 Hb FZ6 Hb D PunjabZ5 Hb SZ4 Hb EZ3 Hb A2Z2 Hb C,HbE,HbS,HbH,34,HbS 变异体, Hb A2 正常值 6 Glu Val,Hb A = 22,Hb S = 2S2,Hb A2 = 22,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,36,异常结果分析,37,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%,38,31 Y Female, Hgb 12.8, Hematocrit 37.4 (II),Alk,Acid,F,S,C,A2,C,S,F,-thalassemia- 轻型,A2,A,A,A,A,C,P,C,P,A=93.4%, F=1.1%, A2=5.5%,Hb A 92.5%Hb F 2.6Hb 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,41,Hb H and Barts - Alpha-thalassemia,H Barts,42,Case 1: 13 Y Male, Hgb 8.1, Hct 24.0 (I),A2= 6.1%S = 82.8 %,43,Case 1:13 Y Male, Hgb 8.1, Hematocrit 24.0 (II),Alk,Acid,F,S,C,S,A2 + F,C,S,F,S,Hgb F/S, A2=4.6% + beta thalassemia minor,A2,A,F,A,C,P,C,P,44,Case 2:65 Y Male, Hgb 9.4, Hematocrit 27.3 (I),Co-migration with A2,A= 62.1 %A2 = 31.7%*,45,Case 2: Hb E Migrates Separately From Hb A226 GluLys,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/CTo 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,N,Ac.,A0 + Lepore,A1,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:,a47 AspHis,Hb Hasharon,Hb A,Hb A2,Hb F,Hb Hasharon,Hb Ha-A2,Hb O-Arab,b121 GluLys,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,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 BartsN 12 : /N 11 : Hb Hope, Gamma chain variants, denatured Hb AN 10 : Hb AN 9 : Denatured Hb FN 8 : /N 7 : Hb FN 6 : /N 5 : Hb D-Punjab, Hb G-Philadelphia, Hb Korle-BuN 4 : Hb S, acetylated gamma chain variantsN 3 : Hb E, Hb F-Ouled Rabah, gamma chain variants, MethemoglobinN 2 : Hb A2, Hb O-Arab, Hb F-Ouled Rabah, gamma chain variantsN 1 : Hb C,正常血样,Birth: 19/01/06Sample collection: 24/01/06Term : 39 w.Weight : 3445 g,输血后检测结果,Birth: 29/01/06Sample collection: 01/02/06Term : 38 w.Weight : 3100 g,F/S homozygote,Birth: 05/01/06Sample collection: 08/01/06Term : 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: 20/02/06Sample collection: 23/02/06Term : 34 w.Weight : 1300 g,F/SC compound heterozygote,Denatured Hb F,Hb F,Hb S,Hb C,Birth: 30/01/06Sample collection: 02/02/06Term : 40 w.Weight : 3900 g,F/AS heterozygote,Hb A,Hb F,Hb S,Birth: 16/12/06Sample collection: 19/12/06Term : 36 w.Weight : 2780g,F/C homozygote,Hb F,Hb C,Denatured Hb F,Birth: 20/12/05Sample collection: 24/12/05Term: 39 w.Weight: 3160 g,F/AC heterozygote,Birth: 18/12/06Sample collection: 21/12/06Term: 40 w.Weight: 3035 g,F/AC in a premature newborn,Birth: 17/10/05Sample collection: 20/10/05Term: 30 w.Weight: 2330 g,F/AD heterozygote,Birth: 29/01/06Sample collection: 31/01/06Term: 37 w.Weight: 3280 g,F/E homozygote,Hb E,Hb F,Denatured Hb F,Birth: 10/11/06Sample collection: 13/11/06Term: 40 w.Weight: ?,F/AE heterozygote,Birth: 10/03/06Sample collection: 13/03/06Term: 39 w.Weight: 3420 g,F/AE in a premature newborn,Birth: 21/07/05Sample collection: 24/07/05Term : 35 w.Weight : 1840 g,F/A0-Arab heterozygote,Birth: 14/02/06Sample collection: 17/02/06Term : 40 w.Weight : 3120 g,F/AKorle-Bu heterozygote:,Birth: 31/10/06Sample collection: 03/11/06Term: 40 w.Weight: 3790 g,Barts in a term newborn,Birth: 05/04/06Sample collection: 08/04/06Term: 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,
