一、实验目的

1.  了解ABO和Rh血型分类系统的背景知识和遗传学原理

2.  掌握微量采血及血涂片的制作方法

3. 学习使用光学显微镜观察和区分红细胞与各种白细胞

二、实验原理

What is Blood

Blood is made of four components: plasma, red blood cells, white blood cells, platelets.

 

http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347852.gifPlasma is a mixture of water, sugar, fat, protein, and potassium and calcium salts. It also contains many chemicals that help form blood the clots necessary to stop bleeding. More than 92% of plasma is water.

 

http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347598.jpgRed blood cells contain a special protein called hemoglobin, which carries the oxygen we inhale with our lungs to all of the parts of our bodies. It then returns carbon dioxide from our body to our lungs so we can exhale it. Hemoglobin is also responsible for making red blood cells red. We have so many red blood cells that our blood itself appears red, even though it contains more than red blood cells.

 

http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347275.jpgWhite blood cells are clear round cells that are bigger than red blood cells. White blood cells produce proteins called antibodies that help our bodies fight infections caused by bacteria, viruses, and foreign proteins.

 

http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347671.jpgPlatelets aren't really cells at all; they are just fragments of cells. When we are injured, platelets gather at the site of the injury and stick to the edges of the wound. They release chemicals that help start the process of blood clotting so that bleeding will stop.

 

What is blood types and Rh statues

Everybody has a blood type. The most common blood type classification system is the ABO (say "A-B-O") system discovered by Karl Landsteiner in the early 1900s. There are four types of blood in the ABO system: A, B, AB, and O. Your blood type is established before you are born, by specific genes inherited from your parents. You receive one gene from your mother and one from your father; these two combine to establish your blood type. These two genes determine your blood type by causing proteins called agglutinogens (a-GLOO-tin-a-gins) to exist on the surface of all of your red blood cells.

There are three alleles or versions of the blood type gene: A, B, and O. Since everybody has two copies of these genes, there are six possible combinations; AA, BB, OO, AB, AO, and BO. In genetic terms, these combinations are called genotypes, and they describe the genes you got from your parents.

In addition to the proteins (agglutinogens) existing on your red blood cells, other genes make proteins called agglutinins (a-GLOO-tin-ins) that circulate in your blood plasma. Agglutinins are responsible for ensuring that only the blood cells of your blood type exist in your body.

Your genotype determines your blood type and statue

The agglutinogen produced by the O allele has no special enzymatic activities. However, the agglutinogens produced by the A and B alleles do have enzymatic activities, which are different from each other. Therefore people whose genotype is OO are said to have type O blood, meaning the agglutinogen on their red blood cells doesn't have any enzymatic activity. People with Type O blood have agglutinins a and b in their blood plasma. Agglutinin a helps the body destroy any type A blood cells that might enter the circulation system. Agglutinin b helps the body destroy any type B blood cells that might enter the circulation system.

The ABO Blood system

 

Blood type (genotype)	Type A (AA, Ai)	Type B (BB, Bi)	Type AB (AB)	Type O (ii)
Red Blood Cell Surface Antigens (Phenotype)	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347789.gif	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347469.gif	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347250.gif	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347120.gif
Plasma Antibodies (Phenotype)	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347509.gif	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115347457.gif	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115348463.gif	http://www.bbioo.com/bio101/UploadFiles/200511/20051126115348429.gif

 

People who have an AA genotype are said to have type A blood because the agglutinogen on their red blood cells has the enzyme activity associated with the A allele. It is important to recognize that people with the AO genotype also have the enzyme activity associated with the A allele, so they are also said to have type A blood. (Remember the O allele doesn't have any enzyme activity associated with it!) People with Type A blood have agglutinin b in their blood plasma. Agglutinin b helps the body destroy any type B blood cells that might enter the circulation system.

Likewise, people with the BB and the BO genotypes are said to have type B blood. These people have agglutinin a in their blood plasma. Agglutinin a helps the body destroy any type A blood cells that might enter the circulation system.

People who have the AB genotype have the enzyme activity associated with both the A and B alleles. These people have no agglutinins in their blood plasma.

The concepts of genotype and phenotype can be easily understood in the case of blood type. Genotype refers the actual genes an individual possesses that determine a particular trait. Phenotype refers to the characteristics of that trait an individual displays. In the case of blood type, both the AA and AO genotypes cause individuals to display the A blood type phenotype. Similarly, both the BB and BO genotypes cause individuals to display the B blood type phenotype. Individuals who are phenotypically type O or type AB have only one possible genotype, OO and AB, respectively.

In different parts of the world, the fraction of individuals with blood type A, B, O, or AB differs. The frequency with which blood types are observed is determined by the frequency with which the three alleles of the ABO gene are found in different parts of the world (allele frequency). Variation in the allele frequency at the ABO gene reflects the social tendency of populations to marry and reproduce within a national, regional, or ethnic group. As people throughout the world intermingle to a greater extent, the distribution of the different blood types will become more uniform throughout the world.

Blood Transfusions

Because there are only four types of blood, it is possible to take blood from one person and donate it to another person in a process called transfusion. In order for a transfusion to work it is essential that the agglutinogens on the surface of the donor's blood cells match the agglutinogens on the surface of the recipient's blood cells. In other words, the blood type of the donor and the blood type of the person receiving the transfusion must be compatible. If the blood types don't match, special antibodies in the recipient's blood, called agglutinins, will attack the donated blood causing blood clots to form in a reaction called agglutination.

If you ever need a blood transfusion, someone will take a sample of your blood in order to determine your blood type and the genotypes of matching blood types. Someone with type A blood can receive blood from people with the AA, AO, and OO genotypes. People with type B blood can receive blood from people with the BB, BO, and OO genotypes.

There are two special genotypes when it comes to blood transfusions: OO and AB.

The first special genotype is OO. People with O blood are said to be universal donors because they can donate blood to everybody. However, people with type O blood can only receive transfusions from other type O donors. Because O blood does not carry either the A or B agglutinogens, the immune system of a person with O blood views these agglutinogens as foreign. People with type O blood have agglutinins in their plasma that will react against the A and B agglutinogens. Therefore, type O blood will undergo agglutination if exposed to A, B, or AB blood.

The second special genotype is AB. People with AB blood are said to be universal recipients because they can receive blood from people with all four blood types. Since in AB blood both the A and B agglutinogens are presented on the surface of the red blood cells, the immune system of a person with AB blood views both of these molecules as part of itself -- not as something foreign. AB blood does not produce agglutinins against either the A or B agglutinogens, and therefore does not undergo agglutination when exposed to A, B, AB, or O blood.

血涂片的显微镜检查是血液细胞学检查的基本方法,应用极广.特别是对各种血液病的诊断有很大价值.但血片制备和染色不良 ,常使细胞鉴别发生困难,甚至导致错误结论.例如,血膜过厚细胞重叠缩小, 血膜太薄白细胞多集中于边缘;染色良好的血片是血液学检查主要基本技术之一.

瑞氏染色法原理

1. 瑞氏染料是由酸性染料伊红和碱性染料美蓝组成的复合染料。

美蓝(又名亚甲蓝mehylene blue)为四甲基硫堇染料,有对醌型和邻醌型两种结构,通常为氯盐,即氯化美蓝，美蓝容易氧化为一,二,三甲基硫堇等次级染料(即天青)市售美蓝中部份已被氧化为天青. 伊红(又名曙红cosin)通常为钠盐即伊红化钠。美蓝和伊红水溶液混合后产生一种憎液性胶体伊红化美蓝中性沉淀, 即瑞氏染料。瑞氏染料溶于甲醇后, 又重新解离为带正电的美蓝和带负电的伊红离子。

2.  细胞的染色即有物理的吸附作用又有化学的亲和作用. 各种细胞和细胞的各种成份化学性质不同对各种染色料的亲和力也不一样. 因此用染色液染色后在同一血片上可以看到各种不同的色彩, 例如血红蛋白嗜酸性颗粒为碱性蛋白质与酸性染料伊红结合染粉红色称为酸性物质, 细胞核蛋白和淋巴细胞浆为酸性,与碱性染料美蓝或天青结合,染蓝色或紫色称为嗜碱性物质, 中性颗粒成等电状态与伊红和美蓝均可结合,染紫红色称中性物质

三、实验材料

1. 实验材料：医用采血针、酒精棉球、载玻片、血推片、消毒牙签

2.  实验试剂：瑞氏染液、抗B血清、抗A血清、生理盐水、蒸馏水

3.  仪器设备：Motic光学显微镜

四、实验步骤  

4.1  ABO血型鉴定

4.1.1  取一块清洁玻片，用记号笔标上记号。

4.1.2  用小滴管吸A型标准血清（抗B）一滴加入左侧，用另一小滴管吸B型标准血清（抗A）一滴加入右侧

4.1.3   以穿刺法自左手无名指指尖取血，在玻片的每侧各放入一小滴血，用牙签搅拌，使每侧抗血清和血液混和。每边用一支牙签，切勿混用。

4.1.4  静置室温下10—15min后，观察有无凝集现象，并据此判断血型。

4.2  血涂片的制作及血细胞观察

4.2.1  取末捎血一滴置于玻片的一端,左手持载玻片,右手以边缘平滑的推片的一端从血滴前方后移接触血滴，血滴即沿推片散开。然后便推片与载片夹角保持30-45度平稳地向前移动,载片上保留下一薄层血膜

4.2.2  血涂片制成后可手持玻片在空气中挥动,使血膜迅速干燥,以免血细胞皱缩.4.2.3  用蜡笔在血膜两侧划线,以防染液溢出,然后将血膜平放在染色架上. 加瑞氏染液2-3滴,使覆盖整个血膜,固定0.5-1.0分钟. 滴加等量或稍多的新鲜蒸馏水,与染料混匀染色5-10分钟.

4.2.4  用清水冲去染液,待自然干燥后或用吸水纸吸干,即可置血涂片于显微镜下进行镜检。

4.2.5  白细胞分类计数:选择涂片的体尾交界处染色良好的区域,在油镜下计数100个红细胞, 按其形态特征进行分类计数,求出各类细胞所占比值。

注：

1. 瑞氏染液:  瑞氏染料1g加甲醇(AR)600ML。将瑞氏染料放在清洁干燥乳钵中,加少量甲醇,充分研磨使染料溶解.将已完全溶解的部分倒入棕色试剂瓶中, 未溶解部分再加少量甲醇研磨,直至染料完全溶解于甲醇为止.

新配制的染料偏碱,须在室温和37℃下一定时间待染料成熟,主要美蓝逐渐增多为天青B后才能使用.贮存时间越久染色效果越好,因此,Deamgilliland 等采用吸光度比值(rA)作为瑞氏染料的质量规格.rA测定方法如下 : 取瑞氏染液15-25ul(视染液浓度而定)加甲醇10ml稀释,混匀后以甲醇为空白管分别以波长650nm,525nm比色,rA=A650/A525

因为美蓝吸收峰为波长650nm,伊红吸收峰波长为525nm,天青B 吸收峰也为650nm,但吸光度A约为美蓝的一半.所以新配制染料的RA接近2,随着美蓝逐渐氧化为天青B,RA也相应下降,RA下降达1.3±0.1即可使用.瑞氏染料在贮存过程中必须塞严,以防甲醇挥发和氧化为甲酸.有人主张配方中加入30ml甘油, 防止甲酸挥发,并可使细胞染色清晰.甲醇必须纯净,如甲醇中丙酮含量过多,染色偏酸,使细胞着色不良.

2.磷酸盐缓冲液(PH6.4-6.8)：磷酸二氢钾0.3g加磷酸氢二钠0.2g 再加蒸馏水至1000ml,配制成磷酸盐缓冲液调整PH,如无缓冲液可用新鲜蒸馏水代替.

3.白细胞分类计数正常参考值
细胞类别 成人
杆状核 0.01-0.05
分叶核 0.50-0.70
嗜酸性粒细胞 0.005-0.05
嗜碱性粒细胞 0-0.01
淋巴细胞 0.20-0.40
单核细胞 0.03-0.08
注意:
1 要避免重复计数,玻片应由血膜边缘向中央依次上下呈曲线移动.
2 白细胞总数超过20×10⁹/L,应分类计数200个细胞,白细胞数明显减少的血片,可检查多张血片。

五        注意事项

1)玻片的清洗：新玻片常有游离碱质,因此应用清洗液或10%盐酸浸泡24 小时,然后再彻底清洗。 用过的玻片可放入适量肥皂水或合成洗涤剂的清水中煮沸20分钟,再用热水将肥皂和血膜洗去,用自来水反复冲洗,必要时再置95%乙醇中浸泡1小时,然后擦干或烤干备用。使用玻片时只能手持玻片边缘,切勿触及玻片表面;,以保持玻片清洁,干燥,中性、无油腻。

2)细胞染色对氢离子浓度十分敏感,在染色过程中玻片必须化学清洁,配制瑞氏染液必须用优质甲醇,稀释染液必须用缓冲液,冲洗用水应近中性, 否则各种细胞染色反应异常,致使细胞的识别困难,甚至造成错误。

3)一张良好的血片,要求厚薄适宜,头体尾分明,分布均匀,边缘整齐, 两侧留有空隙。血片制好后最好立即固定染色,以免细胞溶解和发生退行性变。

4)血膜未干透,细胞尚未牢固附在玻片上,在染色过程中容易脱落, 因此血膜必需充分干燥.

5)染色时间与染液浓度,室温高低,细胞多少有关.染液越淡,室温越低, 细胞越多,所需染色时间越长或应适当增加染液量,因此染色时间应视具体情况而定.特别是更换新染料时必须经试染,摸索最佳染色条件.

6)染液不可过少,以防蒸发干燥染料沉着于血片上难冲洗干净.

7)冲洗时应用流水将染液冲去.不能先倒掉染液,以免染料沉着于血片上.

8)染色过深可用甲醇或酒精适当脱色,最好不复染,必须复染时, 可将染液先稀释好再复染.

9)染色时应注意保护血膜尾部细胞,不能划掉.因为体积较大细胞常在此处出现.
染色结果:在正常情况下血膜外观为粉红色,在显微镜下红细胞呈肉红色；白细胞胞浆能显示各种细胞的特有色彩,细胞核染紫红色,染色质清楚, 粗细松紧可辨。染色结果偏酸,则红细胞和嗜酸性粒细胞颗粒偏红,白细胞核呈浅蓝色或不着色；染色结果偏碱,则所有细胞染成灰蓝色,颗粒深暗，嗜酸性颗粒可染成暗褐色,甚至紫黑色或蓝色。嗜中性颗粒偏粗,偏碱(紫黑色).血片原处呈绿色.

 

苏木精（hematoxylin）氧化成苏木素（hematein）后才能用于染色------成熟