CN1149337A - 网织红细胞的检测 - Google Patents
网织红细胞的检测 Download PDFInfo
- Publication number
- CN1149337A CN1149337A CN95193233A CN95193233A CN1149337A CN 1149337 A CN1149337 A CN 1149337A CN 95193233 A CN95193233 A CN 95193233A CN 95193233 A CN95193233 A CN 95193233A CN 1149337 A CN1149337 A CN 1149337A
- Authority
- CN
- China
- Prior art keywords
- granulophilocyte
- sample
- fluorescence
- feature
- coriphosphine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001995 reticulocyte Anatomy 0.000 title abstract description 6
- 238000001514 detection method Methods 0.000 title abstract description 5
- JRMDFAKCPRMZKA-UHFFFAOYSA-N 6-n,6-n,2-trimethylacridin-10-ium-3,6-diamine;chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=NC3=CC([NH+](C)C)=CC=C3C=C21 JRMDFAKCPRMZKA-UHFFFAOYSA-N 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 37
- 210000004369 blood Anatomy 0.000 claims abstract description 29
- 239000008280 blood Substances 0.000 claims abstract description 29
- 210000004027 cell Anatomy 0.000 claims abstract description 29
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Substances [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052786 argon Inorganic materials 0.000 claims abstract description 4
- 238000000684 flow cytometry Methods 0.000 claims abstract description 3
- 210000003743 erythrocyte Anatomy 0.000 claims description 23
- 239000000975 dye Substances 0.000 claims description 21
- 238000004043 dyeing Methods 0.000 claims description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 7
- 230000003287 optical effect Effects 0.000 claims description 7
- 230000005284 excitation Effects 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 230000006641 stabilisation Effects 0.000 claims description 4
- 238000011105 stabilization Methods 0.000 claims description 4
- 238000000149 argon plasma sintering Methods 0.000 claims description 3
- 241001597008 Nomeidae Species 0.000 claims 2
- 239000007853 buffer solution Substances 0.000 claims 2
- 238000000799 fluorescence microscopy Methods 0.000 claims 1
- -1 argon ion Chemical class 0.000 abstract description 3
- 229920002477 rna polymer Polymers 0.000 description 25
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 8
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 8
- 239000003086 colorant Substances 0.000 description 8
- 210000001772 blood platelet Anatomy 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- ACOJCCLIDPZYJC-UHFFFAOYSA-M thiazole orange Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1.C1=CC=C2C(C=C3N(C4=CC=CC=C4S3)C)=CC=[N+](C)C2=C1 ACOJCCLIDPZYJC-UHFFFAOYSA-M 0.000 description 6
- KPHWPUGNDIVLNH-UHFFFAOYSA-M diclofenac sodium Chemical compound [Na+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KPHWPUGNDIVLNH-UHFFFAOYSA-M 0.000 description 5
- NZYCYASKVWSANA-UHFFFAOYSA-M new methylene blue Chemical compound [Cl-].CCNC1=C(C)C=C2N=C(C=C(C(NCC)=C3)C)C3=[S+]C2=C1 NZYCYASKVWSANA-UHFFFAOYSA-M 0.000 description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000002109 erythrocyte inclusion Anatomy 0.000 description 3
- 210000003924 normoblast Anatomy 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 238000007430 reference method Methods 0.000 description 3
- 230000001235 sensitizing effect Effects 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- INCIMLINXXICKS-UHFFFAOYSA-M pyronin Y Chemical compound [Cl-].C1=CC(=[N+](C)C)C=C2OC3=CC(N(C)C)=CC=C3C=C21 INCIMLINXXICKS-UHFFFAOYSA-M 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- JADVWWSKYZXRGX-UHFFFAOYSA-M thioflavine T Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C1=[N+](C)C2=CC=C(C)C=C2S1 JADVWWSKYZXRGX-UHFFFAOYSA-M 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 1
- 125000000853 cresyl group Chemical group C1(=CC=C(C=C1)C)* 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000010437 erythropoiesis Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 150000002390 heteroarenes Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 210000001768 subcellular fraction Anatomy 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
- G01N15/147—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
-
- G01N15/1433—
-
- G01N2015/012—
-
- G01N2015/014—
-
- G01N2015/018—
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
Abstract
一种用来测定血样中网织红细胞的方法,该方法包括用Coriphosphine O对网织红细胞染色,该方法特别适合于用流式细胞计量术进行的检测。在本方法中用到的流式细胞仪的光学系统(10)包括一个氩离子激光器(12),它作为照射流经流动池(14)的血样的光源。散射光和荧光落到一个用来反射直角散射光(24)并透过荧光(26)的分色镜(22)上。分色镜(30)用来反射绿色荧光(32)并透过红色荧光(38)。
Description
本发明涉及对血样中的网织红细胞进行检测与计数。更具体地说,本发明涉及一种染料,这种染料适合于对核糖核酸(RNA)和核糖核酸聚合物染色,并且特别适合于用荧光流式细胞计量技术检测网织红细胞。
网织红细胞是已经失去了核的未成熟红细胞。已知网织红细胞中含有RNA,对血样中网织红细胞进行检测与计数对临床医师是有价值的。血样中网织红细胞的个数被用作急性出血、溶血性盆血和骨髓移植中红细胞生成活性、诊断及预测值的指示,以及对铁、维生素B12和叶酸疗法的反应的一种量度。本领域中众所周知,网织红细胞是成熟红细胞的前体,因此网织红细胞这一术语包含了细胞的演化和发展过程,在此过程中产生了成熟的红细胞。
过去,血样中网织红细胞是通过人工和自动方法采用适当的染色剂如新亚甲蓝(NMB)、亮甲酚蓝(BCB)、吖啶橙、哌洛宁Y以及噻唑橙来测定的。
用新亚甲蓝这种染料进行的活体染色被认为是确定网织红细胞的参考方法,在使用这种染料时沉淀出RNA。这种方法是人工的,需要在显微镜下统计大量的细胞数目(例如500到1,000),而且缓慢、繁琐且有统计误差。新亚甲蓝是无荧光的,而且真正沉淀的RNA经常难于与沉淀的染色剂区分开。
吖啶橙在用人工及自动方法对网织红细胞进行染色时已有所应用。吖啶橙能沉淀RNA;这就由于潜在的猝灭而阻碍了对RNA含量的定量测定。此外,吖啶橙不会导致被染色细胞的扩散荧光分布。细胞的年龄分布(依据与荧光成正比的RNA含量)就是不可靠的。吖啶橙对流式细胞仪中的塑料管有很大的亲和力,这将导致背景增加,并且延长从流式细胞仪管道清除染料的过程。此外,用吖啶橙染色的细胞也难于同自发荧光的红细胞峰分开,所获得的网织红细胞个数通常要低于用新亚甲蓝获得的值。
使用派洛宁Y需要先用福尔马林固定红细胞;这很不方便而且费时,并且结果一般都不好。而且,派洛宁Y的量子效率非常低,导致荧光信号也非常弱。
使用噻唑橙来检测网织红细胞的一个例子可见于1989年11月28日公布的美国专利No.4,883,867,它是在1985年11月1日申请的申请序号为No.793,813的部分继续申请,现已放弃。
使用硫黄素T来检测网织红细胞的例子可见于1986年2月18日公布的美国专利No.4,571,388。
Shapiro,Howard M.,Practical Flow Cytometry,P.144,AlanR.Liss,Inc.1985,在表7-3列出了用于对DNA和/或RNA染色的三环杂芳族化合物。同时表7-3列出了coriphosphine O(CPO),但它并未将CPO作为一种网织红细胞或RNA的染色剂。
本发明提供一种染料和掺有该种染料的试剂,以定量测定全血中的网织红细胞。
本发明提供了一种定量测定网织红细胞的方法,其中的染料为coriphosphine O。
本发明还提供一种检测网织红细胞的方法,包括用coriphosphine O对样品染色;用激发波长的光激发样品;测量由上述样品中发出的荧光。
还提供一种检测网织红细胞的方法,其中样品受到激发,并且荧光是通过流式细胞仪来测量的。
本发明还提供一种对细胞分类染色的方法,使得受血小板、有核红细胞和Howell-Jolly Bodies的干扰较小。
本发明提供一种对细胞染色的方法,该方法产生一种RNA-染料复合物,它比其它已知的方法产生的复合物更为稳定,持续时间较长,使得由RNA-染料复合物产生的全部颜色都能被看清。
图1所示是可用于实现本发明方法的流式细胞仪光学系统的示意图。
图2所示是来自一个正常供体的流式细胞计量直方图的实例;
图3所示是一种来自异常(高)病人的流式细胞计量直方图的实例;
图4所示是RNA和coriphosphine O试剂的剂量/响应曲线;
图5所示是本发明的CPO方法与噻唑橙方法的相互关系;
图6所示是CPO的稳定性;
图7给出了红细胞和血小板的分析情况;
图8给出了红细胞和白细胞的分析情况。
为方便起见,本发明用于对网织红细胞染色的染料被称作coriphosphine O(CPO),也称为碱性黄7号。coriphosphine O可以购自pfaltz & Bauer,Inc.Division of Aceto Corporation,Waterbury,Connecticut.
申请人已发现coriphosphine O是一种用来对网织红细胞染色的有效染料。网织红细胞染色的作用是为了在流式细胞仪中进行光散射计数而进一步显示出网织红细胞的轮廓。这样,通过使用coriphosphine O作为染色剂就能够对全血样品中的网织红细胞进行检测和计数。coriphosphine O是一种荧光染料,它不会沉淀出网织红细胞的胞内核糖核酸。
coriphosphine O的使用提供了对细胞分类染色的优点,减少了受血小板、有核红细胞和Howell-Jolly Bodies的影响。coriphosphine O还提供了增加RNA-染料复合物稳定性的优点,从而增加了持续时间,人们就能观察到由RNA-染料复合物产生的全部颜色。所产生颜色的稳定时间较之其它技术所用染色剂产生颜色的稳定时间要长得多,例如使用噻唑橙产生的颜色稳定时间约为2小时,而使用coriphosphine O产生的颜色的稳定时间约为8到24小时。
根据本发明,当对血样中的网织红细胞染色时,coriphosphine O优选以水溶液形式使用,优选在一种等渗盐溶液中,最优选在ISOTONRII,美国专利No.3,962,125,Coulter公司,Miami,Florida,CPO浓度大约为0.8到80mg/L,优选为1-40,最优选为5-10mg/L,该溶液可含有微量甲醇。血样可以是全血或血组分,通过血样与coriphosphineO溶液混合来用coriphosphine O溶液染色血样。所用血样及溶液的体积是保证RBC的浓度足以通过仪器。这样该浓度范围就在1∶50-1∶5000,优选为1∶100-1∶1000,最优选为1∶200-1∶800。然后样品保温最少约60秒至大约8小时,优选为30分钟,然后通过一个流式细胞仪。申请人已经发现CPO是一种活体染色剂,因此就不需要有固定措施。
当coriphosphine O从核糖核酸(RNA)上释放出来时,发出很少的甚至不发出红色荧光,并且在大约491.5nm处显示一个强的吸收峰.当coriphosphine O与网织红细胞中的RNA结合时,其光学属性改变十分剧烈.具体说,当coriphosphine O与网织红细胞中的RNA结合时,就显示出很强的红色荧光。激发的最大值大约在491.5nm,辐射最大值大约在630-700nm,得到大约160nm的斯托克斯漂移(Stokesshift)。由于结合的coriphosphine O的激发峰在大约490nm数量级,在使用自动流式细胞仪时光源可以是一个汞灯,其能量线在大约485nm,或是一个氩离子激光器,在大约488nm处有强辐射。尽管在其它波长可以形成激发,用coriphosphine O染色的网织红细胞优选为在大约450nm至大约500nm的波长范围内激发。
当coriphosphine O未与白细胞中的脱氧核糖核酸(DNA)结合时产生很少甚至不产生绿色荧光,但coriphosphine O在与白细胞中的DNA结合时就会显示强的绿色荧光。coriphosphine O染料在未与核酸结合时荧光的缺少提供了一个弱背景,并使操作者可以为自动流式细胞仪选择一个荧光阈(或“门”)。
由于CPO在与RNA结合时发出红色荧光而与DNA结合时发出绿色荧光,CPO的使用就提供了对细胞分类染色而受血小板、有核红细胞、白细胞、以及Howell-Jolly Bodies干扰较小的优点。这就使得人们能够放入(gate-in)红细胞而获得更为精确的计数。
此外,当CPO结合时,绿色荧光的数量或强度根据CPO与“其它”结构的结合情况而与背景的数量或非特异性的染色成比例。这些细胞结构或单元包括DNA和亚细胞小泡,诸如溶酶体,核内体以及颗粒体。这些单元各自与CPO有不同的结合,导致不同数量的荧光。但是,只有单链RNA与CPO结合并且只发出红色荧光。我们将成熟红细胞群所发出的绿色荧光的最大数量既作为红细胞的“阈”又作为网织红细胞的“阈”。所有其它细胞将发出超过这一阈值的绿色荧光。绿色荧光的强度不同提供了对细胞分类染色的优点,使人们能放出(gate-out)非特定的细胞如血小板和白细胞。
coriphosphine O染色剂不沉淀RNA,并因此使coriphosphine O所染色的网织红细胞维持一个相对均一的细胞内RNA的分布,因此,在测得的个体网织红细胞的荧光信号与网织红细胞RNA含量之间有近乎线性的关系。这在临床上就给医生提供了除网织红细胞个数以外的附加信息,因为RNA含量是网织红细胞年龄的函数。因此,使用了coriphosphine O后,临床医师就能获得网织细胞的年龄分布,以及简单的网织红细胞计数。
在使用coriphosphine O对血样中网织红细胞染色时,从染色的网织红细胞发出的荧光信号与从成熟红细胞发出的那些信号很好地分开,其结果可以在自动流式细胞仪上直接读出而无需大量的数据计算。
用CPO染色的网织红细胞、RNA或DNA尽管优选是在一个自动流式细胞仪中计数,但也可以用人工方法或自动的显微镜来计数。
流式细胞计量技术的基本概念从本质上说就是一次一个地将细胞通过特定的敏感区。通过流体动力聚焦,单个细胞通过敏感区,该敏感区由一个聚焦的激光源及用来测量散射光和荧光的检测系统组成。
自动流式细胞仪是本领域中熟知的,本发明并不限于使用任何特定的流式细胞仪。
本发明所用的流式细胞仪的光学系统的一个具体实例将在下文中结合附图1进行说明。图1所示的光学系统用于为测量直角散射光、红色荧光及绿色荧光而设计的流式细胞仪。由10指示的光学系统使用一个氩离子激光器12作为光源,在488nm波长操作,产生一个15mW的输出。从激光器12发出的光由一个圆形透镜16会聚,照射在以常规方式流经流动池14的血样上。
当样品中被染色的红细胞被激光照射时,它们产生散射光和荧光。直角散射光和荧光用一个聚焦透镜18聚光,通过孔20落到分色镜22上。分色镜22反射直角散射光24并透过荧光26。由分色镜22反射的直角散射光24在光电倍增管或光电二极管28中检测。在通过分色镜22的荧光26中,绿色荧光32由分色镜30反射,红色荧光38则透过那面镜。反射的绿色荧光32通过一片滤色镜34,在一个光电倍增管36中检测。透过的红色荧光38通过一个滤色镜40并在光电倍增管42中检测。
这样举个例子,用coriphosphine O染色的网织红细胞就可以在Florida州Miami的Coulter公司所售之COULTERRXL流式细胞仪中检测和计数。在使用这些自动流式细胞仪时,利用样品中的成熟红细胞的位置设置荧光门,这样就设置了荧光门来统计网织红细胞的数目。
使用自动流式计数器对用coriphosphine O染色的网织红细胞进行检测和计数所得到的结果与用一种已知的统计网织红细胞数目的标准方法得到的结果有密切关系,标准方法使用的是亚甲蓝或吖啶橙或噻唑橙。
在自动流式细胞仪中使用由coriphosphine O染色的网织红细胞特别有利之处在于,荧光背景弱而荧光门可以很容易地选择。而且没有胞内网织红细胞RNA沉淀,因而无需固定细胞。此外,在个体网织红细胞的荧光信号之间有一种线性关系,提供了网织红细胞年龄的有关信息。
尽管用coriphosphine O染色的网织红细胞优选是在自动流式细胞仪中计数的,但也可以由人工方法或自动的显微镜来计数。
因此,主题方法包括如下步骤:(a)将待测血样与包括主题衍生物染料成分在内的主题试剂组合物混合,形成细胞悬浮液;(b)将上述细胞悬浮液在不低于2℃不高于25℃的温度下保温不少于一分钟但不超过24小时;(c)在流式细胞仪中测量得到的细胞荧光;(d)绘出经光散射进入的红色荧光与绿色荧光的相关数据直方图(LFS对SS);(e)选择网织红细胞群的荧光阈;并且(f)以网织红细胞百分比×RBC总数(以十亿/ml表示的RBC总数)的形式计算网织红细胞总数,其中RBC总数是从象COULTER STKS(Coulter Corportion,Miami,Florida)这样的血液学分析仪得到的。
下面的非限定性实施例说明本发明的各种特征。下面染色的例子用以获得图4-8所描绘的结果。实施例1
样品收集到三磷酸盐EDTA(K3EDTA)中。将0.002mL患者全血样品加入1.0mL的试剂中。混合样品并在室温下保温至少15分钟,但不超过8小时。然后样品就在到一台校准后的XL流式细胞仪上分析之前再次混合。
图2-4表示用CPO对正常与异常血液进行网织红细胞分析的数据。具体地说,图2所示是一个正常人血液的荧光直方图,显示了用525nm光电倍增管和用630nm光电倍增管所检测到的红细胞分布情况。如图2所示,区域E描绘出与白细胞和血小板分开的网织红细胞。
图3所示是一个异常血液的荧光直方图,显示了用525nm光电倍增管和用630nm光电倍增管检测到的红细胞分布情况。由图3可见,在网织红细胞区(区域E)中数目增加了。如图3所示,CPO特异性地与RNA反应,而样品中RNA数量的增加导致荧光的增强。
图4所示是一张试剂在与数量增加的核糖核酸(RNA)混合时的剂量响应图。RNA加入得越多,荧光强度就越大。
图5所示是本发明的CPO方法与噻唑橙方法(参考方法)的相互关系。如图5所示,CPO方法的结果与参考方法的结果是一样的。这样CPO就是衡量网织红细胞的一种尺度。
图6以时间对网织红细胞百分比的函数绘出了CPO的稳定性。图6显示,在将血液与试剂混合后,大约经过15分钟才建立起平衡。一旦平衡建立,CPO-RNA复合物维持至少8小时的稳定状态。而噻唑橙、吖啶橙和硫黄素T的稳定时间低于2小时。
图7给出了对红细胞和血小板的分析。使用了血小板数目增加的样品。对红细胞和血小板的分析显示,血小板分布与网织红细胞分布是不同的。
图8给出了对红细胞和白细胞的分析。使用了白细胞数目增加的样品。对红细胞和白细胞的分析显示,白细胞分布与红细胞和网织红细胞分布是不同的。
根据上面的教导可以对本发明做各种改动和变化,因此本发明也可以有除上述具体描述外的其它实施方式。
Claims (13)
1.一种检测样品中网织红细胞的方法,其特征在于网织红细胞是通过用coriphosphine O染色;用激发波长的光激发上述样品;以及测量由上述样品发出的荧光来检测的。
2.一种根据权利要求1的方法,其特征还在于样品受到激发,而荧光是通过一台流式细胞仪来测量的。
3.一种根据权利要求1的方法,其特征还在于样品受到激发,而荧光是通过荧光显微术来测量的。
4.一种根据权利要求1的方法,其特征还在于样品是在流式细胞仪中用汞弧灯发出的光激发的。
5.一种根据权利要求1的方法,其特征还在于样品是在流式细胞仪中用氩激光器发出的光激发的。
6.一种根据权利要求1的方法,其特征还在于网织红细胞处于含有全血的样品中。
7.一种根据权利要求1的方法,其特征还在于检测网织红细胞无需对其进行固定。
8.一种根据权利要求1的方法,其特征还在于细胞是分类染色的。
9.一种用于对定量用全血样品中的网织红细胞进行染色的试剂,该试剂的特征在于它包括一种coriphosphine O的水溶液和一种缓冲体系。
10.权利要求9的试剂,其特征还在于该缓冲体系的pH值为6.0-8.0。
11.一种产生稳定的RNA-染料复合物的方法,其特征是将待测血样与包含coriphosphine O水溶液的试剂混合,并使该悬浮液反应足够长的时间以便衍生物能被网织红细胞有效地吸收。
12.一种根据权利要求11的方法,其特征还在于RNA-染料复合物稳定时间约为8到24小时。
13.一种用流式细胞计量术定量全血样品中网织红细胞的方法,其特征在于下列步骤:
(a)将待测血样与含有coriphosphine O水溶液的试剂混合;
(b)使该悬浮液反应足够长的时间,以便衍生物被网织红细胞有效地吸收;
(c)将悬浮液通过一个流式细胞仪;
(d)测量经光散射进入的红细胞群的红色荧光对绿色荧光的强度;以及
(e)根据上述测量确定样品中网织红细胞的量或百分比。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US24737994A | 1994-05-23 | 1994-05-23 | |
US08/247,379 | 1994-05-23 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1149337A true CN1149337A (zh) | 1997-05-07 |
Family
ID=22934708
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN95193233A Pending CN1149337A (zh) | 1994-05-23 | 1995-05-23 | 网织红细胞的检测 |
Country Status (14)
Country | Link |
---|---|
US (1) | US5639666A (zh) |
EP (1) | EP0763201B1 (zh) |
JP (1) | JPH10500776A (zh) |
CN (1) | CN1149337A (zh) |
AT (1) | ATE223044T1 (zh) |
AU (1) | AU2601795A (zh) |
BR (1) | BR9507690A (zh) |
CA (1) | CA2191096A1 (zh) |
DE (1) | DE69527959D1 (zh) |
IL (1) | IL113805A0 (zh) |
NO (1) | NO964982D0 (zh) |
TW (1) | TW296431B (zh) |
WO (1) | WO1995032424A1 (zh) |
ZA (1) | ZA954198B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103827658A (zh) * | 2011-07-22 | 2014-05-28 | 罗氏血液诊断股份有限公司 | 识别和测量网状细胞 |
CN103940793A (zh) * | 2014-03-28 | 2014-07-23 | 北京理工大学 | 荧光检测系统及检测方法 |
WO2019206297A1 (zh) * | 2018-04-28 | 2019-10-31 | 深圳迈瑞生物医疗电子股份有限公司 | 一种血液分析仪及分析方法 |
Families Citing this family (59)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6271035B1 (en) | 1998-10-20 | 2001-08-07 | Coulter International Corp. | Methods and compositions for rapid staining of nucleic acids in whole cells |
US6060322A (en) * | 1998-10-20 | 2000-05-09 | Coulter International Corp. | Method for identification of reticulated cells |
US6197593B1 (en) | 1998-10-20 | 2001-03-06 | Coulter International Corp. | Method for enumerating blood cells |
US6228652B1 (en) * | 1999-02-16 | 2001-05-08 | Coulter International Corp. | Method and apparatus for analyzing cells in a whole blood sample |
DE10024135B4 (de) | 2000-01-28 | 2004-07-08 | Leica Microsystems Heidelberg Gmbh | Mikroskop |
US6368864B1 (en) | 2000-05-05 | 2002-04-09 | Coulter International Corp. | Dyes and methods of reticulocyte enumeration |
US7195919B2 (en) * | 2003-12-19 | 2007-03-27 | Beckman Coulter, Inc. | Hematology controls for reticulocytes and nucleated red blood cells |
CN101349644B (zh) * | 2007-07-20 | 2012-06-27 | 深圳迈瑞生物医疗电子股份有限公司 | 一种白细胞分类试剂和其使用方法 |
EP2522982B1 (en) * | 2007-08-15 | 2015-09-16 | Malvern Instruments Limited | Broad-Range Spectrometer |
US8102161B2 (en) * | 2007-09-25 | 2012-01-24 | Tdk Corporation | Stable output in a switching power supply by smoothing the output of the secondary coil |
CN105440725A (zh) * | 2008-01-04 | 2016-03-30 | 深圳迈瑞生物医疗电子股份有限公司 | 不对称菁类荧光染料,组合物及在生物样品染色中的用途 |
CN101602762B (zh) * | 2008-06-10 | 2013-10-16 | 深圳迈瑞生物医疗电子股份有限公司 | 不对称菁类化合物、其制备方法及应用 |
CN101726579B (zh) * | 2008-10-17 | 2014-06-18 | 深圳迈瑞生物医疗电子股份有限公司 | 血液检测试剂和方法 |
CN101750274B (zh) * | 2008-12-17 | 2014-06-25 | 深圳迈瑞生物医疗电子股份有限公司 | 白细胞分类计数试剂、试剂盒以及白细胞分类计数的方法 |
US9104695B1 (en) | 2009-07-27 | 2015-08-11 | Palantir Technologies, Inc. | Geotagging structured data |
CN101988082B (zh) * | 2009-07-31 | 2015-04-08 | 深圳迈瑞生物医疗电子股份有限公司 | 白细胞分类计数试剂、试剂盒及其制备方法和白细胞分类计数的方法 |
US8085268B2 (en) | 2009-10-20 | 2011-12-27 | Palantir Technologies, Inc. | Techniques for drawing geodetic polygons |
US8564596B2 (en) | 2010-01-12 | 2013-10-22 | Palantir Technologies, Inc. | Techniques for density mapping |
US9501507B1 (en) | 2012-12-27 | 2016-11-22 | Palantir Technologies Inc. | Geo-temporal indexing and searching |
US9123086B1 (en) | 2013-01-31 | 2015-09-01 | Palantir Technologies, Inc. | Automatically generating event objects from images |
US10037314B2 (en) | 2013-03-14 | 2018-07-31 | Palantir Technologies, Inc. | Mobile reports |
US8855999B1 (en) | 2013-03-15 | 2014-10-07 | Palantir Technologies Inc. | Method and system for generating a parser and parsing complex data |
US8903717B2 (en) | 2013-03-15 | 2014-12-02 | Palantir Technologies Inc. | Method and system for generating a parser and parsing complex data |
US8930897B2 (en) | 2013-03-15 | 2015-01-06 | Palantir Technologies Inc. | Data integration tool |
US8799799B1 (en) | 2013-05-07 | 2014-08-05 | Palantir Technologies Inc. | Interactive geospatial map |
US8938686B1 (en) | 2013-10-03 | 2015-01-20 | Palantir Technologies Inc. | Systems and methods for analyzing performance of an entity |
US8924872B1 (en) | 2013-10-18 | 2014-12-30 | Palantir Technologies Inc. | Overview user interface of emergency call data of a law enforcement agency |
US9021384B1 (en) | 2013-11-04 | 2015-04-28 | Palantir Technologies Inc. | Interactive vehicle information map |
US8868537B1 (en) | 2013-11-11 | 2014-10-21 | Palantir Technologies, Inc. | Simple web search |
US9727376B1 (en) | 2014-03-04 | 2017-08-08 | Palantir Technologies, Inc. | Mobile tasks |
CN103852409B (zh) * | 2014-03-18 | 2016-09-14 | 江西科技师范大学 | 用于流式细胞仪中血细胞的成像系统 |
US9129219B1 (en) | 2014-06-30 | 2015-09-08 | Palantir Technologies, Inc. | Crime risk forecasting |
US10372879B2 (en) | 2014-12-31 | 2019-08-06 | Palantir Technologies Inc. | Medical claims lead summary report generation |
EP3611632A1 (en) | 2015-03-16 | 2020-02-19 | Palantir Technologies Inc. | Displaying attribute and event data along paths |
US9460175B1 (en) | 2015-06-03 | 2016-10-04 | Palantir Technologies Inc. | Server implemented geographic information system with graphical interface |
US9600146B2 (en) | 2015-08-17 | 2017-03-21 | Palantir Technologies Inc. | Interactive geospatial map |
US10706434B1 (en) | 2015-09-01 | 2020-07-07 | Palantir Technologies Inc. | Methods and systems for determining location information |
US9639580B1 (en) | 2015-09-04 | 2017-05-02 | Palantir Technologies, Inc. | Computer-implemented systems and methods for data management and visualization |
US10109094B2 (en) | 2015-12-21 | 2018-10-23 | Palantir Technologies Inc. | Interface to index and display geospatial data |
US10068199B1 (en) | 2016-05-13 | 2018-09-04 | Palantir Technologies Inc. | System to catalogue tracking data |
US9686357B1 (en) | 2016-08-02 | 2017-06-20 | Palantir Technologies Inc. | Mapping content delivery |
US10437840B1 (en) | 2016-08-19 | 2019-10-08 | Palantir Technologies Inc. | Focused probabilistic entity resolution from multiple data sources |
US10515433B1 (en) | 2016-12-13 | 2019-12-24 | Palantir Technologies Inc. | Zoom-adaptive data granularity to achieve a flexible high-performance interface for a geospatial mapping system |
US10270727B2 (en) | 2016-12-20 | 2019-04-23 | Palantir Technologies, Inc. | Short message communication within a mobile graphical map |
US10460602B1 (en) | 2016-12-28 | 2019-10-29 | Palantir Technologies Inc. | Interactive vehicle information mapping system |
US10579239B1 (en) | 2017-03-23 | 2020-03-03 | Palantir Technologies Inc. | Systems and methods for production and display of dynamically linked slide presentations |
CN108663502B (zh) * | 2017-03-28 | 2023-09-08 | 常熟常江生物技术有限公司 | 全自动血栓弹力图仪 |
US10895946B2 (en) | 2017-05-30 | 2021-01-19 | Palantir Technologies Inc. | Systems and methods for using tiled data |
US11334216B2 (en) | 2017-05-30 | 2022-05-17 | Palantir Technologies Inc. | Systems and methods for visually presenting geospatial information |
US10403011B1 (en) | 2017-07-18 | 2019-09-03 | Palantir Technologies Inc. | Passing system with an interactive user interface |
US10371537B1 (en) | 2017-11-29 | 2019-08-06 | Palantir Technologies Inc. | Systems and methods for flexible route planning |
US11599706B1 (en) | 2017-12-06 | 2023-03-07 | Palantir Technologies Inc. | Systems and methods for providing a view of geospatial information |
US10698756B1 (en) | 2017-12-15 | 2020-06-30 | Palantir Technologies Inc. | Linking related events for various devices and services in computer log files on a centralized server |
US10896234B2 (en) | 2018-03-29 | 2021-01-19 | Palantir Technologies Inc. | Interactive geographical map |
US10830599B2 (en) | 2018-04-03 | 2020-11-10 | Palantir Technologies Inc. | Systems and methods for alternative projections of geographical information |
US11585672B1 (en) | 2018-04-11 | 2023-02-21 | Palantir Technologies Inc. | Three-dimensional representations of routes |
US10429197B1 (en) | 2018-05-29 | 2019-10-01 | Palantir Technologies Inc. | Terrain analysis for automatic route determination |
US10467435B1 (en) | 2018-10-24 | 2019-11-05 | Palantir Technologies Inc. | Approaches for managing restrictions for middleware applications |
US11025672B2 (en) | 2018-10-25 | 2021-06-01 | Palantir Technologies Inc. | Approaches for securing middleware data access |
Family Cites Families (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3887812A (en) * | 1973-07-02 | 1975-06-03 | Block Engineering | Method and apparatus for detecting and classifying nucleic acid particles |
US3872312A (en) * | 1973-07-02 | 1975-03-18 | Block Engineering | Method and apparatus for detecting and classifying nucleic acid particles |
US3975084A (en) * | 1973-09-27 | 1976-08-17 | Block Engineering, Inc. | Particle detecting system |
US3899297A (en) * | 1973-12-19 | 1975-08-12 | Block Engineering | Biological staining technique and mixture thereof |
US3962125A (en) * | 1975-01-13 | 1976-06-08 | Coulter Diagnostics, Inc. | Multi-purpose diluent for use in blood analysis by electronic instrumentation of the coulter type |
US4447547A (en) * | 1980-04-09 | 1984-05-08 | University Patents, Inc. | Immunoassay for measurement of reticulocytes, and immunoreactive reagents for use therein |
US4332785A (en) * | 1980-04-09 | 1982-06-01 | University Patents, Inc. | Immunoassay for measurement of reticulocytes, and immunoreactive reagents for use therein |
US4325706A (en) * | 1980-08-15 | 1982-04-20 | Ortho Diagnostic Systems Inc. | Automated detection of platelets and reticulocytes in whole blood |
US4336029A (en) * | 1980-08-15 | 1982-06-22 | Ortho Diagnostic Systems Inc. | Method and reagents for quantitative determination of reticulocytes and platelets in whole blood |
US4571388A (en) * | 1983-01-24 | 1986-02-18 | Becton Dickinson And Company | Detection of reticulocytes |
US4707451A (en) * | 1985-09-18 | 1987-11-17 | Becton, Dickinson And Company | Detection of reticulocytes |
US4957870A (en) * | 1985-11-01 | 1990-09-18 | Becton, Dickinson And Company | Detection of Reticulocytes, RNA and DNA |
EP0226272B1 (en) * | 1985-11-01 | 1991-03-06 | Becton, Dickinson and Company | Detection of reticulocytes |
US4883867A (en) * | 1985-11-01 | 1989-11-28 | Becton, Dickinson And Company | Detection of reticulocytes, RNA or DNA |
JPH06100596B2 (ja) * | 1986-09-10 | 1994-12-12 | 東亜医用電子株式会社 | フロ−サイトメトリ−による白血球の分類方法 |
US4822745A (en) * | 1986-12-05 | 1989-04-18 | Albert Einstein College Of Medicine Of Yeshiva University | Method for quantifying human reticulocytes |
JPH0746102B2 (ja) * | 1987-07-31 | 1995-05-17 | 東亜医用電子株式会社 | フロ−サイトメトリ用網状赤血球測定試薬 |
US5360739A (en) * | 1991-12-05 | 1994-11-01 | Miles Inc. | Methods for the identification and characterization of reticulocytes in whole blood |
-
1995
- 1995-05-22 IL IL11380595A patent/IL113805A0/xx unknown
- 1995-05-23 DE DE69527959T patent/DE69527959D1/de not_active Expired - Lifetime
- 1995-05-23 CA CA002191096A patent/CA2191096A1/en not_active Abandoned
- 1995-05-23 EP EP95920628A patent/EP0763201B1/en not_active Expired - Lifetime
- 1995-05-23 BR BR9507690A patent/BR9507690A/pt not_active Application Discontinuation
- 1995-05-23 ZA ZA954198A patent/ZA954198B/xx unknown
- 1995-05-23 CN CN95193233A patent/CN1149337A/zh active Pending
- 1995-05-23 AT AT95920628T patent/ATE223044T1/de not_active IP Right Cessation
- 1995-05-23 AU AU26017/95A patent/AU2601795A/en not_active Abandoned
- 1995-05-23 JP JP7530512A patent/JPH10500776A/ja active Pending
- 1995-05-23 WO PCT/US1995/006537 patent/WO1995032424A1/en active IP Right Grant
- 1995-05-26 TW TW084105347A patent/TW296431B/zh active
- 1995-06-27 US US08/495,771 patent/US5639666A/en not_active Expired - Fee Related
-
1996
- 1996-11-22 NO NO964982A patent/NO964982D0/no unknown
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103827658A (zh) * | 2011-07-22 | 2014-05-28 | 罗氏血液诊断股份有限公司 | 识别和测量网状细胞 |
CN103827658B (zh) * | 2011-07-22 | 2016-04-13 | 罗氏血液诊断股份有限公司 | 识别和测量网状细胞 |
CN103940793A (zh) * | 2014-03-28 | 2014-07-23 | 北京理工大学 | 荧光检测系统及检测方法 |
CN103940793B (zh) * | 2014-03-28 | 2016-05-04 | 北京理工大学 | 荧光检测系统及检测方法 |
WO2019206297A1 (zh) * | 2018-04-28 | 2019-10-31 | 深圳迈瑞生物医疗电子股份有限公司 | 一种血液分析仪及分析方法 |
Also Published As
Publication number | Publication date |
---|---|
EP0763201A4 (en) | 1997-12-17 |
ZA954198B (en) | 1996-11-25 |
MX9605787A (es) | 1998-05-31 |
JPH10500776A (ja) | 1998-01-20 |
AU2601795A (en) | 1995-12-18 |
EP0763201B1 (en) | 2002-08-28 |
BR9507690A (pt) | 1997-10-07 |
EP0763201A1 (en) | 1997-03-19 |
NO964982L (no) | 1996-11-22 |
ATE223044T1 (de) | 2002-09-15 |
IL113805A0 (en) | 1995-08-31 |
NO964982D0 (no) | 1996-11-22 |
TW296431B (zh) | 1997-01-21 |
DE69527959D1 (de) | 2002-10-02 |
US5639666A (en) | 1997-06-17 |
WO1995032424A1 (en) | 1995-11-30 |
CA2191096A1 (en) | 1995-11-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1149337A (zh) | 网织红细胞的检测 | |
US4957870A (en) | Detection of Reticulocytes, RNA and DNA | |
US4883867A (en) | Detection of reticulocytes, RNA or DNA | |
US5296378A (en) | Method for classifying leukocytes by flow cytometry | |
US4882284A (en) | Method for quantitating and differentiating white blood cells | |
KR100258394B1 (ko) | 전혈 속의 망상 적혈구를 동정하고 특성화하는 방법 및 이에 사용하는 시약 조성물 | |
CN1323395A (zh) | 鉴别网状细胞的试剂组合物和方法 | |
US5693484A (en) | Method of classifying and counting cells in urine | |
Lee et al. | Thiazole orange: a new dye for reticulocyte analysis | |
US5559037A (en) | Method for rapid and simultaneous analysis of nucleated red blood cells | |
EP0099266B1 (en) | Limited volume method and apparatus for particle counting | |
US5434081A (en) | Method of classifying leukocytes by flow cytometry | |
US20040018629A1 (en) | Analyzers and methods of analyzing blood | |
US4985174A (en) | Reticulocyte quantitating reagent for flow cytometry | |
EP0634640A1 (en) | Method of counting reticulocytes | |
EP0114462B1 (en) | Detection of reticulocytes | |
EP0226272B1 (en) | Detection of reticulocytes | |
US4707451A (en) | Detection of reticulocytes | |
CA1309327C (en) | Reagent and method for classifying leukocytes by flow cytometry | |
JPH0464588B2 (zh) | ||
MXPA96005787A (es) | Deteccion de reticulocitos |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C01 | Deemed withdrawal of patent application (patent law 1993) | ||
WD01 | Invention patent application deemed withdrawn after publication |