甲基百里香酚兰法直接测定红细胞内镁的实验研究

本文介绍用甲基百里香酚兰法直接测定红细胞内镁的实验方法，并对有关实验条件进行了探讨。该法显示镁浓度在２～１６ｍｍｏｌ／Ｌ内线性良好，ｒ＝０．９９９６。平均回收率为１００．７％；批内ＣＶ＝１．０４％，批间ＣＶ＝５．０３％。作者测定了２０例健康成人红细胞内鲜深度度为１．２９０～２．０５０ｆｍｏｌ／ｃｅｌｌ（＾－Ｘ±２ＳＤ）。     

用8—羟基喹啉作掩蔽剂2—（8‘—羟基喹啉—5’—磺酸—7‘—偶氮）—变色 …

目的：建立２－（８‘－羟基喹啉－５’－磺酸－７’－偶氮）－变色酸（８Ｑ５ＳＡＣ）血清钙测定法。方法 以８Ｑ５ＳＡＣ作显色剂，８－羟基喹啉掩蔽镁，在三乙酸胺缓冲介质中以分光光度法测定血清钙。结果：方法学线性范围０－４．５ｍｍｏｌ／Ｌ，平均回收率１００．８５％，批内ＣＶ０．８４％￣１．２４％，批间ＣＶ１．５２％￣１．５６％，与ＯＣＰＣ法对照，ｒ＝０．９９５，Ｐ〉０．５，与ＭＴＢ法对照，ｒ＝０．９８９，     

全血2,3—二磷酸甘油酸比色测定法

本文介绍了用磷酸甘油酸变位酶测定２，３－二磷酸甘油酸的比色法。探讨了该法的最适反应条件，最适ｐＨ７．５％￣７．６。线性范围可达６．０ｍｍｏｌ／Ｌ。三份不同浓度标本批内ＣＶ１．１％￣２．１％，批间ＣＶ２．５％￣３．９％。６０例南京地区健康成人２．３－二磷酸甘油酸含量为１．６￣２．７ｍｍｏｌ／Ｌ，平均回收率９４％，与Ｓｉｇｍａ试剂盒比较测定结果无显著性差异（Ｐ〉０．０５），相关性良好，ｒ＝０．９８２。     

全血2，3－二磷酸甘油酸比色测定法

本文介绍了用磷酸甘油酸变位酶测定２，３－二磷酸甘油酸的比色法。探讨了该法的最适反应条件，最适ｐＨ７．５～７．６。线性范围可达６．０ｍｍｏｌ／Ｌ。三份不同浓度标本批内ＣＶ１．１％～２．１％，批间ＣＶ２．５％～３．９％。６０例南京地区健康成人２．３－二磷酸甘油酸含量为１．６～２．７ｍｍｏｌ／Ｌ，平均回收率９４％，与Ｓｉｇｍａ试剂盒比较测定结果无显著性差异（Ｐ＞０．０５），相关性良好，ｒ＝０．９８２。     

钼酸铵显色法测定血清过氧化氢酶

介绍一种简便的以钼酸铵显色的血清过氧化氢酶分光光度测定法。该法酶活性在１００ｋＵ／Ｌ内呈良好线性。批内变异ＣＶ＝３．６％，天间变异ＣＶ＝４．５７％。平均回收率为１００．１４％。高ＴＧ、高Ｂｉｌ血清标本，可使Ａ值增加，但不影响Ｃａｔ测定值。ＶｉｔＣ、肝素均可使酶活性减低。由于红细胞内富含Ｃａｔ，溶血标本能显著增加酶活性。血清标本酶活性在－４℃４８ｈ内无变化，在－２５℃能保持５～６周。３８８例体检健康者血清Ｃａｔｘ＝５７．６１ＫＵ／Ｌ，经Ｄ检验，当Ｐ＝０．０５时，属正态分布；Ｐ＝０．１时，属非正态分布。中位数（Ｍｄ）＝５６．２２ＫＵ／Ｌ，百分位数法计算，其９５％范围为２１．５８～１０８．８７（ｋＵ／Ｌ）。初步观察１１９例患者Ｃａｔ活性，显示各类癌症患者Ｃａｔ活性明显减低。     

单一试剂测定血清胆红素探讨

用单一试剂测定血清Ｔｏｔａｌ－Ｂｉｌｉｒｕｂｉｎ和Ｄ ｉｒｅｃｔ Ｂｉｌｉｒｕｂｉｎ ，并进行了实验探讨，血清季ＭＷ＝３．６５％，批间ＣＶ＝４．１８％，ＤＢ批内ＣＶ＝６．８２％，批间ＣＶ＝８．７３％；ＴＢ平均回收率为１０４％。与Ｊ－Ｇ法比较相关显著，相关系数为０．９６９。此法操作简便，快速，稳定。     

红细胞铜锌超氧化物歧化酶的连续监测法

采用黄嘌呤－黄嘌呤氧化酶系统与色原偶联，研制了红细胞铜、锌超氧化物歧化酶测定试剂，并在Ｍｏｎａｒｃｈ２０００Ｐｌｕｓ自动生化分析仪上，改进ＳＯＤ活性测定的连续监测法。结果显示，该法批内ＣＶ２．５％，批间ＣＶ４．０％，回收率９０－１０５％，平均回收率９８．５％；在标准品０．２５－４．００Ｕ／ｍｌ范围内呈线性。方法精密度和准确性均佳，同时还可应用于手工操作的固定时间法。     

流动注射与原子发射光谱法联用测定血清锂

采取流动注射分析技术的优点，将流动注射分析技术与原子发射光谱法联合应用，建立了测定正常人体血清中锂的方法，既可测定正常人体锂含量，又可测定碳酸锂治疗病人血清中锂的含量。该法灵敏度高，最小检出限为０．００６ｍｇ／Ｌ。精密度高，重复性好，批内ＣＶ＝０％，批间ＣＶ＝０．５％。平均回收率为１００．６％。线性范围为０－０．１ｍｇ／Ｌ。     

流式细胞仪测定网织红细胞

目前在我国网织红细胞计数均使用人工显微镜，人为影响因素多，重复性差。采用流式细胞术分析方法测定网织红细胞，与经典的人工方法进行了比较。４份结果高低不同的标本的重复性试验，仪器结果的ＣＶ值大大低于人工方法；５０份标本两种方法结果对比分析，Ｐ＞０．０５，差异无显著意义，相关系数为０．９８。     

脲酶／邻甲酚酞络合剂法测定血浆尿素

本文介绍了一种以邻甲酚酞络合剂为指示剂的酶法测定尿素的新方法。该法批内重复性的ＣＶ分别为５．９４，３．１２和１．１％，日间ＣＶ＝６．８９％。平均回收率１０１％，线性范围可达１００ｍｍｏｌ／Ｌ，ｒ＝０．９９９８。黄疸、脂血、溶血干扰极小，与脲酶／谷氨酸脱氢酶法比较ｒ＝０．９９２９，ｙ（本法）＝１．１０４ｘ－０／５０７３．     

An automated reticulocyte counting method: preliminary observations

We evaluated the counting of reticulocytes in the peripheral blood with a newly developed flow cytometer type of automated counter that performs a single test within 60 s. The volume of sample needed is 100 microliters and the cells are stained with auramine-O in the counter. The mean within-run reproducibility was 4.66% (CV, n = 50), and dilution of blood gave highly linear results with an r value of 0.996. Correlation was good between manual reticulocyte counts and those performed with the counter (r = 0.893). Samples with large numbers of leucocytes, erythrocytes, or platelets did not interfere with the automated reticulocyte counting, and provided accurate and precise data.     

网织红细胞多参数分析在地中海贫血患者中测定的意义

目的调查健康中国成人的网织红细胞多参数分析的参考值范围;并进一步观察在地中海贫血时网织红细胞各参数改变情况及临床意义。方法应用Coulter-GENS.system2型全自动血液分析仪检测了66例健康成人和33例地中海贫血患者7项网织红细胞细胞参数。结果①网织红细胞细胞多参数的正常参考值范围(男女混合)为:RET%1.34±0.48(0.86%~1.82%);RET#0.059±0.023(0.036~0.082);IRF0.3±0.04(0.26~0.34);HLR%0.38±0.14(0.24%~0.52%);HLR#0.017±0.007(0.01~0.024);MRV113±7(106~120);MSCV91±6(85~97)。②地贫患者的7项网织红细胞参数中有多项与正常人有相比有显著性差异,其中RET%、RET#、HLR%和HLR#明显增高(P<0.01),MRV、MSCV明显下降(P<0.01),且变化程度病情的严重程度有一定的关联性。贫血程度较轻的血红蛋白病,网织红细胞各参数变化不大。结论网织红细胞细胞多参数测定提示,地中海贫血时患者网织红细胞细胞中未成熟细胞占的比例增加可能是导致“无效造血”的主要原因之一;网织红细胞细胞多参数分析能在一定程度上提示地中海贫血严重程度;在经典网织红细胞指标RET%,RET#基础上,应用HLR%、HLR#和IRF可以更细微、全面地描述网织红细胞的成熟度,从而更好的评价骨髓造血的功能。     

Lateral flow immunoassay using Europium (III) chelate microparticles and time-resolved fluorescence for eosinophils and neutrophils in whole blood

BACKGROUND: A simple point-of-care method for measuring leukocyte counts in a doctor's office or emergency room could be of great importance. We developed a protocol for measuring cell count by disrupting the cell membrane and analyzing specific proteins within the cells and used it to analyze proteins from eosinophils and neutrophils. METHODS: Lateral immunochromatographic (ICR) assays have been developed for eosinophil protein X (EPX) and human neutrophil lipocalin (HNL) as measures of the concentration of eosinophils and neutrophils. The correlation between the lateral ICR assays and cell counting of eosinophils and neutrophils was performed manually and with an automated cell counter. RIA assays measuring the same analytes were also compared with the results from cell counting and lateral ICR assays. RESULTS: The optimized assays showed analytical detection limits below the clinical ranges of 3.36 microg/L and 2.05 microg/L for EPX and HNL, respectively. The recovery was 114.8%-122.8% for EPX and 94.5%-96.9% for HNL. The imprecision was 3%-17% CV for EPX over the whole range and 5%-16% CV for HNL. The correlation coefficients between manually counted cells and lateral ICR assays were 0.9 and 0.83 for EPX and HNL, respectively. CONCLUSION: The numbers of eosinophils and neutrophils in small amounts of blood can be estimated in the point-of-care setting by means of fast lateral ICR assays of EPX and HNL.     

Analysis of reticulocyte counts using various methods.

The precision and accuracy of manual reticulocyte counts using the Miller disc reticle, other ruled reticle and no reticle are compared with the reticulocyte results from the automated Hematrak 590 instrument. Two slides of each of 50 patient blood specimens were sent to the hematology laboratories of each of six participating hospitals. In addition to between-method comparison (precision), the manual method results using the three different counting techniques were each compared with the Hematrak results to determine if there were significant differences in reported results (accuracy). Statistical analysis revealed that the Miller disc method was the most precise and accurate manual method as compared with the Hematrak. Methods without a Miller disc reported significantly higher reticulocyte counts. Imprecision was also higher among non-Miller manual methods. By using the Miller disc, the accuracy and precision of manual methods may be increased to that of the automated Hematrak method.     

地中海贫血孕晚期妇女网织红细胞参数及网织血小板的变化分析

目的探讨网织红细胞6项参数及网织血小板在地中海贫血（简称地贫）孕晚期妇女中的变化及意义。方法对经基因确诊的39例地贫孕晚期妇女、30例正常孕晚期妇女和30例正常非孕妇女,应用SysmexXE-5000全自动血细胞分析仪测定网织红细胞绝对值（RET#）、网织红细胞百分率（RET%）、未成熟网织红细胞比率（IRF%）、低荧光强度网织红细胞百分率（LFR%）、中荧光强度网织红细胞百分率（MFR%）、高荧光强度网织红细胞百分率（HFR%）6项网织红细胞参数及网织血小板百分率（IPF%）。结果地贫孕晚期妇女、正常孕晚期妇女、正常非孕妇女的六项网织红细胞参数及网织血小板比较,差异有统计学意义（P〈0.05或P〈0.01）,其中地贫孕晚期妇女RET#、RET%、IRF%、MFR%、HFR%、IPF%明显增高,LFR%明显下降。结论地贫孕晚期妇女在生理和病理的双重刺激下骨髓的造血功能更旺盛,检测妊娠妇女的网织红细胞参数及网织血小板,有助于发现地贫患者,减少及控制危重地贫患儿的出生。     

Automated analysis of reticulocytes

An evaluation of the performance of the GEN-S analyzer in examining the parameters of reticulocytes is described. The automated and manual methods of counting the relative quantity of reticulocytes were compared. It was established that the precision of the analyzer significantly surpasses the manual method results. The variation coefficient for the parameters of examination of reticulocytes is lower at a higher content of reticulocytes and the accuracy of the method is better if the content of reticulocytes in blood is higher. The parameters, characterizing the fraction of immature reticulocytes, are least accurate. The study demonstrated that the automated method of analyzing the reticulocytes by using the GEN-S analyzer can be used to ensure a more accurate monitoring of changes of the function of erythropoesis at different anemic conditions and to assess the conducted therapy.     

A general method for concentrating blood samples in preparation for counting very low numbers of white cells

BACKGROUND: To count extremely low levels of white cells (WBCs) in WBC- reduced blood components, a larger volume of sample must be processed. The goal was to develop an all-purpose method for concentrating the samples obtained from WBC-reduced red cells or platelets. The method was designed to be compatible with a variety of counting techniques. STUDY DESIGN AND METHODS: Coded samples of red cell concentrates with an expected WBC concentration of 200, 100, 50, and 10 per mL and of the diluent (undetectable WBCs/mL) were sent to three sites on five occasions and counted by the use of the concentration method, crystal violet stain, and a Nageotte counting chamber. Additional samples were tested by flow cytometry, polymerase chain reaction, and volumetric capillary cytometry. RESULTS: The results from the three test sites showed good linearity, with an overall r2 = 0.9994. The lower limit of accurate detection of the assay was 10 WBCs per mL. The results were biased toward underestimation, particularly at one of the test sites (Site A). There were no significantly different results in Sites B and C. The intra-assay CV was acceptable. Precision (reproducibility) at the three test sites varied. CONCLUSION: This method allows reliable determination of WBC concentrations as low as 0.01 per microL in blood. Despite the use of technologists trained in Nageotte chamber counting, validation testing demonstrated that one test site's performance was significantly different from that of the other two sites, because of both underestimation bias and variation in count results. The sample concentration method, when used in conjunction with an automated assay for WBC identification, should permit larger volume analysis with a greater degree of precision and a lower limit of detection than is found in assays that do not concentrate the sample before counting.     

Automated white blood cell counts in cerebrospinal fluid using the body fluid mode on the platform Sysmex XE-5000

Abstract

Background. The Sysmex XE-5000 offers automated quantification of red blood cells and white blood cells (WBCs) in body fluids, with differentiation of polymorphonuclear cells (PMNs) and mononuclear cells (MNCs). Methods. We evaluated automated WBC counting in cerebrospinal fluid (CSF) using the body fluid mode on the Sysmex XE-5000, comparing it with flow cytometry as the reference method, and also with manual counting by microscopy. Experimental analysis for linearity and limit of detection was performed by diluting isolated WBCs in cell-free CSF. To study the ability to discriminate between PMNs and MNCs, samples were spiked using MNCs separated from peripheral blood. Comparison of WBC counts between a counting chamber and the XE-5000 was performed for 198 CSF samples. Results. In the experimental set-up, within-run (CV 19%) and between-day imprecision (CV 15.3%) in quantitating total number of WBC on XE-5000 was acceptable for WBC counts ≥ 25 × 10⁶/L. Compared with expected cell counts, mean bias was + 2.6% for flow cytometry, + 5.5% for XE-5000 and ? 73.2% for manual counting. Differentiation between PMNs and MNCs was in concordance with flow cytometry. In comparisons of clinical CSF samples, overall agreement between the XE-5000 and manual counting was observed in 81% of the samples, but mean difference in WBC differentiation was higher for PMN (51.1 × 10⁶/L) than for MNC (7.95 × 10⁶/L). Conclusion. Despite limited precision at low WBC counts, XE-5000 could be a favourable alternative to the labour-intensive, time-consuming and less reliable manual counting and cuts turnaround times in routine CSF-based diagnosis.     

Coulter LH 750仪网织红细胞检测参数的临床价值分析

目的探讨BeckmanCoulterLH750全自动血液分析仪检测网织红细胞及其新型参数的方法及临床应用价值。方法用BeckmanCoulterLH750全自动血液分析仪检测248例住院贫血患者抗凝全血标本的网织红细胞百分比(RET％)、网织红细胞绝对值(RET#)、网织红细胞平均体积(MRV)、未成熟网织红细胞组分(IRF)、高散射光强度网织红细胞百分比(HLR％)等五项网织红细胞参数。结果慢性肾功能不全的贫血患者RET#同对照组比较无显著差异,但HLR％显著高于对照组(P<0.05)。失血性贫血的患者网织红细胞各项指标与对照组相比显著增高;肝硬化与正常对照组RET％、RET#和HLR％有显著差异(P<0.05)。结论测定网织红细胞及其相关参数有助于判断红细胞的活动度,对贫血类型的初筛诊断、治疗监测等有重要的实用价值。     

A new platelet storage lesion index based on paired samples, without and with EDTA and cell counting: comparison of three types of leukoreduced preparations.

Platelets derived from whole blood and diverse apheresis procedures come in contact with various artificial surfaces and undergo contact activation, sheer stress-induced shape changes, aggregation and microvesiculation during collection, processing and storage. These dynamic changes are qualitatively reflected in the log-normal platelet size distribution patterns seen, when using modern automated cell counters and can be measured quantitatively by counting the paired samples, without and with added EDTA, and calculating the differences (d) in platelet cellular indices (dPLT/dMPV/dPDW). Reporting the differences instead of the absolute values of cellular indices makes the measurements independent of basic principles used for cell counting (i.e. aperture-impedance or flow-optic). The measurements can be performed either in blood centres, hospital blood banks or nearby patient clinics equipped with a validated cell counter. At least three useful quality indices could be derived simultaneously from this procedure (accurate estimation of platelet yields using the value of the EDTA-containing sample); quantitative assessment of the % of reversible platelet aggregates, an age/pH/temperature-dependent degree of microvesiculation/apoptosis/PS exposure, based on the response to EDTA. This new set of indices correlate significantly with other conventional tests for platelet quality; hence, provide additional supportive evidence for confirming the low pH values and the subjective poor swirling data occasionally seen with stored PC. In the following study, the Sysmex SE 9000 was successfully employed for estimation of platelet cellular indices comparing the platelet storage lesion index of three types of leukoreduced platelet concentrates in current practice. The relationship between this in vitro response to clinical outcome remains to be established.