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微生物与感染
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定量聚合酶链反应评估去白细胞滤器去除血液成分中巨细胞病毒的效果
陆萍1;凌冰1;方风琴2;季育华2

1. 上海市血液中心, 上海200051; 2. 上海交通大学医学院附属瑞金医院检验科, 上海200025

Assessing the effect of removing human cytomegalovirus from blood
components using quantitative polymerase chain reaction

LU Ping1;LING Bing1;FANG Feng-qin2;JI Yu-hua2

1. Shanghai Blood Center, Shanghai 200051, China; 2. Clinical Diagnostic Laboratory, Rujin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

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摘要: 为了评估目前常用的去白细胞滤器去除血液成分中人巨细胞病毒( HCMV) 的效果, 我们在献血员中筛选获得HCMV-IgG、pp65 皆阴性的血液, 分离外周血单个核细胞( PBMC) , 将HCMV 感染的人成纤维细胞与PBMC 共培养, 感染HCMV 的PBMC 经抗人成纤维抗体和抗CD45 磁珠2 次分选纯化后, 将CD45 + 单核细胞加回原全血中, 建立HCMV 潜伏感染的模型, 再用去白细胞滤器过滤。用实时聚合酶链反应( PCR) 定量测定过滤前、后血液样本中的病毒载量, 残留成纤维细胞所含的HCMV 通过定量反转录PCR( RT - PCR) 扩增脯-4-羟化酶exon12a mRNA 来校正。结果显示, 使用去白细胞滤器后, 每单位( 200 ml) 全血血液中, 白细胞的去除率为99. 98% 。全血中平均HCMV 病毒载量从3 742 拷贝/ μl 降至22. 57 拷贝/ μl, 降低2. 50 log10 , 说明去白细胞滤器虽然可明显减少全血中HCMV 病毒的载量, 但并不能完全去除病毒。
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陆萍1
凌冰1
方风琴2
季育华2
关键词 去白细胞滤器成纤维细胞    
Abstract

The purpose of the current study is to assess the effect of removing human cytomegalovirus ( HCMV) by leukocyte reduction filtration ( LRF) in blood center. Peripheral blood mononuclear cells ( PBMCs) from blood
samples negative for HCMV-IgG and pp65 were separated and co-cultured with HCMV-infected human fibroblasts in order to establish infection. The infected PBMCs were purified from fibroblasts by double selection using magnetic beads coated with anti-human fibroblast antibodies and anti-CD45 and then spiked into whole blood units prior to LRF. After LRF, HCMV load in whole blood and in fibroblasts was assessed using real-time quantitative polymerase chain reaction( PCR) for HCMV DNA and quantitative reverse transcriptase PCR( RT-PCR) for mRNA encoding the
HCMV DNA and fibroblast-specific splice variant of prolyl-4-hydroxylase, respectively. After LRF, the rate of leukocyte depletion in whole blood units ( 200 ml) was 99. 98%. After correcting for fibroblast-associated HCMV, the mean HCMV load in whole blood was reduced from3 742 to
22. 57 copies per microliter ( 2. 50 log10 reduction) by LRF. These results suggest that LRF reduces viral load but does not completely remove HCMV from blood components.

Key wordsHuman cytomegalovirus    Leukocyte reduction filtration(LRF)    Fibroblast
通讯作者: 陆萍   
引用本文:   
陆萍1 ;凌冰1 ;方风琴2 ;季育华2 .
定量聚合酶链反应评估去白细胞滤器去除血液成分中巨细胞病毒的效果
[J]. 微生物与感染 , 2009, 4(1): 13-15 .
LU Ping1;LING Bing1;FANG Feng-qin2;JI Yu-hua2.

Assessing the effect of removing human cytomegalovirus from blood
components using quantitative polymerase chain reaction

. Journal of Microbes and Infections, 2009, 4(1): 13-15 .
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http://jmi.fudan.edu.cn/CN/      或     http://jmi.fudan.edu.cn/CN/Y2009/V4/I1/13

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