Department of Clinical Parasitology, Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, London, UK.
Malar J. 2012 Mar 5;11:62. doi: 10.1186/1475-2875-11-62.
All mothers donating umbilical cord blood units to the NHS cord blood bank undergo an assessment for the likelihood of prior exposure to malaria infection. Those deemed at risk due to a history of travel to, or residence in, malaria endemic regions are screened serologically to detect anti-malaria antibodies. A positive result excludes the use of the cord blood for transplant therapy unless a risk assessment can ensure that malaria transmission is extremely unlikely. This paper details the screening of cord blood units from malaria serology positive mothers to detect malaria parasite DNA using a highly sensitive nested PCR.
Uninfected blood from a healthy volunteer was spiked with known quantities of malaria parasites and 5 millilitre and 200 microlitre aliquots were subjected to DNA extraction using QIAamp DNA maxi and DNA mini kits respectively. Nested PCR, to detect malarial SSU rRNA sequences, was performed on the purified DNA samples to determine the limit of detection for this assay with both extraction methodologies. Following assay validation, 54 cord blood units donated by mothers who were positive for anti-malaria antibodies were screened by this approach.
When DNA was purified from 5 millilitres of blood it was possible to routinely detect as few as 50 malaria parasites per millilitre using nested PCR. This equates to a significant increase in the sensitivity of the current gold standard nucleic acid amplification technique used to detect malaria parasites (routinely performed from > 200 microlitre volumes of blood). None of the 54 donated cord blood units from serology positive mothers tested positive for malaria parasites using this scaled up DNA preparation method.
Serological testing for malaria parasites may be overly conservative, leading to unnecessary rejection of cord blood donations that lack malaria parasites and which are, therefore, safe for use in stem cell therapy.
所有向国民保健制度脐带血库捐赠脐带血的母亲都要接受评估，以确定她们是否有可能接触过疟疾感染。那些由于曾经去过疟疾流行地区或居住在疟疾流行地区而被认为有风险的人，会进行血清学筛查以检测抗疟疾抗体。阳性结果排除了使用脐带血进行移植治疗的可能性，除非风险评估可以确保疟疾传播的可能性极小。本文详细介绍了使用高度敏感的巢式 PCR 从疟疾血清学阳性母亲的脐带血单位中筛查疟原虫 DNA 的方法。
用已知数量的疟原虫感染健康志愿者的未感染血液，分别用 QIAamp DNA maxi 和 DNA mini 试剂盒从 5 毫升和 200 微升等分试样中提取 DNA。对纯化的 DNA 样本进行巢式 PCR，以检测疟原虫 SSU rRNA 序列，以确定这两种提取方法的检测限。在进行了方法验证后，用这种方法对 54 个由抗疟抗体阳性母亲捐赠的脐带血单位进行了筛查。
从 5 毫升血液中提取 DNA 时，使用巢式 PCR 可以常规检测到每毫升低至 50 个疟原虫。这相当于目前用于检测疟原虫的核酸扩增技术（通常从 > 200 微升血液体积中进行）的灵敏度显著提高。用这种扩大的 DNA 制备方法，对来自血清学阳性母亲的 54 个捐赠脐带血单位进行检测，均未检测到疟原虫呈阳性。
对疟原虫进行血清学检测可能过于保守，导致不必要地拒绝缺乏疟原虫的脐带血捐献，而这些捐献的脐带血在干细胞治疗中是安全的。
