Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood.
Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood.
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DOI：
10.1038/s41598-022-24550-6
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发表时间：
2022-11-19
影响因子：
4.6
通讯作者：
Howe, Charles L.
中科院分区：
文献类型：
--
作者：
Johnson, Renee K.;Overlee, Brittany L.;Sagen, Jessica A.;Howe, Charles L.
血液等生物标本的当天处理并不总是可行的，这对寻求研究广泛人群或描述罕见疾病患者特征的研究计划提出了挑战。招募中心可能不具备处理血液样本的能力，并且在当地中心分离外周血单核细胞（PBMC）的时间和技术的可变性可能会影响患者间的重现性。一种解决方案是在环境条件下通过隔夜快递将通过常规放血术收集的全血发送到测试中心。确定运输对后续白细胞反应的影响是对运输样本进行任何实验分析的必要先决条件。为此，从健康对照受试者中采集全血，并在模拟运输条件下新鲜处理或在采集和处理后6、24和48 h处理。在终点时，通过全血细胞计数进行全血评估，使用标准化抗体组[HLADR、CD66 b、CD3、CD14、CD16]进行分类和免疫表型分析。从全血中分离PBMC和嗜中性粒细胞，并用脂多糖和热灭活的金黄色葡萄球菌进行离体刺激。细胞因子和趋化因子的刺激释放通过流式细胞仪珠阵列评估。还从PBMC中分离RNA以分析运输诱导的转录变化。全血细胞分类计数显示，在环境温度下保存24 h的运输血液中，大多数参数均保持不变。免疫表型分析表明，在24小时的细胞谱的保存，但与扩大一些人口和经典的单核细胞上的CD 16强度下降。在转录水平上，RNAseq分析鉴定了与源自过夜运输的全血的未刺激的PBMC中的炎症相关的转录因子模块的上调。然而，这些变化在受影响基因的规模和数量上都是有限的。PBMC的离体刺激进一步揭示了从在环境温度下保持24小时的运输血液分离的细胞中的功能应答的保留。然而，此时中性粒细胞应答大部分消失。到48小时，两个细胞群体均未在正常参数内响应。这些发现表明，在过夜运输并在采集后24小时内处理的全血中保持了稳健的免疫表型和PBMC刺激的反应特征，产生的结果代表了静脉穿刺后立即从样本中获得的结果。这种方法对于许多患者招募站点实施是可行的，并且允许对来自大地理区域的患者群体进行复杂的免疫学分析。关于罕见病研究，这满足了招募足够数量的患者进行免疫分析和发现潜在致病机制的普遍需求。
Same day processing of biospecimens such as blood is not always feasible, which presents a challenge for research programs seeking to study a broad population or to characterize patients with rare diseases. Recruiting sites may not be equipped to process blood samples and variability in timing and technique employed to isolate peripheral blood mononuclear cells (PBMCs) at local sites may compromise reproducibility across patients. One solution is to send whole blood collected by routine phlebotomy via overnight courier to the testing site under ambient conditions. Determining the impact of shipping on subsequent leukocyte responses is a necessary prerequisite to any experimental analysis derived from transported samples. To this end, whole blood was collected from healthy control subjects and processed fresh or at 6, 24 and 48 h after collection and handling under modeled shipping conditions. At endpoint, whole blood was assessed via a complete blood count with differential and immunophenotyped using a standardized panel of antibodies [HLADR, CD66b, CD3, CD14, CD16]. PBMCs and neutrophils were isolated from whole blood and subjected to ex vivo stimulation with lipopolysaccharide and heat-killed Staphylococcus aureus. Stimulated release of cytokines and chemokines was assessed by cytometric bead array. RNA was also isolated from PBMCs to analyze transcriptional changes induced by shipping. The complete blood count with differential revealed that most parameters were maintained in shipped blood held for 24 h at ambient temperature. Immunophenotyping indicated preservation of cellular profiles at 24 h, although with broadening of some populations and a decrease in CD16 intensity on classical monocytes. At the transcriptional level, RNAseq analysis identified upregulation of a transcription factor module associated with inflammation in unstimulated PBMCs derived from whole blood shipped overnight. However, these changes were limited in both scale and number of impacted genes. Ex vivo stimulation of PBMCs further revealed preservation of functional responses in cells isolated from shipped blood held for 24 h at ambient temperature. However, neutrophil responses were largely abrogated by this time. By 48 h neither cell population responded within normal parameters. These findings indicate that robust immunophenotyping and PBMC stimulated response profiles are maintained in whole blood shipped overnight and processed within 24 h of collection, yielding results that are representative of those obtained from the sample immediately following venipuncture. This methodology is feasible for many patient recruitment sites to implement and allows for sophisticated immunological analysis of patient populations derived from large geographic areas. With regard to rare disease research, this meets a universal need to enroll patients in sufficient numbers for immunoprofiling and discovery of underlying pathogenic mechanisms.
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