This protocol describes the collection and preservation of blood samples from neonatal and pediatric patients and the application of scRNA-seq, proteomics, and spectral flow cytometry to characterize the immune cell populations from these samples.
方法文章
* These authors contributed equally
This protocol describes the collection and preservation of blood samples from neonatal and pediatric patients and the application of scRNA-seq, proteomics, and spectral flow cytometry to characterize the immune cell populations from these samples.
At birth, the neonatal immune system is abruptly confronted with a radically different environment, rich in microbial and environmental stimuli. Increasing evidence suggests that early-life immunity is not merely immature, but rather uniquely adapted to meet the distinct demands of this developmental window. During this period, the immune system is highly dynamic, undergoing rapid changes to accommodate the shifting external landscape. Understanding this complex and evolving immune landscape requires direct study in neonates. However, a major limitation in such investigations is the low total blood volume in neonates (~100 mL/kg), which restricts the amount that can be safely collected for research, particularly in extremely low birth weight (ELBW) or extremely premature infants. In our recent work, we address this limitation by demonstrating how high-dimensional immune profiling can be performed using minute volumes of neonatal blood. We introduce a set of optimized protocols for longitudinal sampling across early life and apply advanced techniques, including flow cytometry, proteomics, and single-cell RNA sequencing, to maximize the information obtained from minimal input. Together, these methods enable a comprehensive view of the transcriptomic and proteomic signatures of circulating immune cells in neonates, offering new insights into the unique trajectory of early-life immunity.
This protocol is optimized for 100-250 µL blood samples processed within 12 h of collection, either cryopreserved or used immediately. At birth, the neonatal immune system is thrust into a radically different environment, enriched with microbial and environmental stimuli that were previously absent in the protected intrauterine setting1,2. This shift requires the immune system to adapt and respond to a vast array of new antigens, a process that is essential for survival but also poses significant challenges1,2. Increasing evidence suggests that early-life immunity is not simply immature or a miniature version of the adult immune system. Instead, it is uniquely tailored to meet the specific needs of this critical developmental period3,4. During this neonatal window, the immune system's cellular components, including various types of T cells, B cells, and myeloid cells, mature and develop unique characteristics and functions specific to early life5,6,7,8. Despite its importance, neonatal immunity remains difficult to study directly due to ethical and practical constraints. A major limitation is the small blood volume in neonates, averaging ~100 mL/kg9,10. This challenge becomes even more pronounced when collecting blood from extremely premature infants (born before 30 weeks of gestation) and ELBW infants, who can weigh as little as 400 g with only 40 mL of circulating blood. This restricts how much blood can be safely collected, underscoring the need for approaches that maximize data yield from minimal samples. To address this challenge, our work demonstrates optimized protocols for high-dimensional immune profiling using minute blood volumes (100-250 µL), such as those obtained from ELBW infants.
Recent advances in blood collection and processing have enabled the study of immune responses in populations where sample acquisition is traditionally difficult. Microneedle devices such as the Tasso and Touch Activated Phlebotomy systems were initially developed for convenient at-home blood collection in adults. They have since been adapted for use in infants, allowing primary caregivers and clinical teams to collect samples with minimal discomfort. Additionally, they can be used at home without the stress of repeated clinical visits11, facilitating compliance and accessibility. An ethylenediaminetetraacetic acid (EDTA) microtube should be used with the device and shipped overnight to the lab with adherence to appropriate biohazard protocols. The next morning, when samples arrive, they should be processed using red blood cell (RBC) lysis methods to capture circulating immune cells6. Of note, EDTA tubes enable live-cell isolation, but time considerations are important. For functional assays with stimulation, samples must be processed within less than 4 h and ideally less than 2 h from collection. For the non-functional, phenotypic assays presented below, samples should be processed within 12 h to permit overnight shipment while ensuring recovery, as cell viability decreases over time12. Compared to traditional density-gradient separation with Ficoll, lysis is faster, less labor-intensive, and yields higher recovery13. However, peripheral blood mononuclear cell isolation by lysis may leave contaminating granulocytes behind, reducing purity but can be advantageous for studies interested in these cell types14. These tradeoffs highlight how methodological adaptations balance feasibility and accuracy when studying rare or sensitive clinical samples, such as those from neonates. Downstream handling of isolated cells has also benefited from methodological refinements. Cryopreservation with fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) remains a robust approach for immune cells, particularly in fetal and neonatal blood, where cell viability and recovery are critical. Alongside optimizing the blood collection process, we have also adopted downstream techniques that allow for the extraction of maximum information from small sample volumes. We introduce a set of optimized protocols that facilitate sampling across early life, enabling a detailed examination of immune dynamics over time. Our approach leverages advanced techniques such as spectral flow cytometry, proteomics, and single-cell RNA sequencing, which collectively offer an unparalleled view of the proteomic and transcriptional signatures of circulating immune cells in early life. Additionally, spectral flow cytometry offers opportunities to gain functional insight with minimal cell input.
Over the years, several techniques, such as complete blood counts (CBCs), have been used to analyze blood-derived immune cells15, while others have analyzed immune cell ratios in neonatal whole blood samples using DNA methylation16,17. While these techniques have been helpful for learning the proportions of known immune cells, there are still limitations in our ability to gain in-depth insights into cells without a multi-omics system. CBCs provide only broad measures of major blood cell types, making them useful for establishing overall immune status in a clinical setting but limited in their ability to resolve subtypes or functional states. Most recently, studies have begun to address this limitation by employing plasma-derived transcriptomic and metabolomic changes in infants as early as the first week of life18. Mass spectrometry-based proteomics has been adapted to study immune profiles in neonatal samples19. This approach is powerful for measuring a broad range of proteins in an unbiased manner and for identifying potential biomarkers. However, key limitations include the requirement for relatively large protein input and the lack of direct information on cell identity, despite capturing both soluble and insoluble proteins. Targeted multiplex proteomics assay (TMPA) offers alternatives for low-input samples. These methods use targeted antibody-based detection, providing high-sensitivity profiling for samples with limited material. One of these, in particular, has been used to track early-life immune development trajectories longitudinally20.
Flow cytometry enables quantitative analysis of immune cell proteins at the single-cell level by passing fluorescently labeled cells through a focused laser beam and measuring the resulting light scatter and fluorescence signals. In this technique, fluorescently labeled antibodies bind to specific surface or intracellular proteins. This allows for simultaneous detection and quantification of multiple immune cell subtypes and their defining characteristics based on their fluorescence profiles. Spectral flow cytometry extends this approach by capturing the full emission spectrum of each fluorochrome, enabling precise spectral unmixing and the simultaneous detection of over 35 markers in a single assay21,22,23. This enables analysis of many more markers than conventional flow cytometry panels, which are limited to ~8-12 markers. Complementing these cytometric techniques, single-cell RNA sequencing (scRNA-seq) has transformed immunological studies by enabling high-resolution, unbiased transcriptomic profiling from minimal input material24. Despite higher costs and dependence on cell viability, scRNA-seq provides unparalleled insights into early-life immune heterogeneity compared to bulk sequencing. Collectively, these advances demonstrate how innovations in collection, preservation, and analysis are being adapted to overcome the unique challenges of studying fetal and neonatal immunity.
This protocol has been optimized for the analysis of cryopreserved peripheral immune cells isolated via serial RBC lysis. Researchers should consider this protocol if their studies require high-resolution immune profiling from small volumes of blood (100-500 µL), especially when longitudinal analysis is critical. The method is broadly applicable to both term and preterm neonates and is compatible with a wide range of downstream analyses, making it suitable for diverse research questions in neonatal and pediatric immunology.
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Informed parental consent and full compliance with relevant national ethical regulations were ensured in accordance with Yale University's Institutional Review Board (IRB). As specified in the approved IRB protocol, parents were contacted by the research team during the first week following the infant's birth to obtain consent prior to enrollment. No study procedures involving human-derived materials commenced prior to documented ethics approval.
1. Sample collection, processing, and storage (Figure 1)
2. TMPA from dried blood spots (DBS) (Figure 2)
3. Spectral flow cytometry of RBC lysed blood (Figure 3)
4. scRNA-seq of RBC lysed blood (Figure 4)
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We collected DBS cards from 10 extremely preterm infants at three time points: 1 week (n = 12), 1 month (n = 7), and 2 months (n = 3) for TMPA proteomic analysis6 (Figure 2A). These samples were analyzed alongside full-term infant cord blood (TCB; n = 4) and healthy adult blood (AB; n = 5). The normalized protein expression (NPX) of 92 immune-related proteins was quantified using the TMPA platform (Figure 2B). Statistical analysis demonst...
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Obtaining high-quality immunological data from minimal volumes of neonatal and infant blood requires methodological adaptations across multiple steps. In this protocol, we describe four complementary strategies: (i) optimized blood collection and cryopreservation to preserve viable cells for downstream use, (ii) application of spectral flow cytometry for multiparametric immunophenotyping, (iii) scRNA-seq to resolve immune heterogeneity at high resolution, and (iv) TMPA. Together, these approaches maximize data yield whil...
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The authors declare that they have no conflicts of interest.
We thank the following core facilities at Yale: Yale Flow Cytometry core; Yale Center for Genome Analysis. We thank the Yale Center for Clinical Investigation, the Yale Office of Physician-Scientist and Scientist Development, and the Pediatric Critical Care and Trauma Scientist Development Program/NICHD. Respirometry studies were performed by the Chemical Metabolism Core at Yale University. This work was funded by grants to: P01 AI179570 (L.K.), R01AI171980 (L.K.), R01DK129552 (L.K.), R01HL163043 (L.K.), Cystic Fibrosis Foundation (L.K.); K08 AI177743 (N.N.B.), Hartwell award (N.N.B.); K08DK133687 (O.O.), Yale School of Medicine, Department of Paediatrics (O.O.); R01AI123204 (C.R.O). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Figure 1 was created in BioRender. Gawon, K. (2025) https://BioRender.com/7embw10.
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| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| 0.5 M EDTA,pH 8.0 | 赛莫飞世尔科学公司; | 15575-038 | |
| 1x红细胞溶解缓冲液 | Invitrogen | 3177165 | |
| BCA蛋白质检测试剂盒及nbsp; | Abcam | ab287853 | |
| BD Microtainer MAP 微管 | 费舍尔科学公司; | 22-253-270 | |
| 贝尔-阿特低温安全 -1&度;C 冻结控制器 | 米利波尔西格玛 | 41122800 | |
| 牛血清白蛋白(BSA) | 西格玛-奥尔德里奇 | A9647-100G | |
| 《璀璨紫外线496反人类CD3 》; | 赛莫飞世尔科学公司; | 364-0038-42 | |
| Brilliant Ultra Violet 661 反人类 CD11c | 生物科学学士及nbsp; | 612968 | |
| Brilliant Vil750 反人类 CD14 | 生物传说 | 367135 | |
| 辉煌紫罗兰570反人类CD8a | 生物传说 | 301038 | |
| 辉煌紫罗兰711反人类CD56 | 生物传说 | 318336 | |
| cOmplete,迷你型,无EDTA蛋白酶抑制剂鸡尾酒 | 米利波尔西格玛 | 11836170001 | |
| 死细胞清除套件 | 米尔滕尼生物技术公司及nbsp; | 130-090-101 | |
| 二甲基硫酸盐(DMSO)及nbsp; | 赛莫飞世尔科学公司; | BP231-100 | |
| 杜尔贝科磷酸盐缓冲盐水(DPBS),不含钙、镁及; | 赛莫飞世尔科学公司; | 14190144 | |
| 胎儿牛血清(FBS)及nbsp; | Gibco | A5256801 | |
| Fisherbrand 数字漩涡混音器 | 赛莫飞世尔科学公司; | 02215418 | |
| 固定缓冲器及nbsp; | 生物传说 | 420801 | |
| GlutaMAX补充剂 | Gibco | 35050061 | |
| MACS 多站立 | 米尔滕尼生物技术公司及nbsp; | 130-042-303 | |
| 手稿列目 | 米尔滕尼生物技术公司及nbsp; | 130-042-201 | |
| 名称 | 公司 | 目录号 | |
| OctoMACS 分离器 | 米尔滕尼生物技术公司及nbsp; | 130-042-109 | |
| 太平洋蓝反人类 CD4 | 生物传说 | 344619 | |
| 青霉素-链霉素(10,000 U/mL) | 赛莫飞世尔科学公司; | 15140-122 | |
| RB780 反人类 CD33 | BD生物科学 | 755606 | |
| Rierdge 3mm 金属冲击器 | Rierdge | SDD231031PC-01  | |
| RPMI 1640 中型 | Gibco | 11875-093 | |
| Spark UV 387 反人类 CD19 | 生物传说 | 302289 | |
| 塔索收藏套件 | 塔索 | 无 | |
| Thermo Scientific 螺旋盖微管 | 赛莫飞世尔科学公司; | 14-755-287 | |
| Whatman 309 蛋白质保护器 | Cytiva | 41111700 | |
| 僵尸近红外可修复可修复可行工具包 | 生物传说 | 423105 | |
| <强>软件和包 | |||
| Adobe 插画师 | 2024 | ||
| BBKNN | 1.5.1 | ||
| FlowJo | 10.8.1 | ||
| GraphPad 棱镜 | 9.4.0 | ||
| 名称 | 版本 | ||
| 斯坎皮 | 1.9.1 | ||
| 斯鲁布莱特 | 0.2.3 | ||
| <强>商业测定 | |||
| 铬单胞5英尺球衣 | 10X基因组学 | PN-1000263 | |
| Olink 揭示或目标96炎症面板 | 奥林克 | https://olink.com/products/olink-target-96 |
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