Mapping Infant Immunity with Minimal Input: Integrative Single-Cell and Multiomic Profiling

Summary

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.

Abstract

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.

Introduction

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 setting^1,2. 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 challenges^1,2. 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 period^3,4. 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 life^5,6,7,8. 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/kg^9,10. 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 methylation^16,17. 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 assay^21,22,23. 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.

Protocol

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)

1. Inpatient blood collection
   1. Collect and process infant samples after receiving them, store at 4 °C, and process within 12 h of collection.
   2. Collect ~250 µL of whole blood and store in an anticoagulant EDTA blood collection tube. Invert the blood collection tube 8x to mix the anticoagulant.
   3. Proceed to process the whole blood via red blood lysis as described in Section 1.3 (whole blood sample processing).
   4. To collect dry blood spots, use the sterile lancet to make a puncture on the cleaned toe/heel.
      1. Alternatively, pipette ~25-30 µL of blood from the EDTA tube onto the filter paper. Lightly touch the filter paper to the blood drop.
   5. Fill at least one circle with free-flowing blood, one drop per circle. Allow the blood spot to dry. Keep the filter paper open until the blood spots are dry thoroughly.
   6. Label appropriately with time of collection, processing date, timepoint, and specimen Identification.
   7. Place in a clear plastic zipper storage bag. Store blood spot cards at −20 °C or −80 °C for extended stability until ready for proteomic analysis.
2. Outpatient blood collection
   ​NOTE: With approval from Yale University's Institutional Review Board, parents of outpatient participants were given the option to collect blood samples at home using a microneedle self-collection device. This device obtains capillary blood through a lancet applied to the skin. Parents who selected this method received in-clinic training using demonstration kits and were subsequently provided with detailed written instructions and home-use collection kits.
   1. Wash hands and wear gloves before use. Collect all necessary items, including a mirror if only one parent is performing the blood collection.
   2. Remove the device from the tool kit and twist off the cap from a compatible tube.
   3. Press the tube into the device until snug with fill lines facing out.
   4. Remove the infant’s diaper and hold the infant upright.
   5. Follow the heat pack's instructions to activate.
      ​NOTE: If a pack is not available, use some other warming method, such as a hand.
   6. Hold the heat pack on the site (buttock) for 2-4 min to warm and increase blood flow.
   7. Clean the area with an alcohol pad and allow to dry.
   8. Remove the clear cover from the red button. Peel the tab from the device and firmly stick onto the infant's fullest part of the buttocks while holding them upright.
      ​NOTE: Adjust their posture as needed to keep the tube completely vertical for proper blood collection.
   9. Press the button all the way down once and release. Set the timer for 5 min. Watch the tube fill using the mirror as needed.
   10. Blood may not appear for the first 1-2 min. Remove the device after a maximum of 5 total min or sooner if blood fills to the top indicator line before 5 min.
   11. Remove the tube from the device with a slight twist and pull down. Quickly snap the cap fully unto the tube. Invert the tube at least 10x, ensuring the blood touches the cap with each inversion to prevent clotting.
   12. Ship samples overnight after collection.
3. Whole blood sample processing
   1. Process blood samples in a biological safety cabinet.
   2. Carefully transfer the 250 µL of blood into a 50 mL conical tube containing 10 mL of 1x RBC lysis buffer. Rinse the blood collection tube with 10 mL of RBC lysis buffer until empty. Allow the cells to lyse at room temperature (RT) for 15 min.
   3. Once lysis is complete, add ~30 mL of PBS at RT to wash and spin at 430 × g for 5 min at 4 °C. Aspirate the supernatant using a vacuum, without disturbing the pellet.
   4. Continue transferring and rinsing the sample as outlined in step 1.3.2 until the cell pellet appears free of visible red blood cells. Centrifuge at 430 × g for 5 min, then carefully remove the supernatant without disturbing the pellet.
   5. For every 250 µL of blood collected, resuspend the resulting cell pellet in 2 mL of freezing media (10% DMSO in FBS) and split into two cryovials for preservation.
   6. Check cell viability by counting cells resuspended in feezing media with a hemocytometer using trypan blue exclusion.
   7. Transfer 1 mL from step 1.3.5 to each cryovial. Label appropriately with time of collection, processing date, time point, and specimen Identification.
   8. Store cryovial(s) intermediately at -80 °C in a freezing container with isopropanol. After 24 h, transfer the cryovial(s) to liquid nitrogen until ready for analysis.

2. TMPA from dried blood spots (DBS) (Figure 2)

1. On the day of processing, bring the filter paper to RT. Using a 3 mm metal puncher, perforate 2-3 circles in the filter paper where the blood went through, as centrally on the blood spot as possible.
2. Pick up each 3 mm punch with tweezers and add to a well of a 96-well plate or a low-binding PCR tube containing 20 µL of elution buffer (1x PBS, 1x Protease Inhibitor, 0.05% Tween 20). Make sure the punch is covered by elution buffer.
3. Close the tubes or cover the plate with a plastic skirt, and place them on a Digital Vortex rotating at 600 rpm for 60 min at RT.
4. At the end of incubation, carefully transfer the eluate to a new low-binding tube or a 96-well plate. Discard the tubes or plate with the filter punches.
5. Quantitate the protein content using BCA assay.
   1. Take 2 µL of each sample or BSA standard to make serial dilutions of 1:10 and 1:100 in water. Prepare samples and standards in duplicate by adding 15 µL of diluted samples or standard to 200 µL of 1x working solution. Incubate samples for 30 min at 37 °C.
   2. Read the signal at 562 nm on a plate reader. Calculate the final protein concentration based on the BSA standard slope and elution buffer background.
6. Make aliquots of 30 µL for samples at 0.5-1 mg/mL in low-binding 1.5 mL screw cap micro tubes. Freeze samples at -80 °C for later submission to be processed using TMPA service.

3. Spectral flow cytometry of RBC lysed blood (Figure 3)

1. On the day of the experiment, thaw a vial of RBC lysed cells quickly in a 37 °C water bath until one small drop of ice remains.
2. Add the cell suspension to a 15 mL conical tube containing 10 mL of prewarmed (37 °C) complete RPMI (prepared using 500 mL of RPMI 1640, 50 mL of FBS, 5.6 mL of Penicillin-Streptomycin, and 5.6 mL of glutamine supplement). Centrifuge at 400 × g for 5 min at room temperature. Carefully remove and discard the supernatant.
3. Gently resuspend the pellet in 3 mL of complete RPMI and determine cell count using an appropriate method.
4. Spin the resuspended cells at 400 × g for 5 min at room temperature. Aspirate and discard the supernatant.
5.  Adjust the cell concentration to 1 × 10⁶ cells/mL in complete RPMI. Dispense 100 µL per well (0.1 × 10⁶ cells) into a 96-well V-bottom plate. Centrifuge at 764 × g for 2 min at 4 °C and remove the supernatant.
6. Add 100 µL of Live/Dead dye stock in PBS at a 1:2,000 dilution with Fc block and monocyte block (1:100 dilution) to all samples, except the unstained control. Incubate for 15 min at 4 °C.
7. Wash with 100 µL of PBS and centrifuge at 764 × g for 2 min at 4 °C. Carefully discard the supernatant.
   NOTE: It is imperative to use Fc block and monocyte block to prevent any non-specific binding of the antibodies.
8. Add 100 µL of the surface cocktail diluted (1:200 dilution) in FACS buffer (500 mL of PBS, 2.5 g of bovine serum albumin (BSA), 2 mM EDTA, pH 8.5) to all samples, except the unstained control. Incubate for 30 min at 4 °C.
9. Wash with 100 µL of FACS buffer and centrifuge at 764 × g for 2 min at 4 °C. Carefully discard the supernatant.
10. Fix the cells by adding 100 µL of 4% paraformaldehyde to each sample. Incubate for 15 min at RT in the dark.
11. Wash with 100 µL of FACS buffer, centrifuge at 764 × g for 2 min at 4°C, and resuspend in 200 µL of FACS buffer.
12. Prepare ultra-comp beads as single-color controls. Stain the single-color control beads using the same protocol as the samples, including fixation if the samples were fixed.
13. Prepare Fluorescent Minus One (FMO) controls using the same procedure as for the samples.
    NOTE: It is important to treat the single-color controls and the FMOs in the same way as the samples. For example, if the samples are fixed after staining, the FMOs and the single-color controls should be fixed as well.
14. Run the samples, single-color controls, unstained controls, and FMO controls on a conventional/spectral flow cytometer. Analyze the data using software.
15. Set gates based on appropriate isotype and FMO controls. First, gate cells to select the leukocyte population using the forward scatter (FSC) versus side scatter (SSC) plot, which helps exclude debris and select cells based on size and granularity.
16. Following leukocyte selection, gate cells to exclude doublets using a plot of forward scatter area (FSC-A) versus forward scatter height (FSC-H).
    NOTE: This ensures the selection of single cells (singlets), with cells falling along the diagonal considered as singlets, while doublets and clumps are excluded.
17. Gate cells for viability using a viability dye to exclude dead cells, ensuring that the subsequent analysis includes only live cells.
18. After these initial gating steps, further gate live single cells for specific lineage markers to subcategorize different immune cell populations. Divide the live single cells into CD33⁺ myeloid cells and CD33^- lymphocytes based on CD33 expression.
19. Within the CD33⁺ population, differentiate CD14⁺ monocytes and identify CD11c⁺ dendritic cells (DCs) as cells expressing CD11c but not CD14.
20. Within the CD33^- lymphocyte population, gate CD3⁺ T cells and sub-gate to identify CD4⁺ and CD8⁺ T cell subsets.
21. Additionally, identify CD56⁺ natural killer (NK) cells by CD56 expression, and gate CD19+ B cells by CD19 expression.

4. scRNA-seq of RBC lysed blood (Figure 4)

1. Thaw one vial of cryopreserved RBC lysed cells in a 37 °C water bath. Add the sample to 10 mL of T-cell media in a 15 mL conical tube. Centrifuge at 430 × g for 5 min at 4 °C. Discard the supernatant.
2. Add another 10 mL of 1x PBS to the sample. Centrifuge at 430 × g for 5 min at 4 °C. Discard the supernatant.
3. Count the cells and assess viability with trypan blue. If viability is less than 70%, proceed to dead cell removal with Dead Cell Removal Kit.
   1. Dead cell removal
      1. Dilute the 20x Binding Buffer Stock using sterile, double-distilled water to obtain a 1x working solution.
      2. Suspend the cell pellet in 100 µL of dead cell removal MicroBeads per 1 × 10⁷ total cells. Mix thoroughly and incubate for 15 min at room temperature (RT).
      3. Insert the magnetic separation column into the appropriate magnetic stand. Rinse the column with 500 µL of 1x Binding Buffer to equilibrate before use.
      4. Bring the cell volume to 500 µL with 1x binding buffer. Apply the cell suspension to the column and collect the flowthrough in a new 1.7 mL microcentrifuge tube.
      5. Wash 2x with 500 µL of binding buffer. Collect the flowthrough in the same microcentrifuge tube.
      6. Centrifuge the eluate at 300 × g for 10 min. Discard the supernatant.
   2. Count the cells and assess their viability with trypan blue.
   3. Resuspend 20,000 viable cells in 40 µL of PBS with 0.04% BSA in a new 1.7 mL microcentrifuge tube.
   4. Send the cells on ice immediately to a genomic core facility for library generation and sequencing. Collect the sequencing results in the format of Cell Ranger outputs.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH 8.0	Thermo Fisher Scientific	15575-038
1x RBC Lysis Buffer	Invitrogen	3177165
BCA Protein Assay Kit	Abcam	ab287853
BD Microtainer MAP Microtube	Fisher Scientific	22-253-270
Bel-Art Cryo-Safe -1°C Freeze Controller	Millipore Sigma	41122800
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A9647-100G
Brilliant Ultra Violet 496 Anti-Human CD3	Thermo Fisher Scientific	364-0038-42
Brilliant Ultra Violet 661 anti-Human CD11c	BD BioScience	612968
Brilliant Vil750 Anti-Human CD14	BioLegend	367135
Brilliant Violet 570 Anti-Human CD8a	BioLegend	301038
Brilliant Violet 711 Anti-Human CD56	BioLegend	318336
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail	Millipore Sigma	11836170001
Dead Cell Removal Kit	Miltenyi Biotec	130-090-101
Dimethyl Sulfoxide (DMSO)	Thermo Fisher Scientific	BP231-100
Dulbecco’s Phosphate buffered saline (DPBS), no calcium, no magnesium	Thermo Fisher Scientific	14190144
Fetal Bovine Serum (FBS)	Gibco	A5256801
Fisherbrand Digital Vortex Mixer	Thermo Fisher Scientific	02215418
Fixation Buffer	BioLegend	420801
GlutaMAX Supplement	Gibco	35050061
MACS MultiStand	Miltenyi Biotec	130-042-303
MS columns	Miltenyi Biotec	130-042-201
Name	Company	Catalog Number
OctoMACS Separator	Miltenyi Biotec	130-042-109
Pacific Blue Anti-Human CD4	BioLegend	344619
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140-122
RB780 Anti-Human CD33	BD Biosciences	755606
Rierdge 3mm Metal Puncher	Rierdge	sdd231031pc-01
RPMI 1640 Medium	Gibco	11875-093
Spark UV 387 Anti-Human CD19	BioLegend	302289
Tasso Collection Kits	Tasso	N/A
Thermo Scientific Screw Cap Micro Tubes	Thermo Fisher Scientific	14-755-287
Whatman 309 Proteinsaver	Cytiva	41111700
Zombie NIR Fixable Viability Kit	BioLegend	423105
Software and packages
Adobe Illustrator	2024
bbknn	1.5.1
FlowJo	10.8.1
GraphPad Prism	9.4.0
Name	Version
Scanpy	1.9.1
Srublet	0.2.3
Commercial assays
Chromium Single Cell 5’ Kit	10X Genomics	PN-1000263
Olink Reveal or Target 96 Inflammatory panel	Olink	https://olink.com/products/olink-target-96