Paper-based fluorescent assay for blood typing and antibody titer determination using long-term ambient-stored bioengineered RBCs

Domain: chemistry
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
Methods for accurate, rapid blood typing and antibody titration at the point of care are constrained by cold-chain dependent red blood cell (RBC) reagents, time-consuming serial dilutions, and subjective visual interpretation. To overcome these limitations, here we develop a method based on bioengineered red-blood cells. We engineer hemoglobin-depleted RBC membranes that preserve ABO surface antigens, label them with fluorescent nanoparticles, and lyophilize them, demonstrating at least 2 years of stability at room-temperature. We capture hemagglutination on a dual-layer paper pad and quantify it by fluorescence in approximately 8 minutes, eliminating the need for serial dilution. We conduct a registered clinical trial (ChiCTR2300078864) and benchmark performance against the gel microcolumn assay. In a clinical cohort (n=641), ABO typing achieves 100% concordance. In antibody-titration samples, quantitative titers agree with the gel microcolumn assay (coincidence 80.75%, κ=0.8623). In summary, we develop a cold-chain-independent, handheld paper-based assay that enables simultaneous ABO grouping and quantitative antibody titration with clinical-grade performance, supporting scalable manufacturing, cross-site standardization, and transfusion decision-making in emergency and resource-limited settings.

Introduction
Since the early 20th century, the discovery of blood types has formed the foundation of homologous transfusion, which aims to prevent fatal hemolytic reactions and ensure blood safety in allogeneic transfusions1–3. Landsteiner’s rule states that in the ABO blood group system, an individual’s serum will always contain antibodies against the antigens that are absent from their own red blood cells, and will not contain antibodies against the antigens that are present on their red blood cells. This principle underpins the dual verification mechanism of conventional ABO blood typing4–6. In forward typing tests, commercial anti-A and anti-B antibodies are used to detect the presence of A or B antigens on RBCs, while reverse typing employs standard A- or B-type RBCs to identify anti-A or anti-B antibodies.

Antibody titration quantifies the concentration of specific antibodies relative to blood type antigens in a blood sample. This test is clinically valuable for several reasons: (1) Type O blood lacks both A and B antigens but contains anti-A and anti-B antibodies, which can lead to hemolytic transfusion reactions if present at high titers7. (2) In hematopoietic stem cell (HSC) and organ transplants, monitoring ABO blood type changes and adjusting transfusion strategies based on real-time antibody titration can prevent adverse outcomes8,9. (3) High-dose intravenous immunoglobulin (IVIG) can cause hemolysis in recipients with A, B, or AB blood types10. (4) Blood type incompatibility between a mother and her newborn may lead to hemolytic disease of the newborn (HDN)11,12.

Traditional methods for blood typing and antibody titration primarily rely on tube testing and GMA13. Tube testing, though simple, is time-consuming and subject to observer variability. GMA uses gel columns and a centrifuge to assess antibody presence based on RBC sedimentation, offering greater accuracy but requiring complex equipment and skilled operators, making it less suitable for time-sensitive or resource-limited settings, such as emergencies, primary care, or battlefield conditions. Paper-based diagnostics (PADs) have emerged to address portability and speed. Commercial formats include consumer agglutination cards and professional GLIF-type tests, which deliver rapid ABO/Rh results but remain qualitative and restricted to forward typing. Point-of-care (POCT) blood typing has attracted considerable attention, and a number of paper- and fiber-based platforms have already been reported. Li et al. developed a paper-based blood typing device that reports the patient’s ABO/RhD type “in writing,” demonstrating that simple capillary-driven paper cards can support clear visual readout outside conventional laboratories14. Guan et al. later introduced a barcode-like paper sensor for smartphone-assisted ABO typing, showing the feasibility of coupling paper cards with digital imaging for semi-quantitative diagnostics15. On the bioactive paper side, Yeow et al. established a paper-based assay for red blood cell antigen typing using the indirect antiglobulin test, thereby extending paper platforms to secondary blood group phenotyping under IAT conditions16. Hertaeg et al. reported a rapid droplet-wicking paper method for ABO blood typing, further simplifying on-paper handling and reducing assay time17. Manderson et al. developed droplet-based paper assays with laser incubation for blood group antibody screening, and subsequently showed that laser-assisted paper platforms can accelerate the detection of red cell alloantibodies in human samples18,19. Several other groups have proposed related POCT-oriented formats. Songjaroen et al. designed a simple, low-cost portable paper-based ABO blood typing device for point-of-care testing20. Chomean et al. reported a paper-based device combined with image-based high-throughput detection for phenotyping minor blood groups, including C, c, E, e, and Mia21. Min et al. recently presented a microfluidic cotton-thread analytical device for rapid and easily identifiable blood typing22. Collectively, these studies demonstrate that paper and related porous substrates can support POCT-compatible blood group testing, including secondary blood group phenotyping, when used with fresh red blood cell reagents. However, existing platforms generally rely on short-lived cell reagents with limited ambient stability and are not designed to provide long-term preserved red cell reagents that simultaneously support rapid ABO reverse grouping and quantitative IgM antibody titration.

Several research teams have attempted to develop freezing methods for rare blood type RBCs23,24. However, progress has been limited due to the challenge of preventing ice crystal formation in the cytoplasm during freezing, which causes RBC membrane rupture during rehydration. Furthermore, prolonged freezing increases the likelihood of ice nucleation, which can alter the structure of membrane proteins structures, reducing their ability to bind to antibodies25,26. Thus, preserving the antigenicity of RBCs for antibody testing remains a significant challenge.

Antibody titration typically involves serial dilution of serum, followed by mixing each dilution with antigen-positive RBCs to determine the highest dilution at which agglutination occurs4. Despite its use, this method has limitations: (1) Sensitivity and precision, especially when the antibody concentration falls between two dilution points; (2) Resource and time consumption; (3) Cumulative dilution errors.

To address these limitations, we propose a rapid method for detecting IgM anti-A and anti-B antibodies using bioengineered RBCs and fluorescent labeling. The key innovation lies in the modification and freeze-drying of RBCs to create long-lasting RBC membranes that preserve functional antigens. RBCs are converted into RBC membranes using a gentle punching solution, which removes cellular components like hemoglobin while preserving the antigenicity. This process, combined with freeze-drying, allows long-term storage at room temperature. Fluorescent nano-labeling technology enables agglutination to be visualized and analyzed quantitatively using a fluorescence analyzer, eliminating the subjectivity of visual interpretation. We developed a paper-based fluorescent assay (PFA) based on agglutination principles, in which antibody level correlates with the size and number of agglutinated clumps. Blood typing and antibody titration are completed simultaneously in less than 8 min, meeting point-of-care testing needs.

Following establishment of this method, we conducted a clinical trial (ChiCTR2300078864) to assess assay performance. The RBC bioengineering approach is extensible to the storage of rare blood type antigens and is adaptable for membrane protein preservation. These innovations reduce operational complexity and equipment dependency, providing a solution for rapid testing in specialized environments. More importantly, the fluorescent labeling system enables precise quantification, presenting a potential breakthrough for IgG/IgM antibody quantitative detection techniques.

Results
PFA design for BG-Ab testing
Our research focuses on developing a POCT assay for BG-Ab analysis (Fig. 1). Traditional BG-Ab testing relies heavily on fresh RBC reagents, requiring cold chain storage. These reagents induce agglutination of RBCs by specific BG-Ab. Major blood group antigens such as ABO, Rh, Kell, and Duffy are inherent components of the RBC membrane, rather than being adsorbed from plasma onto the RBC surface (e.g., Lewis system)27, and storage-related damage can compromise antigenicity28. RBCs consist of a cell membrane and contents such as hemoglobin and enzymes, but lack nuclei and organelles29, we hypothesized that removing the RBC contents while maintaining the membrane structure would extend the storage life of RBC reagents. To test this, we use a mild punching solution to release RBC contents and prepare RBC biomembranes (RBCbm), preserving the antigenicity of the RBC membrane surface.

Since removing hemoglobin eliminates color indication on RBCs, we label the RBCbm with red FNPs using click chemistry, creating nano-markers to visualize and monitor agglutination. The resulting RBCbm labeled with FNPs is referred to as RBCbm@Fluo. The design allows for the separation of RBC-based clots from free ones, making it possible to directly read the agglutination extent via fluorescent signals. Thus eliminating subjective interpretation and bypasseing the lengthy dilution process (Fig. 1a). Furthermore, freeze-drying enhances the practicality of RBCbm@Fluo. After freeze-drying, the reagent can be stored at room temperature for extended periods, making it ideal for emergency settings, such as ambulances, field environments, aircraft, and ships. It is also suitable for laboratory use. As shown in Fig. 1b, RBCbm@Fluo works based blood agglutination principles. When a test sample containing BG-Ab is added, an antigen-antibody reaction occurs, resulting in visible aggregates or precipitates. The size and number of aggregates correlate with antibody concentration.

To facilitate on-site BG-Ab testing, we developed a paper-based fluorescent assay (PFA) that integrates the RBCbm@Fluo (Fig. 1c). The device, constructed using 3D printing, offers significant advantages for producing cost-effective prototypes. RBCbm@Fluo is freeze-dried onto the reaction zone’s wells, and unaggregated RBCbm@Fluo is removed via on-site filtration before result reading. The filtration membrane consists of two layers: an upper pre-filter that retain larger aggregates, while the lower fine filter membrane that captures smaller aggregates. Additionally, a location well is included for sample positioning and calibration, aiding auto-focusing and image correction. As a result, the concentration of blood group antibodies can be precisely quantified using a portable fluorescence analyzer.

The current focus of the PFA platform is on detecting IgM anti-A and anti-B antibodies (Fig. 1d). Additionally, the platform can be adapted to detect IgG antibodies by adding anti-human globulin (AHG) reagents and incubating at 37 °C. This expanded application is particularly useful for diagnosing hemolytic disease of the newborn (HDN), irregular antibody screening, and direct/indirect Coombs’ tests, thereby providing a broader diagnostic toolset for clinical applications. In future developments, we plan to enhance the PFA’s performance by integrating automation. We aim to integrate a rotating disk with an automated manipulator, which will increase sample processing capacity and improve detection efficiency (Fig. 1e). For instance, a single disk could accommodate 32 samples, enabling up to 240 results per hour—more than a 30-fold increase in efficiency.

Preparation and evaluation of RBCbm@Fluo
Conventionally, RBC bio-membrane processing and extraction methods rely on hypotonic lysis and repetitive freeze-thaw cycles. However, these methods often cause varying degrees of membrane fragmentation, which can damage surface antigens and reduce antigenicity. To address this issue, we have developed an optimized mild method. This approach uses an extremely low concentration of cell-punching reagents (e.g., Triton) to release intracellular components such as hemoglobin while preserving the integrity of the RBC membrane and avoiding antigenicity loss caused by membrane fragmentation. Our experimental results demonstrate that RBC bio-membranes prepared using this mild method exhibit significantly higher agglutination scores (59.60±0.89) when observed under a fluorescent flashlight, compared to the hypotonic lysis method (49.80±1.83) and the repeated freeze-thaw method (41.20±1.64) (Supplementary Table 1). Detailed scoring for different titers is provided in Supplementary Table 2, based on AABB guidelines30–32. Imaging flow cytometry analysis confirmed that RBCbm prepared using the mild punching method maintains a clear and uniform cell-like morphology, similar to that of the original RBCs (Supplementary Fig. 1), demonstrating the superiority of this method in preserving RBC bio-membrane integrity.

The color of RBCs, derived from hemoglobin, is often used as an indicator in clinical tests involving RBC reagents. After hemoglobin release, we introduced FNPs instead of commercially available gold nanoparticles or latex beads to modify the RBC bio-membrane as nano-markers (Fig. 2a). This was aimed to achieving high sensitivity and accurate quantification. We selected 200 nm-sized spherical red FNPs, with carboxyl groups on their surfaces. These FNPs exhibited excellent fluorescence stability with an excitation wavelength of 365 nm and an emission wavelength of 610 nm, ensuring minimal interference from ambient visible light (Supplementary Fig. 2a). Transmission electron microscopy (TEM) analysis revealed a uniformly ordered arrangement of FNPs (Supplementary Fig. 2b). A simple and rapid click reaction formed stable peptide bonds between the RBCbm and FNPs, preserving the physiological and chemical properties of the RBC membrane.

To optimize fluorescence intensity and signal-to-noise ratio, labeling conditions were adjusted (Supplementary Fig. 3). TEM images for RBCbm@Fluo (Fig. 1a) demonstrated that the membrane structure was preserved, with FNPs uniformly distributed on the outer surface, supporting the feasibility of our bioengineering strategy. Supplementary Fig. 5 shows that, when excited by a UV fluorescent flashlight, the particles emit bright red fluorescence, which is also visible to the naked eye at high concentrations (Supplementary Fig. 4).

We conducted a comparative analysis of the morphological characteristics of RBCs, RBCbm, and RBCbm@Fluo using scanning electron microscopy (SEM) (Fig. 2b). RBCs exhibited their typical biconcave disc shape, while RBCbm resembled deflated balloons, with cellular contents released but the membrane structure preserved. Due to the stretching effect of the FNPs, the RBCbm adopted various shapes and sizes. (Supplementary Fig. 5). Atomic force microscopy (AFM) results supported these observations (Supplementary Fig. 6). The phospholipid bilayer provides structural support for the membrane, maintaining cellular stability and integrity. Confocal microscopy analyses confirmed that RBCbm@Fluo preparation introduced visible alterations in the phospholipid bilayer (Supplementary Fig. 7a), membrane proteins (e.g., CD47) (Supplementary Fig. 7b), and cytoskeletal elements such as microtubules and microfilaments (Fig. 2c). Although there was mild structural disruption in the cytoskeleton, the overall cell morphology remained intact.

Dynamic light scattering (DLS) measurements showed that the hydrodynamic diameter for RBCbm and RBCbm@Fluo were 7.04±0.37 μm, 3.40±0.18 μm, and 4.00±0.08 μm, respectively. The slight increase in size for RBCbm@Fluo can be attributed to the attachment of FNPs (Fig. 2d). Zeta potential measurements revealed values of −30.43±1.14 mV for RBCs, −50.63±5.30 mV for RBCbm, and −47.60±5.17 mV for RBCbm@Fluo (Fig. 2e). Flow cytometry analysis indicated that the average recoveries were 93.78±1.24% for RBCbm and 85.23±2.15% for RBCbm@Fluo to the origin RBC (Fig. 2f), indicating the feasibility of large-scale preparation and future applications.

In summary, since our bioengineering approach induces certain changes in the shape, mechanical properties, and size of the RBCs, we successfully developed a RBC bio-membrane-based substitute for standard RBC reagents, providing a reliable, sensitive, and robust platform for blood type antibody detection and other diagnostic applications.

Next, we evaluated the performance of RBCbm@Fluo by examining several key aspects, including antigenicity, specificity, antigen strength, and affinity. Commercial standard human RBC reverse typing reagents were used as the starting reagents for RBCbm@Fluo preparation and served as positive controls. The agglutination induced by the blood type antigen-antibody reaction was similar between RBCs and RBCbm@Fluo (Fig. 2g–i), with the total agglutination score matching closely between the two (Fig. 2j). Detailed scores at different titers are provided in Supplementary Table 3. Notably, the agglutination status was easier to distinguish with RBCbm@Fluo when excited by a fluorescent flashlight and observed with the naked eye. The degree of agglutination varied with different antibody titers, as observed both visually (Fig. 2h) and through fluorescence imaging (Fig. 2i). These results indicate that the antigenicity and specificity of RBCbm@Fluo are at least comparable to those of the RBC reagent. The antigenicity, antigen strength, and specificity of RBCbm@Fluo also comply with the “Technical Review Guidelines for Registration of Human Red Blood Cell Reverse Typing Reagents” issued by the National Medical Products Administration of China, as shown in Supplementary Tables 4–6.

Flow cytometry analysis revealed a reduction in the expression levels of CD47 and blood group A antigen to approximately 60% and 51%, respectively (Fig. 2k, l). We attribute this reduction to two factors: the pore-forming process during RBCbm@Fluo preparation and the labeling of FNPs, which may have led to partial shielding of the surface antigens. Bio-Layer Interferometry (BLI) was used to measure the affinity index (Kd) for antigen-antibody reactions (Fig. 2m and Supplementary Fig. 9). Although significant reduction between RBCbm@Fluo (2.31×10-8) and RBCbm (7.00×10-8) was observed, the fact that they are of the same order of magnitude might indicate that FNPs labeling resulted in a slight injury to the binding affinity. Despite the minor unavoidable damage, the results in Fig. 2 confirm that RBCbm@Fluo retains the necessary characteristics for effective use in reverse typing and antibody titration determination.

To ensure batch-to-batch consistency, imaging flow cytometry was employed to assess RBCbm@Fluo count, membrane surface antigen expression levels, and degree of aggregation as part of quality control, with the coefficient of variation regulated to remain below 15%.

In addition, we validated the feasibility of RBCbm@Fluo for detecting antibodies beyond ABO by testing secondary blood group systems and IgG-class antibodies. We assembled a panel covering Rh (D, C, E, c, e), Kell (K), Duffy (Fya, Fyb), Lewis (Lea, Leb), and MNS (M, N), using commercially available irregular-antibody screening RBC reagents as source materials to prepare RBCbm@Fluo. As shown in Supplementary Fig. 10A, RBCbm@Fluo exhibited the expected antigen profiles. In Supplementary Fig. 10B, agglutination patterns and intensities for the IgG panels (Rh, Kell, Duffy) and the IgM panels (Lewis, MNS) closely matched those of standard commercial reagents, indicating preservation of the relevant antigenic determinants and effective recapitulation of reactivity across multiple systems. Collectively, these results support the feasibility of RBCbm@Fluo for detecting IgG antibodies and secondary blood group systems.

Freeze-dried preservation of RBCbm@Fluo
To further expand the practicality of RBCbm@Fluo, freeze-drying was considered for the long-term ambient storage instead of the typical liquid state used for commercial reagent RBCs (Fig. 3a, b). The mass of RBCbm was measured to be c.a. 44% of the normal RBCs (Fig. 3c), namely, the majority of the H2O might have been removed along with the hemoglobin. This greatly reduced formation of intracellular ice crystals and the membrane rupture during rehydration. A detailed freeze-drying procedure was provided in Supplementary Fig. 11 and Supplementary Table 7. After freeze-drying, RBCbm@Fluo appeared as white, uniform, loose, and power-like substance with an average moisture content reducing to (4.37±0.32)%, meeting the requirements for effective freeze-drying (Fig. 3b). When rehydration by gently shaking in sterile water, it rapidly dissolved into a clear liquid without unexpected aggregation and showed uniform fluorescence under UV light. The microscopic morphology of RBCbm@Fluo after freeze-drying and rehydration is shown in Fig. 3d, e. No noticeable detachment of the fluorescent beads, indicating that the freeze-drying process did not harm the FNPs.

Next, we evaluated the functionality of RBCbm@Fluo post freeze-drying. Nano-flow cytometry revealed that freeze dry did not significant damage to membrane protein expression of CD47 and blood group A antigen (Fig. 3f, g). Agglutination performance of rehydrated RBCbm@Fluo was also assessed, showing no significant differences compared to the original (Fig. 3h). Figure 3i shows that the fluorescence intensity of freeze-dried RBCbm@Fluo remained stable when stored at RT for 1 year, with a decrease of no more than 5% could by the end of the second year.

We further calculated the agglutination scores of RBCbm@Fluo (Fig. 3j and Supplementary Table 8). Results showed the liquid RBC reagent stored at 4 °C became unusable after three months due to severe hemolysis. If RBCbm@Fluo were stored as a liquid at 4 °C, it would experience a c.a. 20% decrease in agglutination scores within three months, agreeing with the quality guarantee period for commercial RBC reagents. Further, it showed a c.a. 70% decrease 6 months later, corresponding to the complete loss of function in agglutination. In contrast, the freeze-dried RBCbm@Fluo maintained stable detection performance throughout a 2-year storage period (Fig. 3j), with less than 10% in agglutination score. In summary, freeze-drying extends the shelf life of RBCbm@Fluo to 2 years, preserving antigenicity and agglutination performance, making it a reliable substitute for commercial RBC reagents.

Establishment of BG-Ab analysis method
We developed a paper-based POCT device for on-site BG-Ab analysis with high accuracy. To optimize the device’s design, we explored three design: baffle type, push-pull type, and flip type (Supplementary Figs. 12 and 13). The flip-type was selected as the optimal choice to minimize operational errors. The device consists of two distinct zones: a foldable reaction zone and a filtration zone (Fig. 4a). The front view (Fig. 4b) shows three reaction wells in the reaction zone and corresponding filtration wells below in the filtration zone. Freeze-dried RBCbm@Fluo is uniformly distributed at the bottom of the reaction wells, where rehydration occurs, and BG-Ab-induced agglutination takes place.

Once the agglutination process is complete, the operator folds the reaction zone, allowing the mixture to flow into the filtration zone under gravity. To eliminate background signals from unaggregated free RBCbm@Fluo, the filtration zone consists of four layers, a filtration membrane, a super-hydrophilic pad, an elastic glass fiber pad, an absorbent pad (Fig. 4c). The first layer separates blood clots from unagglutinated RBCbm@Fluo, while the second layer accelerates filtration. The third layer provides lateral capillary action, and the fourth layer captures the filtrate.

We tested nylon membranes with pore sizes ranging from 1 to 100 μm for refined filtration, selecting a 1–2 μm-sized membrane that effectively captured all visible aggregates based on SEM results (Supplementary Fig. 14). However, a single layer led to clogging due to larger aggregates, so we incorporated a 5 μm pore-size glass fiber pre-filter to trap larger aggregates (Fig. 4d, e). This multi-layer design efficiently separates aggregates relevant for analysis, enhancing sensitivity and precision of fluorescence detection.

The operational process of the PFA is shown in Fig. 4f. First, 60 μL of plasma is added to the reaction zone, followed by gentle shaking to ensure thorough mixing. After five minutes, the operator flips the bottom cover, allowing the liquid to flow into the filtration zone. The liquid passes through both the pre-filter and fine particle filter, retaining aggregates while removing unaggregated RBCbm@Fluo. As shown in Fig. 4g, the fluorescence intensity of the aggregates likely correlates with the antibody concentration, and a fluorescence analyzer measures the total intensity to indicate both the blood group and antibody titer.

We further validated the separation performance of the pre-filter and fine particle filter through confocal microscopy (Fig. 4h). Standard BG-Ab samples diluted in serial dilutions were analyzed using the PFA. The results confirmed that the pre-filter primarily captured larger aggregates, while the fine particle filter trapped smaller ones. This dual-layer filtration design effectively separates aggregates from non-aggregated RBCbm@Fluo, enhancing BG-Ab detection accuracy.

Next, we optimized key parameters, such as dilution factor, RBCbm@Fluo concentration, sample volume, reaction time, and washing buffer volume. Supplementary Fig. 15 gave the direct fluorescence readout using the standard reagent. Taking the titer distribution of the population worldwide into consideration33–36, the dilution factor of 8 was identified to ensure that the linear range would locate in the most needed region, even if there was some loss in sensitivity. Then we used the slope of the linear regression equation to evaluate the optimal parameters, with a higher slope indicating a stronger dependence of fluorescence intensity on the special amounts of antibodies. The variable of sample volume was optimized completely accordingly. And 60 μL was selected out (Fig. 5b). For the dosage of RBCbm@Fluo (Fig. 5a), it was obvious that 200 mg/L gave the higher slope than 100 mg/L, but 100 mg/L was selected for its linear correlation and cost-effectiveness. So was the choice for the volume of washing buffer 150 μL (Fig. 5d). As to the reaction time, 5 min and 10 min gave nearly identical results, so 5 min was selected for efficiency (Fig. 5c).

We tested the PFA’s performance by differentiating standard substance with IgM anti-A antibody ranging from 1:16 to 1:512, yielding a strong linear regression (r2=0.98) (Fig. 5e). Fluorescence intensity correlated with antibody titer within this range, and UV light images provides intuitive evidence (Fig. 5f). So was the case for IgM anti-B antibody (Supplementary Fig. 16). Confocal microscopy images of the agglutinates on the filtration membrane showed that as the titer increased, both the quantity and density of agglutinates increased (Fig. 5g), supporting the superior performance of the PFA within the linear.

We further tested the robustness of the PFA by simulating common interference factors: hemolysis, lipemia, and jaundice. Samples with hemolysis rates of 0.08% (non-hemolytic), 0.8% (hemolytic), and 8% (highly hemolytic). The results showed that hemolysis up to 8% had no significant impact on detection (Fig. 5h). We also simulated lipemia and jaundice by adding cholesterol and bilirubin to the plasma. While lipemia at levels ≥9 mmol/L weakened agglutination (Supplementary Fig. 17), normal cholesterol levels (below 6 mmol/L) did not affect results (Fig. 5i). Of course, such phenomenon also occurs in commercially available RBC reagents. Bilirubin levels below 600 had no significant effect on the test results (Fig. 5j), demonstrating the PFA’s resilience to common clinical interferences.

Clinical evaluation of the PFA assay
We obtained clinical plasma samples from a hospital and employed the as-prepared PFA for blood typing and antibody titer determination. Geographic distributions of the subjects are detailed in Supplementary Fig. 18. Routine blood typing methods used in the hospital include the tube method and the gel microcolumn agglutination (GMA). While the tube method is considered a clinical gold standard, its accuracy and consistency can be influenced by manual operation and subjective interpretation. In contrast, GMA is highly automated, with standardized the gel microcolumn cards, minimizing operational errors and improving reproducibility. To compare the PFA with GMA, a double-blind clinical trial was conducted, registered as ChiCTR2300078864. Our present clinical data mainly reflect Chinese Han donors from Fujian and do not systematically cover rare antigen variants.

Figure 6a presents the representative blood group reverse typing results for Types A, B, AB, and O using the PFA, while Fig. 6b displays images under UV fluorescent light. Threshold values for positive and negative results were established using 216 clinical samples previously determined by the GMA. Receiver operating characteristic (ROC) curve and area under the curve (AUC) analysis determined optimal cutoff values: 998 a.u. for IgM anti-A antibodies and 987 a.u. for IgM anti-B antibodies (Fig. 6c). At these thresholds, specificity and sensitivity reached 100% (Fig. 6d).

Next, 641 clinical plasma samples with unknown blood types were tested, recording fluorescence intensity values. Results revealed 188 Type A, 143 Type B, 48 Type AB, and 262 Type O, yielding a concordance rate of 100% (Fig. 6e and Supplementary Tables 9 and 10). These findings demonstrate that the PFA exhibits excellent accuracy in blood group typing.

To determine thresholds for antibody titer detection, 245 samples were tested to cover each titer interval with a target of at least 50 per interval, confirmed by GMA. These samples were tested using the PFA, and the corresponding fluorescence intensity values were recorded (Supplementary Fig. 19a, d). ROC and AUC analysis yielded cutoff values for IgM anti-A antibodies: 636 a.u. (1:16), 1193 a.u. (1:32), 2757 a.u. (1:64), 4337 a.u. (1:128), and 4896 a.u. (1:256) (Fig. 6f). Similarly, for IgM anti-B antibodies, 599 a.u. (1:16), 1175 a.u. (1:32), 2777 a.u. (1:64), 4467 a.u. (1:128), and 4916 a.u. (1:256) (Fig. 6g).

We then measured antibody titers of 388 clinical samples using both the PFA and GMA in parallel. Fluorescence intensity distributions for total IgM anti-A and anti-B titers are presented in Fig. 6h, i (left), respectively. Minimal overlap between adjacent titer levels highlights the PFA’s precision. The Pearson correlation coefficients between the PFA and GMA were r²=0.8813, r=0.939 (anti-A) and r²=0.8788, r=0.937 (anti-B), indicating strong positive correlations (Fig. 6h, i right).

As shown in Table 1, the PFA demonstrated strong reliability and accuracy, with an overall coincidence rate of 80.75%, Cohen’s Kappa of 0.8623, and a Pearson correlation coefficient of 0.8853. And 100% were within ±1 twofold-dilution step. For anti-A and anti-B titers, coincidence rates were 81.03% and 80.47%, Cohen’s Kappa values were 0.8642 and 0.8601, and Pearson correlation coefficients were 0.8726 and 0.9029, respectively. Concordance rates for different blood types were similarly high: type A (anti-B), 81.37%, type B (anti-A) 74.29%, type O (anti-A) 84.77%, type O (anti-B) 80.79% (Fig. 6j–m and Table 1). At the clinically relevant cutoff of 1:128 (low- vs high-titer), the 2×2 confusion matrix (TP=26, FN=4, FP=12, TN=467) yields a PPA of 86.7% (26/30) and an NPA of 97.5% (467/479) between PFA and GMA (Supplementary Fig. 20).

Concordance differences were largely due to the absence of a fully quantitative clinical reference. Antibody titers were compared against the semi-quantitative GMA, whose discrete dilution endpoints introduce subjectivity near cut-offs. In our dataset, 19.25% of samples at the clinical decision threshold were discordant; 85% of these fell within “threshold ±10%,” consistent with borderline calls inherent to semi-quantitative reads rather than PFA error. Methodological behavior also differed across titer ranges: the PFA detected weak, low-titer agglutination that can be under-recognized by GMA, whereas high-titer endpoints were occasionally assigned one dilution step apart—within the 1–2 step variability reported for traditional titrations. Overall, the PFA achieved high ABO typing accuracy and yielded quantitative, fluorescence-based titers with satisfactory agreement to GMA, reducing subjective interpretation around borderline results.

Methods
Ethical statement
The study was approved by the Institutional Review Board (IRB) of The 909th Hospital (Southeast Hospital of Xiamen University), School of Medicine, Xiamen University (Ethical approval number: L2023002), where all clinical samples were collected. Written informed consent was waived because only leftover clinical specimens were used with no additional risk, and all data were handled under a double-blinded design to protect privacy; the study was approved by the Institutional Review Board in accordance with in vitro diagnostic reagent clinical trial technical guidelines. Written consent for publication was not required.

Preparation of RBCbm
RBC suspensions were obtained from Changchun Bioxun Biotechnology Co., Ltd. The blood was sourced from healthy adult donors who underwent screening for five markers in compliance with national regulations, including transaminase, hepatitis B, hepatitis C, syphilis, and HIV. A1 cells contained both A and A1 antigens, B cells contained B antigens, and O cells contained neither A nor B antigens. The RBC suspension was centrifuged at 1000×g for 10 min to separate the packed red blood cells from the preservative solution. The supernatant was discarded, leaving behind the packed RBCs, which were then subjected to extensive washing and lysis steps to isolate the RBCbm. The RBCbm were prepared using the following three methods: (1) Hypotonic lysis method: 1 mL of RBC precipitate was suspended in 10 mL of 0.25× PBS (Wuhan Servicebio Technology Co., Ltd), causing the cells to swell and rupture, releasing hemoglobin and leaving behind the RBC membrane. The suspension was then washed 3–5 times with an equal volume of distilled water to separate the RBCbm. (2) Repeated freeze-thaw method: 1 mL of RBC precipitate was frozen at −30 °C and then rapidly thawed. This freeze-thaw cycle was repeated 3–5 times, after which the ruptured RBCs were centrifuged. The RBCbm were then washed 3–5 times with 1 mL of distilled water. (3) Mild punching method: 1 mL of RBC precipitate was mixed with 1 mL of punching solution. The mixture was centrifuged to rupture the RBCs, and the RBCbm were separated by washing 3 times with 1 mL of distilled water.

Preparation of RBCbm@Fluo
Take 10 μL of fluorescent nanoparticles (FNPs, MF02, Madenew Biotechnology, China) (solid content: 5.03%, 200 nm, red fluorescence) and disperse them in ultrapure water. Add 200 μL of freshly prepared 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 22981, Thermo Fisher Scientific, US) solution (15 mg/mL) to the dispersion, and sonicate for 1 min to ensure uniform particle dispersion. Incubate the solution at room temperature for 30 min to fully activate the carboxyl groups on the nanoparticle surface. After activation, add 200 μg of RBCbm, prepared using the mild punching method with gentle lysis buffer. Wash with 1 mL of ultrapure water and centrifuge at 6000×g for 5 min to remove unbound materials. Finally, resuspend the engineered RBC bio-membrane (RBCbm@Fluo) in Alsever’s solution (R1016, Solarbio, Beijing, China) and store at 4 °C for future use.

Antigenicity and specificity
Taking A-type RBCs as an example, 50 μL of RBC/RBCbm@Fluo is mixed with 50 μL of IgM anti-A, anti-A1, anti-B, anti-AB, and anti-H antibodies (National Institutes of Food and Drug Control, China). The agglutination strength is then observed and recorded.

Agglutination score
Taking A-type RBCs as an example, IgM anti-A antibodies are serially diluted in a 2-fold series with dilution factors of 2, 4, 8, 16, 32, and 64. For each dilution, 50 μL of the solution is mixed with 50 μL of RBC/RBCbm@Fluo, and agglutination is visually observed at each dilution level. Based on the agglutination strength, a score is assigned following the AABB guidelines 22-24. The agglutination score for each dilution is numerically quantified according to the intensity of the reaction. The scores for all dilutions are then summed to obtain the total agglutination score.

FACS analysis
1×106 cells were harvested by centrifugation at 3000×g for 5 min and then stained with the corresponding antibodies for 20 minutes. The reaction was stopped by washing the cells twice with PBS, and the cells were subsequently analyzed using FACS (BD Biosciences, US). Data were collected and analyzed with FlowJo V10 software.

Characterization of RBCbm@Fluo
The morphology of RBC, RBCbm, and RBCbm@Fluo was observed using Atomic Force Microscopy (AFM, Bruker Dimension ICON, Germany), Transmission Electron Microscopy (TEM, Hitachi H-7800, Japan), and Scanning Electron Microscopy (SEM, Hitachi SU8220, Japan). RBC, RBCbm, and RBCbm@Fluo were stained with DIL dye (Uelandy, D4053) and CD47 (BD Bioscience, 568090), respectively, and images were recorded using a Laser Scanning Confocal Microscope (Zeiss LSM900, Germany). The hydrodynamic size and Zeta potential of RBC, RBCbm, and RBCbm@Fluo were measured using the Zetasizer Nano ZS90 system. The excitation and emission spectra of nanoparticles were characterized using a Steady-State and Transient Fluorescence Spectrometer (FLS980, Shimadzu, Japan), with fluorescence spectra recorded in the range of 200–800 nm. The number of RBC, RBCbm, and RBCbm@Fluo cells in 10 μL samples was counted using flow cytometry (BD Biosciences), and recovery was calculated accordingly.

Cytoskeleton of RBCbm@Fluo
After collecting the RBC, RBCbm, and RBCbm@Fluo samples, 1 mL of 4% paraformaldehyde (Solarbio, P1110) was added, and the samples were fixed at room temperature for 15 min, followed by three washes. Purified mouse anti-β-Tubulin (Abcam, 556321) was then added, and the samples were incubated overnight, with three washes repeated during the incubation. On the following day, FITC Goat Anti-Mouse Ig (Abcam, 554001) and Phalloidin-iFluor 594 (Abcam, 176757) were added, and the samples were incubated at room temperature for 1 h, followed by another three washes. Next, 500 μL of the samples were plated into confocal microscope wells at a density of 1×106 cells per well and incubated at room temperature in the dark for 30 min to allow the cells to settle at the bottom. Finally, cell morphology and cytoskeletal analysis were performed using a confocal laser scanning microscope (Zeiss LSM900, Germany).

Nanoflow cytometry
The expression of CD47 (PE Mouse Anti-Human CD47, BD Bioscience, 568090) and Blood Group A (PE Blood Group A, Abcam, 565385) on RBCbm@Fluo was detected using the Flow NanoAnalyzer (U30E, NanoFCM).

Bio-layer interferometry
The interaction between blood group antigens and antibodies was analyzed using Bio-Layer Interferometry (BLI, GatorPrime, China). The arrangement and procedures for the samples and probes are outlined in Supplementary Fig. 8. A Max plate was prepared, with 250 μL of PBS added to each well, totaling 24 wells arranged in three rows, with one sensor per well. Bio-WGA was diluted to 20 μg/mL. RBC, RBCbm, and RBCbm@Fluo were diluted to a concentration of 1×108 cells/mL. IgM was serially diluted from 1000 μg/mL in a two-fold gradient, resulting in seven different concentrations. After the reaction was completed, data analysis was performed using the built-in Gator software, and the results were extracted.

Observation of RBCbm@Fluo agglutination on paper
RBCbm@Fluo was previously labeled with DiI (Uelandy, D4053). Following agglutination, the pre-filter and fine particle filter of the nitrocellulose membrane were removed, cut into 5.98 mm × 5.98 mm pieces, and air-dried at room temperature for 48 hours. The samples were then placed onto glass slides for confocal imaging. Microscopic images were captured from the side where the sample was introduced. A Zeiss LSM880 Confocal Microscope was used to generate the confocal micrographs, with a 20× dry objective lens employed for imaging. Images of RBCs within the antibody-treated paper were captured as x–y images or as a series of x–y images with stepped variations in the z-direction, which were then used to construct 3D images (or z-stack images). The resolution of all images was 2048×2048 pixels, and the step width for the z-stack images was 100 μm.

Performance evaluation of RBCbm@Fluo
The formation of RBC agglutinates by RBCbm@Fluo at different antibody titers was analyzed using a confocal microscope (Zeiss LSM880, Germany). First, 1×109 A-type RBCbm@Fluo samples were taken, and DIL dye (Uelandy, D4053) was diluted at a 1:1000 ratio. Then, 5 μL of the diluted dye was added to the samples and incubated in the dark for 15 min. Afterward, the samples were washed three times to obtain DIL-labeled RBCbm@Fluo, which was then resuspended in 1 mL of buffer. Standard IgM anti-A antibody solutions were serially diluted in a 2- to 256-fold range. 100 μL of each dilution of IgM anti-A antibody solution was added to separate wells on the confocal microscope slide, with IgM anti-B antibody used as a negative control. After adding 100 μL of DIL-labeled RBCbm@Fluo to each well, the mixture was gently agitated for 5 min at room temperature to ensure thorough mixing. The reaction was then observed, and images were captured using the confocal microscope.

Secondary blood group and IgG antibody detection
Commercially available irregular antibody screening RBC reagents from Changchun Bioxun Biotechnology Co., Ltd were used as source materials to prepare engineered RBCs. Blood group antibodies covering Rh (D, C, E, c, e), Kell (K), Duffy (Fya, Fyb), Lewis (Lea, Leb), and MNS (M, N) systems were tested, with Rh, Kell, and Duffy as IgG-type antibodies, and Lewis and MNS as IgM-type antibodies. Anti-D (IgG), anti-M, and anti-N antibodies were purchased from the Shanghai Blood Center; human-derived anti-C, anti-E, anti-c, and anti-e (IgG) antibodies were kindly provided by the Guangzhou Blood Center; anti-K, anti-Lea, anti-Leb, and anti-Fya antibodies were obtained from CE-ID, and anti-Fyb antibody from Immunocor. For IgG antibody testing, 20 μL of RBCbm was mixed with 60 μL of the corresponding antibody and 50 μL of anti-human globulin (AHG), incubated at 37 °C for 15 min in a tube, and subsequently transferred to a glass slide for agglutination assessment. For IgM antibodies, 20 μL of RBCbm was mixed with 60 μL of the respective antibody and applied directly to a glass slide for agglutination assessment.

Freeze-dry of RBCbm@Fluo
RBCbm@Fluo was centrifuged to remove Alsever’s solution, and the precipitate was dissolved in freeze-dried protective fluid (FDPF). FDPF, provided by Anbio (Xiamen) Biotechnology Co., Ltd., mainly contains BSA, trehalose, Tris-HCl buffer, and antibiotics. The samples were then subjected to phase-wise freeze-drying in a freeze dryer (LGJ-S40, The Fourth Ring, China) for a total of 26 h. The detailed freeze-drying process is described in Supplementary Fig. 5. After freeze-drying, the appearance of the samples was visually inspected to confirm proper formation. An electronic balance was used to weigh an appropriate amount of the plasma freeze-dried preparation. The moisture content of the freeze-dried preparation was determined using a Karl Fischer moisture meter (Mettler Toledo C30, Switzerland) based on the Coulometric principle. The morphology of the freeze-dried RBCbm@Fluo was observed using Scanning Electron Microscopy (SEM, Hitachi SU8220, Japan).

Constructing the paper-based POCT device
(1) Layer-by-layer paper strip assembly in the filtration zone: The paper strip consists of four layers: a filtering membrane, a super-hydrophilic pad (Xinhua 347, China), an elastic glass fiber pad (Boang 1200-015, China), and an absorbent pad (Cobetter, China). The filtering membrane is composed of two components: a pre-filter (Cobetter, China) and a fine particle filter (Munktell MG1502, Sweden). Each material is cut into 29.8 mm wide rectangles using scissors. The materials are then aligned along their edges and bonded at the long edge using a hot glue gun (Deli DL5044, China). Finally, the assembled layers are cut into 29.8 mm × 5.98 mm strips using a CNC high-speed cutter (Jinbiao ZQ3500/ZQ3501, China). (2) Paper-based POCT device: We designed the device for reverse typing and antibody titration using SolidWorks to create a 3D model. The model was then printed with an epoxy resin material using a 3D printer (REMP 3D HD600, China). To prevent interference from the material’s inherent fluorescence, the surface was coated with UV paint to achieve a matte black finish. (3) Fluorescence Analyzer: The matching fluorescence analyzer was designed and manufactured by Anbio (Xiamen) Biotechnology Co., Ltd. It consists of a light source system, a photoelectric detection system, a signal amplifier circuit, a thermal printer, and a display screen.

IgM anti-A and anti-B detection using the PFA
To achieve rapid blood type antibody detection, the following steps are performed. First, 60 μL of plasma is directly added to the sample reaction well without dilution for blood type detection. For antibody titer detection, 10 μL of plasma is mixed with 70 μL of saline to achieve a dilution factor of 8. The subsequent steps are identical for both types of detection. The sample is gently mixed by the operator for 5 min to ensure uniformity. The reaction zone of the detection card is then inverted to allow the liquid to flow into the filtration zone, and 150 μL of saline is added to rinse the free RBCbm@Fluo. Finally, fluorescence intensity is measured using a portable fluorescence analyzer to determine both the blood type and antibody titer.

IgM anti-A and anti-B agglutination titers using GMA
All procedures were carried out according to the manufacturer’s instructions. A 50 μL aliquot of serially diluted serum and 50 μL of a 0.8% RBC suspension of either group A or B were added to the gel card microcolumns (ABO reverse typing card, Bioxun). The card was then centrifuged, and the titer was defined as the highest dilution showing 1+ agglutination.

Clinical evaluation
The study was preregistered in the Chinese Clinical Trial Registry (ChiCTR2300078864; first posted on 20 December 2023, http://www.chictr.org). The clinical evaluation started on 23 April 2024 and was conducted by trained healthcare professionals from the participating clinical institutions and our research team in accordance with institutional guidelines. Routine residual blood samples from adult patients (18–65 years) were eligible, including anticoagulated venous whole blood and/or serum/plasma, collected within 48 h and stored at 2–8 °C prior to testing. Samples were excluded if obtained from infants <4 months or patients >65 years, or if they showed severe hemolysis, lipemia, turbidity, contamination, or originated from patients with severe conditions that may affect blood group antibody detection (e.g., cancer, leukemia, multiple myeloma, Parkinson’s disease, liver disease, tuberculosis). Peripheral blood (~5 mL) was processed by centrifugation at 3000×g for 5 min to separate plasma from RBCs and buffy coat; plasma/serum was used for antibody-related analyses. Each sample was assigned a unique identifier and tested in random order under double-blind conditions. The primary outcomes were sensitivity, specificity, and overall concordance between the POCT method and the standard microcolumn gel card method; secondary outcomes included the kappa coefficient and related agreement statistics. The outcomes reported in the study protocol/registry and in this manuscript are consistent. The preregistered target recruitment was 3000 blood samples and 488 antibody titer samples; however, due to a phased clinical trial design, we enrolled 641 blood samples and 388 antibody titer samples across two phases. This sample-size deviation reflects the phased implementation and availability of eligible residual samples during the study period; hospital approval was obtained to use the corresponding routine clinical results. No other protocol deviations or analysis-plan changes were made.

Statistics and reproducibility
Statistical analysis was conducted using GraphPad Prism version 9.2 (GraphPad Software Inc.) and Medcalc version 23.1.7 (Medcalc Software Inc.). A P value <0.05 was considered statistically significant for all tests. Data are presented as mean values ± standard deviation. Statistical comparisons between groups are described in the figure legends. Each experiment included at least three data sets. Linear regression analysis was used for parameter optimization. Pearson’s correlation test was employed for data analysis. The Pearson correlation coefficient (r) measures the strength of the linear relationship between two variables, where a value of |r|>0.8 indicates a strong correlation. Linear regression and Pearson’s correlation were used to analyze the IgM anti-A and anti-B titers between the two techniques.

The following concordance rates were calculated:

Positive Concordance Rate (Sensitivity) = (True Positive (A) / (True Positive (A) + False Negative (C))) × 100%

Negative Concordance Rate (Specificity) = (True Negative (D) / (False Positive (B) + True Negative (D))) × 100%

Overall Concordance Rate = ((A + D) / (A + B + C + D)) × 100%

The cutoff value was determined based on the Receiver Operating Characteristic (ROC) curve and the optimal Youden index. Cohen’s Kappa (κ) was used to assess the agreement between the two methods.

No statistical method was used to predetermine sample size for laboratory experiments. For the clinical trial, sample size was calculated as follows. To meet the requirements of the study, we first designed the experiment with the following criteria: antibody titers were categorized into six groups (≤1:16, 1:32, 1:64, 1:128, 1:256, ≥1:512), with each group containing at least 30 samples. Using a significance level of 0.05 and a statistical power of 80%, the estimated total sample size required for the experiment was calculated to be between 168 and 180. Therefore, at least 360 clinical samples with unknown blood types were tested to meet the study’s requirements. No data were excluded from the analyses. The clinical trial was conducted under a double-blind design. The experiments in Figs. 2b, c, i, 3d, e, 4e, h, 5g and Supplementary Figs. 5, 6, 14 were independently repeated three times with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors developed a novel paper-based fluorescent diagnostic for blood typing (forward and reverse typing). The\nconcept uses conventional technology but rely on long-life red blood cells (RBC) treated (removal of hemoglobulin),\nflorescent tagged and freeze-dried. This manuscript has 4 sections: 1) Preparation of long life and tagged RBC; 2)\nDiagnostic concept for blood typing, 3) Test optimization and 4) clinical test. The work is impactful and presents elements of\nnovelty. However, at 41 pages of text with 8 composed Figures and 1 Table, this manuscript is way too long and unfocused.\nFurther, the authors are not clear on what is the novelty of their work nor on the current technology of paper diagnostics for\nblood typing.\nThe topic is certainly of interest to the readers of Nature Com, and the work is indeed impactful with some elements of\nnovelty. The manuscript, while long and poorly structured, is well written. I recommend considering a resubmission of this\nmanuscript for further review- once the authors have addressed the following:\n1) Cut length by at least 1/3 and preferably ½. Improve the structure of the manuscript. This is a must. Review has been long\nand painful for an expert. Normal readers will be lost.\n2) Clearly selected and highlight the novelty of this research. For me, this is the long lasting modified and freeze dried RBC\nthat preserve functional antigen, enabling blood typing application. However, shape, mechanical properties and size are\naffected.\n3) Provide an up-to-date review of the current paper diagnostics for blood typing- literature and commercials (there are 2 type\nof commercial diagnostics and a multitude of prototype). Please review the concept and fundamentals. I do not believe this\nstudy brings novelty here.\n4) Rewrite the Discussion. It is very repetitive (cut length by half), self indulging and commercial.\n5) Remove/ justify the AI aspect. While sexy and currently popular, this brings nothing new. Please review the current state\nof image analysis and automation for blood typing in recent systems.\n6) Remain scientific- and remove pre-commercialization development of test.\n7) Improve introduction. Clarify the Landsteiner rule- very confusing as presented. Clarify forward and reverse testing-\nincluding previous paper diagnostics concepts.\n8) 8 min for blood typing is very long. Current paper blood diagnostics (GLIF) do it in less than 2 minute and modern\ncommercial gel card test in 1 min. Please review claims.\n9) Better structure and analysis of secondary group blood test (Duffy, Lewis, Kell, …) and those requiring IgG antibody\n(instead of current IgM).", "reviewer_2": "This study introduces an innovative paper-based fluorescent assay (PFA) for blood typing and antibody titer measurement.\nUnlike traditional methods, it utilizes freeze-dried bioengineered red blood cells, enabling long-term ambient storage. The\nPFA, which delivers results in approximately 8 minutes without complex equipment or subjective interpretation,\ndemonstrated 100% accuracy in ABO blood typing and high concordance with standard methods in clinical tests, promising\nsafer blood transfusions in emergency or resource-limited settings. Its design also facilitates standardization and integration\nwith digital and AI-assisted analysis.\nHowever, there are come comments that could strengthen the paper.\n1. In general, the section related to membrane integrity should be reinforced. Although the results provide strong validation of\nthe system, this aspect would benefit from being revised or presented in a different manner by the authors.\n• In line 272, the sentence “TEM results for RBCbm@Fluo (Fig. 1a) confirmed the integrity of the cell membrane” is not\nentirely accurate. The TEM images provided are not sufficient to support such a statement, and it is recommended that this\nphrase be removed.\n• Line 285: Figure 2f shows significant differences between membranes, and in both the bm and bm-Fluo conditions, a well-\ndefined membrane is not observed as it is in the other samples. Similarly, in the confocal images (Fig. 2g), actin appears\ndisrupted.\n• Line 331: This statement contradicts earlier claims that the membrane remains intact.\n2. Although the paper reports 100% accuracy for ABO blood typing (which is excellent), the 80.75% concordance with the\nreference method for antibody titration is a point that requires further analysis and justification.\nIs this level of concordance acceptable in clinical practice for all antibody titration applications (e.g., in monitoring\npregnancies with Rh isoimmunization, where precision is critical)? What are the implications of the 19.25% discordance?\nAre these due to inherent methodological differences, variability in the reference methods, or limitations of the PFA in certain\ntiter ranges? The discussion should explore the causes of this discordance in greater depth and clarify whether there are\nplans to improve this rate, or if the method is considered \"good enough\" for its intended applications—with a clear rationale.\nComparing this performance with the known intra- and inter-laboratory variability of traditional methods would also\nstrengthen the argument.\n3. The paper predominantly focuses on the detection of IgM anti-A and anti-B antibodies. Although it mentions the potential\nto \"disrupt current antibody detection techniques for IgG/IgM,\" it does not elaborate on the system’s actual capability or future\nplans to detect IgG antibodies, which are critical in many clinical contexts (e.g., hemolytic disease of the newborn, delayed\ntransfusion reactions).\nIs the method equally effective for detecting IgG antibodies? If not, what are the challenges in adapting the system for IgG\ndetection? Is this approach primarily designed for a specific application where IgM detection is more relevant?\n4. While the practicality of ambient-temperature storage is emphasized, the paper does not sufficiently address the\nchallenges or considerations involved in the large-scale production of the bioengineered RBCs and the PFA devices.\nIs the bioengineering process for RBCs easily scalable to meet mass demand? Are there significant regulatory or cost-\nrelated considerations associated with the industrial production of these reagents and devices?\n5. The clinical study was conducted with a considerable number of samples; however, it is not specified whether these\nsamples came from a diverse population or a single geographic region. The prevalence of certain blood groups or antigenic\nvariants can vary significantly across populations.\nWas the method tested on samples from individuals of different ethnic backgrounds or with less common antigenic variants?\n6. Overall, it is recommended that the manuscript be reviewed carefully to correct grammatical errors, spacing issues, and\nother minor formatting inconsistencies."}, "author_response": "Response to Reviewers\nWe would like to express our sincere gratitude for the opportunity to revise our\nmanuscript entitled \"Paper-based fluorescent assay for blood typing and antibody titer\ndetermination using long-term ambient-stored bioengineered RBCs\" (Manuscript\nNumber: NCOMMS-25-43320A). We also greatly appreciate the constructive\ncomments provided by both the reviewers and the editorial team, which have been\ninvaluable in enhancing the quality of our work. We have revised the manuscript\nthoroughly, including structural reorganization, shortening by over 40%, and\nclarifications on novelty, methodology, and limitations. Due to the extensive content\nreduction, to maintain the logical flow and readability, detailed point-by-point\nrevisions are best reviewed in the clean version of the manuscript. Below, we provide\nspecific responses to each reviewer’s comment. All revisions have been incorporated\ninto the revised manuscript, with major changes highlighted in red.\nReviewer 1\nThe authors developed a novel paper-based fluorescent diagnostic for blood typing\n(forward and reverse typing). The concept uses conventional technology but rely on\nlong-life red blood cells (RBC) treated (removal of hemoglobulin), florescent tagged\nand freeze-dried. This manuscript has 4 sections: 1) Preparation of long life and\ntagged RBC; 2) Diagnostic concept for blood typing, 3) Test optimization and 4)\nclinical test. The work is impactful and presents elements of novelty. However, at 41\npages of text with 8 composed Figures and 1 Table, this manuscript is way too long\nand unfocused. Further, the authors are not clear on what is the novelty of their work\nnor on the current technology of paper diagnostics for blood typing. The topic is\ncertainly of interest to the readers of Nature Com, and the work is indeed impactful\nwith some elements of novelty. The manuscript, while long and poorly structured, is\nwell written. I recommend considering a resubmission of this manuscript for further\nreview- once the authors have addressed the following:\n1. Cut length by at least 1/3 and preferably 1/2. Improve the structure of the\nmanuscript. This is a must. Review has been long and painful for an expert. Normal\nreaders will be lost.\nResponse: Thank you for your constructive feedback. We appreciate your\nsuggestion to improve the manuscript's structure and reduce its length. Based on your\nrecommendations, we have made the following revisions:\nFigure Reduction: We have condensed the number of figures from 8 to 6.\nSpecifically, Fig. 2 and Fig. 3 have been merged, as well as Fig. 7 and Fig. 8, to\nstreamline the presentation and reduce redundancy.\nRevised Structure: To enhance clarity and improve readability, we have\nreorganized the manuscript into four distinct sections.\nLength Reduction: In accordance with your request, we have cut the manuscript's\nlength by approximately one-third, ensuring that it remains concise while retaining all\ncritical information and maintaining scientific rigor.\n2. Clearly selected and highlight the novelty of this research. For me, this is the long\nlasting modified and freeze dried RBC that preserve functional antigen, enabling\nblood typing application. However, shape, mechanical properties and size are\naffected.\nResponse: Thank you for your thoughtful and constructive feedback. We\nappreciate your suggestion to more clearly highlight the novelty of our research. As\nyou correctly point out, the key innovation lies in the development of long-lasting,\nmodified, and freeze-dried RBCs that preserve functional surface antigens, enabling\ntheir application in blood typing even under ambient storage conditions. Following\nyour suggestion, we realized that this bioengineering process does introduce changes\nto cell shape, mechanical properties, and size. We have revised the manuscript to\nfurther emphasize the fact concisely as follows:\nIntroduction: We have strengthened the description of the novelty of our\napproach, emphasizing antigen preservation and the unique advantage of long-term\nstorage at room temperature (Line 74, Page 4).\nResults (Figure 2): We now explicitly discuss the observed changes in RBC\nmorphology, mechanical properties, and size. Specifically: Line 165 (Page 7)—\nConfocal microscopy analyses confirmed that RBC @Fluo preparation introduced\nbm\nvisible alterations in the phospholipid bilayer (Fig. S7-A), membrane proteins (e.g.,\nCD47) (Fig. S7-B), and cytoskeletal elements such as microtubules and\nmicrofilaments (Fig. 2c). Although there was mild structural disruption in the\ncytoskeleton, the overall cell morphology remained intact. ……In summary, since our\nbioengineering approach induces certain changes in the shape, mechanical properties,\nand size of the RBCs, we successfuly developed ……\n3. Provide an up-to-date review of the current paper diagnostics for blood typing-\nliterature and commercials (there are 2 type of commercial diagnostics and a\nmultitude of prototype). Please review the concept and fundamentals. I do not believe\nthis study brings novelty here.\nResponse: We thank the reviewer for emphasizing the importance of situating\nour work within the broader development of paper-based blood typing diagnostics.\nComprehensive overviews of this field can be found in the reviews by Then et al.\n(Vox Sanguinis, 2015, 108(2):186–196)[1] and Ebrahimi Fana et al. (Biomedical\nMicrodevices, 2021, 23(3):34)[2], which summarize advances in paper-based\nanalytical devices for blood group phenotyping and highlight ongoing efforts toward\nminiaturization and point-of-care implementation.\nCommercially, two principal categories of paper-based diagnostics are currently\navailable. Consumer-oriented cards (e.g., EldonCard) are widely used for self-testing\nand donor screening, providing ABO/Rh results within minutes through visible\nagglutination. Professional rapid-test formats (e.g., GLIF-type devices) utilize\ncapillary flow and spreading kinetics to achieve ABO/Rh typing in roughly one\nminute. These platforms are rapid, convenient, and well established, but they remain\nrestricted to forward typing, relying on immobilized anti-A/anti-B/anti-D antibodies\nand yielding primarily qualitative results.\nIn the academic literature, various research groups have attempted to improve\nanalytical accuracy, reproducibility, and usability. Wanida Laiwattanapaisal[3]\ncombined wax printing and impregnation techniques to construct a paper-based\nanalytical device capable of simultaneously performing ABO forward/reverse typing\nand Rh determination within ten minutes using whole blood, achieving 85–96%\naccuracy relative to conventional slide testing. Abolfathi[4] optimized a\nnitrocellulose-based matrix, which required smaller sample volumes and provided\nclearer separation between agglutinated and non-agglutinated cells, thereby enhancing\nreadability and reproducibility. Gil Garnier[5] developed a rapid droplet-wicking assay\nbased on capillary absorption, detecting ABO groups within ten seconds and Rh D\nwithin twenty seconds, even with antibody dilutions of 1:2–1:8; their work also\nshowed that lower-density, thicker paper improved sensitivity. Xu[6] introduced a\nswinging microfluidic cotton-thread device, in which vigorous phosphate-buffered\nwashing removed free cells more effectively than passive diffusion, increasing\ncontrast and reducing interpretation bias. Luo Yang[7, 8] employed cotton-pulp paper\ncombined with the colorimetric indicator bromocresol green, enabling real-time\ndetection of ABO/Rh types within 30s through quantifiable color changes supported\nby a calibration algorithm. Their studies provide valuable mechanistic insights into\nhow hydrophilic polymer matrices modulate capillary dynamics and preserve\nantibody bioactivity under ambient storage. However, current efforts remain focused\non ABO forward typing, with reverse typing still dependent on fresh red blood cell\nreagents, and no fundamental technological innovations have been introduced in this\nregard.\nOnce again, we sincerely appreciate the reviewer's highly professional comments.\nThrough the aforementioned analysis, we have come to recognize with greater clarity\nthe innovative nature of our current study: it enables reverse typing and antibody\ntitration using engineered, lyophilized RBCs that preserve antigenicity at ambient\ntemperature. This distinction has now been emphasized in the revised Introduction\n( Line 54, Page 3).\n[1] Then W L, Li M, McLiesh H, et al. The detection of blood group phenotypes using\npaper diagnostics[J]. Vox sanguinis, 2015, 108(2): 186-196.\n[2] Ebrahimi Fana S, Paknejad M, Aminian M. Paper based analytical devices for blood\ngrouping: a comprehensive review[J]. Biomedical Microdevices, 2021, 23(3): 34.\n[3] SONGJAROEN T, LAIWATTANAPAISAL W. Simultaneous forward and reverse ABO\nblood group typing using a paper-based device and barcode-like interpretation [J]. Analytica\nChimica Acta, 2016, 921: 67-76.\n[4] AFSHARI P, ABOLFATHI N. A novel method for blood‐typing using nitrocellulose\n[J]. Biomedical Chromatography, 2017, 31(7): e3908.\n[5] HERTAEG M J, TABOR R F, MCLIESH H, et al. A rapid paper-based blood typing\nmethod from droplet wicking [J]. Analyst, 2021, 146(3): 1048-56.\n[6] MIN S, ZHAN T, LU Y, et al. Rapid and easily identifiable blood typing on microfluidic\ncotton thread-based analytical devices [J]. Lab on a Chip, 2023, 23(21): 4680-9.\n[7] ZHANG H, QIU X, ZOU Y, et al. A dye-assisted paper-based point-of-care assay for\nfast and reliable blood grouping [J]. Science translational medicine, 2017, 9(381): eaaf9209.\n[8] ZHANG H, LIU R, LI Q, et al. Flipped quick-response code enables reliable blood\ngrouping [J]. ACS nano, 2021, 15(4): 7649-58.\n4. Rewrite the Discussion. It is very repetitive (cut length by half), self indulging and\ncommercial.\nResponse: We thank the reviewer for this important comment. We agree that the\nDiscussion in the original version was overly long and at times repetitive, with\nlanguage that could be perceived as promotional. In response, we have substantially\nrevised and condensed the section by removing redundant descriptions, streamlining\ntechnical details, and focusing on key findings in the context of prior work. The\nrevised version is approximately half the original length, highlights the scientific\ncontributions without commercial emphasis, and provides a balanced discussion of\nboth strengths and limitations.\n5. Remove/ justify the AI aspect. While sexy and currently popular, this brings nothing\nnew. Please review the current state of image analysis and automation for blood\ntyping in recent systems.\nResponse: Thank you for your clear and constructive feedback. We fully agree\nthat references to an “AI” component should only be included if they contribute\nsubstantively to the scientific work. As you noted, gratuitous or speculative mentions\nof AI risk distracting from the core technical contributions. In response, we have\nremoved the AI-focused material from the manuscript. Specifically, the AI panel in\nFig. 1e (previously illustrating an AI-based workflow) has been deleted, and the\ncorresponding description has been eliminated from both the figure legend and the\nDiscussion section (first paragraph). Discussion: Deleted the sentences in the first\nparagraph that discussed AI in a speculative manner.\n6. Remain scientific- and remove pre-commercialization development of test.\nResponse: Thank you for your valuable feedback. Based on your suggestion, we\nhave revised the manuscript to remove the discussion related to the\npre-commercialization development of the test. We have restructured the Discussion\nsection to focus more on the scientific aspects of the research, emphasizing the\ntechnical innovations, experimental findings, and potential applications in clinical\nsettings. For example, statements such as \"The prolonged preservation properties of\nour RBC reagent may enable the development of standardized protocols for it as\nblood product preparation and storage, potentially addressing the long-standing\nstandardization challenges of current RBC reagents\" and similar sentences, have been\nomitted to maintain the scientific rigor of the manuscript.\n7. Improve introduction. Clarify the Landsteiner rule- very confusing as presented.\nClarify forward and reverse testing- including previous paper diagnostics concepts.\nResponse: Thank you for your constructive feedback. Based on your suggestion,\nwe have clarified Landsteiner's rule as follows: \"Landsteiner's rule states that in the\nABO blood group system, an individual's serum will always contain antibodies\nagainst the antigens that are absent from their own red blood cells, and will not\ncontain antibodies against the antigens that are present on their red blood cells.\" This\nrevision has been applied in the manuscript on Line 36 in Page 2.\n8. 8 min for blood typing is very long. Current paper blood diagnostics (GLIF) do it\nin less than 2 minute and modern commercial gel card test in 1 min. Please review\nclaims.\nResponse: We sincerely thank the reviewer for raising this important point\nregarding detection time. We appreciate the opportunity to clarify the basis of our\nresults and the rationale behind the reported detection times.\nFirst, it is important to emphasize that the current experimental optimization\nprimarily focuses on antibody titer detection (as shown in the new Fig. 5a–d). The\n8-minute detection time reported in the original manuscript represents the total time\nrequired to obtain both antibody titer results and blood group determination\nsimultaneously. Currently, standard reverse typing using laboratory gel microcolumn\ncards typically requires about 5 minutes of centrifugation, not including preprocessing\nsteps. These preprocessing steps—such as gradient dilution of antibody titers—add\nadditional time to the workflow. Moreover, conventional reagent red blood cells used\nin these assays have a short shelf life and rely on cold-chain transportation, making\nthem less suitable for point-of-care testing. Notably, no commercially available POCT\nreverse typing card currently allows ambient temperature storage and transportation,\nwhich further limits their practical application. In contrast, our detection strategy\nprovides a clear advantage in identifying weak agglutination. By applying an\neightfold dilution, we ensured that the linear detection range falls between 16 and 512,\nallowing simultaneous titer quantification without compromising accuracy. If dilution\nratios are reduced and the amount of engineered red blood cells is increased, the total\ntesting time can be shortened further and the detection efficiency improved.\nInspired by the reviewer’s comment, we additionally conducted experiments\nfocusing solely on blood group determination. Since agglutination-based typing only\nrequires visible onset of agglutination rather than complete agglutinate formation, we\ntested 30 strong-positive and 30 weak-positive clinical samples. We found that strong\nagglutination became visible at approximately 30 seconds, while weak agglutination\nappeared within 2.5mins. Moreover, if blood group determination is set as the sole\ntesting objective in subsequent optimizations, the detection time can be further\nreduced.\nRelevant explanations have been added in the Discussion (Line 362, Page 16) of\nthe revised manuscript.\n9. Better structure and analysis of secondary group blood test (Duffy, Lewis, Kell, …)\nand those requiring IgG antibody (instead of current IgM).\nResponse: Thank you for your insightful feedback. We appreciate your\nsuggestion to enhance the structural and analytical depth of our study by including\nsecondary blood group systems (such as Duffy, Lewis, Kell, etc.) and tests involving\nIgG-class antibodies, rather than focusing solely on IgM-mediated reactions.\nIn the revised manuscript, we have incorporated your advice and conducted\nadditional experiments accordingly. Specifically, we purchased blood group\nantibodies covering the Rh (D, C, E, c, e), Kell (K), Duffy (Fya, Fyb), Lewis (Lea, Leb),\nand MNS (M, N) systems. Among these, Rh (D, C, E, c, e), Kell (K), and Duffy (Fya,\nFyb) are IgG-type antibodies, while Lewis (Lea, Leb) and MNS (M, N) are IgM-type\nantibodies. Commercially available irregular antibody screening red blood cell\nreagents were obtained from Changchun BoXun and used as source materials to\nprepare engineered red blood cells for antibody screening.\nAs shown in Fig. S10-A, we present the antigen profiles of our engineered red\nblood cells used for antibody screening. Fig. S10B demonstrates that agglutination\npatterns and intensities for the IgG panels (Rh, Kell, Duffy) and the IgM panels\n(Lewis, MNS) closely matched those of standard commercial reagents, indicating that\nthe engineered RBCs retained the relevant antigenic determinants and effectively\nrecapitulated reactivity across multiple blood group systems. For IgG antibodies (Rh,\nKell, Duffy), reactions were incubated at 37°C for 30 min and assessed by an\nantihuman globulin (AHG) test to confirm reactivity.\nIn summary, these findings demonstrate the feasibility of using engineered red\nblood cells for the detection of both IgG antibodies and secondary blood group\nantigens. Nonetheless, systematic optimization experiments and more comprehensive\nvalidation studies are warranted to ensure consistency across production batches,\nbefore such methods can be fully adopted in clinical diagnostic workflows. The\nResults (Line 205, Page 9) and Discussion (Line 402, Page 17) have been revised\naccordingly to incorporate these methods and findings.\nReviewer 2\nThis study introduces an innovative paper-based fluorescent assay (PFA) for blood\ntyping and antibody titer measurement. Unlike traditional methods, it utilizes\nfreeze-dried bioengineered red blood cells, enabling long-term ambient storage. The\nPFA, which delivers results in approximately 8 minutes without complex equipment or\nsubjective interpretation, demonstrated 100% accuracy in ABO blood typing and high\nconcordance with standard methods in clinical tests, promising safer blood\ntransfusions in emergency or resource-limited settings. Its design also facilitates\nstandardization and integration with digital and AI-assisted analysis. However, there\nare come comments that could strengthen the paper.\n1. In general, the section related to membrane integrity should be reinforced.\nAlthough the results provide strong validation of the system, this aspect would benefit\nfrom being revised or presented in a different manner by the authors. In line 272, the\nsentence “TEM results for RBC @Fluo (Fig. 1a) confirmed the integrity of the cell\nbm\nmembrane” is not entirely accurate. The TEM images provided are not sufficient to\nsupport such a statement, and it is recommended that this phrase be removed. Line\n285: Figure 2f shows significant differences between membranes, and in both the bm\nand bm-Fluo conditions, a well-defined membrane is not observed as it is in the other\nsamples. Similarly, in the confocal images (Fig. 2g), actin appears disrupted. Line\n331: This statement contradicts earlier claims that the membrane remains intact.\nResponse: Thank you for your thoughtful and constructive feedback. We\nappreciate your suggestions, which have helped us more accurately clarify the\nintegrity of the RBC membranes and highlight the strengths of our approach. We\nwould like to emphasize that when referring to membrane integrity, we are comparing\nthe RBC @Fluo preparation to cell fragments, rather than claiming that no soluble\nbm\nmembrane proteins are lost during the hemoglobin release process. We have revised\nthe manuscript to make our statements more precise and scientifically rigorous. The\nchanges are as follows:\nLine 155 (Revised): TEM images for RBC @Fluo (Fig. 1a) demonstrated that\nbm\nthe membrane structure was preserved, with FNPs uniformly distributed on the outer\nsurface, supporting the feasibility of our bioengineering strategy.\nLine 165 (Revised): Confocal microscopy analyses confirmed that RBC @Fluo\nbm\npreparation introduced visible alterations in the phospholipid bilayer (Fig. S7-A),\nmembrane proteins (e.g., CD47) (Fig. S7-B), and cytoskeletal elements such as\nmicrotubules and microfilaments (Fig. 2c). Although there was mild structural\ndisruption in the cytoskeleton, the overall cell morphology remained intact.\nLine 176 (Revised): In summary, since our bioengineering approach induces\ncertain changes in the shape, mechanical properties, and size of the RBCs.…… We\nattribute this reduction to two factors: the pore-forming process during RBC @Fluo\nbm\npreparation and the labeling of FNPs, which may have led to partial shielding of the\nsurface antigens.……\n2. Although the paper reports 100% accuracy for ABO blood typing (which is\nexcellent), the 80.75% concordance with the reference method for antibody titration is\na point that requires further analysis and justification.Is this level of concordance\nacceptable in clinical practice for all antibody titration applications (e.g., in\nmonitoring pregnancies with Rh isoimmunization, where precision is critical)? What\nare the implications of the 19.25% discordance? Are these due to inherent\nmethodological differences, variability in the reference methods, or limitations of the\nPFA in certain titer ranges? The discussion should explore the causes of this\ndiscordance in greater depth and clarify whether there are plans to improve this rate,\nor if the method is considered \"good enough\" for its intended applications—with a\nclear rationale. Comparing this performance with the known intra- and\ninter-laboratory variability of traditional methods would also strengthen the\nargument.\nResponse: We thank the reviewer for this important observation regarding the\n80.75% concordance with the reference method for antibody titration. First, it is\nimportant to clarify the methodological distinction between our assay and the\nreference gel microcolumn assay (GMA). GMA is inherently a semi-quantitative\nmethod: antibody titration results are interpreted by the position of RBC agglutinates\nin the gel, which reflects discrete dilution endpoints but does not provide continuous\nquantitative resolution. This introduces a degree of subjectivity, especially for\nreactions near the cut-off between two dilution levels, and is well documented to\ncontribute to both intra- and inter-laboratory variability.\nBy contrast, our PFA method provides a quantitative output through fluorescence\nsignal measurement, correlating directly with the extent of agglutination. This\nquantitative approach reduces subjective interpretation and allows more precise\ndiscrimination of antibody levels, particularly around borderline titers where\nsemi-quantitative methods often disagree. In our analysis of the 19.25% of discordant\nsamples at the critical threshold, we found that 85% of the data fall within the range\nof “threshold±10%”. Therefore, a portion of the observed 19.25% discordance may\nbe due not to errors in the PFA method, but rather to the inherent limitations and\nvariability of the semi-quantitative reference method itself.\nWe acknowledge, however, that further optimization is necessary to strengthen\nclinical acceptance. Our ongoing work focuses on refining the dynamic range\ncalibration, validating against larger multi-center datasets, and establishing\nequivalence criteria relative to current standards. We therefore position our method\nnot as a replacement of established assays in all critical contexts at present, but as a\ncomplementary tool offering greater quantitative resolution, reduced subjectivity, and\nthe potential for standardization in the long term.\nIn the revised Results（Line 337, Page 15）and Discussion（Line 383, Page 16）,\nwe have explicitly emphasized this distinction—that the apparent discordance reflects\nin part the comparison of a quantitative method (PFA) with a semi-quantitative\nmethod (GMA)—and have discussed how this impacts interpretation, clinical\napplicability, and future directions for improvement.\n3. The paper predominantly focuses on the detection of IgM anti-A and anti-B\nantibodies. Although it mentions the potential to \"disrupt current antibody detection\ntechniques for IgG/IgM,\" it does not elaborate on the system’s actual capability or\nfuture plans to detect IgG antibodies, which are critical in many clinical contexts (e.g.,\nhemolytic disease of the newborn, delayed transfusion reactions). Is the method\nequally effective for detecting IgG antibodies? If not, what are the challenges in\nadapting the system for IgG detection? Is this approach primarily designed for a\nspecific application where IgM detection is more relevant?\nResponse: We sincerely thank the reviewer for their insightful feedback on our\nmanuscript. The suggestion to broaden the scope by including the detection of IgG\nantibodies is highly appreciated, as IgG antibodies are indeed critical in various\nclinical scenarios, such as hemolytic disease of the newborn and delayed transfusion\nreactions. In response, we have made revisions that address the concerns raised about\nthe detection of IgG antibodies and the challenges in adapting our system for this\npurpose.\nWe have conducted additional experiments, including the purchase of IgG-type\nantibodies for Rh (D, C, E, c, e), Kell (K), and Duffy (Fya, Fyb) systems, and used\ncommercial red blood cell reagents for antibody screening. Fig. S10-A and S10-B\ncompare the antigen profiles and agglutination patterns of our engineered red blood\ncells to standard reagents. For IgG detection, reactions were incubated at 37℃ for 30\nminutes, followed by the AHG test to confirm reactivity. The agglutination patterns\nfor the IgG antibodies tested were comparable to commercial reagents, indicating the\nengineered red blood cells maintained the necessary antigenic properties. It should be\nnoted that when extending the detection to IgG antibodies, heating and the\nintroduction of antiglobulin tests (AHG) are required—a limitation shared by other\nclinical methods and not unique to this approach.\nLooking ahead, we foresee the primary application of engineered red blood cells\nfor IgG antibody testing, especially in Hemolytic disease of newborn and secondary\nblood group systems, in hospital settings. The manuscript has been updated to reflect\nthese revisions, particularly in the Results (Line 205, Page 9), where we discuss the\ndetection of IgG antibody.\n4. While the practicality of ambient-temperature storage is emphasized, the paper\ndoes not sufficiently address the challenges or considerations involved in the\nlarge-scale production of the bioengineered RBCs and the PFA devices. Is the\nbioengineering process for RBCs easily scalable to meet mass demand? Are there\nsignificant regulatory or cost-related considerations associated with the industrial\nproduction of these reagents and devices?\nResponse: We sincerely appreciate the reviewer's valuable feedback.\nScalability of the Bioengineering Process for RBCs: The engineered RBC\nworkflow—membrane isolation, nanoparticle labeling, and lyophilization—is readily\nautomatable and scalable for industrial production. Current optimization focuses on\nbatch consistency, membrane integrity, and antigen stability. The paper-based\nfluorescent assay (PFA) devices can be mass-produced via standard roll-to-roll\nprinting and lamination, enabling high-throughput fabrication. Once molds are\nfinalized, the unit cost is expected to fall below ¥1 per device.\nRegulatory Considerations: All RBCs originate from certified commercial\nsources compliant with existing regulatory standards. Scale-up will require GMP\ncertification and validation of product stability and safety under ambient conditions,\nboth achievable within established biomanufacturing frameworks.\nCost-related Considerations: Main cost drivers are certified RBCs, microspheres,\nand reagents. Ambient storage eliminates cold-chain expenses. The per-test cost lies\nbetween tube and GMA, while the compact fluorescence reader is far cheaper than the\ndedicated special centrifuge system. Substituting fluorescent microspheres with\ncolored beads would enable visual readout and further reduce costs for low-resource\nsettings.\nIn conclusion, while the large-scale production of engineered RBCs and PFA\ndevices does present scalability, regulatory, and cost-related challenges, we believe\nthat the advantages of ambient-temperature storage, combined with optimized\nproduction protocols and cost-effective instrumentation, will enable the method to\nbecome a practical and affordable alternative to conventional blood typing and\nantibody detection methods. These considerations are addressed in greater detail in\nthe revised manuscript (Line 389, Page 17), and we are committed to further refining\nthe process to make it scalable, regulatory-compliant, and cost-efficient.\n5. The clinical study was conducted with a considerable number of samples; however,\nit is not specified whether these samples came from a diverse population or a single\ngeographic region. The prevalence of certain blood groups or antigenic variants can\nvary significantly across populations. Was the method tested on samples from\nindividuals of different ethnic backgrounds or with less common antigenic variants?\nResponse: We sincerely appreciate the reviewer’s insightful comment regarding\nthe diversity of the population from which the samples were collected, as well as the\npotential variability in blood group prevalence and antigenic variants across different\npopulations. In response to your query, we have gathered the geographical\ninformation of the samples included in our clinical study, which is represented in\nFigure a (blood group sample distribution by province) and Figure b (antibody titer\nsample distribution by province). Regarding the blood group testing, the samples were\nprimarily obtained from the clinical setting at The 909th Hospital, School of Medicine,\nXiamen University, which was the ethical-approved unit for the study (Ethical\napproval number: L2023002). As such, most of the blood group samples are sourced\nfrom the local population of Fujian Province. These samples consist of both outpatient\nand inpatient clinical samples, reflecting a general representation of the local\npopulation. For the antibody titer testing, the samples were collected from blood\ndonors, and the collection process was conducted at blood donation stations located in\nhigh-traffic areas, such as tourist sites, which have a diverse demographic. This\nresulted in the inclusion of samples from various provinces. The current data do not\nindicate any significant geographic or regional differences in the antibody titer results,\nbut it is worth noting that future studies could explore potential variations across\nregions, as blood group and antibody distributions do vary by geographic location and\nethnic background. To address this point, we have provided additional details in Fig.\nS19 and incorporated the geographic breakdown into the Results (Line 301, Page 13).\n6. Overall, it is recommended that the manuscript be reviewed carefully to correct\ngrammatical errors, spacing issues, and other minor formatting inconsistencies.\nResponse: We greatly appreciate the reviewer’s suggestion. We have thoroughly\nrevisited the manuscript and addressed all identified issues. Specific attention was\ngiven to improving grammar, ensuring consistent spacing, and rectifying formatting\ndiscrepancies throughout the text.\nYours sincerely,\nXiaohui Wang\nInstitute of Health Service and Transfusion Medicine\nTaiping Road, Haidian District, Beijing 100850\nP. R. China\nTel: +86 10 66931049\nE-mail address: lovechina1980@163.com"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have nicely addressed my previous comments. However, new claims are included in this new version – again-\nignoring the pioneer work performed over 10 years ago by various groups- which is not honest.\n1) If the authors now claim innovation in secondary blood groups typing ( Kell (K), Duffy (Fya, Fyb), Lewis (Lea, Leb),\nand\nMNS), they should review, cite and compare their study to the previous work on Indirect Antiglobulin Paper Test for Red\nBlood Cell Antigen Typing by Flow-through Method and the detection of blood group phenotypes using paper diagnostics\nperformed over 10 years ago by various groups in Australia and the US. Also.\n2) Do the authors need to incubate at 37C antiglobulin tests for IgG based secondary groups? Is so, how?; if not, how come?\n3) Time of detection for RBC (forward typing, 3 min cited ) and antisera ( reverse typing- 8 min cited) analysis should be\ncompared with commercial practice and paper typing testing in the literature. The authors should also precise if the 8 min\nreverse test with antiglobulin includes the 37C incubation, which is usually 15 min, unless heated 5 min by photothermal\nheating by Laser.", "reviewer_2": "Thanks for the substantial improvements. To fully align with our original requests, a few items remain open:\n2. Concordance: Please state clinical acceptability by intended use (including Rh isoimmunization monitoring), define\n“threshold 10%,” re-express discordance in dilution steps (±1, ±2, ≥3), and add quantitative agreement metrics—% within ±1\ndilution, weighted kappa (95% CI), and PPA/NPA with a confusion matrix at the stated cutoff, comparing these to published\nintra-/inter-laboratory variability of GMA.\n3. IgG detection: The new data show IgG reactivity using 37 °C + AHG, but they do not demonstrate that the fluorescent PFA\nitself detects IgG or retains its advantages (≈8 min, no complex equipment, ambient workflow). Please either provide PFA-\nbased IgG performance vs a reference (e.g., sensitivity/specificity or agreement by dilution steps, κ, time-to-result) or state\nexplicitly that the current method is limited to IgM and outline a concrete plan to adapt PFA for IgG.\n4. Scale-up/regulatory/cost: Please add concrete scale-up parameters (batch size/throughput, step yields, lot-to-lot QC\ncriteria, real-time/accelerated stability per ICH), a region-specific regulatory pathway (classification, intended use, required\nclinical validation, biocompatibility/safety testing, GMP controls for human-derived materials), and a quantified cost model\n(BOM, labor/capex assumptions, yields) with sensitivity analysis and numeric comparison vs tube and GMA, including\nreader costs. Also discuss supply-chain risks and mitigation.\n5. Population diversity/variants: The cohort appears largely from a single region. Please report ancestry/ethnicity categories\nand province-level counts, list uncommon antigen variants actually tested (e.g., weak/partial D, DEL, Duffy-null, Kell\nvariants) with n per variant, and provide stratified performance (agreement with 95% CIs) by region/ethnicity/variant. If\nunavailable, state generalizability limits and outline a multicenter plan."}, "author_response": "Response to Reviewers\nWe would like to express our sincere gratitude for the opportunity to revise our\nmanuscript entitled \"Paper-based fluorescent assay for blood typing and antibody titer\ndetermination using long-term ambient-stored bioengineered RBCs\" (Manuscript\nNumber: NCOMMS-25-43320B). Below, we provide specific responses to each\nreviewer’s comment. All revisions have been incorporated into the revised manuscript,\nwith major changes highlighted in red.\nReviewer 1\nThe authors have nicely addressed my previous comments. However, new claims are\nincluded in this new version – again- ignoring the pioneer work performed over 10\nyears ago by various groups- which is not honest:\n1. If the authors now claim innovation in secondary blood groups typing ( Kell (K),\nDuffy (Fya, Fyb), Lewis (Lea, Leb), and MNS), they should review, cite and compare\ntheir study to the previous work on Indirect Antiglobulin Paper Test for Red Blood Cell\nAntigen Typing by Flow-through Method and the detection of blood group phenotypes\nusing paper diagnostics performed over 10 years ago by various groups in Australia\nand the US. Also.\nResponse: We thank the reviewer for this important comment and sincerely\napologize for not adequately acknowledging the pioneering work on paper-based\nsecondary blood group typing in the previous version. We now explicitly cite the key\ncontributions from Gil Garnier, Shen Wei and others [1–9], who developed paper-based\nABO/RhD devices, rapid droplet- and laser-incubated assays, and bioactive paper\nplatforms for secondary blood group phenotyping. Their mechanistic exploration of\nagglutination and flow behavior on paper cards has provided valuable guidance and\nconceptual inspiration for the design and positioning of our own platform.\nThese studies have been added and described in the Introduction (Line 62), as\nbelow: Point-of-care (POCT) blood typing has attracted considerable attention, and a\nnumber of paper- and fiber-based platforms have already been reported. Li et al.\ndeveloped a paper-based blood typing device that reports the patient's ABO/RhD type\n\"in writing,\" demonstrating that simple capillary-driven paper cards can support clear\nvisual readout outside conventional laboratories [1]. Guan et al. later introduced a\nbarcode-like paper sensor for smartphone-assisted ABO typing, showing the feasibility\nof coupling paper cards with digital imaging for semi-quantitative diagnostics [2]. On\nthe bioactive paper side, Yeow et al. established a paper-based assay for red blood cell\nantigen typing using the indirect antiglobulin test, thereby extending paper platforms to\nsecondary blood group phenotyping under IAT conditions [3]. Hertaeg et al. reported a\nrapid droplet-wicking paper method for ABO blood typing, further simplifying on-\npaper handling and reducing assay time [4]. Manderson et al. developed droplet-based\npaper assays with laser incubation for blood group antibody screening, and\nsubsequently showed that laser-assisted paper platforms can accelerate the detection of\nred cell alloantibodies in human samples [5-6]. Several other groups have proposed\nrelated POCT-oriented formats. Songjaroen et al. designed a simple, low-cost portable\npaper-based ABO blood typing device for point-of-care testing [7]. Chomean et al.\nreported a paper-based device combined with image-based high-throughput detection\nfor phenotyping minor blood groups including C, c, E, e and Mia. Min et al. recently\npresented a microfluidic cotton-thread analytical device for rapid and easily identifiable\nblood typing [8-9]. Collectively, these studies demonstrate that paper and related porous\nsubstrates can support POCT-compatible blood group testing, including secondary\nblood group phenotyping, when used with fresh red blood cell reagents. However,\nexisting platforms generally rely on short-lived cell reagents with limited ambient\nstability and are not designed to provide long-term preserved red cell reagents that\nsimultaneously support rapid ABO reverse grouping and quantitative IgM antibody\ntitration.\nWe have also moderated our claims. Our work does not seek to claim conceptual\nnovelty for paper-based secondary group typing, which has clearly been demonstrated\nbefore. Instead, the innovation we emphasize is the bioengineered, freeze-dried RBC\nmembrane-based fluorescent reagent (RBC @Fluo), which (i) maintains antigenicity\nbm\nfor at least two years at ambient temperature, (ii) enables ABO reverse grouping and\nquantitative IgM titration on a single paper device within minutes, and (iii) remains\ncompatible with secondary blood group phenotyping. In this context, secondary blood\ngroups in our study are used primarily as proof-of-concept that RBC @Fluo preserves\nbm\na broad antigen repertoire.\nReferences\n[1] Li M, Tian J, Al-Tamimi M, Shen W. Paper-based blood typing device that reports patient's\nblood type \"in writing\". Angew Chem Int Ed Engl. 2012 May 29;51(22):5497-501. doi:\n10.1002/anie.201201822. Epub 2012 Apr 18. PMID: 22511466.\n[2] Guan L, Tian J, Cao R, Li M, Cai Z, Shen W. Barcode-like paper sensor for smartphone\ndiagnostics: an application of blood typing. Anal Chem. 2014 Nov 18;86(22):11362-7. doi:\n10.1021/ac503300y. Epub 2014 Oct 27. PMID: 25301220.\n[3] Yeow N, McLiesh H, Guan L, Shen W, Garnier G. Paper-based assay for red blood cell\nantigen typing by the indirect antiglobulin test. Anal Bioanal Chem. 2016 Jul;408(19):5231-8. doi:\n10.1007/s00216-016-9617-6. Epub 2016 May 16. PMID: 27185543.\n[4] Hertaeg MJ , Tabor RF , McLiesh H , Garnier G . A rapid paper-based blood typing method\nfrom droplet wicking. Analyst. 2021 Feb 7;146(3):1048-1056. doi: 10.1039/d0an01896a. Epub\n[5] Manderson CA, McLiesh H, Tabor RF, Garnier G. Droplet-based blood group antibody\nscreening with laser incubation. Analyst. 2021 Apr 26;146(8):2499-2505. doi: 10.1039/d0an01993c.\nPMID: 33682869.\n[6] Manderson CA, McLiesh H, Tanner J, Alves D, Manolios J, Garnier G. Laser incubation\nfor the rapid detection of red cell alloantibodies in human blood samples. Vox Sang. 2022\nNov;117(11):1302-1309. doi: 10.1111/vox.13352. Epub 2022 Sep 14. PMID: 36102143.\n[7] Songjaroen T, Primpray V, Manosarn T, Khumchanta W, Sakuldamrongpanich T,\nKulkeratiyut S, Laiwattanapaisal W. A simple and low-cost portable paper-based ABO blood typing\ndevice for point-of-care testing. J Immunoassay Immunochem. 2018;39(3):292-307. doi:\n10.1080/15321819.2018.1486856. Epub 2018 Jun 28. PMID: 29953329.\n[8] Chomean S, Bunnun P, Auttapong J, Kaset C. Phenotyping of minor blood groups (C, c, E,\ne, and Mia) using a paper-based device and image-based high-throughput detection. Anal Chim Acta.\n[9] Min S, Zhan T, Lu Y, Pan D, Chen X, Xu B. Rapid and easily identifiable blood typing on\nmicrofluidic cotton thread-based analytical devices. Lab Chip. 2023 Oct 24;23(21):4680-4689. doi:\n10.1039/d3lc00501a. PMID: 37817672.\n2. Do the authors need to incubate at 37℃ antiglobulin tests for IgG based secondary\ngroups? Is so, how?; if not, how come?\nResponse: We thank the reviewer for this important methodological point. We\nfully agree that clinically significant IgG antibodies against secondary blood group antigens should, in\nprinciple, be detected by a classical 37 °C indirect antiglobulin test (IAT) with ~15 min\nincubation followed by addition of anti-human globulin (AHG). Based on the current\nstate of the research, the PFA platform is formally validated only for IgM-mediated\nABO reverse grouping and IgM titration at room temperature. The IgG secondary-group\nexperiments were included only as proof-of-concept to show that RBC @Fluo retains\nbm\na broad antigen repertoire and can operate under standard antiglobulin conditions. For\nthese experiments, RBC @Fluo were incubated with IgG typing sera at 37 °C for ~15\nbm\nmin and then tested with AHG according to standard IAT procedures.\nWe have clarified this in the revised manuscript by (i) explicitly describing in the\nMethods (Supplementary Information P18) that secondary-group tests used 37 °C IAT\nwith AHG rather than the room-temperature PFA workflow, and (ii) revising the\nDiscussion (Line 436) to state that extension of PFA to a truly rapid IgG assay would\nrequire future integration of on-chip 37 °C incubation and antiglobulin steps.\nThe revised Discussion as follows: In this study, the PFA platform is formally\nvalidated only for IgM-mediated ABO reverse grouping and IgM titration at room\ntemperature. The IgG secondary-group experiments were included solely as proof-of-\nconcept, to demonstrate that RBC @Fluo retains a broad antigen repertoire and\nbm\nremains functional under standard antiglobulin conditions, rather than as a rapid IgG\nPFA assay. In these experiments, RBC @Fluo were incubated with IgG typing sera at\nbm\n37 ℃ for approximately 15 min and then tested with AHG according to conventional\nIAT procedures. Extension of PFA to a truly rapid IgG assay will require future\nintegration of on-chip 37 ℃incubation and an antiglobulin step into the PFA workflow.\n3. Time of detection for RBC (forward typing, 3 min cited ) and antisera ( reverse\ntyping- 8 min cited) analysis should be compared with commercial practice and paper\ntyping testing in the literature. The authors should also precise if the 8 min reverse test\nwith antiglobulin includes the 37C incubation, which is usually 15 min, unless heated\n5 min by photothermal heating by Laser.\nResponse: We thank the reviewer for this very helpful comment regarding time-\nto-result. In routine practice, qualitative manual ABO reverse typing by tube/column\nagglutination usually takes 3 – 5 min, while IgM antibody titration by serial\ntube/column dilutions takes ≈10 min (not counting dilution preparation). In this\ncontext, our timings are: 3 min for qualitative IgM-mediated ABO reverse typing on\nthe PFA platform (plasma + RBC @Fluo at room temperature, agglutination\nbm\ncompleted within 3 min, comparable to tube/column methods), and 8 min for\nquantitative IgM titration ( ≈ 5 min room-temperature incubation + ≈ 3 min\npreprocessing and fluorescence reading), which is similar to the ≈10 min required for\nconventional titration. Although some reported paper-based reverse typing assays can\nprovide a qualitative result within ≈1 min, they typically rely on fresh RBC reagents;\nwe consider a 3 min reverse typing time acceptable for rapid testing given that our\nfreeze-dried RBC @Fluo reagent remains functional after at least two years of\nbm\nambient storage.\nRegarding the second question, we apologize for the earlier ambiguity. The 8 min\nreverse test does not involve an AHG step or any 37 °C incubation; it is purely an IgM-\nmediated ABO reverse grouping/titration at room temperature, without IAT. All\nexperiments involving IgG-based secondary groups were performed using conventional\n37 °C IAT (≈15 min incubation at 37 °C followed by AHG), and were used only as\nqualitative proof-of-concept for antigen preservation on RBC @Fluo, not as an 8 min\nbm\nantiglobulin assay. These clarifications have been added to the Supplementary\nInformation (P18) and Discussion (line 436) of the revised manuscript. The revised\nDiscussion as follows: In this study, the PFA platform is formally validated only for\nIgM-mediated ABO reverse grouping and IgM titration at room temperature. The IgG\nsecondary-group experiments were included solely as proof-of-concept, to demonstrate\nthat RBC @Fluo retains a broad antigen repertoire and remains functional under\nbm\nstandard antiglobulin conditions, rather than as a rapid IgG PFA assay. In these\nexperiments, RBC @Fluo were incubated with IgG typing sera at 37 ℃ for\nbm\napproximately 15 min and then tested with AHG according to conventional IAT\nprocedures. Extension of PFA to a truly rapid IgG assay will require future integration\nof on-chip 37 ℃ incubation and an antiglobulin step into the PFA workflow.\nReviewer 2\nThanks for the substantial improvements. To fully align with our original requests, a\nfew items remain open:\n1. Concordance: Please state clinical acceptability by intended use (including Rh\nisoimmunization monitoring), define “threshold 10%,” re-express discordance in\ndilution steps (±1, ±2, ≥3), and add quantitative agreement metrics—% within ±\n1 dilution, weighted kappa (95% CI), and PPA/NPA with a confusion matrix at the\nstated cutoff, comparing these to published intra-/inter-laboratory variability of GMA.\nResponse: We thank the reviewer for these helpful and professional suggestions\nregarding clinical concordance and interpretation of titers.\nIntended use and clinical acceptability. In this study, the PFA platform is\nintended for IgM anti-A/anti-B titration in settings such as screening out-of-group\nplasma or low-titer group O whole blood, which follows the same interpretive logic as\ntiters used for Rh isoimmunization monitoring. Current guidelines and transfusion\nreviews highlight substantial inter- and intra-laboratory variability in titers and\ngenerally regard a change of >1 twofold dilution as clinically meaningful[1]. Multi-\ncenter comparisons of tube and gel (GMA) methods similarly show that most results\nlie within ±1 dilution, with occasional ±2 dilution differences between methods or\nlaboratories [2]. On this basis, we interpret agreement within ±1 dilution between PFA\nand GMA as clinically acceptable for the intended use.\nDefinition of the 10% threshold. We now clarify that the “10% threshold” refers\nto the relative distance from the ROC-derived fluorescence cutoff (FC) for each GMA\ntiter category: PFA results between 0.9FC and 1.1FC are defined as “within 10% of the\ndecision threshold” and treated as borderline. As noted in the revised Results, most\ndiscordant cases fall within this ±10% zone, in line with known analytical variability of\ntitration and GMA assays [3].\nDiscordance expressed in dilution steps. Following the reviewer’s request, we\nre-expressed all PFA–GMA differences in twofold dilution steps. Among the 388 paired\nmeasurements, 80.75% were identical and 100% were within ±1 twofold dilution; no\nsample differed by ≥2 dilutions. Thus, all samples fall within the range typically\nregarded as clinically acceptable and comparable to tube–GMA inter-method/inter-\nlaboratory variability [2]. According to the Chinese military standard GJB (1:128, low-\nvs high-titer), the 2×2 confusion matrix (TP=26, FN=4, FP=12, TN=467) yields a PPA\nof 86.7% (26/30) and an NPA of 97.5% (467/479) between PFA and GMA (Fig. S20).\nFig. S20 | Confusion matrix of PFA versus GMA for low- vs high-titer classification at the 1:128.\nWhen compared with published multi-center data on GMA and tube titrations—\nwhich show similar or larger inter-laboratory variation (often up to ±1–2 dilutions)\n[2] — these performance metrics indicate that the PFA platform is at least comparable\nto, and in some respects more consistent than, conventional GMA titration for the\nintended IgM anti-A/B applications. By analogy to Rh isoimmunization monitoring,\nwhere >1 dilution change is considered clinically meaningful and titers are ideally\nfollowed in the same laboratory [1], we believe that the observed level of agreement\nbetween PFA and GMA is clinically acceptable.\nThese points have been clarified in the revised Discussion (Line 354 &359).\nReferences\n[1] ACOG Practice Bulletin No. 192: Management of Alloimmunization During Pregnancy.\nObstet Gynecol. 2018 Mar;131(3):e82-e90. doi: 10.1097/AOG.0000000000002528. PMID:\n29470342.\n[2] Kang SJ, Lim YA, Baik SY. Comparison of ABO antibody titers on the basis of the antibody\ndetection method used. Ann Lab Med. 2014 Jul;34(4):300-6. doi: 10.3343/alm.2014.34.4.300. Epub\n[3] Matthew S. Karafin, Robert A. DeSimone, James Dvorak, Ryan A. Metcalf, Monica B.\nPagano, Yara A. Park, Joseph Schwartz, Rhona J. Souers, Zbigniew M. Szczepiorkowski, Lynne\nUhl, Glenn Ramsey; Antibody Titers in Transfusion Medicine: A Critical Reevaluation of Testing\nAccuracy, Reliability, and Clinical Use. Arch Pathol Lab Med 1 December 2023; 147 (12): 1351–\n1359. doi: https://doi.org/10.5858/arpa.2022-0248-CP\n2. IgG detection: The new data show IgG reactivity using 37℃ + AHG, but they do not\ndemonstrate that the fluorescent PFA itself detects IgG or retains its advantages (≈8\nmin, no complex equipment, ambient workflow). Please either provide PFA-based IgG\nperformance vs a reference (e.g., sensitivity/specificity or agreement by dilution steps,\nκ, time-to-result) or state explicitly that the current method is limited to IgM and\noutline a concrete plan to adapt PFA for IgG.\nIgG 检测：\nResponse: We thank the reviewer for this precise and important comment. We fully\nagree that the current IgG data, obtained under conventional 37 °C IAT conditions, do\nnot demonstrate IgG detection by the PFA workflow itself, nor do they document 8-\nmin, ambient, equipment-light performance for IgG. In the present study, the validated\nscope of the PFA platform is limited to IgM-mediated ABO reverse grouping and IgM\nantibody titration at room temperature. The IgG experiments were performed only to\nshow that RBC @Fluo preserves rare antigens under standard 37 °C + AHG\nbm\nconditions and should therefore be interpreted as proof-of-concept for antigen\npreservation, not as evidence of a rapid IgG PFA assay.\nWe have revised the Discussion (Line 436) to state explicitly that the current\nmethod is restricted to IgM applications and does not yet provide a validated IgG PFA\ntest, but that in future the PFA concept could be extended to IgG by incorporating on-\nchip 37 °C heating (e.g., resistive or photothermal) and integrating an AHG step into\nthe fluorescent readout workflow.\n3. Scale-up/regulatory/cost: Please add concrete scale-up parameters (batch\nsize/throughput, step yields, lot-to-lot QC criteria, real-time/accelerated stability per\nICH), a region-specific regulatory pathway (classification, intended use, required\nclinical validation, biocompatibility/safety testing, GMP controls for human-derived\nmaterials), and a quantified cost model (BOM, labor/capex assumptions, yields) with\nsensitivity analysis and numeric comparison vs tube and GMA, including reader costs.\nAlso discuss supply-chain risks and mitigation.\nResponse: We thank the reviewer for these insightful comments on scale-up,\nregulation and cost, which are indeed central to future translation of the PFA platform.\nAs the present work is still at a methodological / clinical proof-of-concept stage, we\ncan only provide an outline based on current data and realistic assumptions, rather than\na full commercial dossier.\nScale-up parameters and QC. In our process, the main bottleneck for large-scale\nproduction is the preparation of RBC . Average recoveries of 93.78 ± 1.24% for\nbm\nRBC and 85.23 ± 2.15% for RBC @Fluo relative to the starting RBCs (Fig. 2f).\nbm bm\nAll downstream steps (fluorescent labelling to obtain RBC @Fluo and lyophilization)\nbm\nare already operated at the 105-test-per-batch scale in our industrial partner—Anbio\n(Xiamen) Biotechnology Co.,Ltd. For RBC preparation, standard industrial\nbm\ncentrifuges would support an estimated batch size of ≥ 104 tests per batch. Lot-to-lot\nQC would follow the same framework used here: we employ imaging flow cytometry\nfor particle counting, antigen expression, and aggregation monitoring to ensure a CV <\n15% during the preparation process. After the product of RBC @Fluo is completed,\nbm\ncommercial monoclonal antibody-based forward grouping reagents or standards are\nused and serially diluted to prepare in-house QC panels, and use these to assess titer,\nlimit of detection (LOD), linear range, and repeatability. The PFA reagents are medical-\ndevice IVDs rather than pharmaceuticals, and thus not directly governed by ICH\nstability guidelines; however, our real-time data already demonstrate >24-month\nambient stability (Fig. 3j). Formal long-term plus accelerated stability studies under an\nIVD registration protocol would be implemented only once the manufacturing process\nis frozen.\nRegion-specific regulatory pathway (China). In China, the intended product would\nbe classified as a Class III in vitro diagnostic reagent for qualitative/quantitative IgM\nanti-A/anti-B detection in human serum/plasma, supporting ABO reverse grouping and\nABO antibody titer assessment to assist transfusion compatibility, and other antibody-\ntiter–based decisions. For such products to obtain approval from NMPA, the clinical\nperformance evaluation must compare the PFA platform in parallel with semi-\nquantitative methodologies such as the slide test, microcolumn gel technique, and tube\nmethod. Statistical analyses, including correlation and linear range assessments, should\nbe conducted to determine their equivalence. A formal Class III registration trial would\ntypically involve ≥3 qualified clinical sites and ≥3000 subjects covering different ages,\nsexes and regions, with prespecified consistency criteria. As a purely in vitro assay, its\nbiocompatibility testing is limited to assessments such as cytotoxicity, sensitization, and\nhemolysis. Human-derived materials must pass standard infectious-disease (HBV,\nHCV, HIV, syphilis, etc.) and pyrogen testing before use to ensure the absence of\npathogenic microbial contamination. Manufacturing would be required to comply with\nmedical-device GMP/quality-system regulations for IVDs (rather than drug GMP),\nincluding specific controls for human-derived materials and post-market surveillance.\nCost-related Considerations: Main cost drivers are certified RBCs, microspheres,\nand reagents. Ambient storage eliminates cold-chain expenses. The per-test cost lies\nbetween tube and GMA, while the compact fluorescence reader is far cheaper than the\ndedicated special centrifuge system. Substituting fluorescent microspheres with\ncolored beads would enable visual readout and further reduce costs for low-resource\nsettings.\nIn conclusion, while the large-scale production of engineered RBCs and PFA\ndevices does present scalability, regulatory, and cost-related challenges, we believe\nthat the advantages of ambient-temperature storage, combined with optimized\nproduction protocols and cost-effective instrumentation, will enable the method to\nbecome a practical and affordable alternative to conventional blood typing and\nantibody detection methods. These considerations are addressed in greater detail in\nthe revised manuscript (Line 389, Page 17), and we are committed to further refining\nthe process to make it scalable, regulatory-compliant, and cost-efficient.\n4. Population diversity/variants: The cohort appears largely from a single region.\nPlease report ancestry/ethnicity categories and province-level counts, list uncommon\nantigen variants actually tested (e.g., weak/partial D, DEL, Duffy-null, Kell variants)\nwith n per variant, and provide stratified performance (agreement with 95% CIs) by\nregion/ethnicity/variant. If unavailable, state generalizability limits and outline a\nmulticenter plan。\nResponse: We thank the reviewer for highlighting the important issues of\npopulation diversity and antigenic variants.\nFirst, regarding geographic origin and ancestry, all participants in this study were\nrecruited in China. The majority of samples originate from Fujian Province, with\nsmaller numbers from multiple other provinces. Ethnicity was not systematically\nrecorded beyond national identity documents; based on the catchment area of our\nhospital and blood centers, the cohort is expected to be predominantly East Asian of\nHan ancestry. We have explicitly stated in the revised Results (Line 328) that our data\nprimarily support performance in this population.\nSecond, our clinical series was not designed to enrich for uncommon antigen\nvariants such as weak/partial D, DEL, Duffy-null or Kell variants, and we did not have\nsufficient, molecularly confirmed cases of these phenotypes to support meaningful\nstratified analyses. For this reason, we cannot provide robust sensitivity/specificity or\nagreement statistics by variant.\nAt present, a systematic evaluation of rare-antigen agglutination has not yet been\nperformed. Future work will need to test RBC @Fluo against rare-antigen red cells\nbm\nfrom diverse sources in a more granular manner—including agglutination strength, lot-\nto-lot stability, and head-to-head comparison with existing RBC reagents—before a\ndedicated multicenter clinical study focusing on rare antigen phenotypes can be\nappropriately designed and implemented.\nYours sincerely,\nXiaohui Wang\nAcademy of Military Medical Sciences\nTaiping Road, Haidian District, Beijing 100850\nP. R. China\nTel: +86 10 66931049\nE-mail address: lovechina1980@163.com"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have nicely addressed my comments. There is now an honest, well structured and written review of paper\ndiagnostics. The authors have also summarized the gap in knowledge and their major contribution in the last sentence of the\nnew paragraph: long lasting reagent red blood cells and their application in blood paper diagnostics. The articles is concise,\nwell structured, has excellent figures- and honestly presents the major innovation of the team, well described in the scientific\ncontext. I recommend to accept as is. Ready to publish.", "reviewer_2": "The revisions have addressed my previous concerns, and the manuscript is now clear and substantially improved. I have no\nfurther comments."}, "author_response": ""}]

Decision letter