The anti-mouse IgG (fab specific)-FITC antibody produced in goal was acquired from Sigma (F5387). All chemical reagents were acquired from Sinopharm Chemical Reagents Co., Ltd., (China). Alexa Fluor TM 647 Carboxylic Acid, Succinimidyl Ester was purchased from Thermo Fisher scientific (United States). Flow cytometer (z2010-254, BD LSRFortessa). In our laboratory, 0.01M PBS (pH 7.2) and hydrogel were prepared. Two lymphocyte hybridoma cells produce the monoclonal antibody against HbF (15-G4, 9-F10). The sample of whole blood was supplied by China-Japan Union Hospital of Jilin University. The blood sample stored at 4°C has been approved by the China-Japan Union Hospital’s Ethics Committee (Approval number: 20220425009).

Blood from healthy adults and umbilical cord blood from newborn fetal umbilical cords were collected and washed three times with saline. Then, added twice volume of distilled water, which is erythrocyte lysate, centrifuged at 10,000 r.p.m for 20 min, collected the supernatant, which was haemoglobin, and determined its concentration. The same concentration of fetal hemoglobin was then mixed with adult hemoglobin in volumes of 1: 1,000, 3: 1,000, and 5: 1,000 to produce fetal hemoglobin with proportions of 0.1%, 0.3%, and 0.5% in the mixed hemoglobin.

Wang’s article described the use of hydrogel to separate small molecular dyes from indicator RBCs. The characteristics of methylene blue and the reaction time of the reactants differed between this experiment and Wang’s. In this experiment, methylene blue (Mw373.90,0.8%), indicator RBCs, and additional reactants were placed on top of hydrogel, incubated, and centrifuged at 285 g for 3 min.

Randomly collected “O” type Rh-positive RBCs, thoroughly washed them, treated them with 0.5% GA-PBS solution at room temperature for 30 min, centrifuged and washed them, then added 0.5% PA-PBS solution to water bath at 37°C for 1 h, centrifuged and washed them, then added 1% PFA-PBS solution to water bath at 37°C for 5 h, and shook them every 30 min. After washing and centrifuging, a 10% suspension of RBCs was made and stored at 4°C for future use.

Chromium trichloride solution (CrCl3.6H₂O) in saline had a concentration of 1.51%. Adjust the pH to 5.0 using 1 mol/L sodium hydroxide prior to use. By using the metal cation immunoglobulin tannic acid method, aldehyde-modified RBCs were made sensitive. In other words, chromium trichloride solution was added to anti-HbF antibody diluted with acetate buffer (0.175 M pH6.4) to achieve a final concentration of 120 μg/mL. After mixing, water-bathing at 37°C for 10 min. To the anti-HbF antibody and 1 mL of hematocrit that had been incubated, 1:2,000 tannic acid solution was added immediately. It is necessary to ensure that the volume ratio of the mixed solution to the tannic acid solution is 3:1, and to incubate the solution in a 37°C water bath for 15 min. After six times of washing, 2% BSA-PBS was added to a 37°C water bath for 2 hours, and a storage suspension was subsequently prepared.

As the second antibody, sheep anti-mouse IgG FITC was used to detect mouse anti-HbF antibody. After 30 min in a 37°C water bath, the cells were washed once and centrifuged before being collected. The hematocrit was then diluted in PBS-BSA at 1%. Using FCM, the fluorescence intensity of negative and positive samples was measured.

Thermo Fisher Scientific Company’s Alexa Fluor TM 647 Carboxylic Acid, Succinimidyl Ester reagent was used to label anti-HbF antibody with F647. Among them, the goat anti-mouse IgG antibody concentration was 16.544 mg, and the labeling time of F647 for goat anti-mouse IgG antibody was 4 h at room temperature, followed by an overnight treatment at 4°C.

The haemoglobin (Hb), fluorescence-labeled antibody, and sensitized indicator RBCs were added to the hydrogel-filled card and centrifuged at a low speed until the immune complex in the positive result and the indicator RBCs in the negative result sank, while the fluorescence-labeled antibody remained on the hydrogel in the negative result (Figure 1).

The hydrogel fluoroimmunoassay substances were detected by flow cytometry calibrated with standard fluorescent microspheres, and the fluorescence intensity of 50,000 cells was determined. The operation steps strictly adhere to the BD LSRFortessa flow cytometry instructions. The flowchart is examined with Flowjo 10.8.1. The distinction between fetal and adult red blood cells is based on the fact that their peaks are distinct (Figure 4). Even though the negative control has only one peak (0.0% fetal hemoglobin), this experiment distinguishes it using the abscissa value of 10³ as the boundary. Flow cytometry values with 0.1%, 0.3%, and 0.5% of fetal hemoglobin are subtracted from negative control values greater than 10³ according to the principle of data processing.

The preceding analysis method was used to analyse and compare the K-B test. The K-B test was conducted in accordance with the AABB technical manual. The experiment involved the observation of 10,000 cells and the counting of fetal RBCs (Athiel et al., 2020). This experiment was repeated three times, and the results were statistically analyzed using GraphPad Prism 9.4.1 to determine if there was a statistically significant difference between the hydrogel fluoroimmunoassay and K-B test for detecting different fetal red blood cells produced by 0.0%, 0.1%, 0.3%, and 0.5% cord bloods.

To demonstrate that the hydrogel is capable of separating indicator RBCs from small molecular proteins not bound to RBCs, we compared the distribution of small molecular substances, RBCs, and immune complexes in the hydrogel medium under different conditions. The substances with the higher density sank to the bottom of the hydrogel medium, whereas the substances with the lower density remained on top of the hydrogel. Figure 2D indicated that 1 h of incubation had no effect on the diffusion of small molecules or RBCs. In Figure 2, (A)₁ and 2 (B)₁ remained unchanged, indicating that methylene blue was not centrifuged to the card’s bottom. Red indicator red blood cells coated with an antibody were added to the top of the 2 (A)₂ card. The top of the card in 2 (B)₂ changed from red to white after centrifugation, and red (indicator RBC-coated antibodies) appeared at the bottom of the card, indicating that the antibody-coated indicator red blood cells were centrifuged to the bottom of the card. The 2 (A)₃ card turns red when the immune complex (indicator RBC-coated antibodies, fluorescently labeled antibodies, and excessive hemoglobin to be detected) is added. The immune complex formed by indicator RBC-coated antibodies—HbF—fluorescently labeled antibodies is centrifuged to the bottom of the card to form red, while hemoglobin at the top of the card is not bound to form an immune complex and is therefore still red. The experimental findings were consistent with Wang’s perspective (Wang et al., 2015). The results demonstrated that hydrogel medium could indeed separate soluble antigens, soluble antibodies, granular substances, and other macromolecules. The centrifugal force of RBCs and small molecules sinking in this experiment was 285 g.

Figure 3A's objective is to determine if the indicator cells were bound to anti-HbF antibody. In comparison to the red line (negative sample), the light blue line (positive sample) is clearly shifted to the right, and the fluorescence value is clearly elevated. The outcomes demonstrated that the anti-HbF antibody was successfully coupled with indicator RBCs, which could be used in the subsequent experiment.

Figure 3B demonstrated that F647-labeled antibody and antibody modification indicated that RBCs could indeed recognise HbF specifically, indicating that the experimental steps had been established successfully. Compared to the red line (negative sample), the light blue line (positive sample) was clearly shifted to the right, and the fluorescence value was clearly increased, indicating that this experiment was able to distinguish between fetal and HbA. As depicted in Figure 5C, this experiment was repeated three times with negative and positive controls. The Hb concentration used in this experiment was 0.608 mg/mL (0.60–0.62 mg/mL). In the traditional FCM detection method, the incubated reactants were required to be washed a minimum of three times; if the washing times were insufficient, the false positive rate would increase, and if they were excessive, fetal RBCs would be lost. It had been reported in the literature that washing times should be minimised as much as possible when detecting fetal bleeding with FCM (Wiley Online Library, 1999). Figure 3B depicted the detection results obtained after a single washing, while Figure 4depicted the separation results obtained using a hydrogel medium. It was discovered that the fluorescence intensity of washing once was demonstrably higher than that of hydrogel separation, indicating that the separation effect of hydrogel was demonstrably more effective than that of washing once. Consequently, the use of hydrogel medium can reduce washing steps, save time, and ensure the accuracy of experimental results.

Figure 4 depicts the detection of mixed haemoglobin in various proportions (the ratio of HbF to HbA) via hydrogel fluoroimmunoassay. In this experiment, 50,000 cells were counted. Figure 4B depicted the global distribution of HbF concentrations of 0.0%, 0.1%, 0.3%, and 0.5%. The fluorescence intensity increased as the ratio of fetal RBCs increased. As shown in the image, the negative result (0.0%) had a peak that represented HbA; there were two peaks with varying proportions of HbF (0.1%, 0.3%, and 0.5%). HbA was represented by the peak on the left, while HbF was represented by the peak on the right. As the fetal RBC ratio increased, the height of the left peak decreased, while the height of the right peak increased. Positive APC-A values for 0.1% HbF were found to be 1.6%, 0.3% HbF was 20.6%, and 0.5% HbF was 23.8%. As the fetal RBC ratio increased, the APC-A positive value increased dramatically. Figures 4C–F represented sub-graphs for HbF concentrations of 0.0%, 0.1%, 0.3%, and 0.5%, respectively. Figure 3C depicted the outcomes of the K-B test on blood samples with varying proportions. Using electron microscopy, 10,000 cells were tallied; Figure 3C displays an upward trend in the proportion of fetal RBCs within adult RBCs. The K-B test and the hydrogel fluoroimmunoassay exhibited a strong correlation. Both were able to reliably and accurately detect the amount of FMH, and their sensitivity can reach 0.1%, which is in line with the clinical treatment standard for 2 mL of FMH. On the basis of these results, a critical concentration of 0.1% is determined. The conclusion reached by Davis is consistent with this one (Davis et al., 2010). Figure 5 was a bar graph of repeated hydrogel fluoroimmunoassay and K-B tests for different hemoglobin concentrations. p < 0.0001 indicated that the fluorescence values of 0.0%, 0.1%, 0.3%, and 0.5% of fetal hemoglobin detected by hydrogel fluoroimmunoassay were statistically significant. Figure 5B demonstrates that the K-B test was statistically significant for detecting fetal red blood cells in 0.1%, 0.3%, and 0.5% of mixed blood (p < 0.0001).