Quantitative Analysis of Solution-Free Blood Cell Staining by the Hydrogel Stamping Methods

Introduction:: Imaging and quantification of stained blood cells are important for identifying blood cells in hematology and for diagnosing diseased cells or parasites in cytopathology. Romanowsky stains have been used traditionally to produce hues in blood cells using anionic eosin Y and cationic methylene blue. While Romanowsky stains have been widely used in cytopathology, end-users have experienced problems with varying results in staining due to several factors affecting the results of diagnosis and the inherent inconsistency of solution-based Romanowsky staining. Here, we demonstrated that staining and destaining of smeared blood are controllable by the contact time of agarose gel stamps used in the ternary mixture of Romanowsky dyes.

Materials and Methods:: Whole blood for sample preparation was approved by the Institutional Review Board (P01-202003-31-007) of the Korea National Institute for Bioethics Policy (Seoul, Korea). To prepare the blood film, 4 µL of whole blood was dropped and smeared in the miLab™ (Noul Co., Ltd.). Then, the blood was fixed for 2 min in methanol. To stain and destain blood cells, 0.7 mL of circular agarose gel (2% w/v) was created and submerged in the ternary mixture solution (EY, 0.0035 mM; MB, 0.07 mM; AZB, 0.14 mM). The circular agarose gel was incubated on the rocker at 150 rpm/37°C for 48 h. After incubation for the adsorption, the circular agarose gel with or without ternary mixture was put on the smeared blood and treated for 10, 30, and 60s for staining or destaining, respectively. In each RBC, the RGB values at the region of interest (ROI), defined as a diameter of 7 µm, were extracted from the split images with each channel using Image J and transformed to values of Hue (H, 1-365).

Results, Conclusions, and Discussions:: In all cases of 10 to 60 s destaininig after staining, H decreased monotonically (Figure 1a-d and e). With 30 s and 60 s of staining, values of H increase up to 100 (lime to kelly green) because of the desorption of MB or AZB from agarose gels. We also evaluated H with different staining-destaining durations. As expected, the longer destaining lasts, the more H decrease (Figure 1f). Data presented in Figure 1 demonstrate that the extent of staining and destaining to RBCs can be quantifiable by assessing values of H from collected micrographs. In addition, we are able to discern colorfulness by values of H. To quantify the actual amount of Romanowsky dye transfer, however, it is imperative to directly measure the concentration changes in the cycle of staining and destaining. While the values of H of the object can be analyzed as a result of staining process, it is difficult to infer the result of how much actual stains came out or entered in the agarose gel.
To better quantify the transferred amount of Romanowski dye from agarose gels, we tested if the transfer is quantifiable by measuring the extent of staining on smeared RBCs. Values of H gave us reasonable estimation, but precise quantification of the transfer needed more quantitative methods with mathematical models. To predict the actual amounts of dyes of the ternary mixture to and from agarose gels, we will need to employ representative models of isotherms (i.e., Langmuir or Freundlich isotherms) and kinetics (pseudo or second-order kinetics) models.

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