Hemoglobin estimation by the classical method should be adhered to for understanding anemia following lead exposure: Old is gold

Dear Editor, Lead pollution is an ongoing problem and shortages of lead chelators are commonly reported following acute lead toxicity.[1] Hence, the development of lead chelators for lead poisoning is in high demand. In this context, a zebrafish model of lead toxicity was recently developed by Guo et al., 2023, in their article “Development of rapid zebrafish model for lead poisoning research and drug screening.” It is indeed a splendid work.[2] However, to understand lead exposure-induced anemia, the authors did not rely on the commonly used method of hemoglobin estimation.[3] The authors stained the gill section of the fish with the o-dianisidine dye. The H2O2 added in the process was expected to generate reactive oxygen by the peroxidase action of hemoglobin, which reacts to form color.[4] Following staining, the authors did the densitometry of the tissue section and concluded that lead exposure in the fish caused anemia. However, it is essential to note that lead can inhibit the peroxidase activity.[5] We performed the color formation test and observed that the addition of lead inhibits the color formation. [Figure 1, for methods, see Appendix A].Figure 1: (a) Spectral scan of different amount of blood with the o-dianisidine solution. (b) Graph representing a reduction in color formation of blood with the o-dianisidine solution in the presence of different doses of lead acetate (**** P < 0.001). (c) Blood smear stained with o-dianisidine dye and Giemsa stain i and iii. Control (blood from healthy individual) (×40 and ×20) ii and iv. Lead acetate treated blood (4 µM) (×40 and ×20)Further, the whole blood stained by the o-dianisidine method produced a very different color/brightness, which is inherently challenging to be commented upon by densitometry. Therefore, we believe that in the published otherwise beneficial lead exposure model, the anemia assessment should be done using the classical method. Ethical consideration This study uses anonymized left-over samples (whole blood) of healthy human volunteers. The sample collection was done in the context of a study approved by Institute Ethics Committee (vide no: INT/IEC/2016/2587, dated: 21.11.2016). Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.