Estimating blood volume on dried blood spots

Archived dried blood spots (DBSs) collected by newborn screening programs represent a potentially valuable resource for assessing chemical exposure of newborns. Moreover, collection, storage, and transport of DBSs are easier and cheaper than standard venipuncture protocols. However, quantitative exposure assessment requires knowledge of the blood volume, which can vary considerably on DBSs. We present a method to estimate the volume of fresh blood originally deposited on DBSs and update the forensic literature regarding the ratio of fresh blood volume to dry blood weight. The weight of the blood residue is determined using the area and areal density of the DBS corrected by an unused section of the same card. Density is determined by cutting out the DBS, scanning and digitizing the image, and applying a mask to improve consistency. Blood volume is calculated as the product of the residue weight and fresh blood volume/dried blood weight ratio for the age-specific hematocrit level. Tests using DBSs prepared with 30 to 100 µL of blood showed excellent agreement. The method was applied to 352 archived neonatal DBSs dating from 2008 through 2023. The estimated fresh blood volume averaged 98 µL, close to the expected value, and followed a lognormal distribution (range: 28 to 199 µL); no differences were found for sex or DBS age. The proposed approach is simple, accurate, and inexpensive, and can facilitate the use of archived DBSs for quantitative and retrospective exposure assessment. Additionally, it is applicable for forensic applications examining bloodstains on cloth, paper, and other materials.