Estimation of Bloodstain Age by Rapid Determinations of Oxyhemoglobin by Use of Oxygen Electrode and Total Hemoglobin.

Abstract

Bloodstain age could be estimated from the ratio of oxyhemoglobin/total hemoglobin (fractional oxyhemoglobin) in the bloodstain by this present method, if the temperature at which the bloodstain had been kept was known. The oxyhemoglobin was determined with an oxygen electrode immersed in water in which the oxygen had been depleted, and the total hemoglobin was determined by conventional colorimetry (cyanomethemoglobin method). Ages of prepared bloodstain samples (within 24 h after bleeding) were estimated by this present method, which requires only 20 microliters of bloodstain and only 5 min for the whole analysis.

A Blue Spectral Shift of the Hemoglobin Soret Band Correlates with the Age (Time Since Deposition) of Dried Bloodstains

“…1). Previous studies on Hb spectral changes with age have concentrated on the α 576 and β 541 bands [17]–[19], [21]–[24]. However, our preliminary studies indicated that neither changes to the absorption values of the α 576 and β 541 bands (or their ratios) nor the Soret band provided a sufficiently high correlation with age to be a useful TSD diagnostic (data not shown).…”

“…The problem is that few reliable and accurate methods exist to approximate the time of deposition of these dried biological stains [16]–[26]. Many of the methods developed to estimate an approximate age of a bloodstain have focused on deteriorative changes to the visible spectrum of hemoglobin (Hb) over time [17]–[19], [21]–[24]. For example, one such method used the α-chain to heme ratio determined by HPLC [19].…”

“…In a subsequent study, a peak designated as “X” was detected only in aged stains, and the area of this peak increased as the age of the stain increased [20]. Other studies have also used HPLC analysis of Hb to estimate TSD [17], [23], [24]. While these studies demonstrated some correlation between the age of a stain and structural changes to the Hb molecule, the reported methods provided inadequate resolution for the time intervals (i.e.…”

Age estimation of blood stains by hemoglobin derivative determination using reflectance spectroscopy

“…Already in the 1930s Schwarzacher attempted to correlate the age of a blood stain with the blood stain's rate of inhibition of solubility in water, which begins rapid and then decreases slowly as the blood stain ages [1]. Over the last two decades, many more techniques have been explored for this forensic quest, including oxygen electrodes [2], RNA degradation [3], atomic force microscopy [4], and electron spin resonance spectroscopy [5]. Although all of these approaches confirm that the physical and chemical properties of blood stains change over time, no technique has yet shown the precision and reproducibility that is needed for age determination in forensic practice.…”

Biphasic Oxidation of Oxy-Hemoglobin in Bloodstains

“…Until now, the ageing of bloodstains has been interpreted by empirical models [2], [17], [18], which hamper understanding of the oxidation decay rates and its dependency on temperature and humidity. We present reflectance spectroscopy measurements of bloodstains on cotton to determine the amount of hemoglobin derivatives in ageing bloodstains.…”