The age of a bloodstain can be used to establish the time when a crime was committed. As blood ages, deoxyhemoglobin (HbO) is converted into methemoglobin (MetHb), which is evident by the color change from red to brown. Human blood from vein and capillary vessels was used in this investigation. Samples were prepared by placing blood on substrates such as gauze and glass. Near-infrared (NIR) spectra of the samples were measured periodically at ambient conditions for one month. Changes in the spectra can be interpreted in terms of chemical changes in the samples. Loss of water plays a significant role in establishing the initial changes in the spectra. After the first hour, a new, broad band appears between 1460 and 1860 nm and grows as a function of aging. This band, due to the formation of methemoglobin and binding of water, continues to increase for ~21 days and can be used to predict the age of a stain. Standard error of prediction for composite data on bloodstains from three sources was 2.3 h over aging periods up to 590 h.

Determining the age of a bloodstain is of great interest in forensic science because the age could enable crime scene investigators to deduce the timeframe in which a crime was committed. The overall goal of our research on bloodstains is to explore the feasibility of developing a portable optical instrument that will be capable of predicting the age of stains found at crime scenes. This hypothetical handheld instrument will measure optical signals (spectra) reflected from a stain and use previously determined calibration data to predict its age. Before the concept of this instrument can be implemented, it is necessary to determine optical properties of blood, which might be used for predicting the age.

The problem of estimating the age of bloodstains has been paramount since the early years of modern forensic methods (1,2). Currently, either a sample of a stain or both the stain and its substrate are sent to a laboratory for analysis. The most widely used analytical method is high performance liquid chromatography (HPLC), which focuses on the relative height of a peak due to a degradation product (3–5). Degradation of RNA has been used successfully to estimate age on the order of years (6); however, this is a time-consuming effort and not adaptable to field applications. Other laboratory instruments such as atomic force microscopy (AFM) (7), oxygen electrodes for measuring hemoglobin (8), and electron paramagnetic resonance (EPR) (9) have been used successfully in the laboratory. However, a portable, field-adaptable instrument for the rapid determination of the age of bloodstains does not exist.

Blood represents about 8% of total body weight and has an average volume of 5 L in women and 5.5 L in men (10,11). It is a transport medium for dissolved or suspended material and travels from the heart to tissues and cells of the body through the distributed blood vessels. Erythrocytes, or red blood cells, are characterized by their primary function of O₂ transport in the blood. Red blood cells are fully packed with hemoglobin, which is an iron-containing molecule that can bind with O₂ loosely and reversibly (10,11). Because O₂ is poorly soluble in blood, hemoglobin is indispensable for O₂ transport. Hemoglobin consists of four highly folded polypeptide chains (the globin portion) and four iron-containing heme groups (protoporphryn molecules), each of which is bound to one of the polypeptides. Each of the four iron atoms found in heme can combine reversibly with one molecule of O₂; therefore, each hemoglobin molecule can pick up four O₂ molecules. Oxygen binds loosely with one of the six coordination bonds of the iron atoms. Hemoglobin is also a pigment that is naturally colored and because of its iron content, it appears reddish when combined with O₂. Red blood cells are about 8 μm in diameter, and there are a million hemoglobin molecules in one erythrocyte.

As blood ages, deoxyhemoglobin is converted into methemoglobin, which is evident by the color change from red to brown (10,11). Deoxyhemoglobin is actually a deoxygenated heme group that lacks O₂ binding to its iron atom. Methemoglobin is a brownish-red form of hemoglobin that occurs when hemoglobin is oxidized during decomposition. The oxidation of Fe²⁺ to Fe³⁺ creates methemoglobin. Oxidized iron lacks an electron to bind oxygen effectively; thus, methemoglobin accumulation can impair tissue oxygenation (11). The Fe³⁺ in methemoglobin binds water rather than oxygen (12,13).

In trying to develop an optical instrument for determining the age of bloodstains, we explored the potential of using the visible, near-infrared (NIR), and mid-infrared (IR) optical regions. There are obvious visible spectral changes as blood ages; the other components of blood such as water and proteins do not have specific bands in the visible region and there is a lack of bands for referencing the magnitude of the color change. The mid-IR region does have very characteristic bands due to proteins and water; however, it was found that the relative spectral changes depended upon the type of blood. For example, blood from veins demonstrated well-defined changes as a function of time, whereas blood from capillaries exhibited very little change other than the loss of water with time. In the NIR region, similar spectral changes were observed for vein and capillary blood samples. The investigations on hemoglobin in the NIR region of 1100–2500 nm has been very limited (14,15). Most investigations of hemoglobin focused on the visible and short-wavelength NIR with the goal of developing oximeters for noninvasive monitor of oxygen transmittal by hemoglobin (16–19).