Determinazione Sangue

[Applications] [Luminol Derivatization Reagents]

Luminol's Test

The forensic luminol test involves the emission of luminol chemiluminescence (CL) in the presence of haemoglobin. It is used at crime scenes as a presumptive test for blood. The major advantage of the luminol test is its high sensitivity, it can detect nanogram traces of blood [14] that are invisible to the naked eye, which makes it up to 20 times more sensitive than any other blood detection test [15]. The limitation with luminol and other blood detection tests is their lack of selectivity. Substances other than blood (2–6) can catalyse the CL reaction that is the central component of the luminol test. One commonly reported interfering catalyst [15, 16, 17, 18, 19] is sodium hypochlorite solution (bleach), a common ingredient in the majority of cleaning agents around most homes and industries. Curiously, there is a lack of quantitative papers on the effect of bleach-based cleaners on bloodstains tested with luminol. Criminals often attempt to clean up blood spills after committing a violent crime, so it is important to know what effect attempted cleaning has on luminol test results. In the present study we apply haemoglobin stains to ceramic tiles and quantitatively observe the effects of repeated washing with water or bleach, on the CL intensity when luminol is applied to the tile. The effect of bleach drying time on luminol CL is also observed.

Non-Chemiluminescent tests

There are also a number of nonchemiluminescent presumptive chemical tests for blood that are used but unfortunately there has been no comprehensive, quantitative evaluation of the merits of all of these tests. Table 1 provides a list of the commonly available presumptive tests for blood. One of these tests is somewhat different from all the others and simply involves the illumination of the suspect forensic area with a bright light of controllable wavelength.Although not strictly a chemical test, this is quite a useful forensic procedure and so-called ‘forensic’ light sources of high intensity are readily available and used in most policing situations. Such light sources usually allow the possibility of identifying blood stains by reflected light or by the emitted fluorescence excited when ultraviolet wavelengths are used to illuminate the forensic sample. The first two tests in Table 1, the benzidine and o-tolidine, are no longer generally regarded as acceptable because of the strong carcinogenic properties of the chemical reagents [20, 21] and have largely been abandoned in recent years. Those tests will not be considered in any further detail here and were not included in the present study. Attention will, however, be paid to possible safety considerations related to the remaining tests which are currently in use.


The luminol method.Standard luminol solution was prepared as described by Creamer et al. [18]. The dilute (15 g/L) haemoglobin solution consisted of 0.15 g haemoglobin (Sigma) dissolved in 10 mL 0.2 mol/L NaOH. The bleach solution used for the tiles was diluted to a concentration of ~ 60 g/L NaClO, while the drying experiments were performed with 125 g/L and 10 g/L NaClO (all dilutions in water). Glazed terracotta tile was used as the test surface. Each tile was sprayed with haemoglobin solution using a hand-held sprayer, and either tested immediately, in the case of the ‘wet’ stains, or left to dry for 1 h. A single wipe across the tile surface with a paper towel soaked in either water or bleach constituted one ‘clean’. The direction of each subsequent clean was parallel to the first and this pattern continued for all cleaning steps. The bleach samples for the drying experiments were prepared as described by Creamer et al. [18], and left to dry for 0, 2, 8 and 16 h. The procedure for measuring CL intensity was as described by Quickenden and Creamer [17]. CL for each tile was re-measured after each stage of cleaning. One researcher tested all the tiles and then two different researchers repeated the same experiments independently.

The leucomalachite green (LMG) method. The LMG reagent stock solution was prepared in a similar manner to the KM reagents. 0.25 g malachite green (Gurr), 100 mL glacial acetic acid (Ajax) and 150 mL distilled water were combined with 5 g powdered zinc in a roundbottomed flask and refluxed until the deep green-blue colour had disappeared and the solution was clear. After cooling, the solution was transferred to a bottle containing an excess of powdered zinc to ensure that it remained in a reduced state, and stored at 4°C. A positive reaction with hemoglobine for the LMG test was signalled by the presence of a blue-green colour developing within 10 s.

The Kastle–Mayer (KM) method. The reagent stock solution was prepared by combining 2.0 g phenolphthalein (Sigma), 20 g potassium hydroxide (Ajax), 30 g powdered Zn (Univar) and 100 mL distilled water in a round-bottomed flask. The mixture was refluxed for 2 h until the solution was reduced (shown by a colour change from deep pink to clear). The solution was then cooled to room temperature and stored in an amber glass bottle. An excess of powdered zinc was added to ensure that the stored solution remained in a reduced state. A ‘working’ KM solution was made by combining 10 mL stock solution with 40 mL absolute ethanol (Ajax) in an amber dropper bottle. Both bottles were stored at 4°C. A positive with hemoglobine reaction for the KM test was signalled by the presence of a bright pink colour developing in the stained area within 10 s

Hemastix® method. The Hemastix® (Bayer) was purchased as a pre-prepared test for blood in urine. The test kit consisted of 50 reagent strips containing 3,3′, 5,5′- tetramethylbenzidine. A reagent strip was simply dipped into a solution and a colour change from orange to bluegreen was observed if haemoglobin was present.


Based on the results presented here, the luminol test is clearly the most sensitive blood detection technique commonly used by forensic investigators. The Hemastix® test was the next most sensitive, followed by the KM and LMG techniques. The Polilight® was by far the least sensitive technique, being 50 000 times less sensitive than the luminol test and 10 times less sensitive that the next least sensitive technique investigated here (LMG). Another interesting finding was that the sensitivity of the KM, LMG and Hemastix® tests decreased considerably when applied to filter paper or cotton swabs of bloodstains. While the amount of blood transferred from a stain to a swab may vary considerably, depending on the investigator, this result clearly shows that it is favourable to test a bloodstain directly. The sensitivity of the luminol test was found to be 1:5 000 000 for both the bloodstained cloth and haemoglobin solutions. This was consistent with previously reported literature values [14, 22]. No literature results were found regarding the sensitivity of either the Hemastix® or Polilight® tests for blood; however, previous literature on the active reagent of the Hemastix® test, TMB, determined its sensitivity to be 1:1 000 000 for diluted haemoglobin solutions [14]. Again, this was consistent with the results of the present study. The results obtained for both the KM and LMG tests varied from previously reported literature values, although it should be noted that the previous literature values fluctuate from paper to paper, possibly indicating inherent variability within the techniques. The sensitivity of the KM test from previous publications ranged from 1:10 000 for blood-soaked cloth [21], 10 times less sensitive than presently reported, to 1:100 000– 1:1 10 000 000 for haemoglobin solutions [14, 20], the present study being consistent with the less sensitive edge of the range. The swab results also varied in the literature, from 1:10 000 [21] to 1:5 000 000 [22], 100 to 50 000 times more sensitive than reported in the present study. The LMG technique showed similar, although less pronounced, variability throughout the literature. The sensitivity of the test ranged from 1:100 (swabs) to 1:1000 (blood-soaked cloth and solutions) within this study, compared with 1:5000 (swabs, cloth and solutions) to 1:100 000 (swabs) from previous publications. The variability of the KM and LMG techniques, both within this research and throughout the literature raise questions about the reliability of the tests. All of the presumptive blood detection techniques investigated here are fairly easy to use, especially the Hemastix® test. While the weight and dimensions of the Polilight® and the darkness required for the luminol test may cause some initial difficulties, these would easily be overcome by regular use.All of the presumptive blood tests examined here are useful to the modern forensic investigator. However, the Kastle–Meyer and leucomalachite green tests use carcinogenic reagents, while the Polilight® exhibits poor sensitivity, making their use undesirable. The luminol test is by far the most sensitive technique, and is easy to perform, particularly when application over a large area is required. When the luminol technique is not appropriate, the Hemastix® test is a simple and effective alternative.

[Applications] [Luminol Derivatization Reagents]