Seventh CSI Lesson: Blood Identification

Blood identification is very essential in crime scene investigation as it helps to identify and match the blood samples with the different suspects, culprits and victimes, thus providing more information to the police for them to crack and solve the case.

Firstly, the context was set:
A man was murdered in the loading bay of a dance club and at the crime scene there were red stains all over: bottles of chili sauce, tomato sauce, fruit juice and dyes. The police were unable to identify which were the blood stains and thus had to bring back all the samples of these stains for blood identification.

There are simple tests used to identify blood:
1.) Add benzidine to a solution made from dried blood and water. The solution turns blue or in the case of luminol, glow in the dark! :) In the lab, we used luminol.

Basic idea of luminol: reveal traces of blood with light-producing chemical reaction between several chemicals and hemoglobin in the blood. The molecules break down and the atoms rearrange to form different molecules. In this reaction, the original molecules have more energy than the products (the resulting molecules). The molecules get rid of the extra energy in the form of visible light photons. This process, is generally known as chemiluminescence, which is the same phenomenon that makes fireflies and light sticks glow.

2.) Kastle-Meyer test uses a substance called phenolphthalein, which is normally colourless, but turns pink in the presence of blood.

Usually, this is how the test is carried out.
A drop of phenolphthalin reagent is added to the sample, and after a few seconds, a drop of hydrogen peroxide is applied to the swab. Sometimes the swab is first treated with a drop of ethanol in order to break open the cells that are present, resulting in increased sensitivity and specificity. This test is nondestructive to the sample, which can then be kept and used in further tests at the lab, such as in DNA analysis. If the swab turns pink right away, it is said to test positive for blood.

However, this test result is presumptive positive. In other words, it is not a conclusive test for blood. Other analyses would be carried out to confirm the presence of blood. This is one thing to note: waiting for a period of time over 30 seconds will result in most swabs turning pink naturaly as they oxidise on their own in the air. Some chemicals and biological fluids that DO NOT contain blood can also cause the colour change. Thus, these substances have to be avoided because they produce false positive results= positive result (colour change) in ABSENCE of blood.

How does this test work?
The Kastle-Meyer test relies on the iron in hemoglobin in the blood to promote the oxidation of phenolphthalin to phenolphthalein. phenolphTHALIN is colourless, but in the presence of blood and hydrogen peroxide, it changes to phenolphTHALEIN, which makes the solution pink.

3.) Add hydrogen peroxide to the blood sample. It will bubble profusely.

Why does it foam/ bubble profusely?
Blood and cells contain an enzyme called catalase. Since a cut or scrape contains both blood and damaged cells, there is lots of catalase present. When the catalase comes in contact with hydrogen peroxide, it turns the hydrogen peroxide into water and oxygen gas.

Catalase does this extremely efficiently-- up to 200 000 reactions per second :O The bubbles seen in the foam are pure oxygen bubbles being created by the catalase. Putting a little hydrogen peroxide on a cut potato will also achieve the same result because catalase in the damaged potato cells reacts with the hydrogen peroxide. Hydrogen peroxide does not foam in the bottle or on skin because there is no catalase to help the reaction to occur. It is stable at room temperature.

We were given 6 different samples to test for blood. Firstly, before we carried out the blood detection test for the first test, we had to fill in our predictions for the samples. After which, we carried out our results and these were our results:

Table of results for first test :) 

The samples tested positive for catalase: 4, 5 and 6

Next, we proceeded onto test 2 using the samples tested positive in the previous test. We were given filter papers and luminol spray. We dripped 2 drops onto filter papers for each sample. Then we placed the filter paper in the open cupboard of our lab bench, where it is darker and we sprayed luminol onto the filter paper. The bluish glow was then observed.

Samples that glow/s in the luminol test: 4 and 5.

Following that, the third test was used to test for the presence of blood for the samples that glowed in the previous test. We were again given filter papers and this time, the Kastle-Meyer Reagent also. We dripped 2 drops of samples onto filter papers individually and 4 drops of Kastle-Meyer Reagent onto each filter paper for each sample. Then, we had to wait 3 minutes and record our observation. After waiting to ensure that no colour develops at this stage (because there may be other external factors that cause the colour change), add two drops of 3% hydrogen peroxide. An intense pink colour is positive test for peroxidise activity, indicative of hemoglobin and thus, blood.

OBSERVATION: SAMPLE 4 TURNED INTENSE PINK :)
sample5 only turned a very light pink!

CONCLUSION: SAMPLE 4 IS BLOOD!!!
sample 5 had a colour change maybe due to other factors such as the presence of other chemicals/ oxidisation that causes the light pink colour to be obtained.

PHOTOS:

Lots of bubbling in sample 4 on the right. Small amount of bubbling in sample 5 on the left :) 

Sample 4 glowing in the dark after luminol was sprayed :)

Reduced phenophthalein! 

Sample 5- light pink! 

QUESTIONS:
Why is it necessary to use more than one test to determine whether the given stain is blood?
These tests are presumptive tests. We cannot conclude directly and accurately based on only just 1 test but we can eliminate the other samples to obtain the final sample that is BLOOD. For example: test 2 is a presumptive test, we would need to carry out other analyses to confirm the presence of blood.

Once you know that a stain is real blood, what else would you do as a forensic scientist?
I would carry out a DNA test to test if the blood matches with suspects or victims. I would also check if it is human/ animal blood.

 With these tests to detect presence of blood, it would be very useful to the forensic scientists as they are able to carry out blood comparison analysis, etc. and aid in progress of the police's work in crime scene investigation.