Forensic Serology: Blood Typing - Mr. Donohue's … · Web viewForensic Serology: Blood Typing Forensic Science Laboratory Syracuse University Chemistry 113 Introduction Serology

Forensic Serology: Blood TypingForensic Science Laboratory

Syracuse University Chemistry 113

IntroductionSerology is an important part of forensic science. Blood

evidence is often found at the scene of violent crimes including assault, murder, and rape. Even a faint trace of blood can lead to a conviction. Dried stains from a shirt were used to convict murderer Ludwig Tessnow, known as “the mad carpenter,” of multiple brutal murders; even after the clothes had been run through the washing machine.

By identifying the blood type of a drop of blood taken from a crime scene, a list of suspects can be narrowed down significantly and DNA testing can be preformed to make a positive identification. Without forensic serology, many cases would remain unsolved or would not have enough physical evidence to convict the perpetrator.

The CrimeTwo men were found a block away from a robbery, running in

the opposite direction of the crime scene. One man’s fist was bleeding and both had blood spatter on their clothes and shoes. The two men were suspected of committing the crime and were taken into custody. The men claimed that one had cut his hand on a knife and they were merely trying to get to the hospital close by. The blood on their clothes was claimed to be that of the one man.

The victim of the robbery was an old woman who heard someone break a window and was shot while trying to investigate. There was blood found on the shattered glass and drops on the carpet leading to the stairs where the victim was shot. The victim’s blood type was A and the suspect with the bloody fist’s blood type was O.

Use this information and the samples collected from the crime scene to help corroborate or contradict the suspects’ story.

BackgroundCrime scenes often contain blood samples, in the form of pools, splatters, or stains. When

a suspicious substance is found at the scene, it should first be tested to confirm the presence of blood, and then tested to confirm that the blood is indeed human in origin. The presence of blood can be tested a few ways. The Kastle-Meyer color test relies on phenolphthalein, a common pH indicator used in titration; and hydrogen peroxide, which when mixed with hemoglobin, will turn a deep pink. This, however, can yield a false positive from potatoes, horseradish, and some other vegetable materials.1

1 Saferstein, R., Criminalistics, 9th ed.; Pearson Education, Inc.: Upper Saddle River, NJ, 2007; pp 353-4.

Forensic Serology 1 Rebecca Coutant 2008

Another common test for blood is the luminol test, which produces light when in contact with blood. The blood can be diluted up to 300,000 times and the test will still come up positive. The surroundings must be dark in order to scan for the emitted light.2

Once the sample is determined to be blood, the precipitin test is the most common test for human origin. When an animal (generally a rabbit) is injected with human blood, the animal’s system develops antibodies to counteract the presence of the foreign substance. When blood is then taken from the animal, it contains these antibodies and can be used to test for the presence of human blood in a sample. This rabbit blood is generally called human antiserum. The suspected human blood can be placed on top of the human antiserum in a capillary tube. A cloudy ring or band will form where the two liquids interact if it is indeed human blood.3

The next step of the process is to identify the blood type. In 1901, Karl Landsteiner noticed that blood from one person would not mix uniformly with blood from another person. Instead, the samples would clump, in a process called agglutination. He categorized blood into four major blood types, A, B, O, and AB. He organized the following proteins and enzymes as found in the human population:found that types A, B, O, and AB

Even the use of just a few of these systems can result in a small percent of possible suspects. For example, if a blood stain was tested to be AB, M, Rh-, PGM-2, AK 1, ADA 2-1. The chances of a suspect having the same blood type would be

3/100 x 30/100 x 15/100 x 6/100 x 93/100 x .2/100 = 150,660/1 x 1012 = 1 per 6,637,461 people

2 Saferstein, R., Criminalistics, 9th ed.; Pearson Education, Inc.: Upper Saddle River, NJ, 2007; pp 354.3 Saferstein, R., Criminalistics, 9th ed.; Pearson Education, Inc.: Upper Saddle River, NJ, 2007; pp 354-356.


Type A Type B Type O Type AB42% 12% 43% 3%

Rh + Rh -85% 15%

MN M N48% 30% 22%

PGM-1 PGM-2 PGM 2-158% 6% 36%

AK 1 AK 2-193% 7%

ADA 1 ADA 2-1 ADA 289.9% 9.9% .2%

With DNA testing, this can be decreased to about 1 in 10,000,000,000. Since no two people have been found with the exact same blood typing and DNA coding, this amounts to individual identification.4

Blood typing works much in the same way as a precipitin test. The term blood refers to a suspension of red blood cells (erythrocytes), white blood cells (leukocytes), and platelets in plasma. On the outside of each red blood cell lie chemical structures called antigens. One specific structure present is called type A, while another is called type B. When both structures are present on the cells, it is called type AB, while a lack of either is called type O. These antigens are unrelated to the transportation of oxygen by the red blood cell and therefore do not indicate any functional difference from person to person. These specific structures of the antigens are what form the ABO and Rh systems, along with many others.5

For each antigen, there is a protein called an antibody, a protein which destroys or neutralizes specific antigens. These antibodies will react only with their specific antigen. For example, Anti-A will react only with Antigen A. Because antibodies have two bonding sites, they can attach themselves to two cells with their corresponding antigen and form a clot, or an agglutinate. A generalized schematic is shown in Figure 1.6

As illustrated by the diagram, the antibody receptors are shaped to bond with certain antigens. In order to prevent a different kind of blood from infiltrating the body, blood contains the antibodies for all antigens not present.7

Blood Type Antigens Present Antibodies Present

A A Anti-B

B B Anti-A

AB A and B Neither

O Neither anti-A and anti-B

4 Meloan, C; James, R.E.; Saferstein, R.; “Experiment 9: Blood Identification and Typing,” Lab Manual;. Prentice Hall, 2007; pp. 71-72.5 Saferstein, R., Criminalistics, 9th ed.; Pearson Education, Inc.: Upper Saddle River, NJ, 2007; pp 347-8.6 http://en.wikipedia.org/wiki/Image:Antibody.svg7 Saferstein, R., Criminalistics, 9th ed.; Pearson Education, Inc.: Upper Saddle River, NJ, 2007; pp 348.


Figure 1. Schematic diagram of Antibody and Antigens, similar to a lock and key mechanism.

http://en.wikipedia.org/wiki/Image:Antibody.svg

The agglutination occurs on a very microscopic level (about 500x magnification is needed). In this experiment, imitation blood will be used to show a macroscopic version of agglutination. The purpose is to determine the blood type using the ABO system in order to substantiate or contradict a theory of a crime.

ObjectivesThe objective of this laboratory is to determine the blood type of individual students to

obtain a class data set, and to test a set of samples from a crime scene and use the information to help confirm or disprove the suspects’ testimony.

Forensic Serology: Blood TypingExperimental Method

Procedure A – Is it blood?

Materials• 11 unknown samples• Pipette• Spray bottle with luminol solution• Paper towels• Scissors and metric ruler• Fume hood or modified box

Safety Luminol is an irritant. Use in a well ventilated area. Wear goggles for eye protection.Wear gloves for skin protection.

Procedure

PART ONE:1) Make qualitative observations about each sample. Record in the data table.

2) Use a pipette to drop 2 drops of each sample onto a 10 cm x 10 cm portion of a papertowel. Label each paper towel with the letter of the unknown sample.

3) Separately place each paper towel into the fume hood or box and spray with luminol. Donot saturate paper towel.

4) Record your observations in the data table. Speculate on the possibility of the substancebeing blood.

5) Dispose of the paper towels into the proper receptacle.

PART TWO:6) Your teacher has some unknown samples on paper towels that have been washed and

rinsed. Record your observations.

7) Test to see if any of them had been blood. Record your observations.


Procedure B – Is it HUMAN blood?1. Expectorate into a small plastic bag. Collect several milliliters of saliva and transfer it to

a labeled test tube by snipping a corner off the plastic bag and dumping the contents.

2. Stand the tube upright in a 400 mL beaker filled 2/3 of the way with water. Place the beaker/test tube onto a hot plate and bring to a boil, boiling for 10 minutes. Remove and allow to cool overnight.

a. Boiling the saliva inactivates enzymes which otherwise might destroy some of the blood group components in several hours.

Procedure C – What blood group does it belong to – Solving the proposed crime…

Materials:• Test tubes and rack• Pipette or graduated cylinder, 10 mL• Stirring rod• Deionized H2O• Anti-A and Anti-B serum (in pipettes or dropper bottles)• Four unknown samples

Safety: Wear goggles for eye protection. Proceed with caution when using glassware.

Procedure:1) Using a glass marking utensil, label two test tubes “anti-A” and “anti-B”.

2) Measure approximately 1 mL of your unknown sample into both test tubes.

3) Using the anti-serums, pipette 20 drops (≈ 1 mL) of each serum into the appropriatelylabeled test tube (i.e. anti-A serum into the anti-A test tube).

4) Stir using the stirring rod (be sure to clean it before using it in a different test tube).

5) Check each test tube for agglutination and record your observations. A positive reactionwould agglutinate and a negative reaction would not agglutinate.

6) Determine the blood type of your unknown sample.


Forensic Serology: Blood Typing IData Sheet

Name: ___________________ Lab section: ______________Instructor: ____________________ Lab period: ______________

Procedures A: PART ONE

Unknown SampleObservations

(describe visual appearanceand reaction with luminol)

Blood?

A

B

C

D

E

F

G

H

PART TWO

Unknown SampleObservations

(describe visual appearanceand reaction with luminol)

Blood?

X

Y

Z


Procedure C

Sample Origin Reaction with anti-A Reaction with anti-B

1

2

3

4

Based on this information, what blood type assignments can you make for each sample?

Sample 1: __________

Sample 2: __________

Sample 3: __________

Sample 4: __________


Forensic Serology: Blood TypingPost-Lab Assignment


1. Explain how you determine which sample(s) was blood.

2. Are all red liquids or stains blood? Explain.

3. If a substance catalyzes the reaction of luminol, is it definitively blood? Explain.

4. If a perpetrator visually cleans up blood after a crime, can the blood be detected?Explain.

3. What is the least common blood grouping (AB, Rh, MN…) of the types mentioned in the background section? What are the chances of a suspect having that same blood type?


Forensic Serology: Blood TypingPre-Lab Assignment


1. Define the following terms:a. antigen:

←b. antibody:

←c. Kastle-Meyer color test:

←d. preciptin test:

2. What does luminol do and why is it advantageous as a test for blood?

3. What are the chances of a suspect matching a blood sample of type B, Rh+, N, PCM 2-1, AK 2-1, and ADA-2?

4. Below is a table with the results of a blood typing test. Identify the blood types based on the reaction with the antisera.

Sample Result with anti-A Result with anti-B Blood Type

1 agglutination no reaction

2 no reaction no reaction

3 no reaction agglutination

4 agglutination agglutination


Forensic Serology: Blood Typing

Synthetic BloodMaterials for 100 mL:

Flour (superfine, self-rising) 3.25 g Sodium chloride 0.25 g Glycerol (glycerin) 0.25 mL Corn syrup (light, Karo ®) 0.25 mL Deionized water 100.0 mL Food coloring (red and blue)

Equipment: 250 mL beaker Hot plate Graduated cylinders (10 mL and 100 mL) Stirring rod or magnetic stirrer and plate Thermometer (Celsius) Pipette

Procedure:1) Measure all powders and liquids.2) Pour 48.0 mL of deionized H2O into a 250 mL beaker. Place on a hot plate and begin

to heat on low. Use the magnetic stirrer to stir or using a stirring rod, stir often.3) Add NaCl to the beaker.4) Pipette 5 drops (0.25 mL) of the glycerol into the beaker.5) Pipette 5 drops (0.25 mL) of the corn syrup into the beaker.6) Slowly add the flour to the beaker.

*Do not add all at once. This will cause clumping.7) Add 5 drops (0.25 mL) of red food dye to the mixture.

*Make sure all of the flour is homogeneously dispersed before proceeding.8) Frequently stirring, heat the “blood” mixture to boiling (approximately 90.00C).

Allow to boil for 1-2 minutes (mixture will be very thick). Remove from hot plateand allow to cool.

9) While the “blood” is cooling, make a solution of diluted blue dye. Using a graduatedcylinder, add 1 drop of blue dye to 10.0 mL of H2O. Use a pipette to dispenseapproximately 45 drops (2.25 mL) to the “blood.” Stir completely.

10) While stirring, add 20 drops (1.00 mL) of concentrated red dye.11) Add 20.0 mL of H2O to the “blood.” Stir until a homogenous consistency is achieved.

Store in a refrigerator for stability.


Is It Blood? Activity: To react with luminol, add 10 mL of 0.08 M CuSO4•5H2O to the synthetic blood mixture. Stir well. The copper will now catalyze the luminol to produce a brilliant chemiluminescence under low lighting.

o To prepare 0.08 M CuSO4•5H2O, add 0.2 g of copper (II) sulfate pentahydrate to 10 mL of deionized water

To prepare suggested unknown samples:o A—catsupo B—spaghetti sauceo C—tomato juiceo D—Tobassco sauceo E—red laytex painto F—beet juiceo G—synthetic blood (recipe included)o H—water colored with red food dye

Place a small amount of each sample into a labeled clear vial or container.To prepare the unknown samples:

o X—repeat of A through F or Ho Y—synthetic bloodo Z—copper (II) sulfate solution (few drops of 0.2 g CuSO4/10 mL H2O)

Stain a 10 cm x 10 cm portion of a paper towel with each sample. After 3-5 minutes, rinse the paper towels with water and allow them to dry.

Suggested preparation for the luminolo 0.2 g luminol powdero 10.0 g Na2CO3

o 180 mL deionized H2Oo 180 mL 3% H2O2

A-B-O Blood Typing Activity: To prepare unknowns (adjust color with red food dye)o #1—synthetic blood to simulate Type O bloodo #2—synthetic blood and milk mixture (1:1) to simulate Type A bloodo #3—synthetic blood and vinegar mixture (2:1) to simulate Type B bloodo #4—two containers—one synthetic blood/milk & one synthetic blood/vinegar to

simulate Type AB blood

This activity will allow students to explore the events of laboratory testing of blood types without the hazards the come with using human blood. The samples are milk, vinegar, and deionized water and they will be used to simulate acceptance/rejection in blood. Milk and vinegar react, causing clumping (agglutination) of the “antigen” and “antibodies.” Students will work cooperatively to determine the blood type of their unknown sample and work collectively to analyze the class results.

Prepare unknowns (adjust color with red food dye)o #1—synthetic blood to simulate Type O bloodo #2—synthetic blood and milk mixture (1:1) to simulate Type A bloodo #3—synthetic blood and vinegar mixture (2:1) to simulate Type B bloodo #4—two containers—one synthetic blood/milk & one synthetic blood/vinegar to


simulate Type AB blood The two containers will “act” as one and both will be treated with each antiserum. You will most likely have to explain this simulated AB to those lab groups that receive

unknown #4.Prepare anti-serums (colored with yellow food coloring)

o Anti-A—vinegaro Anti-B—milk

Synthetic blood recipe included.Suggestion: make the unknowns reflect the percentage of the population having these

blood types. For a class of 7 or 8 groups, 7 Type O, 5 Type A, 2 Type B, and 1 Type AB

After the students have determined the identity of their unknown sample(s), have them

record their information on the board (similar to the table in analysis) to assist ingathering group data. When all data is reported, the students will calculate thepercentages of each blood type (this should reflect the given percentages, and samples will be prepared with that in mind) and create a bar graph.

Procedure and Preparation references:

Blood type recipe from: Regional Technicians Group: Problems and Solutions - Recipes webpage: http://www.rtg.wa.edu.au/solution/probsoln.htm

Imitation blood recipe from: Blood Recipes webpage: http://www.shades-of-night.com/painneck/blood.html#mint

Bloodstained Cloth procedure: Meloan, C; James, R.E.; Saferstein, R.; “Experiment 10: Typing of Dried Bloodstains (Advanced),” Lab Manual;. Prentice Hall, Upper Saddle River, NJ, 2007; pp. 82.


http://www.rtg.wa.edu.au/solution/probsoln.htm

ALTERNATIVE BLOOD TYPINGForensic Serology: Blood Typing

Instructor InformationPreparation instructions:

Blood Types

Type A Type B Type O Type AB

Reagents: 1.66 g NaCO3

in 100 mL water

Reagents: 1.48 Ca(OH)2

in 100 mL water

Reagents: pure water

Reagents:equal amounts of A and B

Antiserums

Type A Type B

Reagents:1.11 g CaCl2 in 100 mL waterPrecipitates – CaCO3

Reagents:1.42 g Na2SO4 in 100 mL waterPrecipitates – CaSO4

Imitation blood preparation:reagents:

1 cup water2 tablespoon cocoa powder8 tablespoons light corn syrup1 to 2 teaspoon red food coloring4 drops yellow food coloring

• Mix the water and the cocoa powder first in order to get the clumps out. Remaining clumps can be skimmed off the surface after the blood is prepared.

• Mix the blood type solutions with the imitation blood just until desired color is achieved. • Vary the blood type of the samples so that for some, the blood type of the suspect with

the cut hand is a match with the glass from the broken window and in some it is not. The splatter should always match the victim’s blood type. The blood type/types from the shirts can either be the suspect’s blood or the victim’s.


Forensic Serology: Blood TypingPost-Lab Assignment Answers


1. What conclusions can you draw about the suspects’ story from the blood types of the samples?

*depends on the group*

2. What further analysis should be done on the samples if the blood typing contradicts the story of the suspects?

Confirm further blood types and do a DNA analysis

3. Why does agglutination occur when antibodies are added to the blood?Agglutination occurs because the antibodies link up with their respective antigens like a lock and key. Each antibody has two bonding sites and each cell has multiple antigens. The cells link up with the antibodies to form a network, or a “clump.”

4. What steps must be taken before a blood type can be determined? Describe them.The sample must be determined to be blood, using A Kastle-Meyers test which involves the use of phenolphthalein and turn pink in the presence of blood, or a luminol test, which will emit light in the presence of blood. Then the blood must be identified as human in origin. This will involve the precipitin test, in which a human antisera, generally from a rabbit, is layered with the blood sample. If the blood is human, a cloudy ring will form where the liquids interact.

5. What is the least common blood group of the types mentioned in the background section? What are the chances of a suspect having that same blood type?

AB, Rh-, N, PGM-2, AK 2-1, ADA-21 per = 100/3 x 100/15 x 100/22 x 100/6 x 100/7 x 100/0.2 = 1 per 120,250,120


Forensic Serology: Blood TypingPre-Lab Assignment Answers


1. Define the following terms:a. antigen: A chemical structure which lies on the outside of a blood cell

←b. antibody: A protein which destroys or neutralizes a specific antigen

←c. Kastle-Meyer color test: A test to determine whether a substance is blood or not using

phenolphthalein.d. precipitin test: A test to determine whether blood is of human origin using human antisera

created by injecting rabbits with human blood.e. agglutination: The process of clumping which occurs when antibodies react with their

specific antigen.2. What does luminol do and why is it advantageous as a test for blood?Luminol emits light when it is in the presence of blood. It does not show false positives like the Kastle-Meyer test and it can detect very dilute quantities.

3. What are the chances of a suspect matching a blood sample of type B, Rh+, N, PCM 2-1, AK 2-1, and ADA-2?

1 per = 100/12 x 100/85 x 100/22 x 100/36 x 100/7 x 100/0.2 = 1 per 884,192

4. Below is a table with the results of a blood typing test. Identify the blood types based on the reaction with the antisera.

Sample Result with anti-A Result with anti-B Blood Type

1 agglutination no reaction A

2 no reaction no reaction O

3 no reaction agglutination B

4 agglutination agglutination AB


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Forensic Serology: Blood Typing - Mr. Donohue's … · Web viewForensic Serology: Blood Typing Forensic Science Laboratory Syracuse University Chemistry 113 Introduction Serology