Immunological Assays in Lab Practices

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the primary purpose of the immunochromatographic assay in identifying saliva?

To detect the presence of α-amylase in the sample by creating a visual indication through a pink line.

Why is proper validation of the testing method important in a laboratory setting?

Proper validation ensures that the method works reliably in that laboratory and prevents interruptions in workflow.

What should employees do if they need to deviate from the established Standard Operating Procedures (SOP)?

They must record the deviation along with reasoning and obtain approval from the lab supervisor prior to implementation.

What are the potential consequences of not following SOP in a laboratory?

It can lead to inaccurate results, loss of credibility, and jeopardize one’s career due to perceived incompetence. Signup and view all the answers

In the context of testing, what creates a pink line at the test line in the immunochromatographic assay?

The binding of the α-amylase in the complex to the immobilized antibody in the test zone. Signup and view all the answers

What is the primary chemical reaction that produces light in the chemiluminescence assay for blood identification?

The oxidation of luminol by heme produces chemiluminescent light. Signup and view all the answers

What happens if KM reagent turns pink during the identification process?

If it turns pink, it indicates a false positive, and the process must be started over. Signup and view all the answers

What is a significant drawback of the chemiluminescence assay regarding the environment in which it operates?

The room must be completely dark to see the emitted light. Signup and view all the answers

In the immunochromatographic assay, what type of protein do antibodies detect to confirm the presence of human blood?

Antibodies detect human glycophorin A (GPA), a protein on human red blood cells. Signup and view all the answers

What is one advantage of using the Blue Star chemiluminescent reagent over traditional luminol?

Blue Star does not require 100% darkness to visualize results. Signup and view all the answers

What is the function of antibodies in the immunochromatographic assay?

Antibodies detect human antigens and confirm the presence and species of blood. Signup and view all the answers

What is the primary purpose of serology?

To identify cell types on an item and/or the type of stain present. Signup and view all the answers

How does the chemiluminescence assay demonstrate cleaned areas at a crime scene?

It can detect small droplets and traces of blood, revealing areas that were cleaned. Signup and view all the answers

What is a presumptive test in serology?

A preliminary test that indicates the stain may be of a certain origin but does not conclusively prove it. Signup and view all the answers

What type of antibodies are produced in the immunochromatographic assay from animal injections?

Polyclonal antibodies containing a mixture of antibodies against various human antigens are produced. Signup and view all the answers

Describe a confirmatory test in contrast to a presumptive test.

A confirmatory test conclusively determines the stain's origin and carries less risk of false positives. Signup and view all the answers

What is a false positive result?

A positive result when the actual result should be negative. Signup and view all the answers

List three body fluids commonly tested in serology.

Blood, semen, and saliva. Signup and view all the answers

What constitutes the majority of blood's composition?

Blood is composed of plasma, red blood cells, white blood cells, and platelets. Signup and view all the answers

What is the lifespan of red blood cells, and what is their primary function?

Red blood cells have a lifespan of three months and primarily transport oxygen to cells. Signup and view all the answers

What are colorimetric assays in the context of serological testing?

Colorimetric assays are tests that involve color changes to indicate the presence of specific substances. Signup and view all the answers

What are the three main parts of a spermatozoon?

Head, middle piece, and tail. Signup and view all the answers

What is the primary role of the acrosomal cap in sperm?

It contains enzymes that break down the outer layer of the egg for fertilization. Signup and view all the answers

Why is microscopy considered a confirmatory test for semen identification?

It allows for the visualization of sperm cells and their morphology under a microscope. Signup and view all the answers

What is the purpose of using a Christmas tree stain in microscopy for semen?

It differentially stains nuclei and cell membranes, allowing for the identification of sperm cells. Signup and view all the answers

What are the main sources of saliva production in the human body?

Submandibular glands (70%), parotid glands (25%), and sublingual glands (5%). Signup and view all the answers

What role does α-amylase perform in saliva?

It breaks down polysaccharides into simpler sugars during digestion. Signup and view all the answers

What types of samples can show amylase activity aside from saliva?

Breast milk, blood, tears, semen, vaginal secretions, sweat, and urine. Signup and view all the answers

What technique is used to visualize saliva stains and what is its limitation?

ALS (Alternate Light Source) can be used, but it is not specific for saliva. Signup and view all the answers

What is the function of the tail in spermatozoa?

It is responsible for the mobility of the sperm cell. Signup and view all the answers

What does the Sperm Hy-Liter technique identify specifically?

It identifies human sperm heads only. Signup and view all the answers

What role do white blood cells play in blood?

White blood cells defend the body against infection and contain a nucleus. Signup and view all the answers

What is the primary function of platelets?

Platelets aid in blood clotting and collect at sites of vascular injury. Signup and view all the answers

Describe the visual appearance of blood when dried on a smooth surface.

Dried blood appears flaky and cracks if on a smooth surface. Signup and view all the answers

What is the significance of a pink result in the Phenolphthalin Assay?

A pink result indicates a presumptive positive for the presence of blood. Signup and view all the answers

What potential sources could lead to false positives in the Phenolphthalin Assay?

Oxidants such as household cleaners and plant peroxidases like horseradish can cause false positives. Signup and view all the answers

In what manner is the Phenolphthalin Assay performed?

The assay involves an oxidation-reduction reaction catalyzed by heme, using phenolphthalin as a reductant and hydrogen peroxide as an oxidant. Signup and view all the answers

What does it mean if blood appears black under an alternate light source (ALS)?

While blood may appear black under ALS, it cannot definitively be identified as blood without further testing. Signup and view all the answers

How sensitive is the Phenolphthalin Assay in detecting blood?

The assay is very sensitive, detecting down to 10^-5 fold dilution. Signup and view all the answers

What is the primary purpose of the immunochromatographic assay in blood identification?

To detect the presence of human blood through the binding of anti-GPA antibodies to GPA in a sample. Signup and view all the answers

How long should the sample sit in the universal buffer during the assay process?

The sample should sit for 10 minutes to 2 hours. Signup and view all the answers

What does a pink line at the control line indicate in the immunochromatographic assay?

It indicates that the test has worked properly and is valid. Signup and view all the answers

What is the significance of the prostate in semen production?

The prostate contributes 30% of ejaculate fluid and secretes acid phosphatase and prostate-specific antigen. Signup and view all the answers

What happens if the test line is very faint in the blood assay?

It may indicate a diluted sample or a possible false negative. Signup and view all the answers

What is a potential consequence of a vasectomy regarding sperm production?

It can lead to aspermia, meaning no sperm is present in the ejaculate. Signup and view all the answers

Describe the role of acid phosphatase (AP) in semen identification.

AP is an enzyme found abundantly in the prostate and is used as a presumptive indicator for semen. Signup and view all the answers

What is the function of the colorimetric assay in semen testing?

It detects acid phosphatase by observing a color change indicating its presence. Signup and view all the answers

Why is it important to read the results of the blood assay within 10 minutes?

Results are reliable only before the 10-minute mark; readings after this time may not be accurate. Signup and view all the answers

How can the presence of flavins be used in semen identification?

Flavins fluoresce under UV light, aiding in the visualization of semen stains. Signup and view all the answers

What indicates a 'high-dose hook effect' in the immunochromatographic assay?

It occurs when high concentrations of antigens result in decreased intensity of the test line. Signup and view all the answers

What range of spermatozoa concentration is typically found in normal ejaculate?

Normal ejaculation contains 10^7 to 10^8 spermatozoa per mL. Signup and view all the answers

What is a common issue when using ALS/FLS for identifying semen stains?

It is not specific for semen as it can also fluoresce in other bodily fluids. Signup and view all the answers

What is the half-life of prostate-specific antigen (PSA) in a dried stain?

The half-life of PSA in a dried stain is approximately 3 years at room temperature. Signup and view all the answers

Signup and view all the answers

Study Notes

Fundamentals of Serology & Microscopy (BIO 440)

The course covers forensic examination of fluids like semen, vaginal fluids, sweat, blood, and saliva.
Serology is used to identify cell types and stains on a sample.
Serology can provide guidance for DNA analysis by determining the amount and components (male/female) of DNA expected.

Two Classifications of Tests

Presumptive Tests:
- Preliminary tests indicating the potential origin of a stain.
- Do not definitively confirm the stain's origin.
- Used to screen large items, guiding what tests to run next.
- Pros: narrows down possible origins
- Cons: high risk of false positives, overly sensitive.
Confirmatory Tests:
- Conclusively determine the origin of a stain.
- May involve one or more procedures.
- Pros: less risk of false positives.
- Cons: more expensive, complex, and time-consuming.

False Results

False Positive: A positive result when the actual result should be negative.
- Example: a pregnancy test.
False Negative: A negative result when the actual result should be positive.
- Example: a pregnancy test.

Fluids Tested in Serology

Body fluids commonly tested: blood, semen, saliva, urine, feces, and menstrual fluid, cerebrospinal fluid, amniotic fluid, pleural fluid etc.

General Classifications of Tests

Visual Examinations:
- Naked eye, alternative light sources (e.g., UV light).
Colorimetric Assays:
- Chemical reactions causing a color change, indicating the presence of a substance.
Chemiluminescent Assays:
- Chemical reactions producing light.
Immunochromatographic Assays:
- Using antibodies to detect specific antigens.

Blood

Serology test results are reported as either presumptive or confirmatory test for blood.
Blood comprises 8% of body weight.
Blood consists of plasma, red blood cells (RBCs/erythrocytes), white blood cells (leukocytes), and platelets.
Plasma is the fluid where blood cells reside, being 90% water and 10% proteins, minerals, and hormones such as antibodies.
Red blood cells live for 3 months, lack a nucleus, and contain hemoglobin, a protein carrying oxygen to cells and carbon dioxide to the lungs.
Hemoglobin is a crucial protein within red blood cells, essential for oxygen transport.

Hemoglobin

Contains heme molecule and iron.
Iron is essential for oxygen binding, and its structure plays a key role in oxygen transport. The structure is vital for oxygen transport.

White Blood Cells & Platelets

White blood cells defend against infections, contain a nucleus, and are responsible for DNA in a blood sample.
Platelets aid in blood clotting and are present in areas of injuries. They lack a nucleus.

Workflow for Blood Testing

Blood stain appears as a presumptive positive test.
Confirmed, DNA profiling or identification can be performed, leading to individualization.
Visual examination, presumptive, confirmatory , and DNA profiling are the steps.

Identification of Blood: Visual Examination

Blood appears candy-apple red or deep red.
Dried blood on a smooth surface shows flaky and cracks.
Appears black under alternative light sources.

Identification of Blood: Colorimetric Assay

Phenolphthalin Assay (Kastle-Meyer Test):
- An oxidation-reduction reaction catalyzed by heme.
- Oxidation is loss of hydrogen, reduction is gain of hydrogen.
- The presence of heme oxidizes phenolphthalin, causing a pink color.
- Very sensitive, detecting very low dilutions of blood.
- Not specific, can give false positives, such as from oxidants.

Identification of Blood: Chemiluminescence Assay

Light is emitted as a product of a chemical reaction when viewed with UV light.
The chemical reaction produces light, presumptively indicating the presence of blood.
Can be sprayed on large areas to detect blood.
Detects small blood droplets and traces of blood.
Can detect blood patterns and prints.

Identification of Blood: Immunochromatographic Assay

Confirmatory test for both human blood and origin of the blood (human or animal).
Uses antibodies to detect human antigens.
Polyclonal antibodies are a mixture of antibodies against various human antigens; monoclonal antibodies target one specific site.
Detects human glycophorin A (GPA).
- GPA is a protein found on human red blood cells.
No cross-reactivity with commonly encountered animals, but with higher primates.
Results (positive or negative) are read within 10 min.

Identification of Semen: Visual Examination (ALS/FLS)

Flavins and choline-conjugated proteins fluoresce under ALS/FLS.
A wavelength of 450-495 nm is used for visualization. Blue and yellow colors are visible on the stains, depending upon the wavelengths.
Semen stains have moderate to high fluorescence. Older stains exhibit moderate fluorescence.

Identification of Semen: Acid Phosphatase (AP)

The prostate is the main source of AP in the body.
AP is also found in lysosomes.
Vasectomies have no effect on AP levels.
Half-life of AP in dried stains is about 3 years at room temperature.
Degradation occurs quicker in wet conditions.

Identification of Semen: Colorimetric Assay

Acid phosphatase (AP) hydrolyzes phosphate esters.
A-naphthyl phosphate is a substrate.
The resulting solution forms a purple precipitate from the presence of brentamine Fast B.
How it is done: transfers the stain to a swab or filter paper, use a water-soluble solution (AP) drop, positive result shows a purple color within one minute. After this time, the results are not conclusive.
The method can be used to map a large stain area.

Semen: Prostate-Specific Antigen (PSA)

PSA/P30 is a major protein in semen, produced in the prostate.
Also found in other body fluids (sweat, perianal, mammary glands, blood etc.).
Half-life of PSA in a dried stain is 3 years at room temperature.
Degradation increases with wet environments.

Identification of Semen: Immunochromatographic Assay

P30 testing cassettes are used which work like the previous cassettes, but targeting P30 rather than a-amylase.
High-dose hook effect.
- Caused by very high concentrations of antigens.
- Unbound antigen can reach the test line before the antibody binding complexes are created.

Semen: Spermatozoa

Spermatozoa (sperm cells) consist of head, middle piece, and tail.
- Head contains nucleus and acrosomal cap.
  - Enzymes in the cap help to enter the egg.
- Middle piece has mitochondria, providing energy.
- Tail provides mobility.
60% of sperm have normal morphology.

Identification of Semen: Microscopy

Cells are transferred to a microscope slide using water and heat-fixed (low heat).
Christmas tree stain is applied.
- Red dye first stains all nuclei.
- Green dye stains the cell membranes, sperm neck, and tail.
A coverslip is added.
Microscopic analysis assesses sperm, epithelial cells, and debris.
Cells can be removed from the slide for DNA analysis.

Identification of Semen: Sperm Hy-Liter

Immuno-fluorescence staining kit for identifying human sperm.
Specific to human sperm heads.
Sensitive and flexible, allowing for use on smear slides or extracts.
Differential staining allows for differentiating between sperm cells and epithelial cells. Staining products and methods are used to differentiate and identify different cells.

Saliva

1.0 to 1.5 L of saliva is produced daily.
70% from the submandibular glands, 25% from the parotids, 5% from sublingual glands.
Saliva consists mainly of water, along with electrolytes, antibodies, buffers, glycoproteins, and enzymes.
Amylase enzyme begins breaking down food in the mouth.
- Cleaves starch into maltose.
Human α-amylase has two isomers: pancreatic and salivary.
Amylase activity is present in breast milk, blood, tears, semen, vaginal secretions, sweat, and urine.
Saliva is stable for a few weeks to a few months.

Identification of Saliva: Visual Examination

ALS can be used for searching potential saliva stains.
Visual Examination using a specific wavelength (470 to 555 nm using colored goggles) can give light fluorescence.

Identification of Saliva: Immunochromatographic Assay

Human α-amylase testing cassettes are used.
- Manufacturer considers these as confirmatory tests.
- Industry considers these presumptive tests.
Procedures are similar to other immunochromatographic assays (for example, those for semen).

Standard Operating Procedures (SOP)

SOP outlines the testing procedures, which do not include the purpose of the testing techniques. Steps, not the purpose are outlined.
Every method or procedure tested has its own SOP.
Method must be properly validated before use
Improper validation leads to a halt in the work stream until the proper validation and approval are complete.
Employees must follow the same procedure.
Deviations from SOP require recordation and supervisor approval.
Following SOP prevents invalid results and maintains procedure competency. Failure to follow the standard procedures are viewed as incompetent actions which can cause job loss and a lack of trust.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

This quiz focuses on the principles and applications of immunochromatographic and chemiluminescence assays in laboratory settings. It explores key concepts such as validation of testing methods, adherence to Standard Operating Procedures (SOP), and the chemical reactions involved in these assays. Test your knowledge on the significance of these methods in identifying biological samples!