Immunological Assays in Lab Practices

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.