Basic Serologic Laboratory Techniques and Investigation of Innate Immunity Related Disorders (P) Flashcards
What is immunology?
1) Study of the molecules, cells, organs, and systems responsible for the recognition and disposal of foreign materials (nonself) in our body
2) Study of the body’s response to infectious diseases
3) Study the desirable and undesirable consequences of immune interactions
4) Study of the ways in which the immune system can be advantageously manipulated to protect against or treat disease
What is serology?
1) Study of serum and its immune components
2) Refers to the diagnostic identification of antibodies in the serum
What are serological tests?
These are diagnostic methods that are used to identify antibodies and antigens in a patient’s sample
What are the purposes of serological tests?
1) To diagnose infections and autoimmune diseases
2) To check if a person has immunity to certain diseases
3) Determining an individual’s blood type
What is serum?
The most frequently encountered specimen (especially in immunology and serology)
How is blood collected?
It is collected aseptically via venipuncture and transferred into a clean, dry, sterile tube
What is the reason why hemolysis should be avoided?
Because it may produce a false positive test (or result)
What should be done to blood for preparation for serologic tests?
Allow the blood to clot at room temperature or at 4 DC and then centrifuge
What should be done to serum in preparation for serologic tests (after clotting)?
It should be promptly separated into another tube without transferring any cellular elements
What type of serum is usually recommended for testing?
Fresh, nonheat inactivated serum
What should be done if testing of sx can’t be performed immediately?
1) Serum may be stored between 2 DC and 8 DC for up to 72 hrs
2) Avoid excessive heat and bacterial contamination to the sx
3) Heat coagulates the proteins and bacterial growth alters protein molecules (avoid the sx to be in contact with heat)
4) Sx should not be inactivated
What will happen if serum complement is not inactivated?
It will promote lysis of the RBCs and other types of cells and can produce invalid results
What should be done if there is an additional delay of testing of the serum?
Serum should be frozen at -20 DC or below
What should be done to serum in accordance to other tests?
The serum complement must first be inactivated
What is the sx where most immunologic tests are done?
Serum
What are the unaccepted sxs for serologic tests?
1) Lipemic serum sample
2) Hemolyzed serum sample
3) Bacterial contamination (turbid serum)
4) Icteric serum
5) Chylous serum
6) Contamination with alkali or acid
Can icteric serum yield valid results?
Yes, it may yield valid results for some tests but may interfere with others
What are the characteristics of lipemic serum?
Serum that appears milky due to high lipid concentration
What are the characteristics of icteric serum?
Serum that appears yellow to deep yellow due to high bilirubin
What are the characteristics of hemolyzed serum?
Serum that appears reddish due to the hemoglobin released by ruptured RBCs
What are the characteristics of chylous serum?
White, opaque, milky serum due to chyle / lipid (chylomicrons)
Which of the following blood sx is acceptable for serologic testing?
Third sx
What is inactivation (of complement)?
The process that destroys the complement activity
What are the effects of complement in the serum sx?
1) It interfere with the reactions of certain syphilis tests and complement components (e.g., C1q).
2) It can agglutinate latex particles and cause a false-positive reaction in latex passive agglutination assays.
3) It may also cause lysis of the indicator cells in hemagglutination assays
How to inactivate complement in body fluids?
Complement in body fluids can be inactivated by heating to 56 DC for 30 mins
What should be done if more than 4 hrs has elapsed since inactivation (of complement)?
Sx can be reinactivated by heating it to 56 DC for 10 mins
What is the frequent error that results to lab accidents?
Improper pipetting techniques
Is mouth pipetting acceptable in the lab?
No
How to operate a (serologic) pipette?
1) After the pipette has been filled above the top graduation mark, removed from the vessel, and held in a vertical position, the meniscus must be adjusted.
2) The pipette should be held so that the calibration mark is at eye level. All readings must be made with the eye at the level of the meniscus
3) The delivery tip is touched to the inside wall of the original vessel, not the liquid, and the meniscus of the liquid in the pipette is eased, or adjusted, down to the calibration mark.
4) Before the measured liquid in the pipette is allowed to drain into the receiving vessel, any liquid adhering to the outside of the pipette must be wiped off with a clean piece of gauze or tissue
What should be done when reading pipettes?
1) Pipette should be held at the eye level
2) All readings must be made with the eye at the level of the meniscus
What is a meniscus?
The curvature in the top surface of a liquid
What are the steps for manual pipetting?
1) Using mechanical suction
2) Wipe off outside of pipette with gauze
3) Adjusting the meniscus
4) Drain into receiving vessel
What are automatic pipettes?
1) Automatic pipettes allow fast, repetitive measurement and delivery of solutions of equal volumes. The sampling type measures the substance in question
2) The sampling-diluting type measures the substance and then adds the desired diluent
How is the sampling type of automatic pipette operated?
It is mechanically operated and uses a piston-operated plunger (adjustable to varying amounts of reagent or sample)
Are disposable and exchangeable tips available for automatic pipettes?
Yes
What should be done to automatic pipettes and micropipettors before use?
These equipment must be calibrated first
What are automatic micropipetting devices?
Allow rapid repetitive measurements and delivery of predetermined volumes of reagents or specimens
What is the most common type of micropipette used in many labs?
Automatic or semiautomatic (micropipettor)
What are piston-operated devices?
Devices that allow repeated, accurate, reproducible delivery of specimens, reagents, and other liquids requiring measurement in small amounts
Are micropipettors continuously adjustable?
Yes
What is the purpose of continuously adjusting micropipettors?
So that variable volumes of liquids can be dispensed with the same device
What are the functions of automatic dispensers or syringes?
1) For adding multiple doses of the same reagent or diluent
2) Used for measuring serial amounts of relatively small volumes of the same liquid
What is the purpose of pipettor setting?
To determine the volume to be dispensed
Are dispensers available?
Yes, these are available with a variety of volume settings
How are most dispensers cleaned?
By autoclaving
What is the purpose of diluter-dispensers?
1) Used to prepare a number of different samples for analysis
2) These devices pipette a selected aliquot of sample and diluent into the instrument or receiving vessel
What are the characteristics of diluter-dispensers?
Primarily of the dual-piston type, with one used for the sample and the other for the diluent or reagent
What is the cellular component of natural immunity having the most abundant population?
Neutrophils (PMN cells) which comprises 40 to 70% of the population of circulating WBCs
What are the characteristics of neutrophils?
1) Spherical cells of about 12-15 μm diameter and with numerous ciliary projections
2) Has a mutilobulated nucleus (3-5 lobes)
3) Has a granular cytoplasm
What are the purposes of neutrophils?
1) They are the first responders of inflammatory cells to migrate towards the site of inflammation or injury
2) The hallmark of acute inflammation
3) Responds to acute phase of bacterial infection
What are monocytes?
These are mononuclear cells and largest cells in the peripheral blood
What are the characteristics of monocytes?
1) Has a diameter that can vary from 12-22 μm (has an average size of 18 μm)
2) Has an ameboid cytoplasm and non granulated
3) Has an abundant cytoplasm that stains a dull grayish blue and has a ground-glass appearance due to the presence of fine dust like granules
4) Digestive vacuoles may also be observed in the cytoplasm
What is the distinguishing feature of monocytes?
An irregularly folded or horseshoe-shaped nucleus that occupies almost one-half of the entire cell
What is the percentage of monocytes in circulating WBCs?
4-10 %
How long does monocytes stay in the peripheral blood?
Up to 70 hrs
Does monocytes migrate?
Yes, these migrate to the tissues and differentiates into macrophages
When and why does monocytes migrate?
Monocytes migrate within approximately 8-12 hrs in response to inflammation
Can monocyte differentiate into dendritic cells?
Yes, monocytes can differentiate to macrophages and dendritic cells
What are the uses of macrophages?
1) Macrophages and their precursors called monocytes play a central role in both innate and acquired immunity
2) They are responsible for detecting, engulfing, and destroying pathogens and apoptotic cells
What are the characteristics of macrophages?
1) They are mononuclear phagocytes
2) Has an irregular shape (ameboid)
3) May assume different morphologic forms, some develop abundant cytoplasm and are called epitheloid cells
What are epitheloid cells?
These are activated macrophage
Where are macrophages found?
1) They are found in sub-epithelial connective tissue
2) Found in the interstice of parenchymal organs
3) Found in the lining of the vascular sinusoids in the liver and spleen and in the lymphatic sinuses of lymph nodes
What are the main functions of monocyte, macrophage, and dendritic cells?
1) Phagocytosis
2) Antigen presentation
3) Cytokine presentation
4) Also capable of killing infected host cells
What is phagocytosis?
The process of using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognize pathogens
What is the meaning of PRR?
Pattern (pathogen) recognition receptors
What is the purpose of PRR?
Recognizing pathogens
How does monocytes kill infected host cells?
Via antibody-dependent cell-mediated cytotoxicity
What are the steps of phagocytosis?
1) The macrophage takes up the antigen by phagocytosis
2) The macrophage breaks down the antigen into fragments in the lysosome
3) A class II MHC protein binds an antigen fragment
4) The MHC protein presents the antigen to a Th cell
What are the purposes of eosinophils?
1) Considered to be a homeostatic regulator of inflammation
2) Plays a role in the host defense mechanism by its ability to kill certain parasites
3) Has the ability to interact with the larval stages of some helminth parasites
What are the characteristics of eosinophils?
1) This proinflammatory WBC generally has a nucleus with 2 lobes (bilobed)
2) Its cytoplasm is filled with approximately 200 large granules
3) Eosinophils have a diameter of 12-17 μm in fixed smears
4) Cytoplasm is packed with large rounded granules
What are the 2 types of eosinophil granules?
1) A few rounded homogenously electron-dense granules
2) Many rounded, elongated or oval crystalloid-containing granules
What is the color of the granules of eosinophils?
The granules stains red-orange with Romanowsky stains
What are the characteristics of basophils?
1) Contains high concentrations of heparin and histamine
2) Contains large cytoplasmic granules which obscure the nucleus when stained
3) If unstained, nucleus is visible and usually has 2 lobes
4) If stained with Giemsa stain, these appear as mononucleated cells of 10-14 μm diameter
5) Its cytoplasm is filled with many rounded or oval, purple, dark granules of variable size, partially covering the nucleus
Where do heparin and histamine located in basophils?
Heparin and histamine contents are located in the granules of basophils
What are the purposes of basophils?
Plays an important role in acute, systemic, and hypersensitivity reactions
When does degranulation occur?
Degranulation occurs when an antigen binds to two adjacent immunoglobulin E (IgE) antibodies located on the surface of mast cells
What happens during degranulation?
1) Increased vascular permeability
2) Smooth muscle spasm
3) Vasodilation
4) In severe cases, this can result in anaphylactic shock
What are the different cellular components in the innate immune system?
1) Neutrophil
2) Eosinophil
3) Basophil
4) Monocyte
5) Lymphocyte
a. T cells (T lymphocytes)
b. B cells (B lymphocytes)
c. NK cells
6) Mast cell
7) Macrophage
8) Dendritic cells
What are the cells that can cause degranulation?
1) Basophils
2) Mast cells
