Serology Flashcards
IgM
- IgM antibodies are produced as a body’s first response to a new infection or to a new “non-self” antigen, providing short-term protection. They increase for several weeks and then decline as IgG production begins.
- Pentamer
- Placental transfer = Negative
- Binds to mast cell surfaces=Negative
- Binds to phagocytic cell surfaces= Negative
- activates complement= Positive
IgD
- the role of IgD is not completely understood and IgD is not routinely measured.
- Placental transfer = Negative
- Binds to mast cell surfaces=Negative
- Binds to phagocytic cell surfaces= Negative
- activates complement= Negative
IgG
- About 70-80% of the immunoglobulins in the blood are IgG. Specific IgG antibodies are produced during an initial infection or other antigen exposure, rising a few weeks after it begins, then decreasing and stabilizing. The body retains a catalog of IgG antibodies that can be rapidly reproduced whenever exposed to the same antigen. IgG antibodies form the basis of long-term protection against microorganisms. In those with a normal immune system, sufficient IgG is produced to prevent re-infection. Vaccinations use this process to prevent initial infections and add to the catalog of IgG antibodies, by exposing a person to a weakened, live microorganism or to an antigen that stimulates recognition of the microorganism. IgG is the only immunoglobulin that can pass through the placenta. The mother’s IgG antibodies provide protection to the fetus during pregnancy and to the baby during its first few months of life. There are four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4.
- Placental transfer = Positive
- Binds to mast cell surfaces=Negative
- Binds to phagocytic cell surfaces= Positive
- activates complement= Positive
IgE
- IgE is associated with allergies, allergic diseases, and with parasitic infections. It is almost always measured as part of an allergy testing blood panel but typically is not included as part of a quantitative immunoglobulins test.t
- Placental transfer = Negative
- Binds to mast cell surfaces=Positive
- Binds to phagocytic cell surfaces= Negative
- activates complement= negative
IgA
- IgA comprises about 15% of the total immunoglobulins in the blood but is also found in saliva, tears, respiratory and gastric secretions, and breast milk. IgA provides protection against infection in mucosal areas of the body such as the respiratory tract (sinus and lungs) and the gastrointestinal tract (stomach and intestines). When passed from mother to baby during breast-feeding, it helps protect the infant’s gastrointestinal tract. Significant amounts of IgA are not produced by a baby until after 6 months of age so any IgA present in a baby’s blood before then is from the mother’s milk. There are two IgA subclasses: IgA1 and IgA2.
- Monomer
- Placental transfer = Negative
- Binds to mast cell surfaces=Negative
- Binds to phagocytic cell surfaces= Negative
- activates complement= Negative
Agglutination test
- Antibody molecules crosslink the particles to form visible patterns of clumping or agglutination
- Definition - tests that have as their endpoint the agglutination of a particulate antigen
- Qualitative agglutination test
- -Ag or Ab
- Quantitative agglutination test
- -Titer
- -Prozone
- Applications
- -Blood typing
- Bacterial infections
- -Fourfold rise in titer
Passive Agglutination/Hemagglutination
-Definition - agglutination test done with a soluble antigen coated onto a particle
-Applications
Measurement of antibodies to soluble antigens
-MHA-TP
Immunoelectrophoresis
-Method
-Ags are separated by electrophoresis
-Ab is placed in trough cut in the agar
Interpretation
-Precipitin arc represent individual antigens
Coombs (Antiglobulin)Tests
- Incomplete Ab
- Direct Coombs Test
- Detects antibodies on erythrocytes
- Indirect Coombs Test
- -Detects anti-erythrocyte antibodies in serum
Agglutination/Hemagglutination Inhibition
Definition - test based on the inhibition of agglutination due to competition with a soluble Ag
Complement Fixation Tests
- Ag mixed with test serum to be assayed for Ab
- Standard amount of complement is added
- Erythrocytes coated with Abs is added
- Amount of erythrocyte lysis is determined
Immunofluorescence Testing
-Direct (DFA)
Ab to tissue Ag is labeled with fluorochrome
-Indirect (IFA)
Ab to tissue Ag is unlabeled
Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab.
Immunoassays
- EIA
- CIA
rapid plasma reagin (RPR) test.
-The RPR test is commercially available as a complete system containing positive and negative controls, the reaction
card, and the prepared antigen suspension. The antigen, cardiolipin-lecithin–coated cholesterol with choline chloride,
also contains charcoal particles to allow for macroscopically visible flocculation.
-100RPM for 8 mins card on the rotators with humidifying cover.
- Test CSF = NO
-Heat treat serum = NO Choline chloride
-Visualize macroscopically = YES
(FTA-ABS)
- fluorescent treponemal antibody absorption test
- is an indirect immunofluorescent antibody test that uses testis from infected rabbits as the antigen and a standard 1:5 dilution of heat-inactivated serum. Before testing, it is absorbed with “sorbent,” which removes antibodies to commensal treponemes that could cause false-positive results. It is sensitive and specific for past or current T. pallidum infection and is needed to confirm any positive nontreponemal serological test before the diagnosis of syphilis can be made.
- The FTA-ABS becomes reactive 4-6 weeks after infection.
- The antigen for the FTA-ABS test is whole bacteria. The bacteria cannot be cultured on laboratory media, so the organisms used are a lyophilized suspension of T. pallidum extracted from rabbit testicular tissue. This is spread over and fixed to a slide. Patient serum is mixed with an absorbent (the “ABS” part of the test) containing an extract of a non-pathogenic treponeme, Treponema phagedenis biotype Rieter. The purpose of the absorbent is to remove anti-treponemal antibodies that are not specific for the syphilis bacteria. The pre-adsorbed patient serum is then added to the slide; if the patient has been infected by syphilis, their antibodies will bind to the bacteria. FITC (a fluorophore)-labeled anti-treponeme antibody and TRITC (another fluorophore)-labeled anti-human antibodies are added as secondary antibodies. The spirochete location is identified using the FITC staining, and the TRITC staining identifies whether the patient has anti-T. pallidum antibodies (binding to the same spirochete).
Affinity
-Strength of the reaction between a single antigenic determinant and a single Ab combining site
VDRL Test
-rotator for 4 mins @ 180rpms
-1 drop VDRL antigen to 1 drop patient serum. Shake observe under the microscope. Look for flocculation reaction. (clumping or not.)
- Test Serum must be heated 56 degrees for 30 mins
-Control serum must be heated 56 degrees for 10 mins
-Venereal Disease Research Laboratory test,
-Test for antibody like protein reagin that binds to the test antigen, cardiolipin-lecithin–coated cholesterol particles, causing the particles to flocculate.
- Reagin is not a specific antibody directed against T. pallidum antigens; therefore the test
is highly sensitive but not highly specific; however, it is a good screening test, detecting more than 99% of the cases of secondary syphilis.
-Cerebrospinal fluid is
not inactivated
-23 to 29 degrees
-Use antigen previously made
for testing
- 18 gauge needle used for
serum tests
-30 ± 1 drops/0.5ml or
- 60 ± 1 drops/1.0ml
- 21 gauge needle used for
testing cerebrospinal fluid
- 50 ± 1 drops/0.5ml
- Rinse needles 3 times with
Milli-Q water following QC
tests
-Determine pH of 0.9%
saline used for dilutions
- pH should be 5.9 ± 0.1
- pH of buffered saline
used to prepare antigen
should be 6.0 ± 0.1
Affinity
-Strength of the reaction between a single antigenic determinant and a single Ab combining site
Specificity
- The ability of an individual antibody combining site to react with only one antigenic determinant.
- The ability of a population of antibody molecules to react with only one antigen.
- (True Negative Rate) refers to the probability of a negative test, provided one does not have the condition (judged negative by the
Gold Standard).
Cross Reactivity
- The ability of an individual Ab combining site to react with more than one antigenic determinant.
- The ability of a population of Ab molecules to react with more than one Ag
Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent Assays (ELISA)
-Determine amount of Ab needed to bind to a known amount of labeled Ag
-Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
-Determine amount of labeled Ag bound to Ab
↓ NH4SO4
↓ anti-Ig
Immobilize the Ab
-Concentration determined from a standard curve using known amounts of unlabeled Ag
- Quantitative
-Most sensitive test
Syphilis Timeline
Infection incubation period= 9 to 90 days until first chancre.
- 6 weeks to 6 months= secondary rash
-Secondary rash until latent period= 18 months
-early syphilis=1 to2 years
-late syphilis = 2 year to lifetime.=“Benign” gummatous,
Cardio-vascular syphilis Neurosyphilis
LATE SYPHILIS
‘Tertiary Syphilis’
-Tissue destruction Lesions develop in skin, bone, & visceral organs (any organ).--- very slowly progressive, -The main types are: -Late benign (gummatous) -Cardiovascular & -Neurosyphilis -Blindness, deafness, deformity, lack of coordination, paralysis, dementia may occur -Late syphilis is noninfectious.
SECONDARY SYPHILIS
-Seen 6 wks to 6 mos after primary chancre
-Usually w diffuse non-pruritic, indurated rash, including palms & soles.
-May also cause:
-Fever, malaise, headache, sore throat, myalgia, arthralgia, generalized lymphadenopathy
Hepatitis (10%)
-Renal: an immune complex type of nephropathy with transient nephrotic syndrome
-Iritis or an anterior uveitis
-Bone: periostitis
-CSF pleocytosis in 10 - 30% (but, symptomatic meningitis is seen in <1%)
PRIMARY SYPHILIS
The Chancre
-Incubation period 9-90 days, usually ~21 days.
-Develops at site of contact/inoculation.
-Classically: single, painless, clean-based, indurated ulcer, with firm, raised borders. Atypical presentations may occur.
-Mostly anogenital, but may occur at any site (tongue, pharynx, lips, fingers, nipples, etc…)
-Non-tender regional adenopathy
-Very infectious.
-May be darkfield positive but serologically negative.
Untreated, heals in several weeks, leaving a faint scar.
Prozone Effect
-The hook effect or the prozone effect is an immunologic phenomenon whereby the effectiveness of antibodies to form immune complexes is sometimes impaired when concentrations of an antibody or an antigen are very high
(TP-PA) test
The Treponema pallidum particle agglutination (TP-PA) test is used as a confirmatory test for samples demonstrating reactive rapid plasma reagin (RPR) test results. Once positive, treponemal antibodies typically remain positive for years in contrast to non-treponemal antibody titers that may decline during late syphilis or with effective therapy. The TP-PA test is performed as a reflex test on reactive RPR samples. It may be performed on non-reactive RPR samples upon request if late syphilis is suspected. Results are reported as reactive or non-reactive. Rarely, an indeterminate result may be obtained.
Serum
- the liquid that remains after the blood has clotted
- 15mins @ 1900 rpms
Plasma
- is the liquid that remains when clotting is prevented with the addition of an anticoagulant.
- EDTA
- centrifuged
Traditional Algorithm
RPR–> TP-PA
Specimen Collection for Syphilis
- Blood sample
- 7 days hold time
- Serum separated from blood cells.
- No hemolysis
- No Glycemia
- Centrifuged down to separate serum from RBC.
- Centrifuged 15 mins @ 1900 rpm
- 0.5mL serum in 12X75mm tube to test with.
- specimens stored at 4 degrees when not testing
Darkfield microscopy for T. Pallidum
-Helical bacterium, 6 to 20 micrometers
long and 0.1 to 0.2 micrometer wide, with
4 to 14 evenly distributed coils.
-T. pallidum is a delicate, corkscrew-shaped organism
with rigid, uniform, tightly wound, deep spirals. The length of the organism is 6 to 20 mm; the width is 0.10 to 0.18 mm. The length of the spiral wave is 1.0 to 1.5 mm, and the spiral depth is 0.5 to 0.7 mm. The characteristic motion of T. pallidum is a deliberate forward and backward movement with rotation about the longitudinal axis. Rotation may be accompanied by
a soft bending, twisting, or undulation of the organism from side to side. When attached to or obstructed by heavier particles, the organism may contort, convolute, or bend and thereby distort the coils, but the organism will snap back to its original form in a coil-like manner.
(DFA)-TP
Direct fluorescent antibody for T. pallidum
- fix treponema to slide. Wash slides with patient serum.
- Antibodies in patient serum stick to bacteria.
- Wash again with fluorescently labeled anti-antibody
- look under a fluorescent microscope for binding of patients’ antibodies.
(DFAT-TP)
Direct Fluorescent Antibody Tissue Test for T. pallidum
(USR)
- Unheated Serum Reagin
- basically, the RPR test except done one a slide to -check for flocculation like that of the VDRL slide test. –Uses EDTA to stabilize antigen mixture
(RST)
Reagin Screen Test
- RPR
- Sudan Black
(TRUST)
- Toluidine Red Unheated Serum Test
- Use of Plasma
- Blood sample with EDTA to create plasma
- can be done on CSF
- The antigen is based on the Venereal Disease Research Laboratory antigen, with EDTA, choline chloride, and toluidine red toner added. Performance of the toluidine red unheated serum test (TRUST) is identical to that of the rapid plasma reagin 18-mm circle card test
- Test CSF = YES
- Heat treat serum = NO
- Visualize macroscopically = YES
(FTA-ABS DS)
- Fluorescent Treponemal Antibody-Absorption Double Staining
- The ZEUS IFA Fluorescent Treponemal Antibody-Absorption Double Stain (FTA-ABS DS) Test System is an indirect fluorescent antibody assay intended for the qualitative detection of antibodies to Treponema Pallidum, and to be used as an aid in confirming the presence of syphilis antibodies.
- FITC, rhodamine
(MHA-TP)
Microhemagglutination Assay for Antibodies to T. pallidum
Reverse Algorithm
CIA–> RPR–> TP-PA
Preparation of VDRL Antigen
Pipette 0.4ml VDRL buffered saline (pH 6.0 ± 0.1) into antigen bottle - Pipette 0.5ml VDRL antigen - Dispense within 6 seconds while rotating bottle in a 2-inch circle - Continue to rotate the bottle for 10 more seconds - Pipette 4.1ml VDRL buffered saline into antigen bottle - Shake the bottle up and down vigorously 30 times within 10 seconds in at least a 1 foot arc
Glass slides VDRL
-used for both serum and CSF tests
- The slide should be at least 2 x 3 inches with either
paraffin or ceramic rings that are 14mm in diameter.
- Rings must be high enough to prevent spillage during
rotation
VDRL Test Controls
-Reactive Serum Positive human sera control made from specimen pool ---20°C -Weakly Reactive Serum --Weak human sera control made from specimen pool ---20°C
-Non-Reactive Serum --Negative human sera control made from specimen pool --20°C
-Reproducibility
Samples: 3 reactive sera + 2 negative sera
-Random patient serum samples from previous
runs 4°C
Test Antigen Suspension VDRL
Use the Reactive, Weak and Negative control sera - Serial dilute the Reactive serum to 1:32 using 50 μl of serum with 50 μl of 0.9% saline - Add 1 drop Ag to each test well - Rotate slide by hand for 5 seconds - Rotate slide for 4 minutes on rotator at 180 RPM - Examine each slide well microscopically - Ag that does not demonstrate standard reactivity must be re-made - Ag demonstrating standard reactivity with R,W, and N controls can then be used to test reproducibility samples and test specimens - Complete testing of reproducibility samples - Record all results on log sheet
VDRL Test for CSF specimens
CSF is tested by both qualitative and quantitative test procedures -CSF specimens are not inactivated -Needle used to add the antigen is either a 21 or 22 gauge needle - QC for the needle should be 50 drops per 0.5ml antigen - Ag prepared for serum assay is used to prepare Ag for CSF tests - 0.6ml Ag + 0.6ml of 10% saline - This diluted Ag is only good for 2 hours
- Test Ag for reactivity with both Reactive and Negative controls - Test Ag with reproducibility sera - Rotation of CSF with Ag is for 8 minutes -Test results are read immediately with microscope - Record results on log sheet -The quantitative test is performed on CSF specimen that are reactive by the qualitative assay - Quantitative test dilutions are made to 1:32 for 6 wells - CSF is tested by VDRL only -Reactive qualitative VDRL tests are re-tested by the quantitative VDRL procedure. - Leaving first well empty, dispense 0.05ml of 0.9% saline to the next 2, 5, or 8 wells - Dispense 0.05ml serum to the first empty well and to the first well that contains saline - Mix up and down 5 times and continue with serial dilution of all wells containing saline
- Dispense 1 drop of antigen to each test well - Rotate the slide by hand for 5 seconds - Rotate the slide for 8 minutes at 180 RPM on the rotator - Read immediately using microscope - Quantitative tests are performed until one of the dilutions reaches a negative endpoint - Record test results on log sheet - Positive tests can be confirmed by TPPA test or FTA-abs test
Sensitivity
(True Positive Rate) refers to the probability of a positive test, conditioned on truly having the condition (or tested positive by the Gold Standard test if the true condition can not be known).
