1	CLSC320 Principles of Immunology Fundamental Concepts in Immunology Program for Clinical Laboratory Science Unit - 12 Agglutination Techniques
2	Unit 12 - Guidelines Reading assignment: Pages 254 - 269 of textbook Learning objectives: Those listed on page 255 of textbook Key terms: Those listed on pages 255 & 256 of textbook
3	Agglutination Reactions Direct Agglutination Reactions Indirect Agglutination Reactions Reverse Passive Agglutination Reactions Agglutination-Inhibition Reactions e e e
4	Features & Applications General features: UAgglutination reactions occur when: \particulate multivalent antigens \bind with bivalent antigen-specific antibody \form stable cross-linked lattices that are visible Clinical applications: Udetection of circulating antibodies using known specific antigens Udetection of natural occuring antigens using known specific antibodies Udetection of soluble antigens using prepared antibody-coated particles (reverse passive agglutination)
5	Two Steps of Agglutination - sensitization -antigen-binding site (paratopes) of Ig binds with single epitope on multivalent ag. -nondetectable unless tagged with label antigen antibody
6	Two Steps of Agglutination - aggregation -cross-linking of free epitopes and paratopes -detectable lattice formations occur UHemolysis - tertiary step if C^/ is activated
7	Enhancement of Agglutination
8	Factors Affecting Agglutination - 1 U class of antibody U charge of the Acarrier@ particles U number of epitopes U concentration of reactants U environmental factors IgM better at cross-linking due to size and number of paratopes than is IgG many antigens carry negative surface charges (zeta potential) and this can be reduced by adding LISS thus allowing Ag=s to come closer together antigens containing multiple, densely distributed epitopes will cross-link more readily
9	Factors Affecting Agglutination - 2 U class of antibody U charge of the Acarrier@ particles U number of epitopes U concentration of reactants U environmental factors generally the higher the concentrations the stronger and faster the reaction may get weak or negative reactions if prozone (>Ab) or postzone (>Ag) exists reactions enhanced by: -proper mixing -proper temperature -proper incubation time -proper centrifugation -proper agitation on previous screen
10	Agglutination – Clinical Applications detection of naturally occuring particulate antigens by using antigen-specific reagent antibodies (antisera) or U detection and quantification of antibodies using known particulate antigens with same specificity
11	Direct Agglutination Reactions General concept: antigens that naturally occur on a particle (RBC, bacteria, etc. can be directly agglutinated by adding antigen-specific antibodies (antisera) RBC +
12	Example of DAT Utyping of blood A type = B type = have A antigens on RBC surface have B antigens on RBC surface RBC A antigens + anti-a antisera
13	Example DAT - 2 Uconfirmation of Salmonella infection: Salmonellae O antigen H antigen Vi antigen + anti-Vi antisera This would indicate S. typhi positive
14	Indirect Agglutination – General Concepts detection of antibodies in serum that are not attached to an antigen detection of antibody uses carrier particles that have been coated with antigen or possess the antibody- specific epitope. U examples of carrier molecules include: \ human red blood cells \ sheep red blood cells \ horse red blood cells \ turkey red blood cells \ latex particles \ charcoal
15	Indirect Agglutination - Procedure patient serum is mixed with reactants and allowed to incubate for a set time period. U during this incubation time antibodies will bind with antibody-specific epitopes agglutination may be visible or an anti-antibody may have to be added in order for reaction to be visualized.
16	Example Indirect Agglutination - 1 Uantibody screening RBC K antigens + pt=s serum incubate @ 37^oC for 15 min. + AHG
17	Example ? Agglutination Uscreening for Group A streptococci infection Latex Streptolysin O antigens + pt=s serum rock slide for 3 min. no agglutination = negative agglutination = positive
18	Indirect Agglutination – Technical Considerations U a prozone reaction may be responsible for false negative results if patient has high antibody titer (serial dilutions may be run to detect this) U high IgM levels may inhibit reactions of other Igs
19	Reverse Passive Agglutination - Concepts General concepts: U differs from passive agglutination in that the antibody is attached to the carrier molecule instead of the antigen Clinical applications: U rapid identification of bacterial and fungal infections Latex antigen-specific Ab=s + Ag no agglutination = negative agglutination = positive
20	Reverse Passive – Technical & Clinical Applications Technical considerations: U if red blood cells are used in place of latex particles the reaction is called Areverse passive hemagglutination@ U detection of Candida albicans Clinical applications: U detection of Mycoplasma pneumoniae
21	Hemagglutination Inhibition General concept: U a two-step procedure in which there is competition between a reagent antigen (antigen-coated particle) and a soluble test antigen (pt=s serum) for antigen-binding sites on a reagent antibody. Procedure: U Step 1 \ Reagent antibody + patient serum added together \ incubation period U Step 2 \ Reagent antigen-coated particles added \ incubation period \ interpretation of reaction made
22	Positive Hemagglutination Inhibition U Step 1 - in a positive test U Step 2 - in a positive test Reagent Ab=s + Pt=s serum nonvisible Ag-Ab reaction + reagent Ag-bound particles no agglutination of reagent ag-bound particles
23	Negative Hemagglutination Inhibition U Step 1 - in a negative test U Step 2 - in a negative test Reagent Ab=s + Pt=s serum no Ag-Ab reaction + reagent Ag-bound particles agglutination of reagent ag- bound particles
24	Blood Bank Applications Antiglobulin tests: U also called ACoomb=s test@ a procedure used to detect antibodies present in patient serum U used to detect antibodies (Incomplete) that are unable to reach between two red cells uses an anti-human globulin (AHG) to bridge the gap between red cells coated with antibodies U the anti-human globulin (AHG) binds with the Fc portion of IgG antibodies forming agglutination U two types of antiglobulin tests: \ Direct antiglobulin test (DAT) \ Indirect antiglobulin test (Screen)
25	DAT & Screen in Blood Bank Direct Antiglobulin tests (DAT): U detection of autoantibodies U investigation of hemolytic disease of the newborn (HDN) U detection of medication-induced hemolytic anemia U investigation of hemolytic transfusion reactions Indirect Antiglobulin tests: (Screen) U typing of red blood cell antigens U antibody screening for possible transfusion U compatibility testing (crossmatch) with known antibody positive patients U identification of detected antibodies (Antibody panel)
26	OSF – Rubella Infection On page 262AOne Step Further@ presents a more in-depth discussion of Rubella Infection. This presentation is contained on a separate slide presentation called A One Step Further #12" The student may call up the slide program OSF-12 later or click on the arrow below to view slides now.
27	OSF - HDNB On page 266AOne Step Further@ presents a more in-depth discussion of Hemolytic Disease of the Newborn. This presentation is contained on a separate slide presentation called A One Step Further #13" The student may call up the slide program OSF-12 later or click on the arrow below to view slides now.
28	End of Agglutination Techniques Press the ESC key to end program
29	One Step Further Rubella Infection OSF - 12 Pages 262 & 263 7click arrow to return to main program
30	Rubella Infection - 2 Diseases associated with rubella virus: econgenital rubella eacute rubella - also called AGerman measles@ Viral characteristics: esingle-stranded RNA eenters via upper respiratory tract by: ]direct contact ]droplet contact Acute rubella characteristics: ebenign and self-limiting eseen in children & young adults emild fever with upper respiratory symptoms etransient rash eenlarged lymph nodes eearly fetus at risk so all pregnant women are tested in early pregnancy
31	Rubella Infection - 3 Congenital rubella characteristics: erubella virus can cross placental barrier emay be fatal to fetus emay be cause ocular and brain damage in the fetus emay cause deafness in the newborn emay cause cardiac and liver damage Rubella testing: emost widely used tests: Indirect Fluorescent Antibody (IFA) Enzyme-linked Immunosorbent Assay (ELISA) Passive-Hemagglutination Assay (PHA) Hemagglutination Inhibition (HI)
32	Rubella Infection - 4 Reasons for rubella testing: eto determine the immune status of the individual to rubella virus eto determine the presence of congenital rubella risk in pregnant women eto determine the presence of acute infection presence of rubella virus specific IgM presence of rubella virus specific IgG only absence of rubella virus specific IgM or IgG ‡indicates current infection to rubella ‡indicates past infection to rubella and patient has immunity to rubella virus ‡indicates no past or current infection to rubella and patient has no immunity to rubella virus
33	Rubella Infection - 5 Reasons for rubella testing: continued eto determine the presence of congenital rubella risk in pregnant women eto determine the presence of acute infection presence of rubella virus specific IgM in newborn=s serum ‡indicates congenital infection to rubella presence of rubella virus specific IgM in serum up to age 6 months ‡indicates congenital infection to rubella four-fold increase in both virus specific IgM and IgG titer within 5 days of diagnosis ‡confirms diagnosis of an infection with rubella virus
34	Rubella Infection - 6 Preventive measures: evaccination program attenuated rubella virus used included with mumps and measeles vaccine and is referred to as AMMR vaccine@ induces both virus specific IgG and IgM can not be given to women who are pregnant
35	Rubella Infection - 7 7click arrow to return to main program
36	One Step Further Hemolytic Disease of the Newborn OSF - 13 Page 266 7click arrow to return to main program
37	HDN - 2
38	HDN - 3 Mechanisms of Disease cont=d: ein the case of ABO HDN: the 1st child may be affected since mom already has the antibodies in her circulatory system i.e. mom is O type and baby is A or B type ein the case of Rh HDN: usually the 1st child is not affected since it is the Rh+ red cells of the fetus that get into moms circulation during delivery and sensitize the mom.
39	HDN - 4 Immunologic testing: ePrenatal evaluation: indirect antiglobulin test (antibody screen) ‡if IgG class antibodies are detected the mother=s antibody titer is monitored during pregnancy ‡increasing titer indicates HDN ‡steady titer indicates antigen comatibility between fetus and mom=s red cells indirect antiglobulin test (antibody screen) direct antiglobulin test (DAT) blood typing ‡performed on mom after delivery ‡if positive the antibody is identified eNeonatal evaluation:
40	HDN - 5 Immunologic testing: continued eNeonatal evaluation: direct antiglobulin test (DAT) blood typing ‡performed on cord blood ‡detects antibodies attached to fetal red cells ‡if positive antibodies are eluted off the red cells and identified and NBIL* is ordered *NBIL = neonatal bilirubin test measures unconjugated bilirubin ‡blood type of newborn is performed using forward typing only (no anti-A or anti-B formed yet to perform reverse typing) ‡if mom is O type and newborn is A, B, or AB it alerts the physician to possible ABO HDN and bilirubin can be monitored
41	HDN - 6 Preventive measures: eRhoGAM women who are Rh-D negative and give birth to a Rh-D positive child are given anti-D (RhoGAM or Rh immune globulin) injection within 72 hours of delivery ‡the anti-D will attach to any Rh-D positive fetal cells in mom=s circulation and these cells will be destroyed before mom=s immune system is sensitized Women who are Rh-D negative and are pregnant may be given RhoGAM during 28th week of pregnancy to have anti-D in mom=s circulation should any Rh-D positive red cells from developing fetus make it into mom=s circulatory system during pregnancy
42	HDN - 7 7click arrow to return to main program