antiglobulin tes1
-
Upload
harnadi-wonogiri -
Category
Documents
-
view
224 -
download
0
Transcript of antiglobulin tes1
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 1/24
AntiglobulintestBy John-Paul Adjah [email protected]
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 2/24
Antiglobulin test
• Also known as Anti Human Globulin test(AHG) or coombs test
•
It is very important in detectingclinically significant unexpectedantibodies either in-vivo or in-vitro
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 3/24
Principle of test
IgG coatedred bloodcells
Complementcoated red bloodcells
Red blood cellscoated withincomplete IgGantibodies orcomplement do
not readilyagglutinate.These cells aresaid to be
sensitized withIgG orcomplement
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 4/24
• For agglutination to occur, an additionalantibody that reacts with the FCportion of the IgG or the C3b or C3dmust be added to system to bridge theantibodies or the C3bor C3d forming
agglutination
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 5/24
Anti uman g o u in reagentproduction
• It is made by injecting rabbits, sheep or goatwith purified human IgG or C3.
• The animal is bled after a specified period and
the reagent purified by absorbing unwantedantibodies• The reagent may be polyspecific or
monospecific• Polyspecific Anti-human Globulin: contains a
blend of Anti-IgG & Anti-C3b, -C3d andsometimes C4
•
Monospecific reagents: contains Anti-IgG aloneor Anti-C3b,-C3d alone
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 6/24
Antiglobulin test andinterpretation
Whether the cells havebeen coated, or sensitized,in vivo or in vitro the final
interpretation is based onthe following:
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 7/24
Incomplete Antibodies
attach to the red cells
•Wash cells 3 to 4 times to remove any unattached antibodies
•Only antibodies attached with the cell remain
Add antihuman globulin
Visible agglutination in
test tube
Positive antiglobulin test
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 8/24
Antibodies arenot attached toantigens duringincubation
Wash cells three to four times to remove any unattached antibodies
Add anti human globulin
No visible agglutination
therefore a negative test
Add coombs control check cells (CCC)
Check cells
agglutinated whilst
original test cells
remained
unagglutinatedAgglutination following addition of CCC verifies negative result
Negative antiglobulin test
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 10/24
• Take 0.5 ml of 5-6 times washed and packed0 Rh (D) +ve red cells in a test tube.
• Add 2-3 drops of IgG anti-D (select adilution (titre 1:4) of anti-D which coats thered cells but does not agglutinate them at
37°C).
• Mix and incubate at 37°C for 30 minutes. Ifthere is agglutination, repeat the procedureusing more diluted anti-D.
• Wash 3-4 times and make 5% suspension in
saline for use.
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 11/24
• Perform a Direct antiglobulin test which
should give a 2+ reaction. If noagglutination occurs, repeat the test byusing less diluted anti-D serum.
• 0 Rh(D) negative sensitized red cellsare also prepared by treating 0 Rh(D)
negative cells in the same manner. Thepreparation should give a negativedirect antiglobulin test (DAT)
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 12/24
False negative and positiveresults in antiglobulin test
•
False-negative reactions can occur whenantigen-antibody reactions have occurredbut WASHING IS INADEQUATE and freeantibody remains when the anti-humanglobulin is added
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 13/24
• False negatives results may be obtainedwhen there is:
1. Inadequate cell washing
2. Delay in adding antiglobulinreagent after the washing step
3. Presence of small fibrin clots
among the cells4. Inactive, or forgotten
antiglobulin reagent
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 14/24
Inadequate washing
Inadequate cell washing will leadto unbound antibody remainingin the red cell suspension thatare available to neutralize the
AHG (Coombs serum) so it willnot react with red cells boundwith antibody
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 15/24
Delay in adding AHG
Delay in adding Coombs serum afterwashing step will lead to antibody
eluting off, detaching from the cellwhile cells are sitting in saline. Nowfree antibody present in the saline
neutralizes the AHG or Coombsreagent so it will not be able toreact with the cells bound with
antibody.
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 16/24
Presence of Small fibrin
clot among cells• Small fibrin clot among the cells
that were not washed away willhave immunoglobulin andcomplement present. The
antibodies and complement inthe fibrin clot neutralizes AHGleading to a negative test.
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 18/24
Types of Antiglobulin test
• There are two types:
a. Direct antiglobulin test (DAT)
b. Indirect antiglobulin test (IAT)
DAT
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 19/24
DAT• Direct antiglobulin test is used to detect in-
vivo sensitization (coating) of red cells withimmune antibody (IgG) or the complementcomponent (C3b or C3d) in :
1. Diagnosis of haemolytic disease of thenewborn (HDN)2. Diagnosis of autoimmune haemolytic anemia
(AIHA)
3. Investigation of haemolytic transfusionreaction (HTR)4. Investigation of drug induced red cell
sensitization
eg. Penicillins, phenacetins, quinidine
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 21/24
IAT• The purpose of the indirect antiglobulin test is
to detect In vitro sensitization of red
cells. This is done when sensitization does notlead to direct agglutinationUses• Weak D's in donor bloods and pregnant
females of individuals who type D (-) at roomtemperature when doing ABO and Rh typing• Screening and identification of unexpected
(irregular) antibodies in serum.
• Compatibility testing• Detection of red cell antigens using specific
antibodies reacting only in antiglobulin test (K,Fya, Fyb, Jkb, etc.)
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 23/24
Factors affecting sensitivity ofIAT
TemperatureOptimal temperature : 37°C.Incubation at higher or lower temperature may give false results.
Serum Cell ratioIncreasing the ratio of serum to cells increases the antibody coating.
Commonly used ratio in saline suspension is 2:1 but in LISS suspendingcells, use equal volume of serum and 2% cell suspension.
Incubation timeSaline, Albumin or enzyme technique : 45-60 minutesLISS suspended cells - Routine 15 minutesEmergency : 5 minutes
Suspension mediumThe sensitivity of IAT can be increased with addition of 22% bovinealbumin, enzyme or by using LISS suspended cells.
8/11/2019 antiglobulin tes1
http://slidepdf.com/reader/full/antiglobulin-tes1 24/24
Mechanism of enhancement
•
LISS: reduces the ionic strength of thereaction medium thereby enhancing uptakeof antibodies by the red cells
• Proteolytic enzymes: cleave sialoglycoproteinfrom red cells thereby reducing net chargebetween red cells
• Albumin: reduces the repulsion charges
between red cells