Coombs test

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Coombs test
MeSH Diagnostics D00329

Coombs test !also kno"n as Coombs' test, antiglobulin test or AGT# refers to t"o clinical $lood tests used in immunohematology and immunology% &he t"o 'oom$s tests are the direct Coombs test !DCT, also kno"n as direct antiglobulin test or DAT#, and the indirect Coombs test !also kno"n as indirect antiglobulin test or IAT#% &he Direct 'oom$s test, is used to test for autoimmune hemolytic anemia% (n certain diseases or conditions an individual)s $lood may contain (g* anti$odies that can specifically $ind to antigens on the red $lood cell !+,'# surface mem$rane, and their circulating red $lood cells !+,'s# can $ecome coated "ith (g* alloanti$odies and-or (g* autoanti$odies% 'omplement proteins may su$se.uently $ind to the $ound anti$odies% &he direct Coombs test is used to detect these anti$odies or complement proteins that are $ound to the surface of red $lood cells/ a $lood sample is taken and the +,'s are "ashed !removing the patient)s o"n plasma# and then incu$ated "ith antihuman glo$ulin !also kno"n as 0'oom$s reagent0#% (f this produces agglutination of +,'s, the direct 'oom$s test is positive, a visual indication that anti$odies !and-or complement proteins# are $ound to the surface of red $lood cells% &he indirect Coombs test is used in prenatal testing of pregnant "omen, and in testing $lood prior to a $lood transfusion% (t detects anti$odies against +,'s that are present un$ound in the patient)s serum% (n this case, serum is e1tracted from the $lood, and the serum is incu$ated "ith +,'s of kno"n antigenicity% (f agglutination occurs, the indirect 'oom$s test is positive%234

Contents

3 5echanism 2 Direct 'oom$s test

o

2%3 6a$oratory method

3 (ndirect 'oom$s test

o

3%3 71amples of clinical uses of the indirect 'oom$s test

3%3%3 ,lood transfusion preparation 3%3%2 8ntenatal anti$ody screening

3%2 6a$oratory method

3%2%3 First stage 3%2%2 9econd stage 3%2%3 &itrations

: 'oom$s reagent ; 7nhancement media < =istory of the 'oom$s test > +eferences 71ternal links

Mechanism

9chematic sho"ing the direct and indirect Coombs tests% &he t"o 'oom$s tests are $ased on the fact that anti?human anti$odies, "hich are produced $y immuni@ing non?human species "ith human serum, "ill $ind to human anti$odies, commonly (g* or (g5% 8nimal anti?human anti$odies "ill also $ind to human anti$odies that may $e fi1ed onto antigens on the surface of red $lood cells !also referred to as +,'s#, and in the appropriate test tu$e conditions this can lead to agglutination of +,'s% &he phenomenon of agglutination of +,'s is important here, $ecause the resulting clumping of +,'s can $e visualised/ "hen clumping is seen the test is positive and "hen clumping is not seen the test is negative% 'ommon clinical uses of the 'oom$s test include the preparation of $lood for transfusion in cross?matching, screening for atypical anti$odies in the $lood plasma of pregnant "omen as part of antenatal care, and detection of anti$odies for the diagnosis of immune?mediated haemolytic anemias%

'oom$s tests are done on serum from venous $lood samples "hich are taken from patients $y venepuncture% &he venous $lood is taken to a la$oratory !or $lood $ank#, "here trained scientific technical staff do the 'oom$s tests% &he clinical significance of the result is assessed $y the physician "ho re.uested the 'oom$s test, perhaps "ith assistance from a la$oratory?$ased hematologist%

Direct Coombs test

&he direct 'oom$s test !also kno"n as the direct antiglobulin test or D8&# is used to detect if anti$odies or complement system factors have $ound to +,' surface antigens in vivo% &he D8& is not currently re.uired for pre?transfusion testing $ut may $e included $y some la$oratories%

Laboratory method
&he patient)s red $lood cells !+,'s# are "ashed !removing the patient)s o"n serum# and then incu$ated "ith antihuman glo$ulin !also kno"n as 'oom$s reagent#% (f immunoglo$ulin or complement factors have $een fi1ed on to the +,' surface in?vivo, the antihuman glo$ulin "ill agglutinate the +,'s and the direct 'oom$s test "ill $e positive% !8 visual representation of a positive direct 'oom$s test is sho"n in the upper half of the schematic#%

Indirect Coombs test

&he indirect 'oom$s test !also kno"n as the indirect antiglobulin test or (8&# is used to detect in?vitro anti$ody?antigen reactions% (t is used to detect very lo" concentrations of anti$odies present in a patient)s plasma-serum prior to a $lood transfusion% (n antenatal care, the (8& is used to screen pregnant "omen for anti$odies that may cause hemolytic disease of the ne"$orn% &he (8& can also $e used for compati$ility testing, anti$ody identification, +,' phenotyping, and titration studies%

Exam les o! clinical uses o! the indirect Coombs test

"lood trans!usion re aration 5ain articles: $lood transfusion and cross?matching &he indirect 'oom$s test is used to screen for anti$odies in the preparation of $lood for $lood transfusion% &he donor)s and recipient)s $lood must $e 8,A and +h D compati$le% Donor $lood for transfusion is also screened for infections in separate processes%

8nti$ody screening

8 $lood sample from the recipient and a $lood sample from every unit of donor $lood are screened for anti$odies "ith the indirect 'oom$s test% 7ach sample is incu$ated against a "ide range of +,'s that together e1hi$it a full range of surface antigens !i%e% $lood types#%

'ross matching

&he indirect 'oom$s test is used to test a sample of the recipient)s serum against a sample of the $lood donor)s +,'s% &his is sometimes called cross?matching $lood% Antenatal antibody screening &he indirect 'oom$s test is used to screen pregnant "omen for (g* anti$odies that are likely to pass through the placenta into the fetal $lood and cause haemolytic disease of the ne"$orn%

Laboratory method
&he (8& is a t"o?stage test% !8 cross match is sho"n visually in the lo"er half of the schematic as an e1ample of an indirect 'oom$s test#% #irst stage Washed test red $lood cells !+,'s# are incu$ated "ith a test serum% (f the serum contains anti$odies to antigens on the +,' surface, the anti$odies "ill $ind onto the surface of the +,'s% Second stage &he +,'s are "ashed three or four times "ith isotonic saline and then incu$ated "ith antihuman glo$ulin% (f anti$odies have $ound to +,' surface antigens in the first stage, +,'s "ill agglutinate "hen incu$ated "ith the antihuman glo$ulin !also kno"n 'oom$s reagent# in this stage, and the indirect 'oom$s test "ill $e positive% Titrations ,y diluting a serum containing anti$odies the .uantity of the anti$ody in the serum can $e gauged% &his is done $y using dou$ling dilutions of the serum and finding the ma1imum dilution of test serum that is a$le to produce agglutination of relevant +,'s%

Coombs reagent
'oom$s reagent !also kno"n as Coombs antiglobulin or antihuman globulin# is used in $oth the direct 'oom$s test and the indirect 'oom$s test% 'oom$s reagent is antihuman glo$ulin% (t is made $y inBecting human glo$ulin into animals, "hich produce polyclonal anti$odies specific for human immunoglo$ulins and human complement system factors% 5ore specific 'oom$s reagents or monoclonal anti$odies can $e used%

Enhancement media
,oth (g5 and (g* anti$odies $ind strongly "ith their antigens% (g* anti$odies are most reactive at 3>C'% (g5 anti$odies are easily detected in saline at room temperature as (g5 anti$odies are a$le to $ridge $et"een +,'Ds o"ing to their large si@e, efficiently creating "hat is seen as agglutination% (g* anti$odies are smaller and re.uire assistance to $ridge "ell enough to form a visual agglutination reaction% +eagents used to enhance (g* detection are referred to as potentiators% +,'s have a net negative charge called @eta potential "hich causes them to have a natural repulsion for one another% Eotentiators reduce the @eta potential

of +,' mem$ranes% 'ommon potentiators include lo" ionic strength solution !6(99#, al$umin, polyethylene glycol !E7*#, and proteolytic en@ymes%

History o! the Coombs test

&he 'oom$s test "as first descri$ed in 39:; $y 'am$ridge immunologists +o$in 'oom$s !after "hom it is named#, 8rthur 5ourant and +o$ +ace%224 =istorically, it "as done in test tu$es% &oday, it is commonly done using microarray and gel technology%

$e!erences
3% % F% +osen and +% *eha, Case Studies in Immunology, 4th ed., *arland 9cience, p%3>3% 2% % 'oom$s ++8, 5ourant 87, +ace ++% A new test for the detection of weak and "incomplete" h agglutinins. ,rit J 71p Eath 39:;/2<:2;;?<<%

External lin&s

'oom$s testing ? (nstitute for &ransfusion 5edicine% 'oom$sD test ? direct ? 5edlineplus%org% 'oom$sD test ? indirect ? 5edlineplus%org% 8cute 8nemia ? emedicine%com Drugs that cause haemolytic anemia ? 5erck 5anual% 'oom$s)F&est at the G9 Hational 6i$rary of 5edicine 5edical 9u$Bect =eadings !5e9=#