Detecting Antibodies The Antibody Screen CLS 422 Clinical Immunohematology I.
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Transcript of Detecting Antibodies The Antibody Screen CLS 422 Clinical Immunohematology I.
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Detecting AntibodiesDetecting Antibodies
The Antibody Screen
CLS 422
Clinical Immunohematology I
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ObjectivesObjectives
Explain the purpose of performing an antibody screen.
Discuss the antigen characteristics important in the composition of screening cells.
Describe the phases of antibody detection. Describe the types of antibodies that can be
encountered in each of the phases of antibody detection.
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ObjectivesObjectives State the difference between an alloantibody and an
autoantibody. List factors affecting the antigen-antibody reaction in
the indirect antiglobulin test. Discuss the action of potentiators. Compare and contrast methods for performing an
antibody screen. Assess sources of error affecting the indirect
antiglobulin test. Interpret the results of an antibody screen.
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The Antibody ScreenThe Antibody Screen
Used to detect “irregular” antibodies. Maximize detection of clinically significant
antibodies Minimize detection of insignificant
antibodies Patient’s serum or plasma is tested
against reagent red blood cells (RBCs) with known antigens (screen cells).
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Allo vs. AutoAllo vs. Auto
Alloantibody – antibody directed against antigens that the individual does not possess Immune Naturally-occurring
Autoantibody – antibody directed against one’s own antigens Auto control – patient’s RBCs tested against
patient’s plasma
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What makes an antibody What makes an antibody clinically significant?clinically significant?
The ability to cause decreased RBC survival. If the antibody activates complement, there may be
intravascular RBC lysis. There may be extravascular destruction of
antibody-coated RBCs by the macrophages of the RES.
Hemolytic Transfusion Reaction (HTR) Hemolytic Disease of the Fetus & Newborn
(HDFN)
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Who needs to be tested?Who needs to be tested?
OB patients – looking for antibodies that may cause HDFN in the fetus.
Patients who need an RBC transfusion – looking for antibodies in the recipient that could destroy the donor RBCs (HTR).
Blood, organ and tissue donors – avoid passive antibody transfer; evaluate as source of anti-serum and rare RBCs.
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An Application of the An Application of the Indirect Antiglobulin TestIndirect Antiglobulin Test
Patient’s plasma (unknown antibody) is tested against reagent RBCs (known antigen). Y
YY Incubated at 37oC to allow antibody to sensitize RBCs. Antiglobulin phase to allow sensitized RBCs to agglutinate.
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Screen Cell CompositionScreen Cell Composition
Sets of 2 to 4 different cells Group O Rh Positive and Rh
Negative cells Other major blood group
antigens are represented
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Antigen Profile Antigen Profile (Antigram)(Antigram)
D C c E e Cw K k Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xg
I + + 0 0 + 0 + + 0 + + 0 0 + 0 + 0 + 0 0 + +
II + 0 + + 0 0 0 + + 0 + + 0 + + 0 + 0 + 0 + 0
III 0 0 + 0 + 0 0 + + 0 0 + + 0 + + 0 + + 0 + +
• If the patient’s serum/plasma contains an antibody directed against one of the antigens on the screen cell, the RBCs will agglutinate (or hemolyze)= positive reaction.
• We can not be certain which antigen is the target of the antibody.
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Phases of TestingPhases of Testing
Immediate spin (I.S.)
(optional phase)
37oC
AHG
Lewis, M, N, P1, cold autoantibodies (I, H, IH)
May see D, E, K, strong cold antibodies
Rh, Kell, Kidd, Duffy, Ss
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SpecimenSpecimen
Plasma Serum
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Antibody Screen Antibody Screen Tube MethodTube Method
37C inc
IDII
IDIIIID
I
√√
√
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Antibody ScreenAntibody ScreenGel MethodGel Method
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Antibody Screen Antibody Screen Gel MethodGel Method
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Ortho ProvueOrtho Provue
Courtesy Ortho-Clinical Diagnostics Raritan, NJ
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Antibody Screen Antibody Screen Solid Phase Adherence MethodSolid Phase Adherence Method
RBC antigens are bound to the sides of a microtiter well.
Different wells possess different antigens, and thus are different “screen cells”.
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Antibody Screen Antibody Screen Solid Phase Adherence MethodSolid Phase Adherence Method
YY
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Immucor AutomationImmucor Automation
ABO and Rh are performed using direct agglutination in a microtiter well.
Screens, panels and DATs are performed using solid phase adherence.
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Reporting ResultsReporting Results
Reporting is “all or nothing”, rather than reporting the results of each cell at each phase.
If all cells are nonreactive at all phases, and the Coombs Control Cells are positive, the screen is reported as negative.
IS 37 AHG CC
I 0 0 0 3+
II 0 0 0 3+
III 0 0 0 3+
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Reporting ResultsReporting Results
If one or more cells react at any phase of testing, the screen is reported as positive.
An antibody panel should be performed to determine the specificity of the antibody present.
IS 37 AHG CC
I 0 0 0 2+
II 0 0 2+
III 0 0 0 2+
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Are We Done Now?Are We Done Now?
NO – all cells should be tested through all phases, even if a positive reaction has already been observed.
IS 37 AHG CC
I 2+
II 0
III 1+
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How Would you Report This How Would you Report This Screen?Screen?
IS 37 AHG CC
I 0 1+ 3+
II 0 0 0 2+
III 0 0 0 2+
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How Would you Report This How Would you Report This Screen?Screen?
IS 37 AHG CC
I 0 0 2+
II 0 0 2+
III 0 0 2+
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How Would you Report This How Would you Report This Screen?Screen?
IS 37 AHG CC
I 2+ 0 0 2+
II 1+ 0 0 2+
III 0 0 0 2+
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How Would you Report This How Would you Report This Screen?Screen?
IS 37 AHG CC
I 0 0 0 0
II 0 0 0 2+
III 0 0 0 2+
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How Would you Report This How Would you Report This Screen?Screen?
(Performed Using Gel)(Performed Using Gel)
IS 37 AHG CC
I 0
II 0
III 0
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How Would you Report This How Would you Report This Screen?Screen?
(Performed Using Solid Phase (Performed Using Solid Phase Adherence)Adherence)
IS 37 AHG CC
I 0
II 2+
III
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Factors Affecting Factors Affecting the Antibody the Antibody
ScreenScreenThese factors will affect ANY
application of the indirect antiglobulin test!
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Antigen/Antibody Ratio Antigen/Antibody Ratio
Y
Y
Y
YY
Y Y
Y
Y
Y
Y
Y
Y
YY
YY
YY
Y
Y
Y
Y
YY
Y
Y
Prozone PostzoneEquivalenceRatio is usually 2 drops of serum or plasma to 1 drop of RBC suspension.
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Reaction TemperatureReaction Temperature
Temp oC ABO Rh Kell Duffy Kidd Ss MN Lewis P1 Ii
45
40
35
30
25
20
15
10
5
0
IG Class
IgM Some IgG
IgG IgG IgG IgG IgG IgM IgM IgM IgM
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Reaction TimeReaction Time
IgG antibodies are incomplete Require incubation at 37oC to react Length of incubation dependant on test
medium
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Test MediumTest Medium
May add potentiators to overcome the effects of shielding and the zeta potential Albumin LISS PEG Enzymes
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Quantity and location of Quantity and location of AntigenAntigen
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pHpH
Optimal 6.5 to 7.5
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Sources of Error Sources of Error in the Antibody in the Antibody
ScreenScreenFalse Negative Results
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Improper WashImproper Wash
Y
Y
Y
Y
Y
Y
YY
Y
Y
YY
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Failure to…Failure to…
Add plasma (the antibody source) Make it a habit to add plasma to
tube before adding RBCs. Add reagents Follow manufacturer’s directions Recognize hemolysis as a positive
reaction
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Work QuicklyWork Quickly
Y
Y
Y
Y
Y
Y
Y
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Other causes of false Other causes of false negative resultsnegative results
AHG reagent neutralized Using expired reagents Under-centrifugation Complement-dependant antibody/plasma
specimen
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Sources of Error Sources of Error in the Antibody in the Antibody
ScreenScreenFalse Positive Results
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False PositivesFalse Positives
Over-centrifugation Contaminated reagents Debris Rouleaux
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Limitations of the Antibody Limitations of the Antibody ScreenScreen
Will not detect ABO incompatibility Will not detect antibodies to antigens that are
not present on the screen cells May not detect antibodies exhibiting dosage May not detect antibodies that are low in titer
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Primary vs. Secondary Primary vs. Secondary Humoral ResponseHumoral Response
First exposure
Second exposure
IgM
IgM
IgG
IgG
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Today’s Lab…Today’s Lab…
The Type & ScreenThe Type & Screen(ABO, Rh, and Antibody Screen)(ABO, Rh, and Antibody Screen)