Ag-Ab Testing/Antiglobin Tests Flashcards

1
Q

In terms of Ag-Ab testing in the lab, state 3 sources of Ags

A
  • Patient cells
  • Manufacturing cells, screening cells, panel cells, A and B cells
  • Donor cells
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2
Q

In terms of Ag-Ab testing in the lab, state 3 sources of Abs

A
  • Patient serum/plasma
  • Manufacturing antisera
  • Donor serum/plasma
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3
Q

In terms of Ag-Ab testing in the lab, patient cells are a ____ source of Ags

A

Unknown

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4
Q

In terms of Ag-Ab testing in the lab, manufacturing cells are a ____ source of Ags

A

Known

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5
Q

In terms of Ag-Ab testing in the lab, donor cells are a ____ source of Ags

A

Unknown

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6
Q

In terms of Ag-Ab testing in the lab, patient serum/plasma are a ____ source of Abs

A

Unknown

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7
Q

In terms of Ag-Ab testing in the lab, manufacturing antisera are a ____ source of Abs

A

Known

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8
Q

In terms of Ag-Ab testing in the lab, donor serum/plamsa are a ____ source of Abs

A

Unknown

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9
Q

Explain the difference b/w direct and indirect agglutination

A

DAT does not require incubation b/c the RBCs are already sensitized (came from patient’s arm)

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10
Q

Explain the principle of an antiglobulin test

A

Antihuman Abs bind to Fc portion of sensitizing Abs and form bridges b/w Ab-coated RBCs, resulting in visual agglutination

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11
Q

Abs found in polyspecific AHG reagent

A

Anti-IgG, anti-C3d, anti-C3b

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12
Q

When to use a polyspecific AHG reagent

A

Detection of IgG Abs

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13
Q

Abs found in monospecific AHG reagent

A

Anti-IgG, anti-C3d

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14
Q

When to use a monospecific AHG reagent

A

Differentiate reactivty when poly-AHG is positive

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15
Q

3 common blood groups capable of binding complement

A
  • ABO
  • Lewis
  • Kidd
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16
Q

Coombs Check Cells

- Composition

A

Group O positive (D+) cells coated by manufacturer w/ anti-D (“sensitized cells”)
- Sensitization occurs by Ab or C’ coating RBCs vai complexing w/ Ag on RBC membrane

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17
Q

Coombs Check Cells

- Purpose of use

A
  • Added to nonreactive tubes as a QC step;

- Used to verify that test system is valid (i.e., AHG was added, AHG not neutralized, proper washing of cells occurred)

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18
Q

Coombs Check Cells

- Expected results

A

MUST be positive, generally about 2+

- Free AHG can now bind to sensitized CCs

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19
Q

Coombs Check Cells

- Causes of unexpected results

A

Incomplete/improper washing

- start over w/ the entire test procedure

20
Q

DAT

- Purpose of test

A

Detects in vivo sensitization of RBCs (IgG, C’)

21
Q

DAT

- Sample/specific requirement

A

EDTA sample only

22
Q

DAT

- Steps of procedure

A
  1. 2 drops of 5% cell suspension
  2. Wash 3x
  3. Add 2 drops AHG
  4. Spin and read
    5a. If positive, end procedure
    5b. If negative,add 1 drop CCs
  5. Spin and read
23
Q

IAT

- Purpose of test

A

Detects in vitro binding (IgG Ag-Ab complexes)

24
Q

IAT

- Sample/specific requirement

A

Can use plasma or serum sample cells from EDTA or plain red top tube

25
Q

IAT

- Steps of procedure

A
  1. 2 drops pt plasma + 1 drop 5% I or II cells
  2. Spin and read
  3. Record results
  4. Add 2 drops LISS; incubate 15 minutes at 37C
  5. Spin and read
  6. Wash 3x
  7. Add 2 drops AHG
  8. Spin and read
    9a. If positive, end procedure
    9b. If negative, add 1 drop CCs
  9. Spin and read
26
Q

4 applications (causes of positive reactions) of DAT

A
  • HDFN
  • Txn run
  • AutoAbs
  • Drugs
27
Q

Why do you incubate the RBCs + serum?

A

Allows for Ag-Ab attachement (sensitization)

28
Q

Why do you perform 3 washes?

A

Removes free globulin

29
Q

Why do you add 2 drops of AHG?

A

Crosslinks Ag-Ab complexes

30
Q

Why do you centrifuge?

A

Accelerates agglutination by bringing cells closer together

31
Q

Why do you examine for agglutination?

A

Interpret test as either “+” or “=”

32
Q

Why do you grade agglutination reactions?

A

Determine strength of reaction

33
Q

Why do you add check cells to a negative reaction?

A

Check for neutralization of AHG by free Igs

34
Q

5 applications of IAT

A
  • Ab detection
  • Ab identification
  • Compatibility testing
  • Ab titers
  • Ag testing
35
Q

Interpret DAT results

A
  • Only test in BB considered to be diagnostic
  • Expected result is negative
    _ If positive, due to one of 4 causes
36
Q

Interpret IAT results

A
  • If using patient cells as unkown source → detects presence or absence of specific Ag being tested
  • If using donor cells as unknown source → detects presence or absnece of specific Ag being sent
37
Q

Correct course of action to take when given positive or discrepant results of an IAT or DAT test

A

Begin entire test procedure over

38
Q

Sources of false negative results in AGT

A
  • Incorrect serum to cell ratio
  • Temp too warm or too cold
  • Incubation time too short
  • Improper enhancement media used
  • Improper storage of cells, serum, or reagents
  • Omission of reagents
  • Improper/incomplete washing (AHG neutralized)
  • Saline pH
  • Delays in addition of AHG
  • Centrifugation time too short
  • Over-shaking tubes
39
Q

Sources of false positive results in AGT

A
  • Test cells have positive DAT
  • Over centrifugation or over reading
  • Bacterial contamination
  • Contaminated AHG (depending on what it’s contaminated with)
  • Dirty glassware
40
Q

Factors that affect AGT

A
  • Complementary fit (anti-D and D Ag)
  • Temperature
  • Enhancement media
  • Time/incubation
  • Concentration of Ag and Ab
41
Q

ABID

  • Ag source
  • Ab source
  • Interpretation
A
  • Ags: mfg cells (known)
  • Abs: patient serum/plasma (unknown)
  • Determination of which Ab is present and if it’s an allo or auto-Ab
42
Q

Ab screen/detection

  • Ag source
  • Ab source
  • Interpretation
A
  • Ags: mfg cells (known)
  • Abs: patient serum/plasma (unknown)
  • Neg: No unexpected Abs detected in patietn’s serum
  • Pos: patient has unexpected Abs in serum → reflex to ABID
43
Q

Compatibility testing

  • Ag source
  • Ab source
  • Interpretation
A
  • Ags: donor cells (unknown)
  • Abs: patient serum/plasma (unknown)
  • Neg: donor is compatible for patient transfusion
  • Pos: donor is unsuitable for patient transfusion
44
Q

Ab titers

  • Ag source
  • Ab source
  • Interpretation
A
  • Ags: mfg cells (known)
  • Abs: patient serum/plasma (known); by the time you do the titer, you already know Ab specificity so it’s no longer unknown)
  • ??
45
Q

Ag testing (weak D)/Ag phenotyping

  • Ag source
  • Ab source
  • Interpretation
A
  • Ags: patient/donor cells (unknown)
  • Abs: mfg antisera (known)
  • Unknown donor cells → detects presence or absence of specific Ag being tested
  • Unknown patient cells → detects presence or absence of specific Ag beign tested (entire phenotype)
46
Q

Coombs reagent = ____ = ____

A

AHG; anti-IgG/C’