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Immunohematology - 4444 > ABO detection and ID > Flashcards

ABO detection and ID Flashcards

1
Q

The test used to detect antibodies is called an

A

antibody screen

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2
Q

Antibody screens are used for

A

Patients needing a transfusion
– Pregnant women
– Patients who have had transfusion reactions
– Blood(red cells) and plasma donors

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3
Q

Antibody Screen

Indirect Antiglobulin Test:

A

Uses the patient’s plasma or serum against reagent
RBCs to detect unexpected antibodies

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4
Q

Indirect Antiglobulin Test:

Unexpected antibodies

A

 found in addition to the expected anti-A or anti-B antibodies
 result of RBC stimulation (transfusion, pregnancy)
 maybe
– Clinically significant (IgG)
– Not clinically significant (IgM)

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5
Q

Other Antibodies

Naturally occurring antibodies

A

may form as a result of exposure to
environmental sources (e.g., pollen, fungus, and bacteria), which have
structures similar to some RBC antigens.

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6
Q

Other Antibodies

Passively acquired antibodies

A

are produced in one individual and then
transmitted to another via plasma-containing blood components or derivatives

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7
Q

Other Antibodies

Autoantibodies,

A

when present, may complicate the detection of clinically
significant antibodies

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8
Q

In blood banking, we test knowns with

A

“unknowns”

KNOWN: UNKNOWN:
Reagent RBC + patient serum
Reagent antisera + patient RBCs

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9
Q

When detecting and/or identifying antibodies, we test the patient

A

 When detecting and/or identifying antibodies, we test patient serum (unknown)
with reagent red cells (known)

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10
Q

Indirect Antiglobulin Test

Incorporates multiple PHASES of testing

A

 IS = immediate spin (not required)
 37oC = after incubation at 37oC with or without potentiators
 AHG = anti-human globulin added (anti-IgG) after washing antibody/antigen reaction

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11
Q

 Coomb’s Control Cells (aka Check Cells) =

A

used with negative reactions to ensure
proper washing technique = must be positive

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12
Q

“IAT”

Antigen Source

A

= commercial red cells

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13
Q

IAT

Antibody Source =

A

patient’s serum/plasma

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14
Q

Autocontrol

Tests a patient’s serum with their

A

own RBCs

(+) mean autoantibody

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15
Q

Autocontrol

Autocontrol is incubated with the

A

antibody screen (or antibody panel)

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16
Q

Autocontrol

The AC and DAT can help in determine if the antibodies are directed

A

are directed against
the patient’s cells or transfused cells (allo- or autoantibody)

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17
Q

Autocontrol

If a laboratory technician uses an autocontrol with a screen and it is positive,

A

the technician may run a DAT (patient cells plus AHG) to detect in vivo coating

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18
Q

If autocontrols (-)

A

then you have a alloantibody

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19
Q

DAT could be negative=

A

look up why

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20
Q

Direct Antiglobulin Test

antigen and antibody sources

A

Only incorporates 1 phase
 AHG

 Antigen Source = patient’s red cells
 Antibody Source = Immunoglobulin attached to patient’s red cells

 IgG or C3 coating patient’s red cells in vivo

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21
Q

Clinically
Significant
Antibodies

A
  • Usually IgG

-React best at 37° C and
during the antihuman
globulin (AHG) phase
(indirect antiglobulin test
[IAT])

  • Clinically significant
    antibodies are associated
    with hemolytic transfusion
    reactions (HTRs) and HDFN
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22
Q

Performing
an Antibody
Screen

A
  • Patient’s plasma or
    serum is incubated with
    screening cells

-After incubation, an IAT
is performed using AHG
reagent

  • Want to detect any
    IgG antibodies
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23
Q

Screening cells are

A

are single or pooled donor group O cells;
however, single-donor vials offer increased sensitivity

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24
Q

Screening Cells

group O cells are used so that

A

Group O cells are used so that anti-A and anti-B antibodies will not
react

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25
Q

Screening Cells

Screening cells come in sets of 2 or 3 vials each

A

● Donor Testing vs Patient Testing
● Each vial (donor) has been phenotyped for each antigen

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26
Q

Screening Cells

how many antigens are required on at least one of the vials

A

18 antigens are required on at least one of the vials:
D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb

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27
Q

 The screen only uses

A

2-3 cells

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28
Q

An antibody panel usually includes at least.

A

10 panel cells

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29
Q

Panel

A

Each of the panel cells has been antigen-typed (shown on
antigram)
 + refers to the presence of the antigen
 0 refers to the absence of the antigen

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30
Q

An autocontrol should also be run with

A

ALL panels

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31
Q

The same phases used in an antibody screen are

A

used in a panel

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32
Q

A tube is labeled for each of the panel cells plus

A

one tube for AC

one drop of panel cells
+
two drops of serum

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33
Q

IS Phase

A

 Perform immediate spin (IS) and grade agglutination; inspect for
hemolysis

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34
Q

Potentiators

A

Used in antibody detection and identification to enhance an antigen–antibody
reaction

Saline
(may only enhance
if incubated for a
long time)

Low-ionic-strength
solution (LISS):
common

Bovine serum
albumin (BSA)

Polyethylene glycol
(PEG)

Proteolytic
enzymes
(can destroy some
antigens)

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35
Q

(LISS) 37°C Phase

A

 2 drops of LISS are added, mixed and incubated at 37oC for 10-15 minutes
 Centrifuge and check for agglutination
 Record results

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36
Q

IAT Phase (or AHG)

were testing whether or not _________

to do this we use the

A

 Indirect Antiglobulin Test (IAT) – we’re testing whether or
not possible antibodies in patient’s serum will react with
RBCs in vitro
 To do this we use the Anti-Human Globulin reagent
(AHG)
 Polyspecific (anti-IgG, C3)
 Anti-IgG
 Anti-complement (anti-C3)

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37
Q

AHG Phase steps

A

 Wash cells at least 3 times with saline (manual or
automated)
 Add 2 drops of AHG and gently mix
 Centrifuge
 Read
 Record reactions

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38
Q

All cells are
negative at
AHG, so
add

A

“Check”
Cells

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39
Q
  • 29 y.o. female admitted to Family Birthing Center for labor
    progression. All laboring patients will have an active blood bank
    specimen during their admission. The patient has been determined to
    be a high risk for hemorrhage. The doctor wants to order 2 units of
    red cells to be kept at bedside.
    Patient T/S Results:

Blood Type: A positive
ABSC: Positive

What needs to happen next?

A
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40
Q

Interpreting Antibody Panels

There are a few basic steps to follow when interpreting
panels

A
  1. “Ruling out” means crossing out antigens that did not react
  2. Circle the antigens that are not crossed out
  3. Consider antibody’s usual reactivity
  4. Look for a matching pattern
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41
Q

An antibody will only react with
cells that have the

A

corresponding
antigen; antibodies will not react
with cells that do not have the
antigen

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42
Q

Ruling out

Can only rule out on

A

completely negative
cells

cannot
use for
rule
outs

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43
Q

Ruling out

cross off on what

A

Cross off on homozygous only only except: K and Lu^a

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44
Q

Ruling out

E could be + on

A

Heterozygous

45
Q

Ruling out

Everytime a Rxn happens

A

Lea is present

46
Q

Ruling out

Le^a is

A

IgM

47
Q

Ruling out

Le^a is normally a

A

Cold-Reacting antibody (IgM), so it makes
sense that we see the reaction in the IS phase of testing;
The E antigen will usually react at warmer temperatures

48
Q

Guidelines:

Autocontrol

A

 Negative - alloantibody
 Positive – autoantibody or DTR (i.e.,alloantibodies)

49
Q

Guidelines

Phases

A

 IS – cold (IgM)
 37° - cold (some have higher thermal range) or warm reacting
 AHG – warm (IgG)…significant!!

50
Q

Guidelines

 Reaction strength

A

 1 consistent strength – one antibody
 Different strengths – multiple antibodies or dosage

51
Q

About reaction strengths……

The strength of reaction may be due to

A

Dosage

52
Q

About reaction strengths……

If panel cells are homozygous,

A

a strong reaction may
be seen

53
Q

About reaction strengths……

If panel cells are heterozygous,

A

reaction may be weak
or even non-reactive

54
Q

About reaction strengths…..

Panel cells that are heterozygous should not be

A

crossed
out because antibody may be too weak to react

55
Q

Matching the pattern

Single antibodies

A

usually shows a pattern that matches one of the
antigens (see previous panel example)

56
Q

Multiple antibodies

A

are more difficult to match because they often
show mixed reaction strengths

57
Q

The rule of three must be met to confirm the presence of

A

the antibody

58
Q

Rule of three

P value and confidence interval

A

A p-value ≤ 0.05 must be observed
This gives a 95% confidence interval

59
Q

Rule of three

Patient serum must be

A

 Positive with 3 cells with the antigen
 Negative with 3 cells without the antigen

60
Q

What if the “rule of three” is not fulfilled?

A

 If there are not enough cells in the panel to fulfill the
rule, then additional cells from another panel could be
used
 Most labs carry different lot numbers of panel cells

61
Q

Patient History

GET THE HISTORY!

A
  • Mixed RBC populations from a previous transfusion can remain for up to
    3 months
    – Patient may have come from another hospital
    – Some diseases are associated with antibodies
    – Some antibodies occur at a higher frequency in some races
    – Learn about the diagnosis, age, and race of the patient
62
Q

Autocontrol is postive or DAT indicates

A

Positive autocontrol or DAT indicates that autoantibody or
alloantibody is present to recently transfused cells

63
Q

Negative autocontrol indicates

A

alloantibody

64
Q

Interpretation Guidelines

Phases

A
  • IgM antibodies react at room temperature or during immediate-spin
    (IS) crossmatching; anti-Lea, anti-Leb, anti-M, anti-N, anti-I, anti-P1
    antibodies
  • IgG antibodies usually react at 37° C or with AHG
  • Reactions at different phases may indicate IgM and IgG
65
Q

Interpretation Guidelines

Reaction strength

A

– Reactions with varying strengths indicate multiple antibodies
– Strength is also affected by dosage

66
Q

Interpretation Guidelines

Ruling out antibodies

A

– Panel cells that are negative in all phases can be used to rule out
antibodies
– Begin with the first panel cell that is negative in all phases and
cross out any antigens present
– Panel cells that are heterozygous should not be crossed out,
because the antibody may be too weak to react

67
Q

Interpretation Guidelines

Matching the pattern

A

– The pattern of reactions matches one of the antigen columns when a
single antibody is present
– Multiple antibodies show varying patterns

68
Q

Interpretation Guidelines

Rule of three

A

– To ensure valid results, 3 antigen-positive cells must react and 3
antigen-negative cells must not react with the patient’s serum or
plasma
– If this does not occur, additional panel cells (selected cells) are used

69
Q

Interpretation Guidelines

Phenotyping the patient

Another way to confirm the presence of antibody is to

A

determine the patient’s phenotype for antigen

70
Q

Interpretation Guidelines

Individuals do not make alloantibodies toward

A

antigens on their
own RBCs

71
Q

Interpretation Guidelines

If the patient has recently been transfused, what techniques should be done

A

RBC separation
techniques should be used before phenotyping is done

72
Q

Interpretation Guidelines

Reagent antisera (of the suspected antibody) is added to

A

to
the patient’s RBCs, a negative reaction should result

73
Q

Multiple Antibodies

Matching the pattern is more difficult when

A

-multiple antibodies are
present
 Varying reactions may also be observed

74
Q

Multiple Antibodies

Special techniques can be used to help identify

A

multiple antibodies

75
Q

Multiple Antibodies

Special techniques can be used to help identify multiple antibodies

A

– Selected cells
– Neutralization P1, I
– Chemical treatment
– Proteolytic enzymes
– Sulfhydryl reagents
– ZZAP

76
Q

Selected Cells

Selected cells are chosen from

A

other panel or
screening cells to confirm or eliminate the antibody

77
Q

Selected Cells

The cells are “selected” from other panels
because

A

of their characteristics

78
Q

Selected Cells

The number of selected cells needed depends

A

on how may antibodies are identified

79
Q

Selected Cells

Every cell should be positive for each of the

A

antibodies and negative for the remaining
antibodies

80
Q

Selected Cells

For example:
 Let’s say you ran a panel and identified 3 different
antibodies: anti-S, anti-Jka, and anti-P1
 Selected cells could help…

A
81
Q

Neutralization

Some antibodies may be neutralized as a way of

A

confirmation

82
Q

Neutralization

Commercial “substances” bind to the
antibodies in the patient’s serum, causing

A

them to show no reaction when tested
with the corresponding antigen (in the panel)

83
Q

Neutralization

The control contains

A

saline and serum (no substance)
and should remain positive

84
Q

Neutralization

A control shows that a loss of reactivity is due to the

A

neutralization and not to the dilution of the antibody
strength when the substance is added

85
Q

Neutralization

Manufacturer’s directions should be followed
and a _________ control should always be used

A

Manufacturer’s directions should be followed
and a dilutional control should always be used

86
Q

Neutralization

Common substances

P1 substances

A

P1 substance (sometimes derived from hydatid cyst fluid)

87
Q

Neutralization

Common substances

Lea and Leb substance

A

(soluble antigen found in plasma and saliva)

88
Q

Neutralization

Common substances

I substance

A

can be found in breast milk

89
Q

Neutralization

Common substances

Sda substance

A

derived from human or guinea pig urine

90
Q
A
91
Q

**You should be aware that many of these substances neutralize

A

COLD antibodies; Cold antibodies can sometimes mask more
clinically significant antibodies (IgG), an important reason to use
neutralization techniques

92
Q

Proteolytic Enzymes

Enzymes are used to eliminate or enhance antibody activity

A
  • Destroyed Antigens: Duffy and MNS
    – Enhanced Antigens: Rh, Kidd, and Lewis, I, and P
93
Q

Proteolytic Enzymes

Several enzymes exist:

A

Ficin (figs) Bromelin (pineapple) Papain (papaya)

94
Q

Proteolytic Enzymes

Procedure Steps
 One-stage test

A

Enzymes, RBCs, and serum are simultaneously incubated

95
Q

Proteolytic Enzymes

Two-stage test

A
  • Panel cells are pretreated with enzymes, incubated and washed
    – Patient serum is added to panel cells and tested
96
Q

Sulfhydryl Reagents cleaves

A

 Cleave the disulfide bonds of IgM molecules and help
differentiate between IgM and IgG antibodies

97
Q

Sulfhydryl Reagents

Good to use when you have both IgG and IgM
antibodies (warm/cold)

A

 Dithiothreitol (DTT) is a thiol and will denature Kell antigens
 2-mercaptoethanol (2-ME)

98
Q

ZZAP

A combination of

A

proteolytic enzymes and DTT

99
Q

ZZAP

Denatures

A

Kell, M, N, S, Duffy and other less frequent blood group antigens

100
Q

ZZAP

Does not denature the

A

Kx antigen

101
Q

ZZAP

Good for adsorption techniques

A

“frees” autoantibody off patient’s cell, so that autoantibody
can then be adsorbed onto another RBC

102
Q

Antibodies to
High-Frequency
Antigens

High-frequency antigens occur
in the population at a
frequency of

A

98%

103
Q

Antibodies to
High-Frequency
Antigens

Suspect an alloantibody to a

A

high-frequency antigen if most
panel cells are positive

104
Q

Antibodies to
High-Frequency
Antigens

Additional testing may be
needed to confirm the
presence of the

A

antibody

105
Q

enzymes to enhance or destroy the _________

DDT to destroy _________

A

*Enzymes to enhance or destroy
antigens
*Dithiothreitol (DTT) to destroy Kell
antigens

106
Q

High-Titer, Low-Avidity Antibodies

react at

not implicated in _______

antibodies to ________

A

 Antibodies to high-frequency
antigens that react weakly
 React at AHG phase
 Not implicated in transfusion
reactions or HDFN

107
Q

Antibodies to
Low-Frequency Antigens

May occur alone or are suspected

A

when the screen is negative and
crossmatching is positive

108
Q

Antibodies to
Low-Frequency Antigens

Only one reactive cell suggests

A

this type of antibody

109
Q

Antibodies to
Low-Frequency Antigens

Common antibodies include

A

– Anti-Cw, anti-Wra, anti-V, anti-Cob, anti-Bga, anti-Kpa, and anti-Lua antibodies

Immunohematology - 4444 (17 decks)

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