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
Screening Cells Screening cells come in sets of 2 or 3 vials each
● Donor Testing vs Patient Testing ● Each vial (donor) has been phenotyped for each antigen
26
Screening Cells how many antigens are required on at least one of the vials
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
27
 The screen only uses
2-3 cells
28
An antibody panel usually includes at least.
10 panel cells
29
Panel
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
30
An autocontrol should also be run with
ALL panels
31
The same phases used in an antibody screen are
used in a panel
32
A tube is labeled for each of the panel cells plus
one tube for AC one drop of panel cells + two drops of serum
33
IS Phase
 Perform immediate spin (IS) and grade agglutination; inspect for hemolysis
34
Potentiators
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)
35
(LISS) 37°C Phase
 2 drops of LISS are added, mixed and incubated at 37oC for 10-15 minutes  Centrifuge and check for agglutination  Record results
36
IAT Phase (or AHG) were testing whether or not _________ to do this we use the
 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)
37
AHG Phase steps
 Wash cells at least 3 times with saline (manual or automated)  Add 2 drops of AHG and gently mix  Centrifuge  Read  Record reactions
38
All cells are negative at AHG, so add
“Check” Cells
39
* 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?
40
Interpreting Antibody Panels There are a few basic steps to follow when interpreting panels
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
41
An antibody will only react with cells that have the
corresponding antigen; antibodies will not react with cells that do not have the antigen
42
Ruling out Can only rule out on
completely negative cells cannot use for rule outs
43
Ruling out cross off on what
Cross off on homozygous only only except: K and Lu^a
44
Ruling out E could be + on
Heterozygous
45
Ruling out Everytime a Rxn happens
Lea is present
46
Ruling out Le^a is
IgM
47
Ruling out Le^a is normally 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
Guidelines: Autocontrol
 Negative - alloantibody  Positive – autoantibody or DTR (i.e.,alloantibodies)
49
Guidelines Phases
 IS – cold (IgM)  37° - cold (some have higher thermal range) or warm reacting  AHG – warm (IgG)...significant!!
50
Guidelines  Reaction strength
 1 consistent strength – one antibody  Different strengths – multiple antibodies or dosage
51
About reaction strengths...... The strength of reaction may be due to
Dosage
52
About reaction strengths...... If panel cells are homozygous,
a strong reaction may be seen
53
About reaction strengths...... If panel cells are heterozygous,
reaction may be weak or even non-reactive
54
About reaction strengths..... Panel cells that are heterozygous should not be
crossed out because antibody may be too weak to react
55
Matching the pattern Single antibodies
usually shows a pattern that matches one of the antigens (see previous panel example)
56
Multiple antibodies
are more difficult to match because they often show mixed reaction strengths
57
The rule of three must be met to confirm the presence of
the antibody
58
Rule of three P value and confidence interval
A p-value ≤ 0.05 must be observed This gives a 95% confidence interval
59
Rule of three Patient serum must be
 Positive with 3 cells with the antigen  Negative with 3 cells without the antigen
60
What if the “rule of three” is not fulfilled?
 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
Patient History GET THE HISTORY!
- 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
Autocontrol is postive or DAT indicates
Positive autocontrol or DAT indicates that autoantibody or alloantibody is present to recently transfused cells
63
Negative autocontrol indicates
alloantibody
64
Interpretation Guidelines Phases
* 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
Interpretation Guidelines Reaction strength
– Reactions with varying strengths indicate multiple antibodies – Strength is also affected by dosage
66
Interpretation Guidelines Ruling out antibodies
– 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
Interpretation Guidelines Matching the pattern
– The pattern of reactions matches one of the antigen columns when a single antibody is present – Multiple antibodies show varying patterns
68
Interpretation Guidelines Rule of three
– 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
Interpretation Guidelines Phenotyping the patient Another way to confirm the presence of antibody is to
determine the patient’s phenotype for antigen
70
Interpretation Guidelines Individuals do not make alloantibodies toward
antigens on their own RBCs
71
Interpretation Guidelines If the patient has recently been transfused, what techniques should be done
RBC separation techniques should be used before phenotyping is done
72
Interpretation Guidelines Reagent antisera (of the suspected antibody) is added to
to the patient's RBCs, a negative reaction should result
73
Multiple Antibodies Matching the pattern is more difficult when
-multiple antibodies are present  Varying reactions may also be observed
74
Multiple Antibodies Special techniques can be used to help identify
multiple antibodies
75
Multiple Antibodies Special techniques can be used to help identify multiple antibodies
– Selected cells – Neutralization P1, I – Chemical treatment – Proteolytic enzymes – Sulfhydryl reagents – ZZAP
76
Selected Cells Selected cells are chosen from
other panel or screening cells to confirm or eliminate the antibody
77
Selected Cells The cells are “selected” from other panels because
of their characteristics
78
Selected Cells The number of selected cells needed depends
on how may antibodies are identified
79
Selected Cells Every cell should be positive for each of the
antibodies and negative for the remaining antibodies
80
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...
81
Neutralization Some antibodies may be neutralized as a way of
confirmation
82
Neutralization Commercial “substances” bind to the antibodies in the patient's serum, causing
them to show no reaction when tested with the corresponding antigen (in the panel)
83
Neutralization The control contains
saline and serum (no substance) and should remain positive
84
Neutralization A control shows that a loss of reactivity is due to the
neutralization and not to the dilution of the antibody strength when the substance is added
85
Neutralization Manufacturer’s directions should be followed and a _________ control should always be used
Manufacturer’s directions should be followed and a dilutional control should always be used
86
Neutralization Common substances P1 substances
P1 substance (sometimes derived from hydatid cyst fluid)
87
Neutralization Common substances Lea and Leb substance
(soluble antigen found in plasma and saliva)
88
Neutralization Common substances I substance
can be found in breast milk
89
Neutralization Common substances Sda substance
derived from human or guinea pig urine
90
91
**You should be aware that many of these substances neutralize
COLD antibodies; Cold antibodies can sometimes mask more clinically significant antibodies (IgG), an important reason to use neutralization techniques
92
Proteolytic Enzymes Enzymes are used to eliminate or enhance antibody activity
- Destroyed Antigens: Duffy and MNS – Enhanced Antigens: Rh, Kidd, and Lewis, I, and P
93
Proteolytic Enzymes Several enzymes exist:
Ficin (figs) Bromelin (pineapple) Papain (papaya)
94
Proteolytic Enzymes Procedure Steps  One-stage test
Enzymes, RBCs, and serum are simultaneously incubated
95
Proteolytic Enzymes Two-stage test
- Panel cells are pretreated with enzymes, incubated and washed – Patient serum is added to panel cells and tested
96
Sulfhydryl Reagents cleaves
 Cleave the disulfide bonds of IgM molecules and help differentiate between IgM and IgG antibodies
97
Sulfhydryl Reagents Good to use when you have both IgG and IgM antibodies (warm/cold)
 Dithiothreitol (DTT) is a thiol and will denature Kell antigens  2-mercaptoethanol (2-ME)
98
ZZAP A combination of
proteolytic enzymes and DTT
99
ZZAP Denatures
Kell, M, N, S, Duffy and other less frequent blood group antigens
100
ZZAP Does not denature the
Kx antigen
101
ZZAP Good for adsorption techniques
“frees” autoantibody off patient’s cell, so that autoantibody can then be adsorbed onto another RBC
102
Antibodies to High-Frequency Antigens High-frequency antigens occur in the population at a frequency of
98%
103
Antibodies to High-Frequency Antigens Suspect an alloantibody to a
high-frequency antigen if most panel cells are positive
104
Antibodies to High-Frequency Antigens Additional testing may be needed to confirm the presence of the
antibody
105
enzymes to enhance or destroy the _________ DDT to destroy _________
*Enzymes to enhance or destroy antigens *Dithiothreitol (DTT) to destroy Kell antigens
106
High-Titer, Low-Avidity Antibodies react at not implicated in _______ antibodies to ________
 Antibodies to high-frequency antigens that react weakly  React at AHG phase  Not implicated in transfusion reactions or HDFN
107
Antibodies to Low-Frequency Antigens May occur alone or are suspected
when the screen is negative and crossmatching is positive
108
Antibodies to Low-Frequency Antigens Only one reactive cell suggests
this type of antibody
109
Antibodies to Low-Frequency Antigens Common antibodies include
– Anti-Cw, anti-Wra, anti-V, anti-Cob, anti-Bga, anti-Kpa, and anti-Lua antibodies