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Advanced Laboratory Operations and Automation > Test 5 > Flashcards

Test 5 Flashcards

1
Q

DAT test freshly washed cells with

A

antiglobulin reagents (anti-
IgG and/or anti-C3d) to detect IN-VIVO coated of RBCs.

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2
Q

DAT

The ___ Fab sites on the antiglobulin molecule bind to the Fc
a portion of sensitizing antibody or complement on ____
adjacent RBCs, bridging gap and causing visible
agglutination

A

2 and 2

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3
Q

DAT

The stength of agglutination is proportional to the

A

amount of bound
protein.

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4
Q

Direct AHG

whats all added

A

Washed (3xs) pateint red cells + coombs sera ———-> visual Red cell agglutination

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5
Q

Reasons to
Perform a
DAT

A

-SCREEN FOR
CLINICALLY
UNEXPECTED
AUTOIMMUNE
PHENOMENA

-DETECT EARLY
MANIFESTATION
OF IMMUNE
RESPONSE TO
RECENT
TRANSFUSION.

-ASSIST IN
DIAGNOSIS OF
HDFN.

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6
Q

Collection of Blood Sample for DAT

A

-To verify in-vivo sensitization
EDTA sample should be used.
-EDTA sample will provide RBCs
for elution if necessary.
-If cold hemagglutinin
suspected keep sample at 37C.

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7
Q

Significance of Positive DAT

Positive DAT does not mean that RBCs will

A

have shortened survival

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8
Q

Positive DAT without clinical problems occurs in

A

1:1,000-1:14,000
blood donors and 1-15% of hospital patients.

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9
Q

Significance of positive DAT

what is coated on RBCs

A

IgG and C3d

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10
Q

Healthy individuals can have ___________ molecules of IgG/RBC and C3d/RBC, this is
below threshold of detection

A

5-90

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11
Q

DAT can detect ________ IgG/RBC and _________ C3d/RBC

A

100-500

400-1100

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12
Q

Causes of a positive DAT

A

Autoantibodies Alloantibodies - HTR
Passively acquired
alloantibodies (plasma,
derivatives)
Maternal alloantibodies
Nonspecifically adsorbed
proteins or membrane
modification
Drug induced antibodies Antibodies produced by
passenger lymphocytes
Complement activation
due to bacterial
infection, autoantibodies
or alloantibodies

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13
Q

Autocontrol (AC) run as part of

A

antibody
work up and is not the same as DAT

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14
Q

DAT cells are taken directly to

A

AHG, no
adding of serum or enhancement, no
incubation detects IN-VIVO sensitization

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15
Q

False
Positive
DAT

A

-Want to detect
IN-VIVO
sensitization
not in-vitro.
-False positive
most often
associated with
using
refrigerated or
clotted samples.
-Any positive
obtained on
clotted sample
should be
confirmed with
EDTA sample.
-EDTA
will provide
RBCs for elution
if necessary.

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16
Q

Patient history

A

Crucial to investigation

  • History of recent transfusion.
  • Administration of drugs previously
    associated with immune hemolysis.
  • History of hematopoietic
    progenitor cell or organ
    transplantation.
  • Administration of IVIG or IV anti-D
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17
Q

Test serum/plasma to detect and identify clinically significant antibodies to

may have to distinguish between

A

red cell antigens,
may have to distinguish auto-
from allo-antibodies, if present.

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18
Q

Prepare and test eluate from DAT positive RBCs to define whether

A

Prepare and test eluate from DAT
positive RBCs to define whether
coating protein has red cell antibody
specificity.

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19
Q

Eluate from complement only coated cells

______________ if present, which may not be
present in patient serum/plasma.

A
  • Eluate from complement only coated cells should be
    tested if clinical evidence of hemolysis.
  • Concentrates IgG, if present, which may not be
    present in patient serum/plasma.
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20
Q

Elution definition

A

Removes antibody from sensitized RBCs and recovers antibody in
usable form.

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21
Q

Elution

Thorough washing of the RBCs essential to ensure that

A

that antibody
detected in eluate is only RBC bound antibody.

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22
Q

Elution

Last wash will detect proper

A

washing and should be negative with
all cells tested.

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23
Q

Elution when to preform

A

whenever DAT is positive
for IgG)

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24
Q

Elution techniques free antibodies form the

A

sensitized red cells so that the antibodies can
be identified

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25
Q

No further testing if

A
  • No unexpected antibodies
    present in serum/plasma.
  • Only autoantibody detected in
    eluate.
  • No recent transfusion
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26
Q

Further studies

Confirm specificity of _______ if present

A

Alloantibodies

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27
Q

If DAT is positive but serum and elution studies are negative suspect _______

A

drug induced
hemolysis – reference lab

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28
Q

ABO incompatible components transfused test for

A

Anti-A and Anti-B

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29
Q

Further studies

If patient is an infant perform

A

appropriate
testing on maternal
sample and elution on
cord cells.

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30
Q

Two Types of Cold Autoadsorptions

A

RABBIT ERYTHROCYTE
STROMA TEST (REST)

COLD AUTO
ADSORPITON

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31
Q

Two Types of Warm Autoadsorptions

A

W.A.R.M

WARM AUTO
ADSORPITON

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32
Q

Rabbit Erythrocyte Stroma Test
(REST)

A

For patients who have been recently transfused.

Rabbit cells have I antigen

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33
Q

REST procedure

A

-Incubate patient serum/plasma with rabbit
stroma at 4C.
-I antibody absorbed out.
-Remove (harvest) serum/plasma
-Test absorbed serum against screen cells.

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34
Q

Rabbit Erythrocyte Stroma Test
(REST)

Use with caution because

A

may adsorb out clinically significant antibodies
to D, E, Vel antigens and IgM antibodies regardless of specificity.

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35
Q

Cold Autoadsorption Test

Cant be performed on

A

recently transfused patients

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36
Q

Cold Autoadsorption Test

Collect ______

A

EDTA sample, keep warm

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37
Q

Cold Autoadsorption Test

Seperate _____ from RBCs

wash _____ with saline

A

Separate plasma from RBCs.
Wash RBCs with warm saline

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38
Q

Cold Autoadsorption Test

Ass aliquot of

A

Add aliquot of plasma/serum to RBCs incubate at 4C for 1 hour.

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39
Q

Cold Autoadsorption Test

Harvest serum/plasma and
test against screen cells

A

Negative – no alloantibody.
Positive and negative – alloantibody present, run panel.
All positive, unsuccessful, repeat with 1X adsorbed sample.

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40
Q

Cold Autoabsorption test

Supernatant used for

A
  • Ab screen
  • Reverse grouping cells
  • Autocontrol
  • Limitation: Will not remove a high titer cold agglutinin completely
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41
Q

Autoimmune hemolysis

Immune hemolytic anemia

A

shortened RBC survival mediated
through the immune response, specifically by humoral antibody

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42
Q

Autoimmune Immune hemolytic anemia types

A
  • Warm autoimmune hemolytic anemia (WAIHA)
  • Cold agglutinin syndrome (CAS)
  • Mixed type – both warm and cold autoantibodies present
  • Paroxysmal cold hemoglobinuria (PCH)
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43
Q

Alloimmune Immune hemolytic anemia types

A
  • Hemolytic transfusion reaction
  • Hemolytic disease of the fetus and newborn
44
Q

Drug induced Immune hemolytic anemia types

A
  • Drug dependent
  • Drug independent
45
Q

Characteristics of Immune Red Cell
Destruction

Diagnosis of hemolysis :

A
  • Possible decreased H&H
  • Increased reticulocytes
  • Increased LDH
  • Decreased haptoglobin
  • Increased unconjugated bilirubin
  • Hemoglobinemia and/or hemoglobinuria may indicate acute hemolysis or
    intravascular RBC destruction.
46
Q

Autoantibodies definition

A

Antibodies that are directed against the individual’s own RBCs
are termed autoantibodies or autoagglutinins.

47
Q

Autoantibodies

Studies in animal models suggest

A

that autoantibodies occur because of
a failure of the immune regulatory responses

48
Q

Autoantibodies

RBC survival may be

A

Shorten

49
Q

Autoantibodies

identification of a autoantibody may explain

A

decreased RBC survival
in vivo.

50
Q

Autoantibodies

If a patient’s RBCs are coated with autoantibody, the patient may
present with:

A
  • ABO discrepancy
  • positive Rh control
  • positive DAT
51
Q

Autoantibodies

A positive DAT, a positive
autocontrol, or serum
autoantibody does not
necessarily confer the
diagnosis of AIHA.

A
  • Approximately 0.1% of normal blood donors and up
    to 15% of hospitalized patients have positive DATs
    with no evidence of hemolytic anemia
52
Q

compensated anemia

A

the rate of RBC production
will nearly equal the rate of RBC destruction.

53
Q

uncompensated anemia

A

the rate of RBC
destruction exceeds the rate of RBC production.

54
Q

Serologic Testing

Diagnostic tests in a symptomatic patient include

A
  • DAT using polyspecific and monospecific antiglobulin reagents
  • Characterization of the autoantibody in the serum and/or eluate
    using standard antibody detection and identification procedures
55
Q

AIHA may be diagnosed and classified as

A

cold reactive,
warm reactive, or drug-induced.

56
Q

Autoantibodies can be characterized by
their

A

optimal temperature of reactivity

57
Q

Autoantibodies optimal temperature of reactivity

A
  • Warm Temperatures (30°C to 37°C) - About 70%
    of cases
  • Cold reactive (4°C to 30°C) - About 18% of cases
  • Drug-induced - About 12% of cases
58
Q

Cold reactive autoantibodies are

Most are not

A

are
frequently encountered in serologic
testing.
* Most are not clinically
significant, but occasionally they
are clinically significant and cause
immune hemolytic anemia.

59
Q

Cold reactive autoantibodies

When testing is performed at
4°C, the most commonly
encountered autoantibody is a

A

benign cold agglutinin that may
be found in the serum of most
normal, healthy individuals

60
Q

Cold Reactive
Autoantibodies

The typical cold agglutinin has a
relatively _____ titer

A

low titer (<64 at 4°C)

61
Q

Cold Reactive
Autoantibodies

Cold agglutinins can interfere
with routine

A

serum and cell
testing performed at RT,
potentially affecting
* ABO/Rh typing
* Direct Antiglobulin Test (DAT)
* Antibody detection and
identification
* Compatibility testing

62
Q

Anti-I, Anti-i

A

 Most cold reactive
autoantibodies have anti-I
specificity.
 The I antigen is fully
expressed on RBCs of most
adults but only weakly
expressed on cord RBCs.
 Anti-i is a relatively
uncommon autoantibody.
 This antibody reacts in an
antithetical manner to
anti-I.

63
Q

Anti-H, Anti-IH

A

 Cold agglutinins found in
the sera of group A 1 and
A 1B individuals (and
occasionally group B) may
have anti-H specificity.
 Group O and A 2 cells react
best because they have
the largest amounts of H
antigen.
 Group A 1 and A 1 B cells
have the least H antigen so
they react weakly.

64
Q

Short cold panel

Incubate patient serum/plasma with

A
  • Type O adult cells (I)
  • Type O cord cells (i)
  • Type A1/B adult cells
  • Autologous
65
Q

Other Cold Reactive
Autoagglutinins

Less commonly encountered cold autoagglutinins
have been described, such as

A

anti-Pr, anti-Gd, and
anti-Sdx (anti-R x).

66
Q

Pathologic Cold
Autoagglutinins types

A

 Chronic condition- no known cause
 Acute condition
= Mycoplasma pneumoniae- Anti-I
= Infectious mononucleosis- Anti-i
= Lymphoproliferative disorder- Anti-i
 Paroxysmal Cold Hemoglobinuria

67
Q

Acute condition

Pathologic Cold Autoagglutinins

A

 Mycoplasma pneumoniae- Anti-I
 Infectious mononucleosis- Anti-i
 Lymphoproliferative disorder- Anti-i

68
Q

Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)

Antibody specificity is almost always

A

anti-I, less
commonly anti-i, and rarely anti-Pr

69
Q

Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)

Laboratory findings in CHD

A

include reticulocytosis and a
positive DAT due to complement only.

70
Q

Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)

The peripheral smear may show agglutinated

A

BCs,
polychromasia, mild to moderate anisocytosis, and
poikilocytosis

71
Q

Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)

May cause false positive

A
  • forward and reverse typing
  • Forward – cells so heavily coated spontaneously
    agglutinate.
  • Reverse cells have I antigen and will agglutinate
72
Q

Cold Hemagglutinin
Disease
(Idiopathic Cold AIHA
or CHD) (cont’d)

Pre-warmed testing may eliminate

A

Reactivity

73
Q

Cold
Hemagglutinin
Disease

DAT

Only what is detected on the

A

RBCs

74
Q

Cold
Hemagglutinin
Disease

Why is complement the only protein present

A
  • Antibody is IgM, which in-vivo binds to
    RBCs in peripheral circulation where
    temperature falls to 32C or less.
  • IgM causes binding of complement in
    the cold.
  • When RBCs return to warmer
    circulation IgM dissociates leaving
    RBCs coated with complement only
75
Q

Cold
Hemagglutinin
Disease

Eluate will be

A

will be negative as only complement
is binding to the RBC

76
Q

Cold Hemagglutinin Disease
(Idiopathic Cold AIHA or CHD)

MOST PATIENTS REQUIRE NO
TREATMENT AND ARE INSTRUCTED
TO AVOID THE

A

COLD, KEEP WARM,
OR MOVE TO A MILDER CLIMATE.

77
Q

Cold Hemagglutinin Disease
(Idiopathic Cold AIHA or CHD) (cont’d)

CONSIDERATIONS FOR TRANSFUSION THERAPY

A
  • Rarely require transfusion.
  • Use prewarmed technique for
    compatibility tests or use cold
    autoadsorbed serum.
  • Transfuse blood through blood warmer
78
Q

Paroxysmal Cold
Hemoglobinuria
(PCH)

Different from

A

Paroxysmal Nocturnal
Hemoglobinuria in which hemolysis is caused by
acid produced during sleep

79
Q

Paroxysmal Cold
Hemoglobinuria
(PCH)

RAREST form of

A

DAT positive AIHA

80
Q

Paroxysmal Cold
Hemoglobinuria
(PCH)

Presents as acute transient condition secondary to

A

viral infections particularly in young children

81
Q

Paroxysmal Cold
Hemoglobinuria
(PCH)

Caused by a

A

biphasic hemolysin which induces
hemolysis after exposure to cold

82
Q

Paroxysmal Cold
Hemoglobinuria
(PCH)

Results in what symptoms

A

hemoglobinuria and hemoglobinemia

83
Q

PCH test

A

Classic antibody
produced: the Donath-
Landsteiner antibody
(an autoantibody with
anti-P specificity)

84
Q

Treatment for PCH

A
  • Chronic: Protection from cold exposure is the only useful
    therapy.
  • Acute: postinfection forms are transient and usually
    terminate spontaneously after the infection resolves.
    Steroids and transfusion may be required, depending on
    the severity of the attacks.
85
Q

PCH –
DAT and
Eluate

DAT

A
  • Autoantibody is IgG which acts as cold agglutinin.
  • IgG binds to RBCs in colder parts of body.
  • Causes complement to be bound irreversibly.
  • IgG elutes off of RBCs in warmer parts of the body.
  • ONLY COMPLEMENT is detected
86
Q

PCH

Eluate

A

Since only complement coats cells eluate is
negative

87
Q

PCH

Antibody specificity

A
  • Anti-P
  • Not necessary to transfuse rare P negative blood
88
Q

PCH –
Serum/Plasma
Testing

Autoantibody described as

A

biphasic
hemolysin”.
* Binds to RBCs at low temperatures.
* Binds complement.
* As cells warm up hemolysis occurs.
* Basis of Donath-Landsteiner test

89
Q

Donath-Landsteiner

A
  • Usually add fresh complement
  • Antibody binds at cold temperatures
  • Test is warmed to 37C.
  • As warming occurs complement is activated and
    lysis of RBCs occurs.
90
Q

Warm
Autoimmune
Hemolytic
Anemia
(WAIHA)

Most common type of

A

Most common type of AIHA and most
difficult problem encountered in the
transfusion service.

91
Q

Warm Autoantibodies

React best at

A

37C

92
Q

Warm Autoantibodies

Not found as often in the random population as

A

the almost universal cold
autoanti-I

93
Q

Warm Autoantibodies

Some are apparently

A

Some are apparently harmless.
Harmless autoantibodies are
serologically indistinguishable from
the harmful ones.
A significant percentage of cases
suffer from an anemia of sufficient
severity to require transfusion.

94
Q

Warm
Autoantibodies

Most patients with warm autoimmune hemolytic
anemia (WAIHA) have both

A

IgG and complement
on their RBCs (67%)

95
Q

Warm
Autoantibodies

The IgG immunoglobulins react best by the

A

indirect antiglobulin technique

96
Q

WARM AUTOANTIBODIES (CONT’D)

Peripheral blood symptoms

A
  • polychromasia and
    macrocytosis - reticulocytosis
  • possilbe nucleated RBCs -
    hyperactive bone marrow
  • spherocytosis and occasionally
    RBC fragmentation, indicating
    extravascular hemolysis
97
Q

WARM AUTOANTIBODIES (CONT’D)

Products of hemolysis

A
  • Increased LDH
  • increased bilirubin (particularly
    the unconjugated indirect
    fraction)
  • Increased urinary urobilinogen
  • depleted serum haptoglobin
  • hemoglobinemia and
    hemoglobinuria
98
Q

Rh typing

A

There can be false positives; however,
with the introduction of monoclonal
antisera, the incidence has decreased

99
Q

DAT testing

A

The DAT can be EGA-treated or CDP-
treated RBCs are negative, it is possible
to use these cells for testing with the
Anti-D, -C, -E, -c, -e, -K, -Jk(a), -Jk(b)
and others
* A positive DAT is expected.
* The majority of the cases have both IgG
and complement coating the cells.
* In rare cases, the DAT may be negative
because it is coated with IgA or IgM

100
Q

Serum/Plasma testin

A
  • May have little free autoantibody, may all be
    on RBCs
  • Once all antigen sites coated, detectable
  • Approximately 50% of WAIHA will have
    autoantibody reactive with ALL CELLS TESTED.
  • DANGER – alloantibody may be present also
101
Q

Eluate

A
  • Usually reactive with all cells tested.
  • Negative reactions due to complement only
    OR presence of drug induced antibody
102
Q

WAIHA – Antibody Specificity

May be very

often intially directly against

A

-May be very complex.
-Often initially directed against Rh antigen complex

103
Q

WAIHA – Antibody Specificity

Specificity against simple

A

Rh or other blood group
antigens occasionally seen

104
Q

WAIHA – Antibody Specificity

If simple specificity, give antigen negative and if high
frequency or complex antibody

A

Antigen negative not practical

105
Q

Warm
Autoantibodies mechinisms

A

Extravascular immune RBC destruction
occurs. Sensitized RBC removed by
reticuloendothelial system (RES) cells of liver and
spleen.

106
Q

Warm
Autoantibodies

A minority have either

A

IgG only (20%) or
complement only (13%)

107
Q

Warm
Autoimmune
Hemolytic
Anemia
(WAIHA)

DAT

A

67% of the
cases, RBCs
are coated
with both IgG
and
complement.
20% of the
cases, RBCs
are coated
with IgG
alone.
13% of the
cases, RBCs
are coated
with
complement
alone

Advanced Laboratory Operations and Automation (17 decks)

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