Test 5 COPY COPY 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
No further testing if
* No unexpected antibodies present in serum/plasma. * Only autoantibody detected in eluate. * No recent transfusion
26
Further studies Confirm specificity of _______ if present
Alloantibodies
27
If DAT is positive but serum and elution studies are negative suspect _______
drug induced hemolysis – reference lab
28
ABO incompatible components transfused test for
Anti-A and Anti-B
29
Further studies If patient is an infant perform
appropriate testing on maternal sample and elution on cord cells.
30
Two Types of Cold Autoadsorptions
RABBIT ERYTHROCYTE STROMA TEST (REST) COLD AUTO ADSORPITON
31
Two Types of Warm Autoadsorptions
W.A.R.M WARM AUTO ADSORPITON
32
Rabbit Erythrocyte Stroma Test (REST)
For patients who have been recently transfused. Rabbit cells have I antigen
33
REST procedure
-Incubate patient serum/plasma with rabbit stroma at 4C. -I antibody absorbed out. -Remove (harvest) serum/plasma -Test absorbed serum against screen cells.
34
Rabbit Erythrocyte Stroma Test (REST) Use with caution because
may adsorb out clinically significant antibodies to D, E, Vel antigens and IgM antibodies regardless of specificity.
35
Cold Autoadsorption Test Cant be performed on
recently transfused patients
36
Cold Autoadsorption Test Collect ______
EDTA sample, keep warm
37
Cold Autoadsorption Test Seperate _____ from RBCs wash _____ with saline
Separate plasma from RBCs. Wash RBCs with warm saline
38
Cold Autoadsorption Test Ass aliquot of
Add aliquot of plasma/serum to RBCs incubate at 4C for 1 hour.
39
Cold Autoadsorption Test Harvest serum/plasma and test against screen cells
Negative – no alloantibody. Positive and negative – alloantibody present, run panel. All positive, unsuccessful, repeat with 1X adsorbed sample.
40
Cold Autoabsorption test Supernatant used for
* Ab screen * Reverse grouping cells * Autocontrol * Limitation: Will not remove a high titer cold agglutinin completely
41
Autoimmune hemolysis Immune hemolytic anemia
shortened RBC survival mediated through the immune response, specifically by humoral antibody
42
Autoimmune Immune hemolytic anemia types
* Warm autoimmune hemolytic anemia (WAIHA) * Cold agglutinin syndrome (CAS) * Mixed type – both warm and cold autoantibodies present * Paroxysmal cold hemoglobinuria (PCH)
43
Alloimmune Immune hemolytic anemia types
* Hemolytic transfusion reaction * Hemolytic disease of the fetus and newborn
44
Drug induced Immune hemolytic anemia types
* Drug dependent * Drug independent
45
Characteristics of Immune Red Cell Destruction Diagnosis of hemolysis :
* 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
Autoantibodies definition
Antibodies that are directed against the individual’s own RBCs are termed autoantibodies or autoagglutinins.
47
Autoantibodies Studies in animal models suggest
that autoantibodies occur because of a failure of the immune regulatory responses
48
Autoantibodies RBC survival may be
Shorten
49
Autoantibodies identification of a autoantibody may explain
decreased RBC survival in vivo.
50
Autoantibodies If a patient’s RBCs are coated with autoantibody, the patient may present with:
* ABO discrepancy * positive Rh control * positive DAT
51
Autoantibodies A positive DAT, a positive autocontrol, or serum autoantibody does not necessarily confer the diagnosis of AIHA.
* Approximately 0.1% of normal blood donors and up to 15% of hospitalized patients have positive DATs with no evidence of hemolytic anemia
52
compensated anemia
the rate of RBC production will nearly equal the rate of RBC destruction.
53
uncompensated anemia
the rate of RBC destruction exceeds the rate of RBC production.
54
Serologic Testing Diagnostic tests in a symptomatic patient include
* 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
AIHA may be diagnosed and classified as
cold reactive, warm reactive, or drug-induced.
56
Autoantibodies can be characterized by their
optimal temperature of reactivity
57
Autoantibodies optimal temperature of reactivity
* 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
Cold reactive autoantibodies are Most are not
are frequently encountered in serologic testing. * Most are not clinically significant, but occasionally they are clinically significant and cause immune hemolytic anemia.
59
Cold reactive autoantibodies When testing is performed at 4°C, the most commonly encountered autoantibody is a
benign cold agglutinin that may be found in the serum of most normal, healthy individuals
60
Cold Reactive Autoantibodies The typical cold agglutinin has a relatively _____ titer
low titer (<64 at 4°C)
61
Cold Reactive Autoantibodies Cold agglutinins can interfere with routine
serum and cell testing performed at RT, potentially affecting * ABO/Rh typing * Direct Antiglobulin Test (DAT) * Antibody detection and identification * Compatibility testing
62
Anti-I, Anti-i
 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
Anti-H, Anti-IH
 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
Short cold panel Incubate patient serum/plasma with
* Type O adult cells (I) * Type O cord cells (i) * Type A1/B adult cells * Autologous
65
Other Cold Reactive Autoagglutinins Less commonly encountered cold autoagglutinins have been described, such as
anti-Pr, anti-Gd, and anti-Sdx (anti-R x).
66
Pathologic Cold Autoagglutinins types
 Chronic condition- no known cause  Acute condition = Mycoplasma pneumoniae- Anti-I = Infectious mononucleosis- Anti-i = Lymphoproliferative disorder- Anti-i  Paroxysmal Cold Hemoglobinuria
67
Acute condition Pathologic Cold Autoagglutinins
 Mycoplasma pneumoniae- Anti-I  Infectious mononucleosis- Anti-i  Lymphoproliferative disorder- Anti-i
68
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD) (cont’d) Antibody specificity is almost always
anti-I, less commonly anti-i, and rarely anti-Pr
69
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD) (cont’d) Laboratory findings in CHD
include reticulocytosis and a positive DAT due to complement only.
70
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD) (cont’d) The peripheral smear may show agglutinated
BCs, polychromasia, mild to moderate anisocytosis, and poikilocytosis
71
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD) (cont’d) May cause false positive
- forward and reverse typing * Forward – cells so heavily coated spontaneously agglutinate. * Reverse cells have I antigen and will agglutinate
72
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD) (cont’d) Pre-warmed testing may eliminate
Reactivity
73
Cold Hemagglutinin Disease DAT Only what is detected on the
RBCs
74
Cold Hemagglutinin Disease Why is complement the only protein present
* 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
Cold Hemagglutinin Disease Eluate will be
will be negative as only complement is binding to the RBC
76
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD) MOST PATIENTS REQUIRE NO TREATMENT AND ARE INSTRUCTED TO AVOID THE
COLD, KEEP WARM, OR MOVE TO A MILDER CLIMATE.
77
Cold Hemagglutinin Disease (Idiopathic Cold AIHA or CHD) (cont’d) CONSIDERATIONS FOR TRANSFUSION THERAPY
- Rarely require transfusion. - Use prewarmed technique for compatibility tests or use cold autoadsorbed serum. - Transfuse blood through blood warmer
78
Paroxysmal Cold Hemoglobinuria (PCH) Different from
Paroxysmal Nocturnal Hemoglobinuria in which hemolysis is caused by acid produced during sleep
79
Paroxysmal Cold Hemoglobinuria (PCH) RAREST form of
DAT positive AIHA
80
Paroxysmal Cold Hemoglobinuria (PCH) Presents as acute transient condition secondary to
viral infections particularly in young children
81
Paroxysmal Cold Hemoglobinuria (PCH) Caused by a
biphasic hemolysin which induces hemolysis after exposure to cold
82
Paroxysmal Cold Hemoglobinuria (PCH) Results in what symptoms
hemoglobinuria and hemoglobinemia
83
PCH test
Classic antibody produced: the Donath- Landsteiner antibody (an autoantibody with anti-P specificity)
84
Treatment for PCH
* 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
PCH – DAT and Eluate DAT
* 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
PCH Eluate
Since only complement coats cells eluate is negative
87
PCH Antibody specificity
* Anti-P * Not necessary to transfuse rare P negative blood
88
PCH – Serum/Plasma Testing Autoantibody described as
biphasic hemolysin”. * Binds to RBCs at low temperatures. * Binds complement. * As cells warm up hemolysis occurs. * Basis of Donath-Landsteiner test
89
Donath-Landsteiner
* 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
Warm Autoimmune Hemolytic Anemia (WAIHA) Most common type of
Most common type of AIHA and most difficult problem encountered in the transfusion service.
91
Warm Autoantibodies React best at
37C
92
Warm Autoantibodies Not found as often in the random population as
the almost universal cold autoanti-I
93
Warm Autoantibodies Some are apparently
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
Warm Autoantibodies Most patients with warm autoimmune hemolytic anemia (WAIHA) have both
IgG and complement on their RBCs (67%)
95
Warm Autoantibodies The IgG immunoglobulins react best by the
indirect antiglobulin technique
96
WARM AUTOANTIBODIES (CONT’D) Peripheral blood symptoms
* polychromasia and macrocytosis - reticulocytosis * possilbe nucleated RBCs - hyperactive bone marrow * spherocytosis and occasionally RBC fragmentation, indicating extravascular hemolysis
97
WARM AUTOANTIBODIES (CONT’D) Products of hemolysis
* Increased LDH * increased bilirubin (particularly the unconjugated indirect fraction) * Increased urinary urobilinogen * depleted serum haptoglobin * hemoglobinemia and hemoglobinuria
98
Rh typing
There can be false positives; however, with the introduction of monoclonal antisera, the incidence has decreased
99
DAT testing
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
Serum/Plasma testin
* 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
Eluate
* Usually reactive with all cells tested. * Negative reactions due to complement only OR presence of drug induced antibody
102
WAIHA – Antibody Specificity May be very often intially directly against
-May be very complex. -Often initially directed against Rh antigen complex
103
WAIHA – Antibody Specificity Specificity against simple
Rh or other blood group antigens occasionally seen
104
WAIHA – Antibody Specificity If simple specificity, give antigen negative and if high frequency or complex antibody
Antigen negative not practical
105
Warm Autoantibodies mechinisms
Extravascular immune RBC destruction occurs. Sensitized RBC removed by reticuloendothelial system (RES) cells of liver and spleen.
106
Warm Autoantibodies A minority have either
IgG only (20%) or complement only (13%)
107
Warm Autoimmune Hemolytic Anemia (WAIHA) DAT
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