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INTRODUCTION:
19-09-2015
IMMUNO
HEMATO
IMMUNOHEMATOLOGY
LOGY
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IMMUNOLOGY:
SEROLOGY:
Study of antigen-antibody
reactions in vitro.
HEMATOLOGY:
Study
of
the
cellular
components of the blood.
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IMMUNITY:
DEFINITIONS:
Three functions:
Defense
Homeostasis
Surveillance
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
Markers of Self
Markers of Non-Self
Markers of Self:
Major Histocompatibility Complex
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Components:
Complement system.
Chemokines.
Cells:
11
B Cells
Antibody
Immunoglobulins
Antibody Genes
T Cells
Cytokines
Complement
IMMUNOHEMATOLOGY:
DETECTION,
IDENTIFICATION, and/or
be involved].
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
DEFINITIONS:
24
BLOOD BANK:
Works
primarily
with
donor blood.
Major
areas
of
responsibility:
Donor recruitment.
Donor screening.
Blood collection.
Testing
(typing,
infectious
disease
screening).
Blood
component
preparation.
Component preservation.
May provide reference
lab services.
May store rare donor
blood.
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TRANSFUSION SERVICE:
IMMUNOHEMATOLOGY FACILITIES:
25
HISTORICAL OVERVIEW
26
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1665: Richard Lower, English Physiologist 1st animal-toanimal [dog to dog] blood transfusion.
1818: James Blundell of England 1st successful human-tohuman blood transfusion. This species specific transfusion
had 50% success rate, the rest resulted in death.
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27
Blood Groups.
Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
Karl Landsteiner:
28
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Discovered
that
the
incompatibility
of
many
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Introduced
the
Immunological
era
of
blood
transfusion
therapy
foundation
IMMUNOHEMATOLOGY as a science.
of
29
system.
foetalis (HDN).
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30
ANTIGENS:
31
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weight
for
stimulation
of
antibody
production.
Proteins
Complex
Lipopolysaccharides.
carbohydrates
32
PROPERTY
Foreignness
Size
Chemical
composition
Complexity
DESCRIPTION
Non-self more likely to stimulate
antibody production
>10,000 M.W.
Proteinbest immune response.
Complex carbohydratesecond best
immune response.
Lipids, Nucleic acid weak immune
response.
More complex molecules produce
better immune response.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
33
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Antigen location:
34
ANTIBODIES
35
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Protein.
36
serum / plasma.
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Immunological principles:
37
Bonding:
Noncovalent bonds.
Physical fit:
Concentration of
antigen
and
antibody:
Temperature:
Time:
pH:
Surface charge:
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Specificity:
38
AGGLUTINATION
CONCENTRATION
39
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2 methods:
Agglutination.
Hemolysis.
Precipitation.
2.
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crosslink.
3.
4.
40
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Grading of Agglutination:
41
42
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45
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46
Each parent
information.
contributes
1/2
of
The
genetic
information
is
on chromosomes composed of DNA
the
genetic
contained
Humans
have
23
pairs
of
chromosomes
a.
22 matched (autosomal) chromosomes and
b. 1pair of sex chromosomes.
System
ABO
Rh
Common Genes
A, B, O
D, C, E, c, e
Located on
Chromosome
9
1
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Basic Principles:
47
Genes
are
the
units
of
inheritance
within
the
chromosomes.
alleles.
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Basic Principles:
48
both
in
homozygous
as
well
as
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Basic Principles:
49
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Basic Principles:
50
51
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HISTORICAL PERSPECTIVE:
52
53
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Also
on
lymphocytes,
thrombocytes,
organs,
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ABO ANTIGENS:
54
Adults
greater
numbers
of
branched
chains
Number of Ag sites
A1 adult
8,10,000 to 1,170,000
A1 cord
2,50,000 to 3,70,000
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ABO ANTIGENS:
55
Simple
Mendelian
fashion
from
an
individuals
parents.
Hh Chromosome 19 HH / Hh / hh.
A, B & O - Chromosome 9.
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INHERITANCE:
56
Gene Combination
Phenotype
AO
AA
BO
BB
AB
AB
OO
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INHERITANCE:
57
GENE
TRANSFERASE
-L-fucosyltransferase
-3-N-acetyl-D-galactosaminyl
Transferase
-3-D-acetyl-D-galactosyl
Transferase
No Transferase.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
58
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
antigen.
59
SECRETOR STATUS
60
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Nonsecretors:
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
Secretors:
61
SUBGROUPS OF A & B
62
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2 major subgroups:
~ 80% A1
~ 20% A2.
Group A2
Qualitative differences:
4+
4+
10,00,000
2,50,000
3,00,000
1,40,000
Quantitative differences:
Positive
Negative
Anti-A1 in serum
Absent
? Present
-3-N-acetyl-D-galactosaminyl
Transferase Activity
Normal
Diminished
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Subgroups of A:
63
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64
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
Subgroups of B:
65
ABO ANTIBODIES:
66
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produces
antibodies
to
antigens
not
67
10 years of age.
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68
Age related:
Immunodeficient
individuals:
Immunosuppresse
d patients:
Congenital conditions
Congenital hypogammaglobulinemia
Congenital agammaglobulinemia
Immunosuppressive therapy
Chronic lymphocytic leukemia
Bone marrow transplant
Multiple myeloma
Acquired hypogammaglobulinemia
Acquired agammaglobulinemia
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
69
Mostly IgM.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
Immunoglobulin class:
70
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
Anti AB:
71
produce anti-A.
This
anti-A
can
be
separated
by
absorption
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Anti-A1:
72
ABO HDFN:
Rh HDFN:
Sensitization occurs in
1st pregnancy and
affects
subsequent
positive pregnancies.
Rh negative mother
Rh positive baby.
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Cause both
HDFN Hemolytic disease of fetus and new born &
HTR Hemolytic transfusion reaction.
HDFN: usually presents itself with a maternal IgG
antibody to an antigen on the surface of the babys
red cells.
73
are
an
ABO
group
incompatible
with
the
As
the
recipient
antibodies
react
with
the
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74
75
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INTRODUCTION:
systems.
76
Chromosome 1.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
GENES:
77
3 systems:
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NOMENCLATURE:
78
2.
3.
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D ANTIGEN:
79
No Rh system antigens.
2 pathways:
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Rh null:
80
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Rh ANTIBODIES:
81
82
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83
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RBC
Glucose
Precursor
Substance
(stays the
same)
Galactose
N-acetylglucosamine
Galactose
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
84
RBC
Glucose
H antigen
Galactose
N-acetylglucosamine
Galactose
Fucose
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
FORMATION OF H Ag
85
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Fucose
N-acetylgalactosamine
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
FORMATION OF A Ag
86
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Fucose
Galactose
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
FORMATION OF B Ag
87
88
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ANTISERUM
89
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Antibody present
Anti-A
anti-A
Anti-B
anti-B
Anti-AB
Anti-D
anti-D
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90
MONOCLONAL ANTIBODIES
Animal inoculation.
antigen
through
transfusion,
pregnancy
injection.
or
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Sources:
91
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Criterias:
Specific: does not cross react, and only reacts with its
own corresponding antigen,
strongly and,
use.
92
The
observable
combination
of
reactions
a
red
resulting
cell
antigen
from
with
the
its
haemolysis.
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93
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Agglutination:
Hemagglutination.
Agglutinogen antigen.
Agglutinin antibody.
Two stages:
1.
Sensitization: Abs
attach
to
the
Ags
on
RBC
Sensitized RBC.
2.
94
Ab Hemolysin.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
Hemolysis:
95
saline media.
2.
3.
Temperature:
Optimum
temperature
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Ab size:
1.
varies
96
5.
6.
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Ionic strength:
4.
97
Enzyme treatment:
8.
9.
7.
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of the antisera.
98
99
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METHODS:
100
Anti-A antibodies.
Anti-B antibodies.
Wooden applicator.
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REAGENTS:
101
Verify
that
patient
information
on
the
sample
Use
Patient
serum
for
confirmation
Reverse
grouping or backtyping.
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102
direction.
2. Always label tubes and slides fully and cleanly.
3. Do not perform tests at temperature higher than
room temperature.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
103
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
or plasma).
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
105
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106
107
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Strong
agglutination
of
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positive
result.
A smooth suspension of
RBCs
at
minutes
the
is
end
a
of
negative
result.
110
Take two tubes, label one tube anti- A and the second
anti- B.
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111
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112
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
113
Interpretation
Anti-A
Anti-B
Cell Ag
ABO Group
No Ag
A, B
AB
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Reaction of cells
tested with
114
115
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of an individual.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
116
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
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118
BLOOD GROUP
A CELL
B CELL
Negative
Positive
Positive
Negative
Negative
Negative
AB
Positive
Positive
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
119
Anti-A
Interpretation
AB
Reaction of cells
tested with
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INTERPRETATION OF BOTH:
120
121
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GEL CARDS:
the antibody. Red blood cells with the specific antigen will
agglutinate
when
combined
with
the
corresponding
122
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123
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PROCEDURE:
124
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MICROPLATE TECHNIQUE:
Incubation,
Centrifugation
Reading of results.
Interpretation of results.
125
126
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Contaminated reagents.
2.
3.
4.
5.
6.
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127
2.
3.
Additional Ab.
4.
Plasma abnormalities.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
128
Age:
2.
Lymphoma.
Leukemia.
Following BM transplantation.
RESOLUTION:
enhancing
reaction
in
reverse
grouping
by
1.
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129
1.
2.
Diseases
Leukemia:
ABO
Ags
greatly
depressed.
6.
transplant recipient.
like
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130
1.
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coated
with
sufficient
Ab
to
promote
spontaneous
agglutination.
[RESOLUTION: Wash RBCs in warm 370C to establish cold Abs. Treat
cells with Chloroquine diphosphate to eliminate bound warm Abs]
1.
Ag.]
131
1.
Increased globulin.
2.
Abnormal proteins.
3.
Whartons jelly.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
133
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Rh antigens.
In performing Rh grouping:
Time &
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134
Interpretation or results:
control.
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135
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136
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Wash cells in both test and control tube 3-4 times with
normal saline.
Gently
suspend
the
cell
button
and
observe
for
agglutination.
137
138
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Incomplete antibodies,
Antibodies
agglutinate
that
RBCs
can
sensitize
suspended
but
in
which
saline
at
fail
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to
room
Complements.
PRINCIPLE:
139
140
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Monospecific reagents:
Anti-C3b,-C3d alone
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
AHG Reagent:
141
Positive Control:
Negative Control:
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
142
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use.
143
preparation
should
give
negative
direct
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
144
1.
2.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
TYPES:
145
HDN,
Transfusion reactions.
Principle:
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DAT:
146
147
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In
vivo
coating
of
red
cells
with
IgG
and/or
This
mechanism
could
be
autoimmunity,
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DAT:
148
Requirements:
Pasteur pipettes
Incubator
Centrifuge
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DAT: Requirements
149
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DAT: Procedure
150
151
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b.
2.
To detect Du Ag.
3.
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1.
IAT:
152
serum
produces
agglutination
in
the
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IAT: Principle
154
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Requirements:
Pasteur pipettes
Incubator
Centrifuge
Reagents:
Antihuman globulin
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IAT: Procedure
155
1.
2.
3.
4.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
IAT: Procedure
156
5.
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IAT: Procedure
6.
7.
157
158
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Agglutination.
Conclusion
Correct test.
PC
No agglutination. Incorrect test, repeat.
Agglutination.
Test
Observation
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IAT: Interpretation
No agglutination. Negative.
159
Temperature:
Incubation time:
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160
1.
2.
after the
washing step.
3.
4.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
161
1.
2.
3.
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Dr MANOJ K PATRO, MD [PATH]; MKCG MC BRAHMAPUR
162
CROSS-MATCHING
163
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2.
However,
cross
match
will
not
prevent
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1.
Purpose:
164
1.
Major.
2.
Minor.
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165
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166
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167
Choice of Blood
1st
2nd
3rd
4th
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AB
AB
A*
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168
1.
4 PHASES:
1.
Saline.
2.
Protein.
3.
AHG.
4.
Enzyme.
Saline tube technique at RT: provides the optimum
temperature and medium for the detection of IgM
antibodies
of
ABO
system
and
other
potent
cold
agglutinins.
2.
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169
3.
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4.
Procedure:
1.
2.
3.
4.
incomplete antibodies.
170
added at this
6.
8.
After the last wash, decant all saline and add two drops of
10.
Gently
re
suspend
the
cells
button
and
examine
Note:
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171
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enzymes:
172