30 2011


1900-
Karl Landsteiner
300

AIMO

SLIDE TEST





, zeta-potential

10

/



.

11


PH

6,5-7,5






..
anti-M
12



24 4
IGM

13

14


IgM
ABH, MN,Lewis,Lutheran
a
IgG
Ss, Kell, Rh(DCEce),
Lutheran b, Duffy Kidd

15

zeta-potential


polybrene
16








Rhesus,Kidd,Lewis,P1,I,ABO

Duffy,MNS,Xga
17



zeta-potential
22% Bovine Serum Albumin




Rhesus
18

POLYBRENE



()






.


-Kell
19
.

POLYETHYLENEGLYCOLE (PEG)



,

antiIgG .
.
37
20

LISS


,

21

 TEST TUBE

22


-1

SCORE

10

4+

10

3+

2+

1+

23


-2
+w

+/- m
m+

Mf

Mixed-field

NA

rouleaux

NA

24

Grading System for

Reactions

25
M. Zaharna Practical Blood Bank 2009

26

27
M. Zaharna Practical Blood Bank 2009

COLUMN TECHNIQUE

28

29

AFFINITY COLUMN TESTAFCT

G,antiIgM,C3d

30

ENZYME- LINKED ANTIGLOBULIN

TEST

405 nm
IgG

31


-1



37



IgG

32


-2

33

Addition of Plasma &

LISS
Add

Plasma

Liss
100 l of
Capture LISS

Red Blood
Cells

50 l of
donor/patient
plasma
or
Anti-Sera if weak D
test or rare antigen
typing is required

34

Incubation
Incubate test strips for
10 to 30 minutes.
Depends on the test
running
(60 minutes max)

35

Second Wash Step removing

plasma/proteins
Wells are washed to remove
proteins
Use phosphate buffered
saline (PBS)
pH 6.5 7.5

36

Indicator Cell
Capture-R Ready Indicator Red
Cell
Anti-D

Group O Rh positive
Human Red Blood
Cell

RBC

Anti-IgG

ADD INDICATOR CELLS

50l to each test wells

37

Second Centrifuge step

Indicator cells are
centrifuged
to indicate IgG presence
Centrifuge parameter:
90 sec.

600g
ramp up 135

38

Final Read

39

40



-IgG ANTI-C3d
:

(anti-Jka)
:


41

 AHG
ANTI-IgG
ANTI-Complement (C3b+C3d /C3d)

42

ANTIGLOBULIN
TEST/COOMBS TEST
Moreshi 1908
Coombs 1945

IGG
,IGG

43

 COOMBS-1
in vivo
IgG /
.

1.
2.

3.
4.
44

 COOMBS-2

3-5%
3
AHG

45

46


DAT

-

PEG

rouleaux
47

 COOMBS ()

48


35%
2-3

37
(15-60min)

3
2

49

50

COOMBS CONTROL

51

(,)

PEG
DAT ,

52

53

 TEST
200-500
IgG C3
DAT
IAT 100-200 IgG
C3

54

55

(,
)

56


panel


11-20
.

Rh , Duffy, Kidd
MNS
profile



57

Antibody panel
panel
11

58



rule out





(K,Kpa,Jsa,Lua)
59

STEP BY STEP

60

o
?

(I,P1,Lea,Leb,Vel,Ch/Rg,Sda)
61


?
-

(gM/IgG)

62

63

I
CONTROL
?

64

65

RULE OF THREE

3

3 3

p<0.05
95%
panels

3
66


LANDSTEINER

67

Panel

68

Panel
panels
autocontrol

69

Panel

antibody screening

70

71

 IS(IMMEDIATE SPIN)

72

73

74

75

 .
check cells
AHG!!!

76

77

78

1.

-

79

2.

80

3.

81

4. ;

! !!!!!
82

83


rule of three

84

MHN

Papain, bromelin,
ficin, and trypsin

Kidd
Rh
Lewis
I
P

Fya and
Fyb
M, N
S, s
85

Anti-Kell

86

 DAT

Y
Y
Positive DAT

Sensitized
RBC

Elution

Frees antibody

Antibody ID

87




o
DAT
HDN, ,

panel.

88


:
30min 2h
.
.
DAT
(acid glycine/EDTA)
O (,)

-

89

-
.
.
90

-1

-
-




-
-

91

-2

92

R1R1
(DCee),R2R2(DcEE0,rr(ccee)
Kell-,
Jka- Jkb

93

Remove
serum and
test for
alloantibody

2
tubes
Wash x3 after
incubation

Centrifuge after
incubating; and transfer
serum to 2nd tube of
treated cells; incubate
and centrifuge again

94

95





g ,






96

TEST

. ,
, .


,



Lewis,P1,Sd

97

Lewis Inhibition Tests

Lewis a b

Lewis



Lewis Lewis



98

P1 INHIBITION TESTS
1
,

99

Sda INHIPITION
Sda

100

-1



Ag


score

101

 SCORE ANTI-D

1:2

1:4

1:8

1:16

2+

2+

1+

8
3+

8
3+

10

10

1:32

1:64

1:128

W+ 0

16

5
2+

2
2+

0
1+

0
0
W+ 0

23
64

43

SCORE

SCORE

SCORE VALUES :4+=12, 3+=10, 2+=8, 1+=5 ,W+=2

102

TIT-2




score 10

.


103

,




104

 HTLA
H
LA (high titer,low
avidity)

,

. 1/2048

anti-Ch, -Rg, -Cs, -Yk, -Kna, -McCa and -JMH.

105



ull phenotypes .
Rh null, Ko, Co (a-b-), Lu (a-b-),
Fy (a-b-), Jk(a-b-).

,

panel.
Western blotting /

106

107


-HTLA
AN

108

109

PCR-RFLP
PCR-SSP
Multiplex PCR
RT-PCR
Sanger DNA Sequencing
Pyrosequensing
Microarray technology
Fluidic microarray system
TaqMan Open Array
MALDI-TOF MS
Mini-sequencing
110

111

112

113

Rhesus D positive
CcD.ee Kell positive

114

115

Microwell-agglutination strength 1

Immediate spin-questionable agglutination with

monoclonal anti-D

116

117

Weak D

118

Rhesus D negative
Ccee Kell negative

119

120

Microwell plate-negative

121

122

123


D VI II
Rhesus
Rhesus

124

125

126

127