Blood%20Group%20reagent%20evaluation

1
Blood Group reagent evaluation

• Dr.Atoosa Shariat Torbaghani
• Reference Health Laboratory

2
Universal precautions

• HIV
• HBV

3
Appearance

• NO.
• hemolysis
• Precipitate
• Particle
• Gel-formation
• in inspection

4
Contamination

• Culture
• must be negative
• Using validate process to produce
• Preservative
• Sterile filter

5
Anti A B

• potency Testing
• Prepare new saline contain 1-2 albumin
• Two fold dilution of reagent with saline- one
  tube neg after it( 1/512)
• Wash cells -at least twice
• 2 suspension RBC with saline(A cell B cell)
• 0.1 ml serum dilution0.1ml RBC (1075 mm tube)
• Shake gently
• Incubate 5 min at RT(20-30c)
• Centrifuge (15 sec /1000 g )
• and grade (1 to 4)
• The greatest dilution which gives 1
  agglutination(small clumps macroscopically)

6
Anti A B

• Acceptable
• 1/128 ? A1 , B , A 1B
• 1/64 ? A 2 B , A 2
• Or Reference Reagent

7
????? Potency

• ???? ???? ???? ???? ?? ???? ?? ???? ??? ?? ????
  ??? ???? ???? ? ??? ?? ???? ???? 12/1 ? 4/1 ? 8/1
  ...... ????? ???.
• ???? ???? ????? ?? ????? ????????? ???? ???????
  2-1 ??????? ???.
• ???? ??????75 mm 12 ?? ?????? ?? ??? ????
• ????????? ?? ?? ???? ?? ?? ????? ???? ????? ?????
  ????
• ????????? ?? ?? ???? ??? ?????? ?? ??? ?????? ?
  ????? ???? ????? ????? ????
• ???? ??????? ?? carry over ???? ??? ?? ??????? ??
  ???? ?? ??? ? ????? ??????.
• ??? ????? ??????? ? ????? ???? ?? ?? ??????? ????
  ???? ??? ?? ??????.

8
???? ?????????? ????? ????

• ?? ???? ??????? ?? ???? ????? ? ??? ??? ?????
  ???? ???? ???? ? ????? ???? ? ?? ???? ? ???? ????
  ??? ???? ????.?????? ??? ?? ?? ??? ? ???? ????
  ????? ????? ??????? ???? ????? ????.
• ???????? ???? ?? ????? ?? ??? ?? ????? ????? ????
  .( ?? ??? ?? ???? ???? ???? ????)
• ?????????? 2 ?? ???? pack ??? ?? ????? ???? 2-1
  ??????? ???? ????.

9
????? ???????? ?? ???? ?????? potency ???? ??????
Reagent Red Blood Cells
Anti-A A1 and 3 different A2B
Anti-B A1 and A1B
Anti-A,B A1 and A2 and B
???? A2B ???? ???? AB ?? ?? anti-A1 ? anti-H
????? ???? ???? A2 ???? ???? A ?? ??
anti-A1 ? anti-H ????? ????
10
??? ????? ??? ???? ????? potency

• 1 ???? ???? ?? ???? ???? ??? ?? ?? ???? ??????
  ????? ? ??? ????? ???? ???? ???? ?? ?? ?? ????
  ?????? 0.1 ???? ???? ???? ??? ????
• 0.1 ???? ???? ?? ?????????? ????? ( ????? 2
  ????? ???? ?? ?? ??????? ???? 2 ??????? ????
  ???) ?? ?? ?? ???? ????? ????
• ??????? ???? ?? ?? ?? ???? ????? ????. 5 ????? ??
  ????? ???? ( 30-20 ???? ?????????) ?????? ????
  ??? ?? ??? rpm 1000(100-125 rcf) ?? ??? ?? ?????
  ? ?? 3400 rpm ?? ??? 15 ????? (900-1000 rcf )
  ????????? ????

11
???? ????? ?????

• ???? ?? ???? ?? ?? ????? ???? ???? ?? ??? ??? ?
  ?????????? ????? ??????.
• ????? ?? ?? ??? ??? ???? ???? ??????
• 4 ???? ????? ?? ???? ?? ??? ???? ?????.
• 3 ???? ????? ?? ??? ???? ???? ??? ???
• 2 ???? ????? ?? ????? ???? ???? ????? ??? ???
• 1 ???? ????? ?? ???? ??? ???? ?????? ??? ???
• .D ???? ????? ?? ???? ??? ??? ???? ???? ???
  ??? ???. ???? ???? ???? ??? ???? ??
  ??? ???? ??? ????? ????? ?? ???? ????? ????.
• ????? ???? ?? ?????? ????? ????? ?? ???? ?????
  ???? ??? ??? ?? ????? ????? ???? ????
• ????? ???? ?? ?? ????? 1 ???? ?? ????? ????
  potency ?? ??? ????? ???
• ???? ???? ?? ????? ? ?? ???? ??? ?? endpoint
  ????? ????? ????
• ???? ???? ??? ???? ???? ???? ???? ???.

12
???? ???? ???? ????

• ???? ??? ??? ABO ?? ??? ????? ????? ???? ????
  ??? ?????? ????? ????

13
FDA WHO

• ???? anti-A ?? ????????? 03/188 ?? ??? 1/8 ??
  ???? ????? ???? ??????? 2 ??????
• ???? anti-B ?? ????????? 03/164 ?? ??? 1/4 ??
  ???? ????? ???? ??????? 2 ??????

14
Anti A B

• Avidity(BOB)
• 10 saline suspension of AB cell (once washed)
• 100? cell100? serum(slide area 25mm)
• Time record beginning agglutination(Tlt25C)
• Serum Cell beginning
  time(seconds)
• Anti-A A1 15
• A2 30
• A1B 30
• A2B 45
• Anti-B B
  15

Macroscopic clump(1mm2) before 3 minutes
15
????? Avidity

• ??? 2/1 ?? ?? ????? ???? ?????? ????? ???? ???
  ???? ?????? ? ??? AB ?? ???? ???? ???? ?????? ?
  ?? ?? ?? ???? ????? ?? ?? ?????? ????? ??? ? ????
  ????
• ????? ???? ?? ?????? ? ????? ?????? ??? ???? ???

16
????? ???????? ?? ???? ?????? Avidity ???? ??????
Reagent Red Blood Cells
Anti-A A1 and A2B
Anti-B B and A1B
Anti-A.B A1 , A2 , B , and Ax

• ???? A2B ???? ???? AB ?? ?? anti-A1 ? anti-H
  ????? ????
• ???? A2 ???? ???? A ?? ?? anti-A1 ?
  anti-H ????? ????
• ???? Ax ???? ???? variant A ?? ???????
  ?????? ????? ???? ???? ???? ?? ???? A ???? ??
  ????? ????

17
????? ??????

• ?????? ?? ?? ???? ??? ???? ??? ? ???? ???? ?? ???
  ?????? ????? ????.
• ????? ?????? ?? ?? ???? ???? ??? ???? ?????? ?
  ?????? ?? ??? ???? ??? ? ??????? ????.

18
????? ??????

• ????? ??? ????????????? ?? ?? ?? ???? ??? ????
  ??? ? ???? ???? ??????? ???? ??? ???? ????? ( ??
  ???? ?????? ???? ??? ??? )???? ?????? ???.
• ???????????? ???? ??? ?? ?? ???? ??? ???? ??? ?
  ???? ????? ??????? ???? ????? ? ?? ???? ???? ????
  ? ?? ?????? ?? ?? ??????? ????? ????

19
Anti A B

• Specificity
• Confirm reaction with A,B,AB
• At least 2 reaction
• B. Confirm absence of reaction with panel
  cell due to Ab contamination D,C,E,c,e,cw,k,R,Kpa,
  Kpb,Fy,Fyb,Lub,Lua,JKa,JKb,M,N,S,s,PI,Xga?

Anti-A A1-A2B(3)
Anti-B A1B(3)-B
Anti-AB A1(3)-B(4)-A2(2)-Ax(3)
20
?????? Specificity

• ????? ???? ??? ?? ???? ???
• ?? ???? 7 ??? ?? ???? ??????? ????? ????
• ?????? ????? ????? ?? ??????? ????? ???? ??
• ?????? ??? ????? ??? ? ????? ?? ???? ???? ??????

21
????? ????? ?? ??????? ????? ???? ??

• ????? ?? 4 ????????? ? ??? ??? ???? ???
• ???? ?? ???? ??? ?? ??? ??????? ???? ???? ???
• ?? ?? ??? ???? ?? ??? ?? ???? ???? ???? ??????
  ??????? ?? ????? ????? ???? ?????? ???.

22
????? ???????? ?? ???? ?????? specificity ????
??????
Red Blood Cells Reagent
A1 (1) and A2B (3) Anti-A
A1B (3) and B (1) Anti-B
A1 (2) and A2 (2) and B (4) And at least 3 different Ax Anti-A,B
23
????? ??? ? ????? ?? ???? ???? ??????

• ???? ??? ?? ?? ???? ??? ??? ????? ???? ? ??????
  ????
• A, B, H, Lea, Leb , I , K, k , Kpa , Kpb , Jsb
  ,Pi , D , C , E , c , e , Cw , M , N , S , s , U
  , Lua , Lub , JKa , JKb , Fya , Fyb , Xga ,
  Doa , Dob , Yta , Ytb , Lan , Coa , Cob , Mg ,
  Wra , Sda ,Vw
• ?? ????? ?? ???? ??? ????????? ???? ? ??? ????
  ????? ????? ? ????? ??? ?????? ????? ??? ?? ?????
  ?? ???
• A , B , H , Lea, Leb , I , K, k , Kpa , Kpb ,
  Jsb, Pi , D , C , E , c , e , M , N , S , s , U
  , Lub, JKa , JKb , Fya , Fyb
• ????? ?? 4 ????????? ? ??? ??? ???? ??? ? ??????
  ?????? ????? ???.

24
???? ????? ????? specificity

• ?? ????? ?? ??????? ????? ?????? ? ?? ???? ????
  ???.
• ???? ????? ???? ???? ?? ? ???????? ???? ????
  ????? 2 ????.??? ??? ?? 4 ??? ???? ?????? ?
  ????? ???? ?? 2 ????? ???? ???? 4 ??? ???? ??
  ?????? ????.?? ??????? ??? ??? ?? ??? 8 ??????
  ????? ????? ???? ?? 2 ????? ???? ? ????? ?? ????
  ???? ???????.
• ?????? ?? ??? ???? ???? ???? ?? ????? ?????? ????
  ??????? ???? ?????

25
Anti-A B

• False Agglutination(BOB)
• 10 saline suspension of cell Rh(once washed
  with 0.15 M saline Unwashed)
• 100? cell 100? serum
• Mix two minutes (room temperature)
• or(slide area 13 in.)
• No agglutination, hemolysis, rouleaus
• formation

26
?????? ?? ??? ???????????? ??? ?? ???

• ?????? ???? ???
• ????? ?? ???? ???? ??? ????
• ???? ??? ?? ???? O ???? ????? ????? ????????????
  ????? ?????
• (???? O ???? ???? ???? ?? c ?? ???? ???? ????
  Anti-c ?? ??? IgG ?????? ???)

27
package container

• Container
• Color of label paper
• Information
• Lettering size
• Visual inspection
• Package label

28
stability control

• Minimum of 1 of each batch
• Should be retained at least 6 months beyond
  expiry date

29

• ??? ???????? ???? ??? ??? ???? ??? ??????

30
??????? ???? ??? ???? ???? Anti-Rh
31
Universal precautions

• HIV
• HBV

32
Appearance

• NO.
• hemolysis
• Precipitate
• Particle
• Gel-formation
• in inspection

33
Contamination

• Culture
• must be negative
• Using validate process to produce
• Preservative
• Sterile filter

34
Anti-Rh(FDA)

• Titration
• Serial two fold dilution (anti-Rh)
• 2 suspension cell Rh
• 100? anti-Rh 100? cell
• Incubation(One hour 37c)
• Centrifuge(45 sec at 1000 g)
• Reading grading
• 1 gt 1/16(FDA) , 1/32(BOB)
• Or reference

35
Anti-Rh(BOB)

• Titer
• Two fold dilution serum in 20 albumin
• salin
• 2 suspension RBC(Rh2) with albumin
• 
  salin
• 0.1 ml serum dilution0.1 ml RBC
• Incubate 37C for 60 minutes
• Centrifuge (2 minures at 1000 rpm)
• a control ? albuminRBC
• ? erum dilution RBC Rh negative
• Observe for agglutination
• 1 gt 1/32

36
????? Potency

• ???? ???? ???? ???? ?? ???? ?? ???? ??? ?? ????
  ??? ???? ???? ? ??? ?? ???? ???? 12/1 ? 4/1 ? 8/1
  ...... ????? ???.
• ???? ???? ????? ?? ????? ????????? ???? ???????
  2-1 ??????? ???.
• ???? ??????75 mm 12 ?? ?????? ?? ??? ????
• ????????? ?? ?? ???? ?? ?? ????? ???? ????? ?????
  ????
• ????????? ?? ?? ???? ??? ?????? ?? ??? ?????? ?
  ????? ???? ????? ????? ????
• ???? ??????? ?? carry over ???? ??? ?? ??????? ??
  ???? ?? ??? ? ????? ??????.
• ??? ????? ??????? ? ????? ???? ?? ?? ??????? ????
  ???? ??? ?? ??????.

37
????? ???????? ?? ???? ?????? potency ???? ??????
Reagent Red Blood Cells
Anti-D Dce ( R0r )
Anti-C dCce ( r r )
Anti-E dcEe ( r r )
Anti-c DCcEe ( R1 R2 )
Anti-e DcEe ( r r )
Anti-cd dCce ( r r ) DCe ( R2 r )
Anti-DE Dce (R0 r ) dcEe ( r r )
Anti-CDE dCce ( r r ) Dce ( R2 r ) dcEe ( r r )
38
??? ????? ??? ???? ????? potency

• ???? ???? ?? ???? ???? ??? ?? ?? ???? ??????
  ????? ? ??? ????? ???? ???? ???? ?? ?? ?? ????
  ?????? 0.1 ???? ???? ???? ??? ????
• ???? ???? ?? ?????????? ????? ( ????? 2 ?????
  ???? ?? ?? ??????? ???? 2 ??????? ???? ???) ??
  ?? ?? ???? ????? ????
• ??????? ???? ?? ?? ?? ???? ????? ????. 15 ?????
  ?? ????? 37 ???? ????????? ?????? ????
• ??? ?? ??? rpm 1000(100-125 rcf) ?? ??? ?? ?????
  ? ?? 3400 rpm ?? ??? 45 ????? (900-1000 rcf )
  ????????? ???? .

39

• ????? ???? ?? ?? ????? 1 ???? ?? ????? ????
  potency ?? ??? ????? ???
• ???? ???? ?? ????? ? ?? ???? ??? ?? endpoint
  ????? ????? ????
• ???? ???? ??? ???? ???? ???? ???? ???.
• ???? ??? ??? Rh ?? ??? ????? ?????
• ???? ???? ??? ?????? ????? ????

40
FDA WHO

• ???? low protein anti-D ?? ????????? 99/836 ??
  ??? 1/3 ?? ???? ????? ???? ??????? 2 ??????
• ???? high protein anti-D ?? ????????? 99/836 ??
  ??? 1/8 ?? ???? ????? ???? ??????? 2 ??????

41
Anti Rh

• Specificity
• Confirm reaction with A,B,AB
• At least 2 reaction
• B. Confirm absence of reaction with panel
  cell due to Ab contamination D,C,E,c,e,cw,k,R,Kpa,
  Kpb,Fy,Fyb,Lub,Lua,JKa,JKb,M,N,S,s,PI,Xga?

reaction With cell
Rh
- Rh-
42
?????? Specificity

• ????? ???? ??? ?? ???? ???
• ?? ???? 7 ??? ?? ???? ??????? ????? ????
• ?????? ????? ????? ?? ??????? ????? ???? ??
• ?????? ??? ????? ??? ? ????? ?? ???? ???? ??????
• ?????? ??? ????? ??? ???? anti-A , anti-B

43
????? ????? ?? ??????? ????? ???? ??

• ????? ?? 4 ????????? ? ??? ??? ???? ???
• ?? ????? ???? ???? ????? anti-CDE ????? 4 ????
  ????? ???? ??? anti-C ? 4 ??? ???? anti-D ? 4 ???
  ???? anti-E ?? ??? ???.
• ???? ?? ???? ??? ?? ??? ??????? ???? ???? ???
• ?? ?? ??? ???? ?? ??? ?? ???? ???? ???? ???? ??
  ??????? ?? ????? ????? ???? ?????? ???.

44
????? ??? ? ????? ?? ???? ???? ??????

• ???? ??? ?? ?? ???? ??? ??? ????? ???? ? ??????
  ????
• A, B, H, Lea, Leb , I , K, k , Kpa , Kpb , Jsb
  ,Pi , D , C , E , c , e , Cw , M , N , S , s , U
  , Lua , Lub , JKa , JKb , Fya , Fyb , Xga ,
  Doa , Dob , Yta , Ytb , Lan , Coa , Cob , Mg ,
  Wra , Sda ,Vw
• ?? ????? ?? ???? ??? ????????? ???? ?
  ??? ???? ????? ????? ? ????? ??? ?????? ????? ???
  ?? ????? ?? ???
• A , B , H , Lea, Leb , I , K, k , Kpa , Kpb ,
  Jsb, Pi , D , C , E , c , e , M , N , S , s , U
  , Lub, JKa , JKb , Fya , Fyb

45
?????? ??? ????? ??? ???? anti-A , anti-B

• ?? ???????? ???? ???? ???? ?? A1 ? B ?? ????
  ???? ????? D ???? ??????? ????.
• ( ?? ????? A1B ?? ???? ??? ??????? ???. )

46
???? ????? ?????specificity

• ?? ????? ?? ??????? ????? ?????? ? ?? ???? ????
  ???.
• ???? ????? ???? ???? ?? ? ???????? ???? ????
  ????? 2 ????.??? ??? ?? 4 ??? ???? ?????? ?
  ????? ???? ?? 2 ????? ???? ???? 4 ??? ???? ??
  ?????? ????.?? ??????? ??? ??? ?? ??? 8 ??????
  ????? ????? ???? ?? 2 ????? ???? ? ????? ?? ????
  ???? ???????.
• ????? ???? ? ???? ????? ???? ???? ???.
• ?????? ?? ??? ???? ???? ???? ?? ????? ?????? ????
  ??????? ???? ?????.

47
Anti-Rh

• Avidity (BOB)
• 40 suspension RBC RH positive in their own
  plasma or serum, or in AB plasma or serum
• 100? cell 50 ? serum (slide area 2040)
• Place on illuminated glass ( Tlt47 C)
• The end of 2 minutes record size of clump
• Beginning before 60 seconds
• Clump at least 1mm/2 min
• RBC ? Dce(Rh1) / Dce(Rh0) / DcE(Rh2)

48
????? Avidity

• ??? 2/1 ?? ?? ????? ???? ?????? ????? ???? ???
  ???? ?????? ? ??? AB ?? ???? ???? ???? ?????? ?
  ?? ?? ?? ???? ????? ?? ?? ?????? ????? ??? ? ????
  ????
• ????? ???? ?? ?????? ? ????? ?????? ??? ???? ???

49
????? ???????? ?? ???? ?????? Avidity ????
??????
Reagent Red Blood Cells
Anti-D DCe ( R1 r )
Anti-C dCce ( r r )
Anti-E dcEe ( r r )
Anti-c dCce ( r r )
Anti-e dcEe ( r r )
Anti-CD Dce ( R0 r ) dCce ( r r )
Anti-DE Dce ( R0 r ) dcEe ( r r )
Anti-CDE Dce (R0 r ) dcEe ( r r )
50

• ????? ??????
• ?????? ?? ?? ???? ??? ???? ??? ? ???? ???? ?? ???
  ?????? ????? ????.
• ????? ?????? ?? ?? ???? ???? ??? ???? ?????? ?
  ?????? ?? ??? ???? ??? ? ??????? ????
• ????? ??? ????????????? ?? ?? ?? ???? ??? ????
  ??? ? ???? ???? ??????? ???? ??? ???? ????? ( ??
  ???? ?????? ???? ??? ??? )???? ?????? ???.
• ???????????? ???? ??? ?? ?? ???? ??? ???? ??? ?
  ???? ????? ??????? ???? ????? ? ?? ???? ???? ????
  ? ?? ?????? ?? ?? ??????? ????? ????.

51
?????? ????? ??????

• ????? ???? ??? ?? ???? ???
• ?? ??? ?? ????? ??? ?? ?? ?????? ????? Rh ????
  ????
• ?? ???? 7 ??? ?? ???? ??????? ????? ????
• ?????? ???? ?????? ?????? ????? ???? ? ????? ????
  ??????? ???? ?????? ?? ???? ???? ??? ?????.
• ?????? ?? ????? ?? ??? ?? ????? ????? ???? ??
  ???? ???? ???? ???

52
??????? ???????? ???? ?????? ??????
Reagent Red Blood Cells
Anti-D DCce ( R1 r )
Anti-C DCcEe ( R1 R2 )
Anti-E DCcEe ( R1 R2 )
Anti-c DCce ( R1 r ) DCcEe ( R1 R2 )
Anti-e DcEe ( R2 r ) DCcEe ( R1 R2 )
53
????? ??????

• ???? ?? ???? ? ?? ???? ?????? ????? ????. ??? ??
  ???? ????? 15 ????? 30 ????? 60 ?????
  ?????
• ?????? ?????? ?? ???? ??? ?? ?? ?? ???? ??????.
• ?? ?? ???? ???? ????? ????
• ???? ?? ????? ???? ? ?? ?? ?? ??? ????? ? ??????
  ?? ???? ????? ?????? ????
• ??? ??? ?????? ? ????????? ????

54

• -??? ???? ????? ?? ?? ?? ???? ????? ??? ? ?? ??
  ????? ????? ????? ??? ? ????? ?????? ???? ?????
• -?? ??????? ?? ?????? ???? ?????????? ? ?????
  ????? ???? ???? ???? ? ?????? ???? ????
• ????? ????? 2 ?? ????? ???? ?? ???? ?????? ???

55
??? ??? ???? ?????? ??????

• ?? ??? ???? ( ?? ?? ??? 5 ???? ) ????? ????.
• ????? 1 ????? 1 ????? -3 ????? 5 ????? 10
  ????? ??? ?? ??? ?? ???? ?? ?????
• ?? ?? ??? ?? ???? ?? ???? ??? ???? ??? ? ?? ???
  ??? ???? ??? ???? ??? ??????
• ?? ?? ???? ????? ?????? ?????????? ????? ?????
  ????
• ????? ???? ? ?? ???? ??? ?? ? ??? ??????? ?
  ?????? ????
• ??? ?????? ????????? ????
• ??? ???? ????? ?? ???? ??? ???? ??? ????? ?? ????
  ??? ???? ???? ??? ? ????? ?????? ???? ????
• ??? ???? ????? ?? ??? ??? ???? ??? ????? ? ??
  ?????? ?? ???? ??? ???? ???? ??? ? ?????? ????
  ?????.

56
Anti-Rh(BOB)

• false agglutination
• Three test tube (0.25 ml serum)
• 0.25ml (2 cell suspension once washed AB-
• Incubate ? 37C (1 hour) ? RT( 2 hour)
  2-10 C ( ) ?
• RT( 3 hour)
• No agglutination, hemolysis, rouleaux formation

57
Anti Rh

• 6-Reaction with Du
• Antihuman Globulin test

58
stability control

• Minimum of 1 of each batch
• Should be retained at least 6 months beyond
  expiry date

59
package container

• Container
• Color of label paper
• Information
• Lettering size
• Visual inspection
• Package label

60

• ??? ???????? ???? ??? ??? ???? ??? ??????

61

• THE END