cross transfusion and compatibility

Ahmad A. Al-Qudah

LM755 - ADVANCED DIAGNOSTIC IMMUNOHEMATOLOGY AND BLOOD BANKING

Cross transfusion and Compatibility

Cross transfusion and Compatibility

- Pretransfusion Testing .- Pretransfusion testing elements : - ABO & Rh - Antibody Screening - Cross Matching - Antibody Identification .

- Special Techniques in Ab Identification : - Elution - Hemagglutination inhibition - Titration

Pretransfusion TestingHistory

- After Landsteiner described the ABO .

- Crossmatch :

- Major Crossmatch : Recipient Serum + Donor RBCs.

- Minor Crossmatch : Recipient RBCs + Donor Plasma (not required by AABB)

- RT ( IS Phase ) IgM , then Rh (IgG) and detection at various

Temperatures with or without enhancements .

Pretransfusion Testing

- To select blood components that will not cause harm to the

recipient and will have acceptable survival when transfused.

- ABO compatibility , Rh compatibility , clinically significant

Antibody Screening and Identification .

Indirect Antiglobulin Test Phases

- Immediate Spin ( IS ) Phase at Room Temperature .Detection of ABO incompatibility and Cold Antibody (M,N, Lea+b,P1)

- 37° with Enhancement media (LISS)Allows IgG and other complement to bind to RBCs . - Anti Human Globulin Phase ( AHG )Detection of IgG or complements binds to RBCs

- Check Cells : Approve the Results

ABO & Rh

- ABO & Rh typing for (recipient ) sample .

- ABO & Rh typing for Donor sample .

Antibody Screening

- Autoantibody : is an antibody manufactured by the immune

system that is directed against one or more of the individual's

own cells, Causes Many autoimmune disease.

Antibody Screening

- Alloantibody : is an immune response to foreign antigen,

Formed from previous transfusion or pregnancy.

- Antibody Screening test performed to detect auto-antibodies

/allo-antibodies using DAT (Direct Anti-human Globulin Test)

Antibody Screening- Inclusion of auto control helps to rule out :

Auto antibodies Allo antibodies

- Auto control :Negative - alloantibody .Positive – autoantibody .

Antibody Screening

- Antibody Screens use 2 or 3 Screening Cells to “detect” if

antibodies are present in the serum.

- If antibodies are detected, they must be identified .

Antibody Screening

Cross Matching

- Cross match is done to ensure there are no clinically significant

Abs in patient blood ( Serum ) against donor RBCs and so prevent

hemolytic reaction during or after transfusion .

Cross Matching

- Major cross-match test, consisting of mixing the patient’s

serum with donor RBCs.

- Minor cross-match test, consisting of mixing the donor’s

plasma with patient’s RBCs.

- Minor cross-match test has been completely eliminated in

most blood banks according to AABB SOPs since 1960.

Cross Matching

Prepare 5% suspension of donors RBCs

Cross Matching

Patient serum

2 drops

Donor RBC

1 drop, 5%

Immediate centrifuge

ABO incompatibility

22oC

• Detects only IgM antibody, reactive at 22oC.

• Clinically significant IgG antibody reactive at 37oC not detected

Cross Matching

Patient serum

2 drops

Incubation37oC, 1 hr

Donor RBC

1 drop, 5%

3 washes

Centrifuge

2 drops AHGMix properly

No agglutination = compatible

Detects clinically significant (IgG) antibody

Agglutination = incompatible

Antibody Identification

- It is a set of commercially screening cells with different antigenic

expression corresponding to the most commonly encountered Abs.

Antibody Identification - Screening Cells and Panel Cells are the same with minor differences:

- Screening cells

- Antibody detection

- Sets of 2 or 3 vials

- Panel cells

- Antibody identification

- At least 10 vials per set.

- Panel test is just an extended version of an antibody screen


- 11 panel cells of Group O , last one is auto control (Patient RBCs + Patient serum) .


- Each of the panel cells has been antigen typed .

Antibody Identification - Immediate Spin Technique (IST)

Patient serum

2 drops

Panel Cell

1 drop

Immediate centrifuge

incompatibility

22oC


2+

00

2+

002+

0

02+

0

Record results


Incubation37oC, 15 min

Centrifuge

2 drops LISSMix properly

Agglutination

No agglutination

Detects clinically significant (IgG) antibody

(LISS) 37°C Phase

Patient serum

2 drops

Panel Cell

1 drop

Antibody Identification IAT Phase

Incubation 37oC Centrifuge

2 drops AHGMix properly

Agglutination

No agglutination

Patient serum

2 drops

Panel Cell

1 drop

Wash 3 timessaline


2+

00

2+

002+

0

02+

0

000

00

00

0

00

0

0000

00

00

000

0 0 0

- Record Results , Grade of Reaction .- Add Check cells to any negative AHG .

Antibody Identification - Read Results , Interpreting Antibody Panels


Phase-1: IS , Rx. at room temperature (24°C)

- Cold reactive antibodies (Ex. Anti-H, I, i, P)

Phase-2: LISS Rx at 37°C:

- Rh incompatibility

- wide thermal range IgM antibodies (Anti-Le, I, P)

Phase-3: AHG Rx. (at 37°C)

- will detect the vast majority of IgG antibodies


- Basic steps to follow when interpreting panels :

• “Ruling out” means crossing out antigens that did not react

• Circle the antigens that are not crossed out

• Look for a matching pattern

• Rule of Three .


- The rule of three must be met to confirm the presence of the

antibody.

- A p-value ≤ 0.05 must be observed.

- This gives a 95% confidence interval.

- Patient serum MUST be:

- Positive with 3 cells with the antigen

- Negative with 3 cells without the antigen

Antibody Identification - Rule of three :


- P value = Num# of Positive RBCs! * Num# of Negative RBCs

Total Num# of RBCs used

P value = 3! * 4!

P value = (1*2*3) * (1*2*3*4)

(1*2*3*4*5*6*7)

P value = 2.85 , P value is ≤ 0.05

7

Special Techniques in Ab Identification

- Elution

Elution techniques “free” antibodies from the sensitized red

cells so that the antibodies can be identified and measured .

Y

Y

YYSensitized

RBC

Positive DAT

Elution YYY Y

YY

Frees antibody Antibody ID


- Elution

• Acid elution (glycine acid)

- Most common

- Lowers pH, causing antibody to dissociate

• Organic solvents (ether, chloroform)

- Dissolve bilipid layer of RBC

• Heat (conformational change)

• Freeze-Thaw (lyses cells)


- Haemagglutination inhibition:

based on the neutralization of certain antibodies with its

corresponding soluble substances (antigens).


- Haemagglutination inhibition :


Antibody titration

• semiquantitative determination of Ab’s concentrations• serial 2-fold dilutions of tested serum are prepared• serum testing against corresponding antigens• TITER: is the reciprocal of the highest serum dilution that gives

macroscopic agglutination


Antibody titration

Thank You

cross transfusion and compatibility

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