1. Compatibility Testing (cross matching)
Read out the slide

2. Compatibility testing
Purpose
Selection of safest blood components for transfusion
With acceptable donor’s red cell survival rates
Without destruction of recipient’s red cells
Compatibility testing
Confirms ABO compatibility
Detects clinically significant unexpected antibodies
Compatibility testing is also called as pre transfusion testing. The purpose of the compatibility testing is to provide an appropriate and safe unit of blood to the patient which will not cause any harm to the recipient.
The procedure involves confirmation of ABO and Rh groups of both donor and the recipient. Secondly, it involves detection of not only ABO antibodies but also other clinically significant unexpected antibodies using AHG phase.

3. Importance of cross matching
Routine blood grouping involves only ABO and Rh.
Other clinically significant blood group systems not matched routinely
Though, antibodies to minor antigens are of rare occurrence, they can cause transfusion reactions
Cross matching between patient’s serum and donor’s cells will detect antibodies to other blood groups, if present.
It is essential to perform compatibility testing before issue of blood. It will detect all types of clinically significant antibodies making transfusion safer to the recipient

4. Steps in pre-transfusion testing
Identification of patient & its sample
patient full name & hospital registration #
name of requesting physician
date & time of sample collection
initials of phlebotomist
information clearly written on requisition form and sample
ABO & Rh of recipient and donor blood
Test for clinically significant red cell antibodies on patient serum
This slide illustrates the steps involved in compatibility testing.
It begins with proper collection of blood sample with careful identification and labeling.
Remaining slide can be read out as it is self explanatory

5. Steps in pre-transfusion testing
4. Selection of appropriate unit of blood
ABO & Rh compatible
Expiration date
Component as per need of patient
PRBC, FFP, PC, Cryo
5. Performance of serological cross match
6. Labeling of component with patient identification details
7. Issue after verification of patient identity along with compatibility report & reaction form

6. Pre-transfusion testing procedure
Donor Unit Testing
ABO grouping: Forward and Reverse
Rh grouping: Rh (D) including weak D (Du)
Recipient Testing
Rh grouping, Weak D (Du) not required
IAT testing: Antibody screen
Cross match: Major & Minor
Read out the slide

7. Serological cross match
Major crossmatch: Test donor cells with recipient’s serum to detect antibodies in patient serum
Minor crossmatch: Test donor serum with recipient’s red cells to detect antibodies in donor serum
Inclusion of autocontrol helps to rule out
Auto antibodies
Allo antibodies
Rouleaux formation
Traditionally, cross match can be major cross match and minor cross match.
It is important to include autocontrol in the cross match procedure

8. Preparation of donor cells for cross-matching
Select appropriate unit of blood from inventory
Check for
Donor ID
Blood Group
Expiry date
Hemolysis / leakage
Detach a segment from the blood bag, cut ends of segments and pour the contents in a labeled test tube
Wash red cells with saline 3 times and prepare 5% suspension
It is very important to check the unit for donor ID, blood group, expiry and any evidence of hemolysis or bacterial contamination. In case of bacterial contamination, the color of the unit will be dark violet with foul smell and there may be reddish discoloration of the supernatant plasma.
It is also important that main unit of blood is not opened for taking out the sample. Donor samples can be obtained by detaching the segment of the tubing attached to the unit.

9. Donor cells are taken from segments that are attached to the unit itself.
Segments are sampling of the blood and eliminate having to open the actual unit.

10. Clinically significant Abs
Abs regarded as always being potentially clinically significant;
ABO, Rh, Kell, Duffy, Kidd & S s U
Abs that may sometimes be clinically Significant;
Lea, p, Lua, Lub & Cartwright.
Other Abs are rarely if ever, clinically significant.

11. Cross Matching Procedure
Cross matching should be performed at following phases
Saline phase at room temperature
AHG phase
Cross matching can be performed using conventional test tubes or by using newer technologies such as
Column Agglutination Technology
Solid Phase Technology
Electro Magnetic (EM) Technology

12. Immediate Spin Technique (IST)
Detects only IgM antibody, reactive at 22oC.
Clinically significant IgG antibody reactive at 37oC not detected
2 drops
Patient serum
22oC
IST also called as Saline Cross Match detects only IgM antibodies reacting at RT such as ABO antibodies.
Clinically significant antibodies such as anti-D will not be detected by IST.
Take 2 drops of patient serum in a clean test tube to which add 1 drop of 5% suspension of donor red cells. Centrifuge at 1000 RPM for one min and look for agglutination
Immediate centrifuge
ABO incompatibility
1 drop, 5%
Donor RBC

13. Conventional AHG-crossmatch
Detects clinically significant (IgG) antibody
2 drops AHG
Mix properly
2 drops
No agglutination = compatible
3 washes
Patient serum
Conventional AHG cross match is the ideal method as it detects clinically significant antibodies.
Take 2 drops of patient serum in a clean test tube. Add 1 drop of 5% suspension of donor red cells.
Incubate the test tube at 37C for 30 min.
Wash red cells three times using normal saline
After the last wash, add 1 drop of polyspecific AHG to the dry red cell button.
Mix well and centrifuge for 1000 RPM for one min and look for agglutination.
Absence of agglutination indicates no red cell antibodies detected.
1 drop, 5%
Incubation
37oC, 1 hr
Centrifuge
Agglutination = incompatible
Donor RBC

14. Serological cross match
Phase
Detects
IS phase
ABO incompatibilities
AHG phase
Rh, Duffy, Kidd, others
Points to remember:
Preserve recipients serum & donor red cell segment for a week.
However, fresh sample of the patient is needed after 48 hrs of
transfusion
Do not withdraw sample from the IV line
Infuse red blood cells within 4 hours
It is very important to carry out cross match at 37 C using AHG. Immediate spin cross match in saline phase will detect only ABO incompatibility. Other clinically significant antibodies such as Rh, Kell, Kidd or duffy antibodies will be detectable in AHG phase only.

15. Cross matching Procedure
If antibodies ARE detected:
Antigen negative units found and X-matched
All phases are tested: IS, 37° & AHG
This termed as COMPLETE CROSSMATCH

16. Cross matching for platelets & plasma
No compatibility testing required for platelets and
plasma components
Only ABO matching is required for fresh frozen plasma
No need for compatibility, ABO and Rh matching for platelet
concentrates and cryoprecipitate
Exceptions
Neonates, alloimmunized patients –
preferably ABO & Rh matched platelets
This is for the center with component facility
Since plasma components and platelet concentrates do not contain red cells, there is no need to perform cross match unless these components are grossly contaminated with red cells
Similarly, since there are no naturally occurring Rh antibodies in FFP, there is no need to label it for Rh group

17. Problems In Cross Matching
Incomplete requisition forms
Hemolyzed samples
EDTA and clotted sample
Plasma prevents detection of complement dependent antibodies
Fibrin clots, which may form, can be mistaken for agglutination
One may come across clerical / technical problems which may affect the result of cross matching.
Requisition form should have all details with regard to age of the patient, diagnosis, transfusion history.
Ideally for cross matching, both EDTA as well as Clotted sample.

18. Incompatible crossmatches
Antibody screen
Cross match
Cause
Resolution
Pos
Neg
Antibody directed against antigen on screening cell
ID antibody, select antigen negative blood
Antibody directed against antigen on donor cell which may not be on screening cell OR donor may be DAT positive
ID antibody, select antigen negative blood OR perform DAT on donor unit
Antibodies directed against both screening and donor cells
Antibody ID, select antigen negative blood
If the cross match is incompatible, the causes for incompatibility need to investigated. This Table shows some of the common possibilities leading to incompatible cross match and resolution their of

19. Normal sample Hemolyzed
Samples in the syringes or hemolyzed samples must be rejected.
Normal sample
Hemolyzed

20. Technical Problems Expired / contaminated reagents
Improper cell concentration (cell : serum ratio)
Labeling errors
Improper washing of red cells before forward grouping
Equipment – Improper centrifugation
Dirty glass wares
Reading and recording reactions
Grading of reaction strength
Hemolysis – positive result
Auto-agglutination
These are some of the examples of technical problems that can cause cross match incompatible.
That includes improper reagents, identification errors, equipment not validated etc.

21. Group O Rh neg Packed RBCs
Cross matching: Special Circumstances
Clinical urgency
Immediate
Within an hour
Minutes
Group O Rh neg Packed RBCs
ABO & Rh D type
Group specific blood
Complete crossmatch
If units are issued without X match – take written consent of physician,
complete X match after issue

22. Neonatal (< 4 months) Transfusions
Cross Matching: Special circumstances
Neonatal (< 4 months) Transfusions
Only ABO and Rh grouping; no serum grouping
Antibody screen with maternal serum
Exchange transfusion:
WB or PRBCs within 7 days of collection
ABO and Rh D compatible with maternal sample
Irradiated unit for infant weight < 1500 gm

23. Transfusion in AIHA Avoid transfusion as far as possible in Warm AIHA
There could be problems in blood grouping
Spontaneous agglutination of red cells on addition of antisera in WAIHA
Non specific agglutination of reagent cells during serum grouping in cold AIHA
Difficult to find absolutely compatible blood for such patients.
In emergency, consider the least incompatible blood.
Blood unit showing minimum strength of reaction in terms of titer designated as the ‘least incompatible’.
Blood unit must be compatible with the patient's auto-absorbed-serum.
Transfusion should be done under strict medical supervision.

24. “Dangerous group O donor”
In emergency situations group ‘O’ donor blood is used as universal donor where group identical blood is not available.
This is an outdated concept in major blood banks. Certain donors possess in their plasma potent ABO antibodies, which are dangerous to the recipients’ red cells.
These are anti A and anti B haemolysins, titer of which is > 32
Such donors are called as “dangerous O donor”
Therefore, if group O blood is to be used as universal donor, it should always be plasma depleted (packed red cells)

25. Red blood cell compatibility table  
AB+
AB- B+ B- A+ A- O+ O-
Donor
Recipient

26. Plasma compatibility table
Recipient
Donor O A B AB AB    A       B       O            

27. IAT Patient plasma and O negative pooled cell
30-60 min incubation in 37ºC
Wash 3 times
Add PS-AHG
For more precision in negative results please check with:
Microscope, check cell and elution
PH, time, ionic power, Ab: Ag ratio are important
LISS, PEG, Polybern, 22-30% albumin can be used for special purposes

28. IAT PEG enhances the reaction of anti kidd with low titers
22-30% Albumin enhances the reaction of anti RH and anti P1 in 37ºC
Anti K doesn’t have good reaction in LISS solution

29. DAT 2-5% suspension of RBCs should be prepared
EDTA anticoagulated blood
Wash 2-3 drop of blood for 3-4 times
Wash with press of saline between each centrifuge : cell pellet will be suspend and negative result wont appear.
In multiple myeloma and WM wash 6 times
Immediately add 2 drops of PS-AHG and spin(30 seconds in 3000 rpm)
If agglutination observed : DAT positive
If the result was negative : incubate 5-10 min in RT(C3d)
If the result was negative : add2-3 drops of CC

30. Check cell preparation
1 drop of 1:4000 of diluted serum can corrupt 1 drop of AHG
Then any contamination with serum may lead to AHG negative result
preaper 5% suspension of Rh pos cells
You can use Anti D or RhoGam
Dilute the anti body
Add 250 micro liter suspension to each tube of .5 ml RBCs
Incubate in 37ºC for 30 minuets
Then centrifuge at 3000 rpm for 1 minuete

31. Check cell preparation
Select the first negative tubes and wash 3 times. For example 1:32 and 1:64 and 1:128
Add AHG and centrifuge
+2 agglutination should be noticed