Compatibility Testing (cross matching)

Compatibility testing

PurposePurpose Selection of safest blood components for transfusion With acceptable donor’s red cell survival rates Without destruction of recipient’s red cells

Compatibility testing Compatibility testing Confirms ABO compatibility Detects clinically significant unexpected antibodies

Importance of cross matchingImportance 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.

1. Identification of patient & its sampleo patient full name & hospital registration #o name of requesting physiciano date & time of sample collectiono initials of phlebotomisto information clearly written on requisition form and

sample

2. ABO & Rh of recipient and donor blood3. Test for clinically significant red cell antibodies

on patient serum

Steps in pre-transfusion testingSteps 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

Steps in pre-transfusion testingSteps in pre-transfusion testing

Pre-transfusion testing procedure

Donor Unit Testing Donor Unit Testing ABO grouping: Forward and Reverse Rh grouping: Rh (D) including weak D (Du)

Recipient TestingRecipient Testing ABO grouping: Forward and Reverse Rh grouping, Weak D (Du) not required IAT testing: Antibody screen Cross match: Major & Minor

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

Serological cross matchSerological cross match

Preparation of donor cells for cross-matching

Select appropriate unit of blood from inventory Check for

o Donor ID o Blood Group o Expiry dateo 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

• 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.

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.

Cross Matching Procedure

Cross matching should be performed at following phaseso Saline phase at room temperatureo AHG phase

Cross matching can be performed using conventional test tubes or by using newer technologies such aso Column Agglutination Technologyo Solid Phase Technologyo Electro Magnetic (EM) Technology

Immediate Spin Technique (IST)Immediate Spin Technique (IST)

Detects only IgM antibody, reactive at 22oC.

Clinically significant IgG antibody reactive at 37oC not detected

Patient serum

2 drops

Donor RBC

1 drop, 5%

Immediate centrifuge

ABO incompatibility

22oC

Conventional AHG-crossmatchConventional AHG-crossmatch

Patient serum

2 drops

Incubation37oC, 1 hr

Donor RBC

1 drop, 5%

3 washes

Centrifuge

2 drops AHGMix properly

Agglutination = incompatible

No agglutination = compatible

Detects clinically significant (IgG) antibody

Serological cross match

PhasePhase DetectsDetectsIS 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

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

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

Incomplete requisition forms

Hemolyzed samples

EDTA and clotted sample

oPlasma prevents detection of complement

dependent antibodies

oFibrin clots, which may form, can be mistaken for

agglutination

Problems In Cross MatchingProblems In Cross Matching

Incompatible crossmatches

Antibody Antibody screenscreen

Cross Cross matchmatch

Cause Cause ResolutionResolution

Pos Neg Antibody directed against antigen on screening cell

ID antibody, select antigen negative blood

Neg Pos 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

Pos Pos Antibodies directed against both screening and donor cells

Antibody ID, select antigen negative blood

Normal sampleHemolyzed

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

oGrading of reaction strengthoHemolysis – positive resultoAuto-agglutination

Technical ProblemsTechnical Problems

Clinical urgency Clinical urgency

Immediate Within an hour Minutes

Group O Rh neg Packed RBCs

ABO & Rh D type

Group specific blood

ABO & Rh D type

Complete crossmatch

Cross matching: Special CircumstancesCross matching: Special Circumstances

If units are issued without X match – take written consent of physician, complete X match after issue

Neonatal (< 4 months) Transfusions Only ABO and Rh grouping; no serum grouping Antibody screen with maternal serum Exchange transfusion:

o WB or PRBCs within 7 days of collectiono ABO and Rh D compatible with maternal sampleo Irradiated unit for infant weight < 1500 gm

Cross Matching: Special circumstancesCross Matching: Special circumstances

Transfusion in AIHA Avoid transfusion as far as possible in Warm AIHA There could be problems in blood grouping

o Spontaneous agglutination of red cells on addition of antisera in WAIHA

o 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.

o Blood unit showing minimum strength of reaction in terms of titer designated as the ‘least incompatible’.

o Blood unit must be compatible with the patient's auto-absorbed-serum.

Transfusion should be done under strict medical supervision.

“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)

Red blood cell compatibility table

                       AB+

           AB-

           B+

     B-

           A+

     A-

     O+

O-

AB+AB-B+B-A+A-O+O-

DonorRecipient

  

Plasma compatibility table

O

B

A

AB

AB

BAO

DonorRecipient

IATIAT

• 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

IATIAT

• 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

DATDAT

• 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

Check cell preparationCheck cell preparation

• Check cell: • 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

Check cell preparationCheck 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