CROSS-MATCHING (Compatibility testing)

 This is a laboratory procedure to determine serological compatibility between a blood donor and an intended
recipient before blood is transfused.
 Unknown serum is tested with known red cells in vitro to detect antibodies.
 Negative results are always taken to indicate compatibility while positive results indicate incompatibility.

Types of cross-match.

(1) Major cross-match

 In this procedure, the recipient serum is sampled against donor washed red cells to detect antibodies in
the recipient’s serum which may damage the red cells of proposed donor.

(2) Minor cross-match

 In this procedure, the donor’s serum is sampled against recipient’s washed red cells to detect antibodies
in the donor’s serum which may damage the red cells of intended recipient.

Importance / purpose of Cross-match

1) It detects irregular antibodies in the recipient’s serum that are directed against donors cells (major) r
donor’s antibodies directed against recipient’s cells (minor)
2) It detects errors in ABO grouping
3) It prevents transfusion reaction
4) It ensures maximum benefit for the recipient of the compatible blood.

What the Cross-match will not do

 Can not ensure normal survival of donor’s cells in the recipient’s circulation
 Cannot prove absence of irregular antibodies n the donor or recipient’s serum
 Cannot prevent Iso-immunization of the recipient (patient)
 Cannot detect all the errors in ABO grouping
 Cannot detect all errors in rhesus typing.
 Phases of Cross – Match
 There are 4 phases of cross-match each of which is important for identifying specific classes of antibodies by
their reaction properties.

1) Saline room temperature phase (SRT)

 Detects cold natural antibodies (IgM), cold auto agglutinins & allo antibodies e.g. Anti P, Anti Lu,
anti Le, MNSs, It also detects ABO incompatibility.
2) Saline 37oC phase (S37oC)
 Detects warm saline antibodies (IgG) immune antibodies.
 Detects some cold antibodies with wide thermal range (4-37oC)
 Distinguishes cold antibodies that reacts at RT but fail at 37oC
3) Albumin 37oC phase (S37oC)
 Detects warm immune antibodies with small molecules that fail to react in saline medium.
 Detects most of the rhesus system antibodies.
4) Coombs37oC phase (S37oC)
 Detects warm incomplete antibodies
 Detects most antibodies associated with acquired hemolytic anemia
 Detects antibodies of other blood groups e.g. Anti kell, Duffy, Kidd
 Detects blocking sensitizing antibodies.

Major cross-matching technique

Principle:
When recipient serum is incubated with donor cells at various temperatures and conditions, any observable
reactions indicates presence of reacting antibodies therefore incompatibility. While no reaction at all indicates
absence of reacting antibodies therefore compatible.
Purpose:
 To ascertain serological compatibility between an intended recipient & proposed donor.
Requirements:
 Recipient’s clotted blood of known blood group
 Donor blood pack (s) of similar blood group
 Test tubes (75mm by 10mm)
 Centrifuge tubes
 Pasteur pipettes
 Normal saline (in wash bottles or beakers)
 Microscope & slides
 Albumin 20% & AHG
 Water bath at (37oC)

Procedure:
(1) Four Tube method
 Wash donor cells 3 times in large volume of normal saline
  Make a 4% saline cell suspension of washed donor cells labeled with the last 3 digits of the donor pack
number.
 Label 4 test tubes appropriately as – SRT, S37oC, A37o C & C37oC.
 Put 2 drops of recipient’s serum in each tube followed by 2 drops of 4% donor’s cells.
 Add 2 drops of 20% albumin to A37oC tube.
 Mix all the tubes gently and incubate at respective temperatures for 15 min t0 1 hr.
 Centrifuge all tubes at 1000rpm for 2 min and examine for agglutination or hemolysis macroscopically
(SRT) and microscopically (37oC) tubes.
 If no reaction, wash C37oC tube in large volume of saline 3-4 times and decant the supernatant in the
last wash completely.
 Add 2 drops of AHG into the tube mix gently and centrifuge again at 1000rpm for 2 min.
 Shake lightly and examine for agglutination or hemolysis macro & microscopically and record your
results

Results:
SRT S37oC A37oC C37oC. comment

Donor I - - - - Compatible
Donor II - - - + Incompatible

(2) Two Tube method

 Wash donor cells 3 times in large volume of normal saline
 Make a 4% saline cell suspension of washed donor cells labeled with the last 3 digits of the donor pack
number.
 Label 2 test tubes appropriately as – SRT & S37oC.
 Put 2 drops of recipient’s serum in each tube followed by 2 drops of 4% donor’s cells.
 Centrifuge SRT immediately at 1000rpm for 2 min and look for agglutination macroscopically.
 If no agglutination, add 2 drops of albumin in SRT tube , mix then incubate at 37oC water bath for 15
min – 1 hr.
 Centrifuge both tubes and check for agglutination or hemolysis macro & microscopically.
 If no reaction, wash S37oC tube 3-4 times in large volume of saline discarding the supernatant in the
last wash completely.
 Add 2 drops of AHG into the tube, mix gently and centrifuge again at 1000rpm for 2 min.
 Shake lightly and examine for agglutination or hemolysis macro & microscopically and record your
results

NB:
 The 4 tube technique is the standard method but 2 tube technique is valuable if the number of the tubes are
limited.
 When cross-match is done for more than one recipient and more than one donor, it is must that each of the
tubes be labeled with recipient’s number at the top and donor number below the phase labeled e.g.

Recipient no. 021 021

A37oC
371
 Phase A37oC
Donor no.: 371

 The longer the incubation time, the better the results

Emergency Cross-match
 There seems to be no way of selecting compatible blood rapidly with perfect safety
 ABO incompatibility & large amount of immune antibodies can be detected after incubation for as little as 15
min
 In urgent cases, it is recommended therefore, that same method of cross-match are employed as in non-urgent
cases but reading be carried out within 15 min and 1st pint of blood issued out if no reaction is observed.
 However, more time must be allowed for compatibility test in subsequent pints the patient is to receive.
 The use of LISS in cross-match is of particular value in an emergency since a short incubation time can be
used while maintaining a high degree of sensitivity in C37oC.
 There’s absolutely no time for cross-match in desperate cases.
 A unit of blood group O rhesus –ve can be issued if the patient is a woman
 If a man, O rhesus +ve blood can be given unless he is known to have been previously transfused with O
rhesus positive when he should have received O rhesus –ve blood.
 If patient blood group is known with certainty, he can be given blood of his own group.
 Any blood which has not been cross-matched must be grouped before issued.
 In all this, the degree of emergency must be weighed against the risks involved, if there is no sufficient time
for the normal cross-match.

Causes of Automatic Incompatibility

 There are times when a patient may be found to be incompatible with all donors
 Such cases are found in
a) Patient with auto – hemolytic anemia
 Solution:- select least incompatible blood
b) Presence of cold auto agglutinins
 This reacts more at room temperature but weakly or non-reactive at 37oC.
 In case of auto-hemolytic anemia from several pints of blood direct coombs test is done.
SRT S37oC A37oC C37oC
Donor I +++ + - -
Donor II +++ - - +
Donor III +++ + + +
Solution
 Evaluate all the phases of cross-match and select the one with fewer or no reaction at 37oC e.g.
Donor I.
 Discover case of auto-hemolytic anemia by performing DCT on patient’s washed red cells.
 Choice of blood for transfusion
 If possible, patient should receive own blood group e.g. A blood group receives A blood group.
 The rhesus –ve patient must be given rhesus negative blood of the same group or O rhesus +ve with low titre