This is a simple and a rapid method. However,

incompatibilities due to weak saline agglutinins and all

incomplete immune antibodies will not be detected.

The period of incubation should not be reduced or else

agglutination may not occur. Almost all ABO group

incompatibilities will be detected by this method. Rh

incompatibilities will never be detected.

Saline Tube Method

1. Label one tube for the patient’s 2–5% saline cell

suspension. Label one tube for each donor’s saline cell

suspension. Label two tubes for each cross-match—

one marked P with patient’s number and donor’s

number, and one marked D with donor’s number and

patient’s number.

2. Add saline to the cell suspension tubes and make the

cell suspensions.

3. To the patient’s tubes (P. major side), add one drop of

patient’s serum and one drop of donor cell suspension.

4. To the donor’s tubes (D. minor side) add one drop of

donor’s serum and one drop of patient cell suspension.

5. Mix the cells and sera by gently tapping the tubes.

6. Let stand at room temperature for 30 minutes.

7. Examine for agglutination both macroscopically and

microscopically.

This method needs a longer incubation period but has

the advantage of detecting weak reactions more often.

However, it still will only detect complete agglutinins—not

immune antibodies of the Rh type.

Immediate-spin and thermal incubation modification:

1–5. As in the previous method.

6. After 2–3 minutes at room temperature, centrifuge at

500 to 1000 rpm for 2 minutes.

7. Remove and examine macroscopically, holding the

bottom of the tube over a concave mirror (a microscope mirror is good enough) so that you can observe

the button of cells at the bottom when you tap the

tube gently to resuspend the cells. If the cells, show

agglutination, check them microscopically by placing

a drop on a slide. This indicates incompatibility and

the cross-match result can be recorded. If the cells

resuspend without any clumps, then proceed to the

next step.

8. Incubate the tube for 60 minutes at 37oC.

9. Examine the cells for agglutination, both, macroscopically and microscopically. Centrifugation brings

the cells into close proximity and hastens agglutination

where it is going to occur—the immediate spin.

Incubation at 37oC may detect some complete

warm antibodies active in saline but missed in room

temperature incubations.

Albumin Tube Method

1. Set up the tubes as for saline method.

2. Incubate the cells and serum for 60 minutes at 37°C—

thermal saline incubation.

Blood Banking (Immunohematology) 369

3. Allow a drop of albumin to run down the inside of

each cross-matching tube so that it forms a layer

between the cells and serum (human/bovine albumin,

22–30%).

4. Incubate at 37°C for an additional 30 minutes.

5. Dislodge the cells gently and look for agglutination

microscopically. This method will pick up some

incompatibilities not detected in saline due to

incomplete, immune antibodies, such as Rh antibodies.

Coomb’s Cross-matching

1. Wash the patient’s and donor’s cells three times in

saline. Then make 5–10% suspension of each in saline.

2. To labeled patient’s tubes (P. major side) add 2 drops of

patient’s serum and 2–4 drops of donor cell suspension.

3. To labeled donor’s tube (D. minor side) add 2 drops of

donor’s serum and 2–3 drops of patient cell suspension.

4. Incubate tubes at 37°C for one hour.

5. Centrifuge, decant the serum and wash the cells thrice

in saline.

6. Add Coombs’ serum to each tube and after letting

stand for 5 minutes at room temperature, centrifuge

at 500–1000 rpm for 2 minutes.

7. Examine the cells macroscopically and microscopically.

The Coombs’ serum in the cross-match as in the indirect

Combs’ test, will detect the presence of incomplete,

immune antibodies such as Rh antibodies and show any

Rh incompatibility. Remember that not all Rh negative

persons have anti-Rh (D) antibodies—in fact only very

few do—so that not all cross-matches of Rh-negative

recipients with Rh-positive donors__or vice versa__will be

incompatible. Transfusion of the blood may be perfectly

safe. However, the Rh-positive transfused cells may

sensitize the recipient so that he or she develops antiRh antibodies. On subsequent cross-matches (after 2–3

weeks), an incompatibility would be demonstrated. Thus,

where Rh-negative individuals have been previously

transfused, it is important to do a more sensitive crossmatch, since there is a good chance that the previous

transfusion may have been with Rh-positive blood.

It is obvious that cross-matching of bloods of the same

groups generally gives a compatible result (major and

minor). Cross-matching of O group blood with any other

group gives a compatible major side (hence, O group

people are called universal donors) but always gives an

incompatible minor side. Any group of blood may be

given to AB group of patient with a compatible major

cross-match (universal recipients) but again there is an

incompatible minor cross-match.

Given on next page are the diagrammed results of

the cross-matching of the different groups of blood (+ =

agglutination, — = no agglutination).

Blood which shows a major incompatibility should

never be transfused (in the patient’s body the ‘major side’

would be the full plasma volume—about 2,500 mL of

antibodies and the transfused donor cells—200–250 mL).

The minor cross-match is important but not as important

as the major side, (in the patient’s body, the minor side

would be the donor plasma volume 250–300 mL__of

antibodies and the large number of his own cells—about

2,500 mL. The donor plasma becomes diluted in the much

larger volume of the patient’s plasma, and thus, the donor

antibodies become diluted and dispersed so that although

a few cells may be coated or agglutinate, there is no major

reaction).