Procedure number: 202
(Blood Bank Name and address)
Page 1 of 3
Subject: Preparation of Red Blood Cells for Analysis
PREPARATION OF RED BLOOD CELLS FOR ANALYSIS
PURPOSE
To prepare red blood cell suspensions to be used for serologic testing.
PRINCIPLE
A proper ratio of plasma to red cells is important for accuracy in antigen-antibody reactions; too heavy a
concentration of cells might result in a weak or false negative reaction. Also, abnormal patient albumin:globulin
ratios may cause pseudoagglutination, so adequate washing is important in the preparation of the cell suspension.
RBCs can be obtained by "defibrinating" (mashing) clotted (red top) specimens. This method is particularly useful
for obtaining the large number of cells necessary for adsorption and elution techniques, and is also described below.
SPECIMEN
The preferred specimen is an EDTA (lavender top) tube of venous blood. Un-anticoagulated glass (red top) tubes
may be used if completely clotted. Prior to preparation of a cell suspension, the specimens should be centrifuged
except cord blood specimens and specimens incorrectly provided in “serum separator” tubes. Patient plasma/serum
is separated from the red cells and placed into an appropriately labeled tube with the patient's name and medical
record number or date of birth, and the date the sample was drawn.
RBCs are removed directly from cord blood specimens without centrifugation. For serum separator tubes, remove
RBCs prior to centrifugation and wash them as below. Then centrifuge the tube, and obtain the serum specimen. If
this sequence is neglected, RBCs can be recovered from beneath the gel in a serum separator tube, but this may
cause a false positive DAT.
PROCEDURE
Washed 3-5%patient or donor red cell suspension for tube tests starting with packed cells
1.
Properly label a 12 x 75 test tube.
2.
With a pipette, remove approximately 3-5 drops of blood from the patient or donor sample and add to the test
tube. For donor tubing segments, cut open the segment and squeeze 1-2 drops of blood into the test tube.
NOTE: Since antibody coated RBCs are heavier than uncoated cells and settle to the bottom of the sample the
contents should be mixed well before blood is removed. If a clotted specimen is used, blood should be
removed from the bottom of the clot for the same reason.
3.
Fill the test tube approximately 3/4 full with saline by positioning the tip of the wash bottle directly over the
tube and squeezing it. This will mix the blood and the saline, increasing the efficiency of washing.
4.
Centrifuge the tube for the appropriate time calibrated for cell washing.
5.
When the centrifuge stops, remove the test tube and decant the supernatant saline.
6.
Shake the cells loose from the bottom of the tube.
7.
Add the volume of saline necessary to prepare 3-5% suspension, approximately 40 drops.
Procedure number: 202
(Blood Bank Name and address)
Page 2 of 3
Subject: Preparation of Red Blood Cells for Analysis
Washed patient or donor 0.8% red cell suspension for gel tests starting with packed cells
1.
Label two 12x75 mm test tubes with the patient/donor identifier(s).
2.
Place 100 µL of patient cells in one of the tubes.
3.
Wash the cells with saline and decant.
4.
Remove 10 µL of washed cells and place into the second tube.
5.
Add 1 mL of gel test diluent.
Preparation of a 0.8% Suspension Reagent Cells for Gel Tests starting with a 3-5% Suspension
Starting with 1mL of reagent cells this procedure yields enough cells for approximately 60 tests. A starting volume
of 100 µL yields enough for 5 or 6 tests.
1.
Label a 12 x 75 mm tube for each reagent cell. Also record the manufacturer, lot # and date and time of
expiration, which is 24 hours from the time of preparation.
2.
Thoroughly mix the cells and place 1 volume (typically 1 mL or 100µL) of each reagent cell in the respective
labeled tube.
3.
Centrifuge the tubes for 1 minute and decant the supernatant.
4.
Add 3 volumes (typically 3 mLs or 300 µL respectively) of gel diluent 2 to each tube.
Method to defibrinate a clot
Materials
1.
Sieve and pestle
2.
Clotted blood specimen
Procedure
1.
Spin the specimen until a good separation occurs between the clot and serum (3,500 rpm for 5 minutes).
2.
Pipette the serum into a clean test tube labeled with the patient's name, account number, and date of the
sample.
3.
Place the sieve over a Petri dish.
4.
Pour the remaining clot onto the sieve and mash well with the pestle. Rinse the sieve with saline.
5.
Transfer red cells suspended in saline from the Petri dish into clean 13x100 mm test tubes labeled with
patient's name and date of sample. Rinse the Petri dish and transfer the saline to the labeled tubes.
6.
Spin these tubes at 3,500 rpm for l minute. Aspirate the supernatant saline and pool the packed cells, rinsing
remaining cells from the tubes with saline.
7.
Continue pooling and centrifugation until all the cells are contained in one test tube.
Page 3 of 3
Procedure number: 202
(Blood Bank Name and address)
Subject: Preparation of Red Blood Cells for Analysis
8.
Wash the cells until the supernatant is free of hemoglobin.
NOTE: Failure to rinse tubes with saline results in loss of many cells due to their adherence to the glass.
Preparation of a Standard Cell Suspension
Generally, a 3-5% red cell suspension concentration is used for testing patient and donor red cells in the form of a
crossmatch, a DAT, or an antigen typing procedure. A standard may be prepared (see table below) for use as a
comparison.
Final Concentration
RBC volume, mL
2%
3%
4%
5%
Final volume, diluted with saline, in mL
0.1
5
3.3
2.5
2
1.0
50
33
25
20
Adopted
Reviewed
Reviewed
Reviewed
Reviewed
Reviewed
Reviewed
Reviewed
Reviewed
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Reviewed
Reviewed