(Blood Bank Name and address)

Procedure number: 202 Page 1 of 3

Subject: Preparation of Red Blood Cells for Analysis
PREPARATION OF RED BLOOD CELL FOR ANAL!I
P"RPOE
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 antigenantibody reactions! too "ea#y a
concentration of cells mig"t result in a $eak or false negati#e reaction. Also% abnormal patient albumin:globulin
ratios may cause pseudoagglutination% so ade&uate $as"ing is important in t"e preparation of t"e cell suspension.
'B(s can be obtained by )defibrinating) (mas"ing) clotted (red top) specimens. T"is met"od is particularly useful
for obtaining t"e large number of cells necessary for adsorption and elution tec"ni&ues% and is also described belo$.
PECI#EN
T"e preferred specimen is an *+TA (la#ender top) tube of #enous blood. ,nanticoagulated glass (red top) tubes
may be used if completely clotted. Prior to preparation of a cell suspension% t"e specimens s"ould be centrifuged
e-cept cord blood specimens and specimens incorrectly pro#ided in .serum separator/ tubes. Patient plasma0serum
is separated from t"e red cells and placed into an appropriately labeled tube $it" t"e patient1s name and medical
record number or date of birt"% and t"e date t"e sample $as dra$n.
'B(s are remo#ed directly from cord blood specimens $it"out centrifugation. 2or serum separator tubes% remo#e
'B(s prior to centrifugation and $as" t"em as belo$. T"en centrifuge t"e tube% and obtain t"e serum specimen. 3f
t"is se&uence is neglected% 'B(s can be reco#ered from beneat" t"e gel in a serum separator tube% but t"is may
cause a false positi#e +AT.
PROCED"RE
$as%ed 3&'(patient or donor red )ell s*spension for t*+e tests startin, -it% pa).ed )ells
4. Properly label a 45 - 67 test tube.
5. 8it" a pipette% remo#e appro-imately 97 drops of blood from t"e patient or donor sample and add to t"e test
tube. 2or donor tubing segments% cut open t"e segment and s&uee:e 45 drops of blood into t"e test tube.
N;T*: Since antibody coated 'B(s are "ea#ier t"an uncoated cells and settle to t"e bottom of t"e sample t"e
contents s"ould be mi-ed $ell before blood is remo#ed. 3f a clotted specimen is used% blood s"ould be
remo#ed from t"e bottom of t"e clot for t"e same reason.
9. 2ill t"e test tube appro-imately 90< full $it" saline by positioning t"e tip of t"e $as" bottle directly o#er t"e
tube and s&uee:ing it. T"is $ill mi- t"e blood and t"e saline% increasing t"e efficiency of $as"ing.
<. (entrifuge t"e tube for t"e appropriate time calibrated for cell $as"ing.
7. 8"en t"e centrifuge stops% remo#e t"e test tube and decant t"e supernatant saline.
=. S"ake t"e cells loose from t"e bottom of t"e tube.
6. Add t"e #olume of saline necessary to prepare 97> suspension% appro-imately <? drops.
(Blood Bank Name and address)
Procedure number: 202 Page 2 of 3
Subject: Preparation of Red Blood Cells for Analysis
$as%ed patient or donor 0/0( red )ell s*spension for ,el tests startin, -it% pa).ed )ells
4. @abel t$o 45-67 mm test tubes $it" t"e patient0donor identifier(s).
5. Place 4?? A@ of patient cells in one of t"e tubes.
9. 8as" t"e cells $it" saline and decant.
<. 'emo#e 4? A@ of $as"ed cells and place into t"e second tube.
7. Add 4 m@ of gel test diluent.
Preparation of a 0/0( *spension Rea,ent Cells for 1el Tests startin, -it% a 3&'( *spension
Starting $it" 4m@ of reagent cells t"is procedure yields enoug" cells for appro-imately =? tests. A starting #olume
of 4?? A@ yields enoug" for 7 or = tests.
4. @abel a 45 - 67 mm tube for eac" reagent cell. Also record t"e manufacturer% lot B and date and time of
e-piration% $"ic" is 5< "ours from t"e time of preparation.
5. T"oroug"ly mi- t"e cells and place 4 #olume (typically 4 m@ or 4??A@) of eac" reagent cell in t"e respecti#e
labeled tube.
9. (entrifuge t"e tubes for 4 minute and decant t"e supernatant.
<. Add 9 #olumes (typically 9 m@s or 9?? A@ respecti#ely) of gel diluent 5 to eac" tube.
#et%od to defi+rinate a )lot
#aterials
4. Sie#e and pestle
5. (lotted blood specimen
Pro)ed*re
4. Spin t"e specimen until a good separation occurs bet$een t"e clot and serum (9%7?? rpm for 7 minutes).
5. Pipette t"e serum into a clean test tube labeled $it" t"e patient1s name% account number% and date of t"e
sample.
9. Place t"e sie#e o#er a Petri dis".
<. Pour t"e remaining clot onto t"e sie#e and mas" $ell $it" t"e pestle. 'inse t"e sie#e $it" saline.
7. Transfer red cells suspended in saline from t"e Petri dis" into clean 49-4?? mm test tubes labeled $it"
patient1s name and date of sample. 'inse t"e Petri dis" and transfer t"e saline to t"e labeled tubes.
=. Spin t"ese tubes at 9%7?? rpm for l minute. Aspirate t"e supernatant saline and pool t"e packed cells% rinsing
remaining cells from t"e tubes $it" saline.
6. (ontinue pooling and centrifugation until all t"e cells are contained in one test tube.
(Blood Bank Name and address)
Procedure number: 202 Page 3 of 3
Subject: Preparation of Red Blood Cells for Analysis
C. 8as" t"e cells until t"e supernatant is free of "emoglobin.
N;T*: 2ailure to rinse tubes $it" saline results in loss of many cells due to t"eir ad"erence to t"e glass.
Preparation of a tandard Cell *spension
Denerally% a 97> red cell suspension concentration is used for testing patient and donor red cells in t"e form of a
crossmatc"% a +AT% or an antigen typing procedure. A standard may be prepared (see table belo$) for use as a
comparison.
2inal (oncentration 5> 9> <> 7>
'B( #olume% m@ 2inal #olume% diluted $it" saline% in m@
?.4 7 9.9 5.7 5
4.? 7? 99 57 5?
Adopted 'e#ie$ed 'e#ie$ed
'e#ie$ed 'e#ie$ed 'e#ie$ed
'e#ie$ed 'e#ie$ed 'e#ie$ed
'e#ie$ed 'e#ie$ed 'e#ie$ed