PRACTICAL

BLOOD BANKING

Dr.MarwanA.Ibrahim
AssistantProfessorofAppliedPhysiology
DepartmentofMedicalLaboratories
CollegeofAppliedMedicalSciences
MajmaahUniversity

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ABO grouping: It is one of the most important tests carried out in daily routin
of blood banking.It is performed on all potential transfusion recipients and
blood donors. The imprtance of the test is due to the nature of the ABO
system.un like other blood group system the antibodies (Abs) are present in
the serum of all the adults where the corresponding antigen (Ag) is absent.

ABO procedure is devided in to:

Forward
(Red Cell grouping)

(Ab)

(Ag)

Serum

Cell

Reveres
(serum grouping)

Performed in
bench1&2

Performed in bench
2

Non

Rh grouping: The Rh grouping system consists of a number of antigens (D, C,

E, c and e), the most important of these is Rh (D) Ag, and it is the prsence or
absence of this Ag that defines whether a person is termed Rh (D) negative or
Rh (D) positive.
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P/ts red blood cells (RBCs) tested againest known antisera.

-Slide & plate test.
-Tube method.

Tube test:
Anti-A, Anti-B, Anti-AB *(not used), Anti-D, and normal saline tubes), must be
prepaired and kept in rack before the sample are received to save time for BG,
and X-matching.

PROSEDURE:
1- Give the sample seq. number (the same number must be on its request)
2-Wash the blood 3 time to remove unwanted Ag, and other substances
which may interfer with agglutination.
Wash is important >> Why?
-help to remove non specific unwanted Ags, Abs.
-Get sure (specific) results.
3- make suspension sus (2- 5%), by adding approximatly 2 drops of blood +
20 drops 2 ml of Normal saline (N.S).
Suspension is important to:
Make the Ag/Ab reaction more clear, and to avoid false ve or +ve results.
Very heavy suspension may give false +ve , very light give very weak view
and so, false ve or giving doubtful view

need microscopical examination

more time is wasted .

4- Move 2 drops of suspension to previously prepaired (Anti-A, and Anti-B)
tubes 1:1, and 2:1 for Anti-D. Ratio is important to get good, readable, clear
and sure results.
5-Centrifuge for 15 sec. High speed (or 20 sec at 3000rpm)
6- Dislodge the cell button and observe aggl. (Macroscopic examination).
7- Give score, and record it.
In case of Anti-D tube;if:
-Agglutination.
-No aggl

Group+

Group

see under microscope look for any clumps (not just sticking)

e.g, A+ = ABO grouping = A (mean the p/t have A Ag on his RBCs)

Rh grouping=+ (mean the p/t have rhesus monky Ag on his RBCS), and vice versa.

Microscopical examination: It used with Rh ve group befor and after testing for D
Ag.

-ve D tube

microscopic examination

albumin incubation 20mins

if still ve

wash 2 time

Add

add coombs reagent

centrifuge and read,

- If ve

gp is ve.

In D -ve blood bags they use 2 tube loaded with Anti-C & Anti-E (C & E are
weak Rh Ags)
- If +ve

centrifuge

read agglutinantion.

gp D +ve (weak Rh Ag).

To make
suspension

N.S

Anti-D

Anti-A

Anti.B (2 drpos of each antisera)

Figure: Tubes loaded with antis and normal saline ready to be uesed for blood
grouping during BG and X-matching prosedure.

Table: Forward ABO blood grouping. + = agglutination, - = no agglutination.

Anti-A

Anti-B

Anti-D

Blood group

A+

B+

O+

AB+

Table: Grading Table.

Grade
4+
3+
2+
1+
_+
(+)
0
Hs
H
Hw
mf

Score
12
10
8
5
3
1
0
Strong
hemolysis
Moderate
hemolysis
Trace
hemolysis
Mixed field

Notation of serological reaction

One solid clump, no free cells,clear supernatant
Several large clumps ,clear supernatant
Many medium-sized clumps,cluody red suepertanten
Many small clumps cluody red supernatant
Many very small clumps
Apear negative, but visible microscopicslly
-ve macro-, and microscopically
Red supernatant, few or no intact RBCs
Pink supernatant ,some intact RBCs
Pink-tinged supernatant,many intact cells
Small, tightly agglutinated clumps, againest a background of
free cells microscopically, the clumps refractile

Slide test (rapid test):

1. Whole (not susbended blood) can be used.
2. Mix with wooden applicator.
3. Gently rotate the slide for a peroid not exceeding 2 minutes. Agglutination
occurs within 30-60 sec.

+#

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Here the p/ts serum tested againest known A, B, O cells.

Test cell
A1

A2

Blood group

AB

PROSEDURE:
1-take samples from any bags with known (group A, B and O)
2- Wash the bag cell

makes suspension.

3- Centrifuge the p/t sample

serum.

4-add 2 drops of serum on blood suspension.

5- Centrifuge (better to incubate15-30 mins at room temp. 1st )
6- Dislodge the cell button and observe the agglutination.
In case of group O serum

continue.

7-Incubate at 37C /1 hr.

8-centrifuge

look for haemolysis in tube A & B.

if we have agglutination

high Anti-A.Anti.B titer

dangerous O

only can be given to group O p/ts.

As shown in table (O) tube (tube with O cells) is always give ve results in
normal people.
In Bombay Phenotype p/ts, O tube give +ve results because of the presence
of Anti-H Abs in his serum (not present in normal people).
-agglutination in O tube

mean high Antibody titration

given to O blood

group p/t Only- and the blood bag labeled (only for O), dangerous O group.
Actions affect test Quality Control:1. No wash before performing the procedure, this may affect the specificity
and sensetivity of the test, because washing help to remove many unwanted
Ags this step point is missing in all blood bank porsedure.
2. Suspention have no standard concentration, (desired 2-5%), and that
make it hard to reading and comparing the tubes.
3. As known anti-A and anti-B,antiAB antibodies are IgM Abs and its activity
are reduced delayed- in high temperature like body temperature 37C, with
this some time Incubation of the plate in the incubator for 5-10
reading the agglutenation directly are sometimes done!(false ve ).

mins

then

4. Ag/Ab ratio is variable and that may adversly affect their reaction, like
adding 1 drop of anti-A with 3 or 4 drops of blood suspension(with antiA,and
B usually 1:1 is the best zone of equivalence).
5. Rh grouping: Wash before adding coombs reagent in detecting (D) some
time ignored.

-#

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The function of X-match is to prevent ABO incompatibility ( between the p/t

and blood bag) , particularly when A or B cells are transfused into a group O
person, with a high titer of anti-A/anti-B, it is important to note that in addition
to agglutination,haemolysis is also a sign of incompatibility. The actual
procedure followed will depend on the clinical circumstances, scince
emergency situation the testing protocol may have to be modified in
order to provide blood as quickly as possible to the patient (P/t).
Major cross-match = p/ts serum + blood bag RBCs sus.
Minor cross-match = blood bag serum + p/ts RBCs.
PROSEDURE:
If blood transfusion request form received,
-Check the p/ts name and file number.
-Check diagnosis, haemoglobin level, blood group if known.
-Check blood unit type and number.
-In performing Major cross-match prosedure we have 3 steps (phases), ve
result of the 1st moving to the next step:
1). Saline phase
2). LISS or 22% Bovin albumin phase
3). Coombs (anti-human globulin) phase.
-In blood bank we put step 2 and 3 in one step and check for agglutination once.
e.g 1 RBC or (whole blood) unit is wanted:

1. Give the sample seq. number (the same number must be on its
request form).
-The 1st sample in morning shif labeled

M1,.. and so on.

2. Lable 2 test tube with (AC1) and (SP1), and 3rd extra tube to
save the remaining serum and seal it with the sample tube with
parafilm.
3. Centrifuge the tube sample D5BB
(AC)= Autocontrol

(Sp) = Serum protein

P/ts serum
+

p/ts serum

p/ts Cells

Centrifuge machine

+
Bag cells

Make sus. From the sample and blood bag

blood grouping for both of them

(even if known).
- if the p/ts group unknown

wait for BG and then take the same group from the

blood bags (or other blood component).

4-Add 1 drop of sus. Into Ac, SP tubes.
5-add 2 drpos of serum in to Ac, Sp tubes.
6-Centrifuge

15 sec/3000 rpm

7-Look for haemolysis +agglu. ((SALINE PHASE))

8-if ve

add 2 drpos albumin

9-incubate (20 mins)

10-wash 2 time at least.
11-add 2 drops of coombs reagent.
12-centrifuge
13- read

-ve

microscopical exam.((COOMBs PHASE))

14- Record the result.

AC
M1

Serum remainig

SP
M1

serum+RBC

sus.

Anti-D

Anti-A

Anti-B

Cross-matching
- URGENT cases: request for blood transfusion need to give blood directly
and do not wait for donation from p/ts relatives has high priority on other
requests like:

R.T.A ( Road Traffic Accident)

CCU (Cardiac Care Unit).

CRF (Chronic Renal Failur).

Other wise we check the haemogolin (Hb) level to asses whether the crossmatching must be done immediatly or can wait.
The doctor ask for blood in EMERGENCY TRANSFUSION REQUEST
uncross matched, urgently crossmatched, only ABO & Rh (same as p/t), or
group O. other wise we check the haemoglobin level to deside whether the
p/t need the blood now or can wait for a donation.
Actions affect Cross-matching Quality Control:1.Using different suspention concentration make it hard to compare between
tubes from the first look, AutoControl(AC) test tube (p/ts cells and serum)
used to detect any auto Abs and usually as ve control in normal people for

other test tube contains sample from blood bags want to be X-matched with
p/ts serum , but if we use different suspension concentrations in these tubes
the role of AC tube is reduced because it is hard to compare especially if the
reaction is weak.
2. Microscopical evaluation is not always performed on ve results, and rarly
used by some tec. while some other use it frequently in X-matching ve tubes.

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To detect already sensetized RBCs e.g Haemolyitc disease of new born.

1st pregnancy
Rh ve mother
Rh +ve baby

The mother produce

Ab againest Rh Ag.

2nd pregnancy
Rh ve mother
Rh +ve baby

PROSEDURE:
1- Take 5 drops of blood sample.
3-make sus (2-5%)
4-add cooms reagent (2drops).
5-centrifuge
6-dislodge the cell button and observe agglutinatiom.
Actions affect DCT Quality Control:There are differences between the procedure followed by each technician.
Some important steps are missed like:
1-Wash before adding Coombs reagent.
2-Coombs control check cells are not available (CCCC), coombs control
not used, but some staff use tube with Rh+ve O group + anti-D and add

coombs reagent and run it with our DCT sample as +ve control and to
insure that coomb;s reagent are working.
3- +ve results of DCT are not followed with other step to identefy the Abs:
(elution and then titration).
Elution: (wash out material) to remove one substance from another, usually an adsorbed material from an adsorbent surface, by washing it out
with a solvent

1. Normal Saline (0.9% NaCl):

To maintain osmotic balance and prevent the RBC from getting shrinking (if
NaCl is less than 0.9%), or swelling up and get burst, which will interfer with
aggl. reaction.

2. 22% -- 30% Bovin Albumin:

Help to reduce the charge arround the RBCs, which may cuase a repulsion
between the cell and the Abs and also between one cell and another. And
so, make the agglutination easier to occur, and help it to be more clear.
Usage:
-ve tubes of incombatibility test (X-matching) before adding coombs reagent.

3. Coombs reagent (Anti-Human Globulin) or anti-antibody:

IgG anti-body or incomplete antibody, have y shape (2 hands only)

make

very small clot (not easy to see or detect), unlike the IgM class Abs with 5
hands

make visible clot or agglutination.

So, if we have weak +ve agglutination (IgG attached to Red cell surface), we
need to add coombs reagent to be attched to that IgG on the cell surface
and make a big visible clot.

Negative AHG reaction

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Fig: AHG by binding to IgG Abs on cell surface, bring the cells close to each
other and so the reaction is more visible.
Usage:
In tests detecting IgG class Abs.
1- DCT.
2- IDCT (X-matching, Antibody screening).
3- with ve Rh group to detect Du (weak D or Rh Ag).

4. Store the platelets at RT (Room Temperature):

Platelets when stored at refregerator show higher activity but removed quickly
from the circulation.

5. Anti-AB;
- To be sure of the results obtained with anti-A, Anti-B.

It helps to identify group A2, which give ve reaction with anti-A and anti-B
alone.
That mean if we get no agglutination with anti-A and anti-B that does not
mean necessary that the blood group is O, it may be group A2 by the using
of Anti-AB we can decide.

6. Incubator;
Anitibody classes are (IgG, IgA, IgM, IgE, and IgD).
In blood bank we deal with IgG (e.g Anti-D) and IgM (anti-A and Anti-B).
IgG= act better in hight temperatur (37C) incubator.
IgM= Cold antibody act well in room temperature (RT), and so if incubated in
37C that may delay the reaction.

7. Anti-H:
Major Ag in human RBCs are:
A antigen (in A group)
B antigen (in B group)
H antigen (in all blood group but in different persent) the greatest amount is
present on red cells of O group, intermediate amounts are present on red
cells of A1 and A1B. Blood with out H antigen is extremely rare (Bombay).
And so used to distenguish between normal O group and Oh (Bombay
group).
No agglutination with anti-H)

Fig: Simple chemical strucure of ABO antigens.

!
Whole
Blood (WB)

Platelets
concentrat
e
Packed
RBCS

In blood bank the following manner are used in labeling the bags:

[Donor intials and number]

Date of donation

Date of Expiry

[unite type (RBCs Plt..etc)]

[Blood group (+ or -)]

signature