PRACTICAL

BLOOD BANKING Dr.MarwanA.Ibrahim AssistantProfessorofAppliedPhysiology DepartmentofMedicalLaboratoriesCollegeofAppliedMedicalSciencesMajmaahUniversity

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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:

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.

Forward (Red Cell grouping)

Reveres (serum grouping)

Performed in bench1&2

Performed in bench 2

Non

Serum Cell

(Ab) (Ag)

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. Group+

-No aggl 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 if still ve Add

albuminincubation 20mins wash 2 time 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) centrifuge read agglutinantion.

- If +ve gp D +ve (weak Rh Ag).

N.S Anti-D Anti-A Anti.B (2 drpos of each antisera)

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

Anti-A Anti-B Anti-D Blood group

+ - + A+

- + + B+

- - + O+

+ + + AB+

To make

suspension

Figure: Tubes loaded with antis and normal saline ready to be uesed for blood

grouping during BG and X-matching prosedure.

Table: Grading Table.

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 B O Blood group

- - + - A

+ + - - B

+ + + - O

- - - - AB

Grade Score Notation of serological reaction

4+ 12 One solid clump, no free cells,clear supernatant

3+ 10 Several large clumps ,clear supernatant

2+ 8 Many medium-sized clumps,cluody red suepertanten

1+ 5 Many small clumps cluody red supernatant

_+ 3 Many very small clumps

(+) 1 Apear negative, but visible microscopicslly

0 0 -ve macro-, and microscopically

Hs Strong

hemolysis Red supernatant, few or no intact RBCs

H Moderate hemolysis

Pink supernatant ,some intact RBCs

Hw Trace

hemolysis Pink-tinged supernatant,many intact cells

mf Mixed field Small, tightly agglutinated clumps, againest a background of free cells microscopically, the clumps refractile

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 mins then

reading the agglutenation directly are sometimes done!(false ve ).

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 +

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.

Centrifuge machine

AC

M1

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 cross-

matching 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

Serum remainig

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.

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

1st pregnancy

Rh ve mother

Rh +ve baby

2nd

pregnancy

Rh ve mother

Rh +ve baby

The mother produce

Ab againest Rh Ag.

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

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.

! ! ! !

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

Platelets concentrat

e

Whole Blood (WB)

Packed RBCS