By :

FAISAL ALMUTAIRI
SAMPLE RECEIVING:

• Plain tube (red cap): SERUM for cross matching

and ICT, Reverse ABO
• EDTA tube ( lavender tube ): whole blood for ABO,
cross matching, DCT

Test in blood bank lab :

• ABO (BLOOD GROUP ) TEST

• Weak D Testing (Du) Testing
• direct coombs test (DCT)
• indirect coombs test (ICT)
• cross matching
• antibody identification

1. ABO (BLOOD GROUP ) TEST

There are four major blood groups and eight different blood types. Doctors call this
the ABO Blood Group System.
The groups are based on whether or not you have two specific antigens -- A and B:

• Group A has the A antigen and B antibody.

• Group B has the B antigen and the A antibody.
• Group AB has A and B antigens but neither A nor B antibodies.
• Group O doesn’t have A or B antigens but has both A and B antibodies.

There’s also a third kind of antigen called the Rh factor. You either have this antigen
(meaning your blood type is “Rh+” or “positive”), or you don’t (meaning your blood type
is “Rh-” or “negative”). So, from the four blood groups, there are eight blood types:

• A positive or A negative
• B positive or B negative
• AB positive or AB negative
• O positive or O negative
Method to identify ABO grouping:

• Full Automated by machine ( tango machine , ortho autovue)

• Semi-Automated by Gel card
• Manual method by tube method or slide method

A- Full Automated by machine ( tango machine , ortho autovue)

Ortho autovue innova machine :

Principle :
The ORTHO BioVue system column agglutination technology standardizes blood
typing and compatibility testing in a 6-microcolumn cassette preloaded with diluent
and/or reagent and glass beads. Test red cells, with or without serum, are placed in the
chamber above the column. Upon centrifugation, red cells are forced through the bead
column where agglutinated cells are trapped, while unagglutinated red cells travel to the
bottom of the column, forming a discrete pellet.
B- Semi-Automated by Gel card.

• Prepare cell suspension 200 ul normal saline + 10 ul blood

• Take 10 ul from cell suspension and add to A, B,AB,AND CONTROL
• For Reverse ABO take 10ul from A cell and B cell and 40 ul serum Add to A1
and B1.
• Centrifugation 10 minute
• Read result

Forward Reverse
Ag A Ag B Ag AB A cell B cell
Group A +ve -ve +ve -ve +ve
Group B -ve +ve +ve +ve -ve
Group AB +ve +ve +ve -ve -ve
Group O -ve -ve -ve +ve +ve

C- Manual method by tube method or slide method

• cells Wash three times

• Make cell suspension 3-5 %
• Take four tube for A,B,AB,D add two drops of anti A,B,AB,D and
• two drops of cell suspension
• Centrifugation
• Read result
slide method:

1. On the section of slide labeled anti-A place one drop of antibody A.

2. On the section of slide labeled anti-B place one drop of antibody B.
3. On the section of slide labeled anti-AB place one drop of antibody AB.
4. On new slide labeled anti-D place one drop of antibody D.
5. Place one drop of cells in each antibody containing circle.
6. Carefully mix each solution with a separate applicator stick.

7. Tilt slowly for one minute, then observe for the agglutination.

Reverse ABO ;

• Back or reverse type with A and B cells

• Commercially available A and B cells are used.
• Patient serum added to the known cells.
• Used as confirmatory for the forward method.
• Reverse should be oppositely forward blood group

A and B cell preparation :

• Take segment from donor bags known blood group A and B

• Add segment to labeled test tube A cell tube and B cell tube
• cells Wash three times
• Make cell suspension
 2. Weak D Testing (Du) Testing.

Some red cells possess the D antigen but it is expressed so weakly that the cells are
not agglutinated directly by anti-D sera. An indirect antiglobulin test is necessary to
identify patients with the Weak D (formerly known as Du )phenotype. Weak D testing
is done on all prenatal patients and candidates for Rh immune globulin. Weak D
testing is also done on Rh negative donors to ensure they are truly D negative. It
may or may not be done routinely on Rh negative candidates for transfusion,
depending on the policy of the transfusing institution. If routine weak D testing is
done, weak D positive patients should receive Rh positive blood

Procedure of (Du) Testing.

• If the Rh test is negative ?
• Prepare a washed, 3% suspension of patient cells
• In test tube Add one drop of suspension and two drops anti D and two drops
bovine albumin .

• Incubate tube at 37oC for 15 to 30 minutes.

• Wash three time
• Add coombs reagent
• Centrifugation 30 sec
• Read result under microscopy
• When result positive Rh+
• When result negative add coombs control cells (CCC) and Centrifugation 30 sec
• After add (CCC) result should be positive
• If result negative repeat test

Other blood group systems

❖ The MNSs System

This system was discovered by injecting animals with human red cells. MNSs genes on
chromosome 4.There are two loci: M/N and S/s. The antigens are M, N, S, and s. There
are naturally occurring (IgM) antibodies to all these antigens. Anti-S and anti-s
commonly develop immune characteristics (IgG class) as a result of pregnancy or
transfusion.
 ❖ The Lewis System

This system was focuses on a single locus with two antigens, Le a and Le b. These
antigens do not form an integral part of the red cell membrane, but are soluble antigens
which may be present in body fluids and secretions. They are adsorbed on to the
surface of red cells if they are present in the plasma in sufficient amounts. There are
only three phenotypes: Le(a-b-); Le(a+b-); and Le(a-b+). Lewis phenotypes may change
during pregnancy. Examples of Le(a+b+) are only transient. Lewis antibodies are only
found in Le(a-b-) individuals, and are almost entirely IgM. They are the only Blood group
antibodies which have never been implicated in HDN (hemolytic disease of the
newborn.)

❖ The Kell System

In this system there are four antigens at two loci: K (Kell) and k (cellano), and Kpa and
Kpb. The Kp(a+) phenotype and the Kp(a-b-) phenotype are both rare. The Knull
phenotype K- k- Kp(a-b-) is associated with chronic granulomatus disease (CGD), an
inherited defect in the bacterial capacity of neutrophils. Antibodies to Kell system
antigens are IgG. Named for the family of the antibody producer Mrs. Kellacher.

❖ The Lutheran (Lu) System

This system is a single locus system, with antigens Lua and Lub. The Lu(a) negative
phenotype is very rare. Antibodies to Lutheran antigens are IgG. The genes of the
Lutheran group are linked to the genes responsible for the secretion of ABH
substances.

❖ The Duffy System

The Duffy system is also a single locus with two antigens, Fy a and Fy b. The only rare
phenotype is Fy(a-b-), which has a higher frequency in countries where there is a high
incidence of Plasmodium falciparium malaria. This phenotype gives a degree of
immunity to the disease because the malarial parasite requires Duffy antigens to enter
the red cells. Duffy antibodies are almost exclusively IgG. This system is named after
the family of the antibody producer, Duffy.

❖ The Kidd (Jk) System

Another single locus system, two antigen system (Jka and Jkb). There are four possible
phenotypes: Jk(a-b-); Jk(a+b-); Jk(a-b+); Jk(a+b+). Jk(a-b-) is a rare phenotype.
Antibodies to the Kidd antigens are almost exclusively IgG.
Incompatible transfusion or pregnancy can lead to the formation of antibodies to all
these Blood groups, if the recipient/mother lacks the relevant antigen. It is possible to
detect all red cell antibodies using an antibody detection panel and different detection
techniques. (Some antibodies, usually IgM class, react best at room temperature or
cooler, and some work best at 37 degrees entigrade). If an antibody is detected in a
serum the red cells from that patient are tested for the presence of the antigen. Antigen
detection techniques also vary according to the nature of the antibody-antigen
interaction. The presence of a particular antibody specifically excludes the patient from
carrying that antigen.

❖ The P System

This system was also discovered by injecting animals with human red cells. P1 is the
most common antigen which has variable strength of expression. Anti-P1 may be
naturally occurring. It is most often an IgM antibody

❖ The I system

This system found on RBC membranes as well as in plasma, milk, and amniotic fluid
These antigens may be I or I .They form on the precursor chain of RBC Newborns have
strong i antigen and Adults have strong I antigen

3. direct coombs test (DCT)

What is this test?

This test detects immunoglobulin and/or complement bound to red blood cells surface
antigens in vivo.. Immunoglobulin and complement are involved in the immune system.
This test is used to evaluate for antibody-induced hemolysis (destruction of red blood
cells)
You may need this test if you have:

• Autoimmune hemolytic anemia

• Cold autoimmune hemolytic anemia

• Hemolytic disease of fetus OR newborn due to ABO immunization

• HUS - Hemolytic uremic syndrome

• Paroxysmal cold hemoglobinuria

• Transfusion reaction due to blood group antibody-antigen mismatch

Procedure of DCT .
• cells Wash three times
• Make cell suspension 3-5 %
• In test tube add one drops from suspension and two drops bovine albumin
• Centrifugation 30 sec
• Read result .when result negative ? keep tube at room temperature for 10 minute
• Centrifugation 30 sec
• When result negative add coombs control cells (CCC) and Centrifugation 30 sec
• After add (CCC) result should be positive

4. Indirect coombs test (ICT)

The indirect Coombs test (also known as the indirect antiglobulin test or IAT) is used to
detect in-vitro antibody-antigen reactions. It is used to detect very low concentrations of
antibodies present in a patient’s plasma/serum prior to a blood transfusion. In antenatal
care, this test is used to screen pregnant women for antibodies that may cause
hemolytic disease of the newborn. The IAT can also be used for compatibility testing,
antibody identification, RBC phenotyping, and titration studies.
Procedure of ICT .
• Label 3 tubes as cell 1, cell 2 and cell 3.
• In the tube labeled as cell 1, take 2 drops of serum. And 1 drops of cell 1
• In the tube labeled as cell 2, take 2 drops of serum. And 1 drops of cell 2
• In the tube labeled as cell 3, take 2 drops of serum. And 1 drops of cell 3
• Add 2 drops of bovine albumin to each tube.
• Incubate all the tubes at 37°C for 45 – 60 min
• Wash the cells 3 times with normal saline
• Add 2 drops of Anti Human Globulin to each tube
• Centrifugation 30 sec
• When result negative add coombs control cells (CCC) and Centrifugation 30 sec
• After add (CCC) result should be positive

5. Cross Matching (Compatibility test).

Cross Matching is a procedure performed prior to a blood transfusion to determine

whether donor blood is compatible (or incompatible) with recipient blood. Compatibility
is determined through matching of different blood group systems, the most important of
which are the ABO and Rh system, and/or by directly testing for the presence of
antibodies against a sample of donor tissues or blood.
Cross-matching will detect incompatibilities between the donor and recipient that will not
be evident on blood typing. There are two types of cross-matches: Major cross-match
and Minor cross-match.

The major cross match involves testing the patient’s serum with donor cells to
determine whether the patient has an antibody which may cause a hemolytic
transfusion reaction or decreased cell survival of donor cells. This is the most important
cross-match.
The minor cross match involves testing the patients cells with donor plasma to
determine whether there is an antibody in the donor’s plasma directed against an
antigen on the patient’s cells.

• Selection of Appropriate Donor Units.

Red cell compatibility.

It is a general principle that red cell components of identical ABO group and RhD type
as the recipient should be used for transfusion. O Rh negative is the universal red cell
donor blood that can be given to all patients. This is common practice when a patient’s
blood group is unknown and in emergency situations especially for women of child-
bearing age.
Plasma compatibility
Plasma contains anti-A and anti-B antibodies depending upon the blood group. Our
body also has antibodies to A and/or B antigens according to our blood group. Patients
should only receive plasma that does not contain an antibody which could attack the
antigens present on their own red cells.
Group A recipients have A antigen on their red cells, so they can’t receive group O or
group B plasma as the anti-A will attack their red cells. Group B recipients have B
antigen on their red cells, so they can’t receive group O or group A plasma as the anti-B
will attack their red cells.
Group AB recipients can only receive group AB plasma. Group O recipients do not have
either A or B antigen, so can safely receive plasma of any blood group type.

Platelet Compatibility
ABO identical platelets are usually preferred. However, in some circumstances, the
need for other special requirements may be more important than providing the same
ABO group.

Cross Matching procedure.

Tube method

• Prepare a washed, 3% suspension of donor cell

• Label a test tube. Add two drops from the patient serum and two drops
from the appropriate donor cell suspension.
• Mix the tubes and incubate at 37°C for about 45 minutes.
• Wash the cells 3 times with normal saline
• Add two drops of AHG (Antihuman globulin) and mix.
• Centrifugation 30 sec
• Read macroscopically and microscopically and record the results
• If result compatible “no reaction” add CCC and Centrifugation 30 sec
to confirm your result
• After add (CCC) result should be positive.
 Gel card method
• prepare a donor cell suspension 200 ul normal saline + 10 ul blood
• take 40 ul serum and 10 ul donor cell suspension and 50ul Bliss (normal
saline)
• incubation for 15 min
• Centrifugation 5-10 min
• Read result

Immediate-spin cross-matching (ISCM)

Immediate-spin cross-matching is an abbreviated form of cross-matching that is faster,
less expensive but also less sensitive. It is an immediate test that combines the patient's
serum and donor's red blood cells at room temperature. No agglutination indicates a
negative test reaction, or compatible match. Indications for ISCM are dependent on the
circumstances of the patient and it can be used in place of a full cross-match or
performed as a preliminary test.
6. antibody identification

principle:
Patient serum/plasma is tested against an Identification panel of reagent red cells that
are fully antigrammed for the antigens of the major and minor B.G systems using the
same technique with which Ab detected in screening or cross matching (for eluate from
DCT positive cells additive techniques are held). The positive and negative reactions
should be compared with the panel profile in conjunction with the screening
results. Determining the specificity of an unexpected alloantibody is important in pre-
transfusion and prenatal testing. If the antibody specificity is known, it is possible to test
donor blood for the absence of the corresponding antigen. Antibodies should also be
identified in donor blood so that this blood is not transfused to antigen-positive
recipients.
PROCEDURE
The patient's sample should first be tested with Screening Cells I and II. If one or both of
these show agglutination, proceed with the antibody identification.
1. Select a cell panel and the corresponding antigen matrix. Be sure the lot number
on the cell panel matches the lot number on the antigen matrix.
2. Fill in all known patient information on the cell panel worksheet.
3. Number as many tubes as there are cells in the panel. Include the patient's
initials on each tube.
4. Using a controlled drop dispo pipette held at a consistent angle, add 2 drops of
patient serum to each tube.
5. Gently invert the panel cells several times to resuspend.
6. Add one drop of the appropriate panel cells to each corresponding numbered
tube.
7. Set up an autologous control, if none was run with the antibody screen:
 8. To a tube labeled with the patient's initials and "auto", add 2 drops patient serum
and one drop of a washed 3% suspension of patient cells.
9. If the Screening Cells were negative at the Immediate Spin phase, proceed
directly to step 11. If they were positive at Immediate Spin, centrifuge the tubes the
calibrated time for saline and continue with step 10.
10. Gently resuspend and examine for agglutination using the lighted agglutination
viewer.
11. Record all reactions under a column headed IS (for Immediate Spin) on the panel
worksheet.
12. Add 2 drops PEG to all tubes, shake to mix, and incubate 10 - 30 minutes at
37oC. Note that you must incubate at least 15 minutes if using a dry heat
block. (or add 2 drops of LISS & incubate for 15 - 20 min)
13. Wash all tubes three or four times with physiologic saline and add one
drop of Polyspecific Anti-Human Globulin to each tube.
14. Shake to mix and centrifuge the time appropriate to the Coombs spin calibration
in the serofuge (if using gel system).
15. Gently resuspend and examine for agglutination using the lighted agglutination
viewer.
16. Record all reactions under a column headed AHG on the panel worksheet.
17. The auto control may be positive or negative. If it is positive, do a DAT and get a
recent transfusion history and medications list.
18. Confirm all negative reactions with Coombs Control Cells. No agglutination after
centrifugation following the addition of Coombs Control Cells invalidates the cell
panel results

Autocontrol.
Patient RBCs + Patient serum
INTERPRETATION
• To determine antibody specificity, use the following protocol:
• Look at each negative cell and cross off all antigens that are present (positive) on
that cell. Use X for homozygous cells, and / for heterozygous cells.
• Eliminate antigens along the top of the cell panel by crossing off all that have
been crossed off at least three times in the antigen matrix, ideally with at least
one of them homozygous for the antigen. Do not eliminate antigens based on
only one or two heterozygous cells crossed off, especially if you are getting
different strength reactions on different cells.
• Exceptions to the above policy include:
• you may rule out Kell based on 3 heterozygous cells - no need for a homozygous
cross-off
• you may rule out low frequency antigens (Cw, V, VS, Kpa, Jsa, Lua) based on
only one cross-off, whether homozygous or heterozygous
• you may also rule out low-frequency antigens if there are no cells positive for
them on the panel
• if anti-D is present, you may rule out anti-C or anti-E based on three
heterozygous cells (r'r for C and r"r for E)
• From the antigens not crossed off, look for a pattern of agglutination matching
the pattern you got in the test. This should identify the antibody specificity.
• At this point there still may be one or more low-frequency antigens not crossed
off. If the cell that has the low-frequency antigen also has the antigen that
corresponds to the antibody you believe you have identified, you may now cross
off this low frequency antigen, because the positive reaction on this cell is most
likely due to the antibody you have identified.
• Often you are unable to rule out the possibility of a second or third antibody
because the corresponding antigens are all present on the same cells. For
example, you have identified Anti-Jka but can't eliminate Anti-Kell because
all Kell positive cells are also Jka positive. Check other cell panels and find 3
other cells that are Kell positive and Jka negative. A negative result when testing
these cells with the patient's serum eliminates that antibody; a positive result
confirms the second antibody.
• When the antibody(ies) have been identified, be sure there are at least 3 cells
that are possess that antigen and give a positive reaction, and at least 3 cells
that are lack that antigen and give a negative reaction. Always check the results
of the screening cells. You may have to test more cells from other panels.
• As a final confirmation of the antibody specificity, if the autocontrol is negative or
only weakly positive, type the patient's cells for the antigen. The result should be
negative, unless the patient has been recently transfused. The result will then be
a mixed field.
• If you get different reactions at different phases of testing; if you get different
strengths of reactions on different cells, or if your results don't form a pattern
corresponding to one of the antigens on the matrix, consider multiple antibodies.
See (6) above.
 • If you get a pattern of reactivity not matching any of the antigens on the matrix,
the antibody may be showing dosage and reacting only with homozygous cells.
This is most often seen in the MNS and Kidd systems, but can be seen in other
systems. Eliminate only the non-reactive homozygous cells; the pattern of
reaction may then match an antigen present only on the remaining homozygous
cells.

Confirmation:
Q: how to confirm such a situation?
Sample antibody ID problems
1-A 27-year-old female comes in for a tonsillectomy. She’s never been transfused or
pregnant. The above panel is performed. What is the antibody and what should you do?
Answers to Panels

1. Anti-Leb

Single cold-reacting antibody. Anti-Leb reacting at these temperatures is not clinically

significant, so no specific interventions will be necessary. You might consider using
prewarmed crossmatches to eliminate the antibody activity.
2-A 19-year-old G3P1Ab1 female comes in at 28 weeks for routine prenatal exam.

2. Anti-D
Note that this is likely a gel or solid-phase panel (though it could be a liquid panel only
recording the IAT reactions). Single warm-reacting antibody. Fairly straight-forward
identification. Check the clinical situation (and don’t forget to ask about recent RhIG
injection or infusion!).
3. A 45-year-old male with a myelodysplastic syndrome and history of multiple
transfusions.

3. Anti-K and anti-E

After your cross-offs, no single antibody explains all of the reactions, so you should try
to fit two antibodies (again, this panel only shows IAT results, so no concern about
different “phases” here). Anti-K and anti-E is the best fit. Note the slightly weaker
reactions in cell 6 due to dosage.
 ➢ Preservation and storage of Blood component

Blood component Storage temperature Shelf life

Red cells: 42 days
Red cells 2–6 ºC Paediatric red cells: 35 days
Washed red cells: 28 days

Platelets 20–24 ºC 5 days

Fresh frozen plasma.

cryoprecipitate At or below –25 ºC 12 months

➢ Anticoagulant used in blood bank

➢ CPD AND CPD-A1

Citrate-phosphate-dextrose (CPD) and citratephosphate-dextrose-dextrose (CP2D).

Allows 21 days of RBC/Whole Blood storage.
Citrate-phosphate-dextrose-adenine (CPDA-1) has extra adenine compare to CPD or
CP2D as a source of ATP, which allows 35 days of RBC/Whole Blood storage.

➢ SAGM

SAGM is a combination of constituents as additive solution to give the red cell optimum
viability. It actually stands for
- Sodium Chloride: provides isotonicity
- Adenine: maintains ATP for red cell viability
- Glucose: supports red cell metabolism
- Mannitol: helps reduce red cell lysis

Preservation of red cells in SAGM solution can keep the life of red cells to up to 42
days, longer than using CPD Solution and CPDA solution alone.
➢ Sample rejection slip
o Specimen hemolyzed

o Specimen clotted

o Quantity not sufficient (QNS)

o Incomplete request form

• No doctor sign or stamp

• No diagnosis

• Test not recommended

o Mismatched file name, number on request and tube

• Specimen unlabeled

• Specimen mislabeled

• Specimen inadequately labeled

o Wrong collection tube

: ‫❖ مجهود شخصي‬

" ‫ "كل ما احتاجه دعواتكم‬... ‫❖ إن أصبت فمن هللا وإن اخطأت فمن نفسي والشيطان‬