MLS 411: blood group serology

MODULE 2

BY

MLS A.M USMAN ([Link] Immun. BUK, [Link] UDUS, PDE)

Email: abbilusud81@[Link]
08039501900 or 08052838810
 Flow of Presentation
2

• Collection and Preparation of Samples

• Collecting Patient Samples
• Donor Samples
• Selection of Appropriate Donor Units
• Testing of the donor sample
• Testing of the patient sample
• Cross-match
• Summary of Pre-transfusion Testing
 COMPATIBILITY TESTING
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 As the knowledge of new blood group system increased, so did the search for more
sensitive pre-transfusion compatibility testing methods. Pioneer blood bankers
mixed the patient’s serum and the donor’s red cells and observed for direct red
blood cells lysis, agglutination, or both. This became known as the major cross-
match test.

 The term compatibility test and cross-match are some times used interchangeably,
they should be clearly differentiated.

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 COMPATIBILITY TESTING CONTD…
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 The compatibility testing or pre-transfusion testing refers to a set of procedures

required before blood is issued as being compatible. It include the following;
• Review of patients blood bank history and record.
• ABO and Rh typing
• Antibody screen for unexpected antibodies (ICT and DCT)
• Cross matching (major and minor)
• Extended antigen typing (e.g kell, Duffy etc.)
• Molecular Typing (DNA-based)
• Electronic Cross-matching (ECM)
• Gel testing
 COMPATIBILITY TESTING CONTD…
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 The cross-match became part of a series of pre-transfusion test known as
compatibility testing. The compatibility test includes an ABO and Rh grouping
performed on the donor and recipient samples, screening of the donor’s and patient’s
sera for unexpected antibodies, and a cross-match.

 The purpose of pre-transfusion or compatibility testing is to ensure the best possible

results of a blood transfusion.

 The transfused red cells will have an acceptable survival rate, and there will be no
significant destruction of the recipient’s own red cells.

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 COMPATIBILITY TESTING CONTD…
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 Collection and Preparation of Samples.
o Positive Patient Identification
• The major cause of transfusion associated fatalities have been clerical errors
resulting in incorrect ABO grouping

• The most common cause of clerical errors and thus transfusion accidents is
misidentification of the patient involved in the transfusion.

• Confusion in identification of the patient when the blood sample was drawn, a
mixed up samples during handling in the lab, and error in identification of the
patient when the transfusion was given.

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 COMPATIBILITY TESTING CONTD…
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 To prevent collection of samples from the wrong patient, the blood request form
must be used to confirm the patient’s identity before phlebotomy is performed. The
request form must state the intended recipient’s full name, and unique hospital
identification number.

 Other information such as age and date of birth, address, sex, and name of
requesting physician can be used to verify patient identity further but is not
required on the form.

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 COMPATIBILITY TESTING
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 The patient’s wrist band identification must always be compared with the
requisition form.

 If the patient does not have a wristband or if the patient’s identity is unknown,
some form of positive identification must be attached to the patient before
collection of samples.

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 COMPATIBILITY TESTING
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 Collecting Patient Samples
• Hemolyzed samples can not be used for testing Because of hemolysis caused by
activation of complement

• Serum or plasma may be used for pre-transfusion testing. Most blood bank
technologist prefer serum because plasma may cause small fibrin clots to form
which may be difficult to distinguish from true agglutination.

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 COMPATIBILITY TESTING
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• When a specimen is received in the lab, a blood bank technologist must confirm
that the information on the sample and requisition form agree.

• All discrepancies must be resolved before the sample is accepted, and if any doubt
exists, a new sample must be drawn.

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 COMPATIBILITY TESTING
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 Donor Samples.
• Donor testing samples must be taken when the full donor unit is drawn. Depending
on the method used for testing, clotted sample, anti-coagulated samples, or both,
are obtained.
• Donor information and medical history card, the pilot samples for
processing, and the collection bag must all be labeled with the same unique
number code before starting the phlebotomy, and the numbers must be verified
again immediately after filling.

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 COMPATIBILITY TESTING
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• Donor and recipient samples must be stored for a minimum of 7 days following
transfusion.

• The samples should be stoppered and refrigerated at 1-6°C, carefully labeled, and
adequate in volume so that they can be re-evaluated if the patient experiences an
adverse response to the transfusion.

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 COMPATIBILITY TESTING PROTOCOLS.
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 Selection of Appropriate Donor Units.

• In almost all cases, blood and blood components of the patient’s own ABO and Rh
group should be selected for transfusion.

• When blood and blood components of the patient’s type are unavailable or
when some other reason precludes their use, units selected must lack any Ag
against which the patient has a significant Ab.

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 SELECTION OF APPROPRIATE DONOR UNITS.
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• When transfusion of an ABO group different from the recipient must be given,
packed red cells must be used rather than whole blood which contains plasma Abs
that are incompatible with the patient’s red blood cells.

• Group O packed red blood cells can be safely used for all patients, however,
conservation of a limited supply of group O blood should dictate its use for
patients of other AB types only in special circumstances.

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 SELECTION OF APPROPRIATE DONOR UNITS.
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• If ABO-specific blood is not available or is in less than adequate supply,
alternative blood groups are chosen as summarized in the following table;

Patient ‘s BG Alternative BG given as packed cells

O NONE
A O
B O
AB A, B, O
only one of the three should be used for a given patient

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 SELECTION OF APPROPRIATE DONOR UNITS.
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• Rh-negative blood can be given to Rh-positive patients, however, good inventory
management again should conserve this limited resource for use in Rh-neg
recipients.

• If Rh-neg units is near expiration, the unit should be given rather than wasted.

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 SELECTION OF APPROPRIATE DONOR UNITS.
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• Rh-pos blood should not be given to Rh-neg women of

childbearing age.

• Transfusion of Rh-neg male patients and female patients beyond

menopause with Rh-pos blood is acceptable as long as no
performed anti-D is demonstrable in the sera.

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 COMPATIBILITY TESTING PROTOCOLS.
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 Testing of the donor sample.
• According to the Code of Federal Regulation (CFR) and the American Association
of BB (AABB) standards, ABO and Rh grouping (including a test for weak D) and
tests intended to prevent disease transmission must be performed on a sample of
blood taken at the time of collection of the unit of blood from the donor.

• A screening test for unexpected antibodies to red blood cell Ags is required by
AABB standards on samples from donors revealing a history of prior transfusion
or pregnancy.

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 TESTING OF THE DONOR SAMPLE
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• The transfusing facility is required by AABB standards to confirm
the ABO cell grouping on all units and Rh grouping on units
labeled Rh-neg.

• Tests for weak D (Du) are not required to be reported. The

transfusion facility does not need to repeat any other testing
procedure.

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 TESTING OF THE PATIENT SAMPLE.
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• A record of all results obtained in testing patient samples must be maintained.
• Identification number should be assigned each time a patient is admitted for
treatment.
• Any discrepancies between previous and current results must be resolved before
transfusion is initiated.
• A new sample should be collected from the patient, if necessary to resolve the
problem.
• ABO and Rh grouping results should be included in the file.
• Also, notations concerning unusual serologic reactions and the identity of
unexpected Abs in the patient’s serum should be included.

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 TESTING OF THE PATIENT SAMPLE.
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• ABO and Rh grouping and Ab screening of the patient’s serum
can be performed in advance of or at the same time as the cross-
match.

• If the patient has had a transfusion or has been pregnant within

the last 3 months or if the history is unavailable or uncertain, the
sample must be obtained from the patient within 3 days of
scheduled transfusion.

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 TESTING OF THE PATIENT SAMPLE.
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 ABO Grouping.
• Determination of the patient’s correct ABO group is the most critical pre-
transfusion serologic test.

• If the cell and serum grouping results do not agree, additional testing must be
conducted to resolve the discrepancy.
• If the patient’s ABO group cannot be satisfactorily determined and immediate
transfusion is essential, group O packed red blood cells should be used.

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 PRINCIPLE OF ABO GROUPING TECHNIQUE

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 The principle of ABO grouping is based on a specific antigen on red cells and
1gM antibodies in the typing serum (which can be either forward or serum
grouping).
 It has the following methods

• Glass slide or white porcelain tile method

• Glass test tube

• Micro well plate or micro plate.

PROCEDURE OF REVERSE GROUPING USING TUBE METHOD
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•Spin test sample to separate serum

•Set up 6 tubes correctly labeled with donor/patient number.

•Prepare once washed 2-5% suspension of the test cells.

•Add 1 drop of standard A cells in tube labeled A, B cells in tube labeled B.

 ABO TYPING (Back or reverse type with A and B cells)

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Commercially available A and B cells are added to two tubes of plasma

AB B
Back or reverse type with A and BA O
cells
A B A B A B A B
 CELL AND SERUM ABO GROUPING
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 CELL AND SERUM ABO GROUPING
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 Controls are run correspondingly with tests for comparison.

• Key
+ = Agglutination
- = No Agglutination
 PRACTICAL ASPECTS OF ABO TYPING

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• ABO grouping tests should be done at room temperature (200c - 240c) or lower,
testing in hot environment weakens the reaction.

• Routine ABO grouping must include both cell and serum testing as each serves as
a checker on the other.

• Antisera used in the ABO grouping must be as per the manufacturer’s instructions.
 INTERPRETATION OF ABO TYPING

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 TESTING OF THE PATIENT SAMPLE.
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 Rh Grouping.
• This is another type of grouping system in which the presence of Rhesus antigen
on the Red cells surface indicate the Rhesus positive and absence of which indicate
Rhesus negative.

• Rh grouping is performed using anti-D blood grouping serum. Tube or slide tests
should be performed according to the manufacturer’s directions for the reagent,
which may or may not include the use of a suitable diluents control.

• Control must be run in parallel with Rh grouping tests performed on patient’s

samples, to avoid incorrect designation of Rh neg, patient as Rh positive.

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 PATIENT RED CELL WITH –D AG.

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 If Rh –ve do
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 MERITS AND DEMERITS OF ABO/Rh TYPING
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 MERITS OF ABO TYPING
 Rapid and simple
 High sensitivity and specificity
 Essential for transfusion safety
 DEMERITS OF ABO TYPING
 Limited scope (only detects ABO/Rh incompatibility)
 TESTING OF THE PATIENT SAMPLE.
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• Direct antiglobulin test (DAT) should be performed on the patient’s red blood
cells to determine whether uptake of autoantibody, (alloantibodies, if the patient’s
has been recently transfused) is responsible for the positive control result.

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 TESTING OF THE PATIENT SAMPLE.
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• If the Rh group of the recipient can not be determined and transfusion is essential,
Rh negative blood should be given.

• The test for Du is unnecessary when testing transfusion recipients. Individuals

typing as Rh neg in direct testing should receive Rh-neg blood and those typing as
Rh pos in direct testing should receive Rh pos blood.

• As Du are considered Rh pos and may receive Rh pos blood during transfusion.

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 TESTING OF THE PATIENT SAMPLE.
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 Antibody Screening.
• The patient’s serum or plasma must be tested for unexpected Abs.
• The aim of the Ab screening test is to detect as many clinically significant Abs as
possible.
Clinically significant Abs refers to Abs that are reactive at 37°C or in the DAT or both
and are known to have caused a transfusion reaction or unacceptably short survival
of the transfused red blood cells.

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 TESTING OF THE PATIENT SAMPLE
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 Antibody Screening.
The role of antibodies screening include
 Detection of alloantibodies using(DAT and IAT)

 Detect the presence of clinically significant antibodies in the patient plasma.

 In order to select antigen negative unit to avoid immediate or delayed transfusion

reaction and ensure that transfused red cells survive optimally in the patient
circulation.
 If the test is positive, antibodies identification is done using panel of cells.
 COMPATIBILITY TESTING USING IAT METHOD
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 2 drops of patient serum is added in to a clean tube

 2 drop of 3% suspension of pool group O cells.

 Mix and incubate at 370C for 45minute.

 Wash 4 times in physiological saline.

 Centrifuge lightly and finally observe for agglutination.

 Add 2 drops of AHG reagent and centrifuge at 100rpm for 15 seconds

 Read for agglutination or haemolysis.

 COMPATIBILITY TESTING USING IAT METHOD
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Controls should be included

 To a known Rh D positive cells, add anti D, Incubate at 370c for 30min, add AHG,
This will give(+++) agglutination.

 To a known AB serum, add Rh D Positive cells, AHG and this will give no
agglutination
 MERITS AND DEMERITS OF DCT AND ICT
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 MERITS OF DCT
 Detects Abs/complement attached to RBCs
 Diagnose autoimmune hemolytic anemia
 It is rapid (15-30mins)
 DEMERITS OF DCT
 False positive due to drug-induced antibodies
 Limited sensitivity for certain antibodies
 MERITS AND DEMERITS OF DCT AND ICT
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 MERITS OF ICT
 Detects free antibodies against RBC Ags
 Screens for potential transfusion reactions
 It is a rapid method (15-30mins)
 DEMERITS OF ICT
 False positives due to residual plasma
 Limited sensitivity for certain antibodies
 COMPATIBILITY TESTING FOR HIGH TITRE HAEMOLYSIN
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 Procedure for high titre haemolysin test
o Centrifuge 5mls of donors whole blood sample and separate the plasma

o Prepare 1 in 56 dilution of the donors plasma with saline

o Prepare 5% suspension of A2B red cells in saline

o Add 2 drops of donors 1in 56 diluted plasma to 1 drop of 5% A and B Red cells suspension

o Centrifuge lightly and observe for agglutination

o Agglutination indicate high titre of anti A and B.

 TESTING OF THE PATIENT SAMPLE.
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• Abs Regarded as always being potentially clinically significant
• ABO Rh Kell Duffy Kidd Ss U

• Abs that may sometime be clinically Significant

• Lea p Lua Lub Cartwright.

• Abs that rarely, if ever, are clinically significant

• Leb Chido/Rodgers (Cha/Rha) York, Sd Xg & Bg

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 TESTING OF THE PATIENT SAMPLE.
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• Correct ABO grouping results are much more critical to transfusion safety than
Ab screening.

• Most Abs, other than anti-A and anti-B do not cause severe hemolytic transfusion
reactions. Thus the vast majority of patients would not suffer grave consequences
if transfused with blood from ABO group compatible donor without the benefit of
Ab screening tests.

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 TESTING OF THE PATIENT SAMPLE.
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• Detection of unexpected Abs is important, however, for the selection of donor red
blood cells that are likely to survive maximally in the patient circulation.

• Weakly reactive Abs that are capable of reacting with their Ags at 37°C can cause
decreased survival of transfused incompatible red cells.

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 TESTING OF THE PATIENT SAMPLE.
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• Because large numbers of Ab molecules are present in the patient’s circulation
compared with the number of red cells in a unit of blood, incompatible donor cells
are highly vulnerable to destruction by patient Abs.

• Abs screening offers several advantages over direct cross-matched testing for
detection of Abs;
• Testing is performed using selected group O red cells that are known to carry
optimal representation of important blood group Ags.

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 TESTING OF THE PATIENT SAMPLE.
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• Testing can be performed well in advance of the anticipated
transfusion, allowing ample time for identification of unexpected
Ab and location of suitable donor units lacking the corresponding
Ag.

• Methods used to detect Abs in patient’s sera must demonstrate

all significant coating, hemolysin, and agglutinating Abs active at
37°C.

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 CROSS-MATCHING

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 This refers to the complex testing performed prior to blood transfusion, to

determine if the donors blood is compatible with the blood of an intended
recipient.
 It is categorised into:
• (a). Major cross matching and

• (b). Minor cross match.

 CROSS-MATCHING

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 CROSS-MATCH
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 The two main functions of the cross-match test can be cited as,

1- It is a final check of ABO compatibility between donor and patient.

2- It may detect the presence of an Ab in the patient’s serum that will react with Ags
on the donor RBCs but that was not detected in the Ab screening because the
corresponding Ag was lacking from the screening cell.

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 CROSS-MATCH
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 Major and Minor cross-match tests
• Major cross-match test, consisting of mixing the patient’s serum with donor
RBCs.

• Minor cross-match test, consisting of mixing the donor’s plasma with patient’s
RBCs

• The minor cross-match test has been completely eliminated in most blood banks,
because donor samples are screened beforehand for the more common Abs.

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 CROSS-MATCH
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 Method for major cross-match tests.

 Cross-match methods can be categorized by the test phase in which the procedure
ends.
• Saline technique (Immediate spin (IS) and saline at 37°C ) (Abbreviated Cross-
match)
• Albumin
• Antihuman globulin(AHG)
 When no clinically significant Abs are detected nor are there previous record of
such Abs, a serologic test to detect ABO incompatibility is sufficient.

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 CROSS-MATCH
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• In IS (the patient’s serum with donor cell are centrifuge immediately) absence of
hemolysis or agglutination indicates compatibility.

• False reaction may be seen in the presence of other IS reaction (auto -I). In patient
with hyper immune ABO Abs, when the procedure is not performed correctly
(delayed in centrifugation or reading) when rouleaux is observed, or when infant’s
specimens are tested.

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 CROSS-MATCH
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• PROCEDURE FOR IMMEDIATE SPIN (IS)

• Label 1 tube for each donor sample to be cross matched
• Add 2 drops of recipient’s serum to each tube
• Add 1 drop of 2-5% saline suspension of donor’s red cells from each donor unit to
appropriate tube.
• Spin the tubes at 100rpm for 1 minute
• Look for agglutination
• No agglutination should occur if the donors and recipient’s blood are compatible
 CROSS-MATCH
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 ALBUMIN LAYERING TECHNIQUE
 PROCEDURE

• Place 2 drop of patient serum in a labeled tube

•Add 1 drop of 2-5% test red cell saline suspension

•Incubate at 370c for 45-60 minutes.

 CROSS-MATCH
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 ANTIGLOBULIN CROSS-MATCH
• The procedure begin in the same manner as the IS cross-match,
continues to 37°C incubation and finishes with AHG test.

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 COMMON DIFFICULTIES IN CROSSMATCHING
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 It include the following:

• Rouleaux formation this occurs in protein abnormalities such as multiple

myeloma, this is called pseudo agglutination and can be distinguish from true
agglutination by adding a drop of normal saline and observe microscopically.

• Autoagglutinin it occurs in patient with lymphoma,leukaemia, systemic lupus

erythromatosus and other autoimmune diseases.
 MERITS AND DEMERITS OF CROSS-MATCHING (MAJOR AND MINOR)

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 MERITS OF CROSS-MATCHING
 Detects recipients antibodies against donor RBCs
 Identifies potential for immediate hemolytic reaction
 Comprehensive assessment of compatibility
 DEMERITS OF CROSS-MATCHING
 Time-consuming (30mins to1hour)
 Requires donor and recipient samples
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 SUMMARY
60 PRE TRANSFUSION TESTING
Is to provide safe, compatible blood for transfusion to each individual patient. The
steps necessary for safe transfusion are:
1. Accurate ABO and Rh typing of the patient.
2. Accurate ABO and Rh typing of the donor.
3. Screening tests for antibodies in the donors and patients serum.
4. In the presence of patient antibodies, selection of appropriate units for each patient.
5. Compatibility Testing - (Major)
6. Accurate completion of paperwork and labels

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 PRE TRANSFUSION TESTING
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 Compatibility Testing
 Each compatibility test is a unique experiment in which an unknown (patient)
serum and (donor) red cells are tested for the detection of unexpected antibodies
which are directed against antigens found on the cells. Negative results indicate
compatibility.
 This is one of the most important tests performed by a transfusion service.

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 PRE TRANSFUSION TESTING
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 The purposes of compatibility testing are:
1. To detect irregular antibodies in the recipient serum that are directed against the
donor’s cells.
2. To detect errors in ABO grouping.
3. To detect errors in labeling, recording, or identifying donor’s or recipient’s
samples. 4. To Prevent haemolytic transfution reaction(Both immediate and delay
type).
5. Rule out transfusion transmissible infections(TTI) e.g HBV,HCV,HIV and malaria.
6. Prevent transfusion of high titre anti A and anti B.
7. It also prevent HDN.
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 PRE TRANSFUSION TESTING
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 Compatibility testing does not:
1. Ensure normal survival of donor red cells.
2. Prove that donor and or recipient serum is free of antibodies. Prevent immunization
of the recipient.
[Link] ALL ABO typing errors.
4. Detect errors in Rh typing of either recipient or donor unless the recipient’s serum
contains an Rh antibody.
5. Detect ALL error of identification.
6. Pre-transfusion testing of the recipient must include an ABO and Rh typing,
antibody screen, and a cross-match with all donor units.

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 END OF MODULE QUIZ
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Q1. In a clear term, define Compatibility testing and state its clinical significance to
transfusion medicine?
Q2. Clearly demonstrate the clear cut between compatibility testing and cross-
matching?
[Link] clearly the clinical relevance of negative Du Antigen testing and how it
impacts safe transfusion practice?
Q4. Differentiate between DCT and ICT and highlight the major distinguishing
attributes.

THANKS FOR LISTENING