BENITO JUÁREZ AUTONOMOUS UNIVERSITY OF

OAXACA

Faculty of Chemical Sciences

Immunology Laboratory

Starter: QB. “Antonio Girón

PRACTICE REPORT N. 1
”Cross Tests”

TABLE #4

Students:
Cruz Hernandez Diego Sait
Patricio Jiménez Victor Eduardo
Rivers Rivers William de Jesus
Ruiz RíosBárbara
Zárate Ramos Rosa Andrea

6th
Semester Group: “A”

School Year: 2014 – 2014

March 2014, Oaxaca; Oaxaca
INTRODUCTION .

Crossmatch tests are relatively simple but very careful procedures, the
purpose of which is to guarantee serological compatibility (in vitro) between the
recipient's blood sample and the unit of blood that is intended to be transfused.

The validity of the cross-match tests lies in the correlation that exists between
the compatibility tests performed in-vitro and the survival of the red blood cells in
the patient's circulation.

As we mentioned, the objective of compatibility testing is to prevent the

transfusion of incompatible blood (we define transfusion compatibility as the lack of
immune reaction between antigens (Ag) and antibodies (Ab) of donor and
recipient, where compatibility does not guarantee identity. between both, it only
indicates that, at that moment, there will be no decrease in transfusion
performance due to an immune cause)

From this point of view, cross-testing must guarantee:

 The ABO and Rh compatibility between the recipient and the unit to
be transfused.

 That there are no clinically important antibodies against the donor's

erythrocyte antigens in the recipient's serum.

 That in the plasma of the blood unit there are no antibodies of clinical
importance against the recipient's erythrocyte antigens.

Among the transfusion safety measures are those aimed at avoiding the acute
hemolytic reaction, that is, those that ensure compatibility between donor-recipient.

GOALS .

Perform cross-match blood compatibility tests to:

 Guarantee that the erythrocytes to be transfused are ABO compatible with

the recipient.
 Detect antibodies in the recipient's serum that are directed against antigens
present on the donor's erythrocytes.
BASIS .

Blood compatibility tests are routinely carried out in 0.85% saline solution, in
this medium the antibodies that agglutinate are mainly of the IgM class.

There are two crossover tests and they receive this name because they react
in a tube:

 The recipient's plasma or serum with the donor's red blood cells
(major test).
 The donor's serum or plasma with the recipient's red blood cells
(minor test)

These tests serve to ensure that all transfused red blood cells are compatible
with the antibodies contained in the patient's plasma. As well as, to avoid
stimulating the production of new antibodies against red blood cells in the receptor,
especially anti-Rh D.

Cross tests are divided into:

a) MAJOR PROOF:
It consists of reacting the recipient's serum with the donor's
erythrocytes. In the major test, the patient's antibodies (plasma or serum)
that are found in large volume, against the recipient's red blood cells, are
verified.
b) MINOR PROOF:
It consists of reacting the donor's serum with the recipient's
erythrocytes. This test is performed when plasma is to be transfused.

The use of bovine albumin facilitates the detection of IgG class antibodies.
Since these are very frequently hindered in their agglutinating effect by the Z
potential.

These tests are a fundamental requirement, because it is the best way to

detect antibodies that can damage the transfused red cells and cause a hemolytic
transfusion reaction.

PROCEDURE .
A. Necessary Equipment, Materials and Reagents

Reagents
The reagents must be of analytical grade
 Isotonic saline solution 0.9%
 22% bovine albumin solution

Materials
 4 test tubes of 13x100
 Disposable Pasteur pipettes
 Parafilm
 Vacutainer tubes with and without EDTA
 Ajugas for venipuncture
 alcohol swabs
 Tourniquet

Biological Material
 Donor and recipient serum.
 Suspension of 5% erythrocytes from donor and recipient

Apparatus and instruments

 Centrifuge.
 Water bath at 37 °C

B. Practice Development

Preparation of Biological Samples

1. Extraction of a venous blood sample.

a) Extract a whole blood sample by venipuncture in a
tube with EDTA and one without anticoagulant.
Both samples must be obtained for both the donor
and the recipient.

b) Wait 15 minutes for the sample without additive to coagulate

completely.

c) Centrifuge the sample without

anticoagulant to obtain serum for 5
minutes at 2500 rpm.
 d) Once the centrifugation process is completed, verify that the separating gel
is between the two phases of the whole blood and that the serum is
transparent and does not present hemolysis.

2. Preparation of the erythrocyte suspension.

a) From the well-mixed whole blood with EDTA, place 10 drops in a test tube,
add isotonic saline solution up to ¾ of the test tube, place parafilm at the
mouth of the tube, mix gently by inversion.
b) Centrifuge for 3 minutes at 1500 rpm.
c) After the centrifugation time has elapsed, carefully decant the supernatant
and discard it.
d) Add isotonic saline solution again as in section a and repeat the same
procedure as in b and c so that a total of 3 washes must be performed.
e) Once you have the package of washed erythrocytes, resuspend it in isotonic
saline solution to obtain an approximate concentration of 5%.

Procedure.

Phase I. Quick saline.

1. In a tube marked PM (Major Test), place two drops of the donor's
erythrocyte suspension and two drops of the recipient's serum. Mix gently.

2 Drops of 2 Drops of
Donor Receiver
Erythrocyte serum
Suspension

2. In a second tube, marked pm (minor test), place two drops of the recipient's
erythrocyte suspension and two drops of the donor's serum. Mix gently.
2 Drops of
2 Drops of
Receiver
Donor
Erythrocyte
serum
Suspension
 3. Centrifuge both tubes at 1000 rpm for 60 seconds.

4. Observe the supernatant from both tubes for

hemolysis. Gently resuspend the erythrocyte button
to look for agglutination. Write down the results.

Phase II. Albumin.

5. If agglutination is not observed in the phase I tubes,

add two drops of the 22% bovine albumin solution to
both tubes. Mix gently.
6. Incubate at 37°C for 15 minutes.

7. After the incubation period, centrifuge both tubes at 1000 rpm for 60
seconds.
8. Gently resuspend the RBC button and look for the presence of
agglutination. Write down your results.
RESULTS .

Within the development of the practice we used two different blood groups
belonging to the AB0 system, and the tests were carried out as follows:
Quick Saline Test. 00
Donor (Victor): Group 0
Receiver (Saúl): Group 0
Erythrocyte
Serum.
Package.
Receiver.
In the Major Test (PM) Donor.
Group 0
Group 0
between groups 00 there is
no agglutination. The
antibodies and antigens are
compatible and therefore
agglutination will not occur.

Serum. Erythrocyte
Donor. Package.
In the minor test (pm)
Group 0 Receiver.
among the 00 groups, no
Group 0
agglutination occurred. The
same case occurs as with
PM, however the albumin test
will be performed on PM and
PM.

Quick Saline Test. A0

Donor (Victor): Group 0
Receiver (Alan): Group A

In the Major Test (PM) between

Erythrocyte
Serum. A0 groups there is no
Package.
Receiver. agglutination. Group 0
Donor.
Group A erythrocytes do not present
Group 0
antigens that interact with group
A serum antibodies and
therefore there is no
 In the minor test (pm) between Serum. Erythrocyte
the 0A groups , agglutination Donor. Package.
was present . This is because Group 0 Receiver.
the antigens present in the Group A
erythrocytes of group A interact
with the antibodies present in the
serum of group 0, since group 0
has Anti-A and Anti-B antibodies,
thus causing agglutination in the
erythrocytes.

Proof. Albumin. Groups 00

The Albumin test was performed on the PM and PM tubes of groups 00, where
there was no agglutination.

2 drops of albumin are added

to each tube, placed in a water
bath at 37ºC for 15 min, and
centrifuged at 1000 rpm for 1
min.
 P.M There was no agglutination Not presenting
agglutination means that
the blood of the recipient
p.m There was no agglutination and the donor are
COMPATIBLE.

DISCUSSION .

Blood group determinations are high-demand tests in any clinical laboratory,

whether for simple knowledge of blood type or for a specific purpose, without
following adequate quality control algorithms. These cases, without being
approved, the simple techniques used can go unnoticed.
In blood banking this should not be overlooked. In practice, the possibilities of
the different antigen-antibody reactions that can occur in blood have been
analyzed when blood fluid (both plasma and blood) from different individuals are in
contact; It has been established that even individuals with blood that initially appear
similar may not be compatible and therefore cause significant health problems. For
this reason, emphasis should be placed on quality control in laboratories through
the application of cross-matching techniques (cross-match testing) to avoid
undesirable reactions.
Cross-testing must be mandatory, carrying out all the corresponding
methodology. A negative result in a reaction between individuals of two different
blood types cannot be considered a secure compatibility. Factors such as the Z
potential, the variability of antigenic groups present in the membrane, the
imbalance between the Ag-Ac concentration (postzone and person) generate
ambiguous results.
The immunological reactions between antigens and antibodies are not simple
agglutinations like those analyzed in practices, within the body, these trigger
serious pathologies and that is why they are so important.

CONCLUSION .

The immune response generated by the body against an antigen is based on

antibody reactions against these antigens. The presence of antigens on the
erythrocyte membrane allows us to obtain a clear view of these responses, when
there is an interaction of these erythrocytes with xenobiotic antibodies or vice
versa.
 Antigen-antibody reactions can be determined by basic and typical laboratory
tests, such as common blood group tests. These tests allow us to qualitatively
quantify immunological responses to antigens of the ABO system, observing
agglutination or hemolysis reactions. The analysis of these agglutinating and/or
hemolysis reactions illustrates an interaction between an antigen and an antibody,
which is determined by an immune response. When these characteristics are not
observed, we can initially imagine that there is no such immune response.
Speaking in terms of a blood transfusion, these reactions can translate into
compatibility or non-compatibility of blood between donors and recipients.
In transfusion practices it is important not to make errors in the determination
of these reactions, as well as in their assessment.
This is why it is important to perform cross-match tests, where, knowing the
different interactions that will result in non-compatibility and therefore agglutination
and/or hemolysis, we must have absolute certainty of compatibility. Two individuals
with the same blood type cannot be ruled out incompatibility. The erythrocyte
membrane presents a variety of antigens that can affect the antigen-antibody
reaction, or antigens that are not potentially immunogenic; There may be factors
such as the Z potential, which alter the reaction to a certain degree. For this
reason, cross-matching tests are mandatory techniques for quality control in a
transfusion laboratory.

QUESTIONNAIRE .

1. Mention at least four blood group classification systems.

 ABO system.
 Rh system.
 MNS system.
 Lutheran System.
 Diego System.
 Duffy system.
2. Explain the concept of Z potential applied to erythrocyte membranes.

The surface of red blood cells has a negative electrical charge due to sialic
acid molecules in the membrane. When red blood cells are suspended in solutions
containing free ions, the cations are attracted to the negative charges on the
surface of the red blood cell, so that a positive ionic cloud is formed around the red
blood cells, which, because it is made up of electrical charges, of the same sign,
will create a repulsion between the erythrocytes. This repulsion is known as the
Zeta potential.

3. How is an immunoglobulin made?

An immunoglobulin (Ig) molecule or also called an antibody is a glycoprotein that

is produced by B lymphocytes or their derived cells, plasma cells; It has a
symmetrical nuclear structure composed of two identical light chains and two
identical heavy chains; These chains contain a series of homologous repeat units,
approximately 110 amino acids long, which fold into a globular structure called the Ig
domain.

An Ig domain has two  -fold sheets, both held together by a disulfide bond, and
helices adjacent to each - sheet are connected by short loops. The heavy and light
chains consist of variable amino terminal regions (V) that participate in antigen
recognition and constant carboxyl terminal regions (C); the C regions of the past
regions mediate effector functions.
BIBLIOGRAPHY .

 Abbas, Abul K. Cellular and Molecular Immunology. 7th Edition, ELSEVIER

Editorial, Barcelona, Spain, 2012.
 Murphy Kennet, Travers Paul, Walport Mark. Janeway Immunobiology. 7th
Edition, McGraw-Hill Publishing. Spain
 Roit. Immunology, Fundamentals. 10th Edition, Editorial Medica
Panamericana, Barcelona, Spain.
 Kubby. Immunology. 6th Edition. McGraw-Hill Publishing House, Printed in
Mexico, 2007.