Cross matching was developed to identify recipients who are likely to develop rejection of a graft from a given donor

1. Complement Dependent
Cytotoxicity CrossMatch
(CDC XM)

2. Contents
• Introduction
• History
• Principle
• Reagents Required
• Complement System
• Procedure
• Scoring based interpretation
• False Positive and False Negative Reactions
• Limitations of CDCXM
• AHG

3. Introduction
• Cross matching was developed to identify recipients who
are likely to develop rejection of a graft from a given donor.
• During transplantation there is a production of antibodies
which is specific for foreign antigens, especially Major
Histocompatibility Antigens (MHC molecules) known as
Human Leukocyte Antigen (HLA)in human in the graft
and causes Graft Rejection.

4. Alloantibodies or HLA
Antibodies
• Antibody that reacts with an antigen from genetically
different member of the same species.
• There are three common methods in which
alloantibodies are developed-:
1. Blood transfusion (express class I and class II
antigens)
2. Platelets transfusion (express Class I antigens)
3. Number of Pregnancy (express Class I antigens)
4. Previous transplantations.

5. HLA Antibodies
• Transplant recipients may develop anti-HLA and non-
HLA antibodies after transplantation.
• Although these antibodies may be donor-specific or
non–donor-specific, their presence may increase the
risk for acute rejection and chronic rejection.
• Before transplants, patient must be tested for these
HLA antibodies.

6. History
• First successful kidney transplant was done by
Dr.Joseph Murray in 1954 .
• Ronald Herrick gave his kidney in his identical
twin brother Richard.
• In 1964, Terasaki developed the “microcytotoxicity
test’’, a tissue-typing test for organ transplant donors and
recipients that required only 1 microliter each
of antisera used to identify human leucocyte
antigene (HLA).

7. Principle
• Anti-HLA antibody binds to the antigen present on
cell membrane of lymphocyte of donor.
• Complement is then added, results in the formation of
antigen-antibody complex which leads to cell lysis.
Lysed cells stained with eosin dye. Then analyzed under
the inverted microscope
• A negative cross match means that there is no
antibodies against lymphocytes cell and it is minimal
risk of acute rejection.
• A positive cross match means that there is antibodies
against lymphocytes cell and it is higher risk of hyper
acute rejection.

8. Reagents Required
• Two conical flasks with
10-12 glass beads.
• Distilled water
• McCOY’s media (PH
7.4)
• I N NaOH
• Lymphoprep.
• Terasaki trays
• Paraffin oil
• Patient serum
• Negative control and
Positive control
• Rabbit compliment
• Eosin dye (4%)
• Formaline (35-42% and
PH 7.4)
• Cover slide
• Bright field inverted
microscope

9. Glass beads:
• Glass beads are used for Defibrinate the blood by
moving the conical flask on 60° angle for 10-15 minutes.
• When extended for more then 20 min at room
temperature it caused haemolysis.
• The main disadvantages of this method is the loss of
carbon dioxide from the blood ,CO2 affects the pH of
medium also responsible for frothing .

10. Media Preparation
• McCOY’s media is used for the propagation and survival of
lymphocytes cells.
• It is a combination of Inorganic Salts (Calcium Chloride,
Magnesium Sulfate, Potassium Chloride ,Sodium Bicarbonate,
Sodium Chloride and so on) ,Amino Acids (L-Alanine,L-
Arginine ,L-Asparagine,L-Aspartic Acid,L-Cysteine,L-Glutamic
Acid,L-Glutamin), Vitamins(Ascorbic Acid,Folic
Acid,Riboflavin,Vitamin B12), also L-Glutamine
but without sodiumbicarbonate (NaHCO3 ).
• It should be store at 2°C to 8°C.
• Add I N NaOH in it untill its pH becomes 7.4.Check the pH of
media with pH paper.
• Filter the media with the help of a filter paper & keep it in a bottle
or store at 2°C to 8°C.

11. Lymphoprep
• Lymphoprep is a density gradient solution designed for cell
separation.
• It is combination of sodium diatrizoate and polysaccharide.
• The polysaccharide, which contributes to the overall density of
the medium, also aggregates the erythrocytes to enhance their
rate of sedimentation.

12. Terasaki tray
• Tray wells have a special polystyrene surface treatment
to ensure enhanced mixing of reagents and samples in
each well, even when frozen and thawed.
• Treatment ensures a limited affinity to antibodies even
in high concentrations and limited protein adsorption.
• The optical clarity of the trays allows them to be
viewed with an inverted microscope. It have 72 wells
each of 10 micro ltrs.

13. Liquid paraffin oil
• Liquid paraffin oil is a mineral oil, and is a by product
of petroleum distillation.
• It is a transparent, colorless, odorless and tasteless and
non toxic oil.
• It prevents dehydration and cells drying during
incubation period of lymphocytes cells.

14. Negative control
• Negative control is obtained by normal “AB serum”
from a “non transfused” male individual donors
lacking lymphocytotoxic activity.
• Fetal calf serum can be used as an alternative.

15. Positive control
• Positive control is obtained by pool of antisera that
react with high frequency against donor lymphocyte
cells.

16. Rabbit compliment
• Rabbit compliment is a complex group of “nine serum
protein (C1-C9)” which act upon an antigen antibody
complex causing cell lysis.
• It is highly sensitive for human lymphocyte cells.
• Complement should be store at - 20 ° c .
• It can be stored at 4° c (maximum of one hour).

17. Eosin dye (4% )
• Eosin is a fluorescent and acidic dye.
• It is used to stain dead lymphocyte cells for examination
under the microscope.
• Eosin shows cytoplasm stained pink-orange
and nuclei stained darkly.
• The cell lysis is detected by the entry of dye into the
dead cells .
• Dead cells are usually dark while live cells are much
brighter than dead cells.

18. Formalin (35-42%) and PH 7.4
• It is Used for fixation.
• Formalin is useful for masking the epitopes sites and
thus prevent further antibody-antigen reactions.

19. Bright field inverted microscope
• Bright field inverted microscope is used for
reading/analysis the trays.
• Cytotoxic reaction(Lymphocytes cells) are analyzed at
10 x zoom.

20. Complement System
• The complement system is a Biochemical Cascade that attacks the surfaces of
foreign cells.
• Large group of serum proteins that participate in the lysis of foreign cells.
• Three mechanisms are required for complement activation:
1. Classical Pathway: Activation is Ab-dependent. Initiated by an immune
reaction of antibodies.
2. Alternative Pathway: Activation is Ab-independent. Initiated by direct
interaction of complement proteins with microbial endotoxins.
3. Lectin pathway: Activation is Ab-independent; it occurs when lactin binds to
mannose on surface of pathogens (bacterial cell walls, yeast walls, or viruses)

21. Complement System

22. The Membrane Attack Complex (MAC)
• The membrane-bound complement component C5b is
bound by the next complement molecule, C6. After
binding of c6 molecule it binds to the c7 and then c8
and c9. These combined molecules gives the name
"membrane attack complex"
• The MAC creates a transmembrane pore leading to the
lysis of the target cell.

23. Procedure
• Take two clean and sterilized conical flasks containing
8-10 glass beads of 2 mm diameter and label them.
• Collect 15 ml of whole blood in Red top vacutainers
and put it in the flask labeled for Donor.
• Defibrination is done by rotating the flask at 45 degree
from the surface for 10-15 min till the clumps of fibrin
are seen.

24. Preparation of McCoy's media :
• Weigh 1.2 gm of McCoy's powder for a single cross
match and add 100 ml of double distilled water.
• Adjust the pH to 7.2-7.4 by using 1N NaOH solution
• Sterilize immediately by membrane filtration & transfer
in clean tight cap bottle or flask.

25. • Defibrinated blood of the patient and donor diluted
with and equal volume of freshly prepared McCoy's
media.
• Arrange 6 falcon tubes (15 ml) on a test tube stand.
• Label above 2 test tubes as P (Patients Cells), 3 tube as
D(Donor cells).
• Add 4 ml of lymphoprep in each test tube.
• Distribute equal amount of patient's diluted blood
sample with the help of pasteur pipette in the 2 tubes
each labeled as P & Donor's diluted blood sample in 2
tubes labeled as D.
• Centrifuge all the tubes at 800 g for 25 min at 22 ºC.
Acceleration and Deacceleration should be 1.

26. • After centrifugation, isolate the lymphocytes which
forms Buffy coat layer (white cloudy ring) with help of
1000 ul pipette & transfer in respective fresh labeled
tubes as P cells & D cells.
• Add McCoy's media in all the above tubes upto 15ml
& mix gently.
• Centrifuge all the tubes for 10 min at 800 g.
• After Centrifugation discard the supernatant. Same
procedure is to be followed with all four tubes.
• Re suspend the cell pellet in 800ul of McCoy's media
to each tube.
• Determination of Cell concentration and viability

27. • Cells concentration is counted using the Neubauer
chamber. Introduce 10μl of sample into the neubauer
chamber and count the number of cells in the all four big
squares.
• Calculate the concentration of cells using formula
• Concentration ( cells/ml) = Number of cells
Volume of sample (in ml)
• Adjust the final concentration to 2 x 10 3 cells/ul
• Cell viability is checked using Trypan Blue dye.
• Add 0.1 mL of trypan blue stock solution to 1 mL of cells.
• Load a hemacytometer and examine immediately under a
microscope at low magnification.
• Count the number of blue staining cells and the number
of total cells. .

28. • % viable cells = [1.00 – (Number of nonviable cells ÷
Number of total cells)] × 100
• The final volume of lymphocyte suspensions used should have
viability 90%.
• Take 2 Blank Tissue Typing Trays & label as R-T and 37 degree
along with their names layering of each well is done with paraffin
oil.
• Prepare the serial dilution of the patient's serum in,1:2,1:4, 1:8,1:16
and 1:32 ratio.
• Dispense 1 ul of appropriate controls and test serum in appropriate
wells as illustrated below with the help of Repeater.
• Add 1ul of cell suspension to each well.
Preparation of Crossmatch tray:

29. 12 11 10 9 8 7 6 5 4 3 2 1
NS+
D
NS+
D
1:2+
D
1:4+
D
1:8+
D
1:16
+D
1:32
+D
EMP
TY
M+
D
NC+
D
EMP
TY
PC+D
NS+
D
NS+
D
1:2+
D
1:4+
D
1:8+
D
1:16
+D
1:32
+D
EMP
TY
M+
D
NC+
D
EMP
TY
PC+D
NS+
D
NS+
D
1:2+
D
1:4+
D
1:8+
D
1:16
+D
1:32
+D
EMP
TY
M+
D
NC+
D
EMP
TY
PC+D
NS+
P
NS+
P
1:2+
P
1:4+
P
1:8+
P
1:16
+P
1:32
+P
EMP
TY
M+
P
NC+
P
EMP
TY
PC+P
NS+
P
NS+
P
1:2+
P
1:4+
P
1:8+
P
1:16
+P
1:32
+P
EMP
TY
M+
P
NC+
P
EMP
TY
PC+P
NS+
P
NS+
P
1:2+
P
1:4+
P
1:8+
P
1:16
+P
1:32
+P
EMP
TY
M+
P
NC+
P
EMP
TY
PC+P
Loading of Terasaki trays

30. • Incubate trays at their respective temperatures like, 37
degree C & room temperature for 75 mins.
• After incubation,add 5 ul of rabbit complement in each
wells of Tissue Typing Trays.
• Again incubate for 75 mins on respective temperatures.
• Dispense 5 ul of Eosin Dye (5%) in each well of Blank
Tissue Typing Trays..
• After 5 min add 3 ul of formaldehyde (pH 7.2-7.4) to
each well.
• Carefully lower the cover slips on wells.
• Now the trays can be used for the observation and
analysis under an inverted microscope.

31. • Trays are read with a Bright field inverted microscope.
• Scoring for cross match is done by the identification of % of
dead cells as well as % of live cells.
• Scoring chart:-
Scoring based Interpretation
Interpretation Dead cells(%) Score
Negative 0-10 1
Doubtful
Negative
11-20 2
Weak Positive 21-50 4
Positive 51-80 6
Strongly
Positive
81-100 8

32. Positive Crossmatch
Negative Crossmatch

33. False positive and False negative
reactions
False positive due to:
• Poor viability of the lymphocyte
cells.
• Contamination of positive control
with patient serum.
• Excess incubation.
• Incorrect dilution of serum.
• Incorrect incubation temp.
• Effect of patient drug treatment on
cells.
False negative due to:
• High no. of platelet.
• Low incubation temp.
• Shorter incubation time.
• Missing antiserum or complement in
the test.
• Incorrect dilution of serum.
• Missing fixative.
• If too many cells are added, antigen
excess can cause false negative
reactions because antibody is
insufficient.
• Defective batches of complement
• Effect of patient drug treatment on
cells.
• Too much oil in the well .

34. Limitation of CDC-Crossmatch
•The test is less sensitive compared with other assay.
•Only complement dependent antibodies are detected and not all
antibodies.
•A low HLA antigen expression on lymphocytes cause false negative
reaction.
•Non-HLA auto antibodies also give false positive results.
•Both HLA and non-HLA antibodies are detected.
•Some IgG isotype (IgG2 & IgG4) & IgA do not fixed with
complement and gives negative reaction.

35. Increase the sensitivity of CDC by using
AHG
• The AHG-CDC crossmatch is a modified version of CDC
(complement dependent cytotoxicity) performed by addition of
antihuman globulin to increase the sensitivity, as AHG can
induce cross-linking of antibodies and increase the visual
cytotoxicity.
• Lymphocytes separated from the donor are treated with the serum
of the recipient. Antibodies, if present, bind with the
complimentary epitopes of the antigens, forming an antigen-
antibody complex. Complement activation will be difficult when
inadequate numbers of antibodies are present. Addition of goat
Anti-Human Kappa Light Chain Immunoglobulin (AHG) can
resolve this constraint as the AHG binds to the light chain of the
antibody in turn binding complement with a high affinity.