gel technology ppt

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GEL TECHNOLOGY
By Dr Neelam Shah
• Different methods for Antibody detection.
• #Tube tests
• #Column agglutination technology . includes
•
-Gel technology
•
- In another column agglutination technology, a
column of glass microbeads in a diluent is used instead
of gel. As with the gel test, the beads may either entrap
agglutinated cells.
• Immunofluorescence
• Solid-Phase Red Cell Adherence Tests
• Enzyme-Linked Immunosorbent Assay
Agglutination Reactions
Stage 1
 Sensitization: attachment
of Antibody to Antigen on
the RBC membrane.
Stage 2
• Lattice formation:
formation of bridges
between the sensitized
red cells to form the
lattice that constitutes
agglutination.
Ab molecule cross link RBCs forming lattice
INTRODUCTION
• Gel technology system developed by Dr.Yves
Lapierre of France,gives more reproducible
and standardied test results.
• This technology utilizes the differential
migration of RBC agglutinates through a small
microtube containing Dextran Acrylamide
gel,size exclusion gel column.
Fully automated system
USES
• For any immunohematolgical tests that has
hemagglutination as its endpoint.
-ABO and Rh typing
-Typing for other blood group system-Antibody screening and identification
-Compitability testing including cross matching
ADVANTAGES OF GEL TECHNLOGY
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Simple ,reliable ,rapid , very sensitive.
No need to multiple washing of red cell before adding into
Antihuman globulin (AGH) serum. and no need to add
sensitized controlled cell to all negative AGH tests.
Less specimen volume needed.
Greater uniformity between repeated tests.
No variations among technologist in reading and grading
agglutination.
Provision of centrifuge calibarted to optimal speed for fix
and corrected length of time , reduces potential error
during this phase.
The cards can be digitally photographed , downloaded as
permanent laboratory data.
DISADVANTAGES
• Special centrfuge to accommodate the
microtube card.
• Special incubators to incubate microtube
cards.
• Pipettes to dispose serum and red cell
suspention.
• Expesive.
Centrifugation of microtubes
ABOUT GEL CARD
• Gel held in a microtubes contain in a plastic card.
• Each microtubes contain cephadex(dextran
acrylamide) gel prepared in a buffer solution such
as LISS or Saline.
• Preservatives –Sodium azide
• Specific reagents –Agh or RBC specific antisera is
added.
• Sedimenting agent is bovine serum albumin.
• Reagent is uniformly dispersed throughout gel
column.
ABO/Rh(D) Group Card
• Human red blood cell antigen can be divided
into four groups A, B, AB and O depending on
the presence or absence of corresponding
antigens on the red blood cells.
• The Anti-A, Ant-B reagent are used to detect
the presence or absence of corresponding
antigens on red blood cells.
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Gel-card of Rh system
• In Newborn's the antigens are not completely
developed, thus weaker reaction may be
shown when compared to adults , when
tested.
• In adults the antigens and respective
antibodies are present, but in newborns the
antibodies appear after 4 to 6 months of birth.
• Also human red blood cells are classified as Rh
(D) positive or Rh (D) negative depending
upon the presence or absence or of Rh (D)
antigen.
• The D antigen and weak D (Du) are fully
developed at birth. If the mother is Rh (D)
negative, it is very much important to
determine the D antigen and weak D of the
newborn.
PRINCIPLE
• Serum and cell reaction takes place in a microtube
consisting of a reaction chamber that narrower to
become column.
• The card containing red blood cells is centrifuged under
specific conditions, the red blood cells possessing
corresponding antigen will agglutinate in the presence
of the antibody directed towards the antigen in the gel
matrix and will be trapped in the gel column.
• The red cells, which do not react, are not trapped in
the gel column and get settled at the bottom of the
column. The reactions are visually read and graded
according to their reactivity pattern.
• REAGENTS
• The Matrix ABO/RhD Group Card contains six microtubes ,
pre-filled with gel in a suitable buffer containing specific
Monoclonal Anti-A, Anti-B and Anti-D antibodies.
• SAMPLE COLLECTION
• No special preparation of the patient is required prior to
sample collection.
• Samples should preferably be tested as soon as possible. If
delay in testing occurs sample should be stored at 2-8°C or
may be used within 7 days of collection. Preferably, blood
sample should be drawn into citrate, EDTA or CPD-A
anticoagulant. Do not used hemolysed or contaminated
samples or those contaning clots.
SAMPLE PREPARATION
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FOR FORWARD GROUPING
Prepare a 5% suspension in LISS
1.bring Liss at room temp before testing.
2.dispense 0.5 ml of Liss into a clean glass test tube.
3.Add 50 µl of whole blood or 25 µl of pack cell and mix gently.
FOR REVERSE GROUPING
Prepare a 0.8% suspension in Liss
1.Collect known A1 and B cells.
2.Wash the cells twice with 0.9% normal saline and discard the
supernent.
• 3.dispense 0.5 ml of Liss into a clean glass test tube.
• 4.Add 5 µl of pack cell and mix gently.
SAMPLE PREPARATION
• PLASMA OR SEUM FOR REVERSE GROUPING
• If serum is used instead of plasma , the serum
must be cleared by centrifuging at 1500g for 10
min , so as to avoid presence of fibrin residues ,
which might interfere with test results.
• If delay in testing occurs sample should be stored
at 2-8 degree c. after separation and may be used
up to 48 hrs or stored frozen a -20 to -80 degree
c.
TEST PROCEDURE
• 1. Allow samples and reagent to reach room temperature.
• 2. Label the appropriate microtubes of the Gel card with patient
name / identification number.
• 3. Pipette 50 μl of 0.8% A1 cell suspension to the microtube 5.
• 4. Pipette 50 μl of 0.8% B cell suspension to the microtube 6.
• 5. Pipette 50 μl of patient’s plasma or serum to the microtube 5
and 6.
• 6. Take care to ensure that the micropipette tip does not touch the
reagent in the microtube.
• 7. allow the card to incubate 10 min at room temp.
• 8. Pipette 10 to 15 μl of 5% patient’s cell suspension to the
microtube 1 to 4.
• 9. Centrifuge the cards for 10 minutes in the card centrifuge.
• 10. Read and record the results.
INTERPRETATION OF RESULTS
• Positive Reaction : Agglutinated red blood
cells form a clear line on the surface of the gel
or get dispersed in the gel.
• Negative Reaction: Unagglutinated red blood
cells settle at the bottom of the microtube.
• Reading and interpretation of results must be
done after centrifugation process only.
• The reaction strength may be recorded as
follows:
Strength of reaction
• 4+ Agglutinated red blood cells form a solid band at
the top of the gel column.
• 3+ Most agglutinated red blood cells remain in the
upper half of the gel column.
• 2+ Agglutinated red blood cells are observed
throughout the length of the column. A small button of
red blood cells may also be visible at the bottom of the
gel column.
• 1+ Most agglutinated red blood cells remain in the
lower half of the column. A button of cells may also be
visible at the bottom of the gel column.
Strength of reaction
• ± Most agglutinated red blood cells are in the
lower third part of the column.
• Negative (0) All the red blood cells pass through
and form a compact button at the bottom of the
gel column.
• Mixed field agglutination Agglutinated red blood
cells form a band at the top of the gel and nonagglutinated red blood cells form a compact
button at the bottom of the gel column.
• H Hemolysis
Strength of reaction
Readin & grading of hemagglutination in
gel system
EXPECTED REACTIVITY PATTERN FOR
ABO GROUPING
Anti-A
Anti-B
Blood Group
1+to 4+
Negative
A
Negative
1+to 4+
B
1+to 4+
1+to 4+
Ab
Negative
Negative
O
EXPECTED REACTIVITY PATTERN FOR
Rh(D) TYPING
Anti-D
Rh(D) type
1+ to 4+
Rh positive
Negative
Rh negative
REACTION FOR REVERSE
GROUPING
A1
B
Blood group
1+ to 4+
Negative
B
Negative
1+ to 4+
A
1+ to 4+
1+ to 4+
O
Negative
Negative
AB
• Human red blood cells that shows weak reaction
with anti-A and anti-B probably indicates
subgroups of A and B and further testing is
recommended.
• Very weak D/partial D type human red cells may
give negative reaction . such cells should be
retested with coombs card.
• The microtube control must show a negative
reacion . if control is positive , then the ABO
determination is not valid . repeat the procedure.
REVERSE GROUPING CARD WITH
AUTOCONTROL
• Card contain neutral gel in all microtubes.
• 0.1% sod azide as preservative.
• All procedure is same as mention early.
AHG (Coombs) Test Card
AHG (Coombs) Test Card
• Generally antibodies involved in transfusion reactions
are of two types, namely the complete and the
incomplete.
• The complete antibodies agglutinate in vitro human
red blood cells in saline medium.
• The incomplete type of antibodies sensitize red blood
cells without agglutination.
• Usually IgM class of antibodies and IgG1 and IgG3 type
of IgG antibodies fix complement.
• Cell lysis , in vivo, is mediated through the complement
system and the complement C3b is metabolized to
C3d.
• In the direct antiglobulin test, Anti Human
Globulin reagent is used to detect antibodies
adsorbed to the red blood cells in vivo.
• In the indirect antiglobulin test, Anti Human
Globulin reagent is used to detect antibodies
adsorbed to the red blood cells in vitro.
• Anti Human Globulin reagent is useful for
compatibility testing, antibody detection,
antibody identification, umbilical cord red blood
testing and the detection of weak D/partial D.
• REAGENTS
• AHG (Coombs) Test cards contain six microtubes prefilled with gel
in a suitable buffer containing anti-human IgG and monoclonal antiC3d.
• PRINCIPLE
• As the gel card containing red blood cells is centrifuged under
specific conditions, the red blood cells sensitized with antibody will
agglutinate in the presence of the Anti Human Globulin reagent in
the gel card and will be trapped in the gel column.
• The red cells, which do not react are not trapped by the gel matrix
and are pelletted to the bottom of the microtubes. The reactions
are visually read and graded according to their reactivity pattern.
• SAMPLE COLLECTION
• (1) For DAT blood dawn in EDTA is preferred but
citrated whole blood may be used.
• (2) For IAT serum no more than 48 hours should
be used . donor units may be tested up to the
end of their dating.
• (3) To avoid presence of fibrin residues, serum or
plasma may be cleared by centrifuging at 1500 g
for 10 minutes
• (4) Do not use hemolysed or contaminated blood
samples, or those containing clots.
• SAMPLE PREPARATION
• Preparation of 0.8 % red blood cell suspension is as
follows:
• 1. Bring the Liss to room temperature before testing.
• 2. Add 0.5ml of Liss into a clean glass test tube.
• 3. Add 5 μl of packed cells to the Liss and mix gently.
• 4. The red blood cell suspension so obtained should be
used for testing.
• 5. Set up as many gel cards as may be required
A. DIRECT ANTIGLOBULIN TEST (DAT)
1. Label the appropriate microtubes with
patient name or identification number.
Remove the aluminium foil carefully.
2. Pipette 50 μl of the 0.8% patient’s red cell
suspension.
3. Centrifuge immediately for 10 min; read and
record the results.
A. DIRECT ANTIGLOBULIN TEST (DAT)
• INTERRETATION
• Negative reaction indicates absence of
detectable IgG or C3d component on the red
cells.
• Positive reaction indicates that red blood cells
are sensitised with IgG or complement
componant C3d.
B. ANTIBODY SCREENING IAT
• 1. Label the appropriate microtubes with patient
name or identification number. Remove the
aluminium foil carefully.
• 2. Pipette 50 μl of the 0.8% red cell suspension.
• 3. Immediately add 25 μl of patient/donor serum
or plasma. The interval between cell and
serum/plasma transfer should not exceed 10
minutes.
• 4. Incubate 15 minutes at 37 C in an incubator.
• 5. Centrifuge for 10 min; read and record the
results.
B. ANTIBODY SCREENING IAT
• INTERRETATION
• Negative reaction indicates Absence of
detectable iregular antibodies in the patient’s
or donor’s serum/plasma.
• Positive reaction indicates Presence of
irregular antibodies.
• Further testing is recommended to
identifybthe antibody specificity.
FOR COMPATIBILITY TEST
• 1.Pipette 50 μl of the 0.8% of donor red cell
suspension.
• 2.Pipette 50 μl of the 0.8% of patiet’s red cell
suspension added to other microtube,this serves
as an autocontrol.
• 3.add 25 μl of patient serum or plasma. The
interval between cell and serum/plasma transfer
should not exceed 10 minutes.
• 4. Incubate 15 minutes at 37 C in an incubator.
• 5. Centrifuge for 10 min; read and record the
results.
FOR COMPATIBILITY TEST
• INTERPRETAION
• The auto control should negative to validate
results.
• Negative reaction indicates Compatibility of
donor blood with the recipient.
• Positive reaction indicates incompatibility of
donor blood with the recipient.
• After incubation hemolysis observe in upper
portion of column , it should be interpreted as
positive reaction.
• PERFORMANCE
• Evaluation of AHG (Coombs) Test cards for
Antibody Screening / Antibody Identification
and Autocontrol was performed by an
external Immunohaematology Laboratory in
comparison to a Reference method.
• 1.Total 551 samples were tested for Antibody
Detection..
Test card
Reference method
Sensitivity:
92.9%
97.6%
Specificity:
97.2%
98.8%
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• 2. Total 544 samples were tested for
Autocontrols
Test card
Reference method
Sensitivity:
94.5%
97.6%
Specificity:
94.5%
96.2%
• 3. Total 43 samples were tested for Antibody
Identification
Sensitivity:
Test card
Reference method
96%
94%
REMARKS
• (1)Freezing or Evaporation of the gel card due to exposure
to heat may impede the passage of unagglutinated red
blood cells through the gel card.
• (2) Gel cards showing damaged aluminum foil should be
discarded.
• (3) Bubbles in the gel card may interfere the passage of
unagglutinated red blood cells. gel cards with bubbles
entrapped in the gel may be centrifuged before testing. gel
cards with resisting bubbles should be discarded.
• (4) Gel cards showing decreased volume or cracked gel
should be discarded.
• (5) Usage of red blood cell concentrations other than those
described may interfere the test results.
• (6) Usage of hemolysed samples may interfere the final
interpretation of results.
• (7) Bacterial or other contamination of reagents during
use may cause false positive or false negative results.
• (8) Fibrin residue in the serum or red cell aggregation
in the red cell suspension can trap non agglutinated
cells presenting a pink line on top of the gel when most
cells pellet to the bottom after centrifugation.
• (9) Do not use lipemic, icteric and hyperproteic
samples.
• (10) Gel card that exhibit drying if used ,can lead to
erroneous result.
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LISS SOLUTON
• The antigen-antibody interaction in blood group
serology is dependant on antigen density,
concentration of antibody, pH, ionic concentration of
reaction medium and temperature.
• Reducing the ionic concentration of the reaction
medium especially enhances the uptake of weak
antibodies by the red blood cell antigens.
• Also, usage of LISS (Low Ionic Strength Solution) is
helpful in detection of weak antibodies during cross
match techniques, antibody screening and antibody
identification.
Liss
• REAGENT
• LISS is a buffered low ionic strength solution of
appropriate sodium chloride molarity useful in
serological applications.
• STORAGE AND STABILITY
• Store the reagent at 2-8°C. Do not freeze.
• The shelf life of the reagent is according to the
expiry date indicated on the label. Do not use
beyond expiry date
LISS(LOW IONIC STRENGTH SALT
SOLUTION)
• Contain 0.2% Sodium chloride.
• Consist of 0.17M saline180 ml
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0.15M phosphate buffer-20 ml
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0.3M sodium glycinate,pH 67-800ml
• All anibodies are not equally sensitive to liss.anti A and
antiB remains unaffected.
• To prevent lysis of red cells at such a low ionic strength, a
nonionic substance such as glycine is incorporated in the
LISS
• Most laboratories use a LISS additive reagent, rather than
LISS itself. These commercially available LISS additives may
contain albumin in addition to ionic salts and buffers.
liss
• PRINCIPLE
• In blood group serology, the ionic concentration of reaction
medium is largely dependant on the concentration of
sodium and chloride ions contributed by isotonic saline.
• When optimum concentration of antibody is present,
antigen-antibody interaction occurs even though the
sodium and chloride ions are present in sufficient quantity.
• But when weak antibodies are present, sodium and
chloride ions may interfere with binding of antibody to the
antigens present on the red blood cell membrane.
• By lowering the ionic concentration of salt, the ionic
strength is reduced which increases the rate of antibody
uptake by red blood cells.
liss
• PERFORMANCE
• The performance of LISS was evaluated on over
100 samples (from donors, patients and
neonates) drawn on recommended
anticoagulants.
• The evaluation demonstrated 100% specificity
and sensitivity of the reagent versus the expected
results with common known ABO , Rhesus
phenotypes, Cross match, DAT and Autocontrol.
liss
• REMARKS
• 1. Aged or stored red blood cells may show weaker
reactivity than freshly collected cells.
• 2. Usage of red blood cell concentrations other than
those described may interfere the test results.
• 3. Usage of hemolysed samples may interfere the final
interpretation of results.
• 4. Bacterial or other contamination may cause false
positive or false negative results.
• 5. Red cell aggregation in the red cell suspension may
interfere the passage
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References
WHO manual
AABB manual
Henry’s clinical diagnosis and management
www.DiaMed.com
• THANK YOU……….
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