Column agglutination gel techniques.pptx

MUHAMMAD SALMAN
BLOOD BANK
AKUH

CONTEN
TS
Historical Aspects
Basics of
immunohaematology
Principle of Gel Technology
Grading of reaction
Applications / Uses
Literature
Advantages /

Historic
aspects
Ancient Egyptians bathed in blood and
aristocrats consumed blood so as to cure
disease.
1616:Concept of circulation of blood Sir
William Harvey
1665: First canine transfusion Royal
Society of London
1667: Animal to human transfusion J Denis
1818:Human to human transfusion James
Blundell

• 1869:Use of nontoxic anticoagulant-
sodium phosphate: Braxton Hicks
• 1901:Discovery of ABO blood groups and
transfusion reactions : Nobel prize awarded
Karl Landsteiner
• 1916:The first anticoagulent preservative
(citrate- glucose) was used to preserve
rabbits blood for 2 weeks.
Rous-Turner’s solution was used for storage
of blood during the First World War.

•1939:Discovery of hemolytic disease of
newborn: Levine and Stetson.
•1940: Discovery of Rh factor and anti Rh:
Landsteiner and Weiner
•1940: discovery of human antibody
similar to antiRhesus : Wiener and
Psters
•World War II stimulated blood preservation
research and developed techniques in blood
transfusion.
•The pioneer work of Dr. Charles Drew during
World War II led to the establishment of a world
wide system of blood banks

•1950: Freeze drying of plasma New
concept of component therapy
•1959: First attempt of Bone Marrow
Transplant in France
•1978: CPDA-1 was developed, ↑ shelf
life of RBCs for 35 days
•1988: First umbilical cords blood
transplant
•1999: NAT for HIV and HCV

BasicConceptsin
Immunohematology

14 nm 35 nm
24 nm
IgG IgM
Intracellular distance

IgM – Large pentameric
structures can connect
RBCs

Effects of NormalSaline
The RBCs are surrounded by an electron cloud
Require longer incubation time for cells suspended in saline
medium.
Weak & incomplete antibodies get repelled and hence are
unable to cause agglutination

Incubation time is
15 min Only.
LISS Methods

Gel Technology
• Innovative approach to red cell serology.
• Invented by Dr.Yves Lapierre of France in 1988.
• Developed to minimize problems associated
with conventional techniques of blood
grouping.
• Addresses the issues of standardization and
documentation with unmatched sensitivity,
specificity and efficiency.
• DiaMed-ID Microtyping system from DiaMed AG,
Switzerland in assocition with Dr. Lapierre, is
based on the Gel Tecnology.

• Controlled centrifugation of RBCs through a gel
column
• Serum and cell reaction takes place in a microtube
.
• Six microtubes in a plastic card – easy handling.
• Microtube consists of a reaction chamber that
narrows to become a column with a conical
bottom.
• Reaction chamber is designed to allow prior
incubation of test serum and RBCs.
• Each column contains Sephacryl gel

Add Reactants
Serum/plasma/
red cells
Principle
Reaction
Chamber
Gel and
Reagent
6 Microtubes in Plastic Card

• Sephadex gel matrix acts as a sieve.
• Large aggutinates remain on or near the top of gel
interface.
• Smaller agglutinates pass partway through gel ,
depending on size.
• Unagglutinated cells pass to base of microtube to form a
button.
• Cells are always added prior to serum so that
serum does not come into contact with gel –
this eliminates the ‘WASH PHASE’ as in
conventional technique.
• Grading of reaction depending on the

INTERPRETATION OF
GEL TEST
4+
Solid band
of red cells
at top of
gel
3+
Agglutinated
red cells in
upper half
2+
Red cell
agglutinates
through
length
1+
Aggl. red
cell in
lower half
of gel col.
NEGA
TIVE

Uses of Gel Technology
Any immunohaematology test that
has haemagglutination at its end
point:
ABO-Rh typing, typing for other blood
group systems.
Antibody screening and
identification. Compatibility testing
– crossmatching. DAT/IAT, other
Coombs phase test.
Antibody classification- IgG, IgM, IgA,
complement…

Forward & Reverse typing
• The process of identifying an individual’s bld. grp.
Involves testing of red cells with known antisera
(FORWARD TYPING) and plasma with known
group red cells (BACK/REVERSE TYPING)

Reagents required
• i. ID DiaClon ABO/D + Reverse typing cards
containing monoclonal anti- A, anti- B & anti- D
suspended in the gel. The tube labeled “Ctl” is
the negative control. Two tubes with “neutral” gel
serve for reverse grouping with A1 and B cells.
• ii. ID NaCl/Enzyme card which contains six
microtubes with neutral gel.
• iii. ID – Diluent 2: modified LISS for red cell
suspension.
• iv. Test cell reagents: ID DiaCell A1, B, O (0.8±

• For ABO/D determination (forward typing)
• Prepare a 5% red cell suspension in ID – Diluent 2
(LISS) as follows:
• Allow the Diluent to reach room temperature
before use.
• 1. Dispense 500μl of ID – Diluent 2 (LISS) into a
clean test tube.
• 2. Add 25μL packed cells & mix gently.
• For Reverse typing
• Use Plasma or serum

50 µl
Diacell
A1
50 µl
Diacell
B
50 µl
Patient
Serum
50 µl
Patient
Serum
10 – 12.5µl Patients RBC
suspension 5%

Forward and Reverse typing
• Incubate the card at room temp. for 10 minutes
• Centrifuge the card for 10 minutes.

• Determination of
ABO/Rh- antigen and
DAT.
• DAT on newborn blood
samples has become a
standard procedure,
since it is important to
know if the newborn’s
red cells have been
sensitised with
maternal antibodies in
utero.
ID-Card “ABO/Rh for newborns”

Cross Matching
Reagents required
• i. ID LISS/Coombs cards with six microtubes
containing polyspecific AHG (Anti- IgG+ C3d).
• ii. ID – Diluent 2: modified LISS for red cell
suspension.
Prepare a 0.8 -1.0% red
cell suspension in ID–
Diluent 2 (LISS) as
follows:
i.Dispense 1 mL of ID –
Diluent 2 into a clean
test tube.
ii.Add 10μL of packed

Direct and Indirect Coombs Test
• Detection of Ig & complement bound to RBCs is
critical in diagnosis of immune mediated
hemolytic anaemia.
• DAT is used in inv. of Autoimmune &/
Alloimmune Hemolytic anaemia in which RBCs
are coated with in vivo antibodies &/
complement.
• IAT detects alloantibodies in serum of blood
donors, prospective transfusion recepients
&prenatal patients.

Direct Coomb’s Test
Patient’s RBC
suspension Add 50 µl of
above soln.
Results

DAT +ve: Further options
DC-Screening I
•DAT + with polyspecific
AHG indicates RBCs
coated with
Ig/complement.
•To differentiate the
reaction, monospecific
AHG reagents-anti IgG,-
IgA,
-IgM, C3….
•Results define a
clinical condition :
•WAIHA- IgG ± C3
•CAD- C3 ± IgM/IgA

DAT +ve: Further options….and more
DAT IgG1/IgG3
•Risk of hemolysis by
AIHA
/HDN depends on
amount of IgG &/
complement coated on
RBCs as well as on IgG
subclasses involved.
•IgG1 & IgG3
activate
complement.
•In DAT IgG1/IgG3 card
2 dilutions of both anti-
& anti-IgG3 are added to
gel to differentiate betwn
low &high risk for

DAT +ve: Further options….and more
DAT IgG-Dilution
•DAT IgG – Dilution card
prvides an indication of
clinical importance of DAT
positive results.
•The number of IgG
molecules per cell
influence the red cell
destruction seen in
AIHA,HDN and
Transfusion reactions.

Indirect Coomb’s Test
O cell
suspension Add 50 µl of
above soln.
Results
Add 25µl of
patient serum
Centrifuge Incubate

Review of Literature
• Kaur et al at Christian Medical College and
Hospital presented their experience with gel
based DiaMed ID microtyping system for
incompatibility testing over a year period and
noticed a startling 65 fold rise in the reported
no. of incompatibe units in 1 yr. which rose from
4 (0.02%) to 260 (1.6%) thus making
gel technology more sensitive than
CTT for crossmatching.

• Role of Gel based technique for Coomb’s test
• M Jaiprakash, PK Gupta Harsh Kumar
• Dept. of Transfusion Medicine, AFMC, Pune
• AIM: Compare CTT & GT for Coomb’s test & to
evaluate their sensitivity & specificity.
• Result
s:
POSITIVITY CTT GTT
GT Sensitivity Specificity NPV
DATDAT 100% 6.1%97.3% 8
1
.
6
0
%
0
%
IAT IAT 100% 5.4%97.6% 6
1
.
6
0
%
0
%
• Conclusion: Gel Technology is a better
alternative to CTT.

• Gel card in diagnosis of autoimmune
haemolytic anaemia.
• Renu Saxena et al at AIIMS, New Delhi.
• Aim: To compare the efficacy of Gel card
Coomb’s test with conventional Coomb’s test in
diagnosis of AIHA.
• Results: 13 out of 50 cases showed positive ICT
& DCT by both CTT & GT. Out of 13 cases 3
showed (+)DCT & (-)ICT by CTT but GT showed
(+)DCT & (+)ICT in all 3 cases.
• Conclusion: Simple, reliable and quick method
for detection of autoantibodies, antibodies

Antibody Screening and
Identification
•
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Various Gel Cards
Rh-Subgroups Single Antigen
Single Antigen Single Antigen

Particle Gel ImmunoAssay (PAGIA)
• Gel Technology now adopted with use of inert
polymer particles for detection of:
• Syphilis
• Paroxysmal Nocturnal Haemoglobinuria
• Leishmaniasis
• Sickle Hb Screening

Paroxysmal Nocturnal Hemoglobinuria
• Patients with PNH have a defective gene called
PIG-A, involved in the biosynthesis of glycosyl-
phosphatidylinositol (GPI).
• Without GPI, important regulatory proteins (e.g.
CD55 or “DAF”, and CD59 or “MIRL”) cannot
bind to the cell surface and protect blood cells
from attacks of complement.
• This may result in a break down of erythrocytes
and release of hemoglobin which causes the
urine to turn dark during an episode (or
“paroxysm”) of hemolysis, though this is not
found in all cases.

Paroxysmal Nocturnal Hemoglobinuria
Reagent1:
ID-PNH-Gel card with rabbit antiserum against
mouse immunoglobulines.
Reagent 2:
Monoclonal antibodies directed against DAF and
MIRL respectively, and negative control, ready-
to-use, 1.4 mL.
Additionally required: Diluent 2.

ID-PNH-Gel card with rabbit
antiserum against mouse
immunoglobulines.
Monoclonal
antibodies
directed against
DAF and MIRL
respectively, and
negative control,
ready-to- use,
1.4 mL.

Principle of PNH Gel Technology
• Monoclonal anti-DAF and anti-MIRL are incubated
with RBC suspension of suspected PNH cases
and then centrifuged through a microtube
containing rabbit antimouse antibody suspended
in a gel column.
• After centrifugation through the gel, cells
carrying antibodies, confirming the presence of
MIRL or DAF, will show a positive reaction.
This denotes that the patient does not have
PNH.
• Negative results or double population confirm the
presence of PNH.

ID-PAGIA Syphilis Antibody Test

Advantages of Gel Technology
Improved sensitivity and
specificity Easy to use, simple
to read
No wash phase in IAT
Minimal training required
Reliable, reproducible
results
Easy storage and long shelf life of
reagents Easy disposal of
biodegradable cards

Disadvantages of Gel Technology
Special centrifuge to accommodate the
microtubes cards.
Special incubators to incubate the microtube
cards. Pipette to dispose 25ul of serum.
Expensive.

INTRODUCTION
 Process whereby an analyticalinstrument
performs many tests with only minimal
involvement ofanalysts.
 - mechanization of stepsin aprocedure
 Started in 1940; to reduce manual errors due to
fatigue/erroneous sampleidentification.
 Consolidate chemistry & immunoassaysystems
on oneplatform.

INTRODUCTION
 Factors necessitatingLaboratory
automation
 Turnaround times demands
 Staff shortages
 Lessmaintenance
 24 /7uptime
 Throughput of samples
 ↑ no. of analytes & methods on one system.

Aut om
at ed i dent i f i cat i on m
et hods
Barcodes
 A barcode comprises a series of vertical bars and
spaces arranged in various combinations to
represent different characters.
 There are different barcode systems, each with
different rules governing the representation of the
characters. eg. CODABAR system with ABC
symbols
 By combining the numbers, letters and other
characters, a series of barcodes can be built up to
represent donation numbers, blood groups and
various blood products.
 In each instance an eye readable number or
description is included with the machine- readable
code.
 Device which will interpret barcodes pass a beam of
light across the code making use of 2 levels of

Bl ood bank i nf or m
at i on syst em
s
 Blood bank information systems are computer
systems that have been developed specifically to
assist the blood bank professionals in management
of the patients, donor and blood component
information.
 Helps to assesstrends and decide future policies by
accessing the statisticalinformation.
 Helps to correlate the laboratory data with donor
records and help to trace the donor records
following transfusionreactions.

 BBISconsists of :
 Hardware
 Software: application software,operating
software and interfacesoftware
 Users
 FDA,AABBandCPAhave specific regulatory and
accredation requirments for BBIS

Stages in Laboratory Investigations
 Pre –analyticalstage
 Analytical stage
 Post –analyticalstage

PRE – ANALYTI CAL
STAG
ES IN LAB.
 Sample Delivery-
 Blood drawers or runners / courier facility
 Pneumatic tube deliverysystem
 Conveyers or tracksystem
 Mobile robots
 Sample processing –Threephases
 Pre-centrifugation :- all measurements in <45min.
 Centrifugation :- blood (Plasma/Serum),Urine &
Other body fluids (conc.particulate matter)
 Post-centrifugation

PRE ANALYTICAL MODULES
 Labeling –Bar code labelsystem.
 Sorting –stopper color, size,tests ordered,
instrument designrequirements,
 Decapping
 Aliquoting
 Recapping
 Storage / retrieval

CENTRIFUGATION &
VO
LU
M
E D
ETER
M
I N
ATI O
N

ANALYTI CAL STAG
E
 Tasksincluded –
 Sample introduction & transport to
cuvet or dilutioncap.
 Addition ofreagent
 Mixing of sample& reagent
 Incubation
 Detection
 Calculation
 Readout & result reporting

PO
ST – AN
ALYTI C
AL STAG
E
 Data processingi.e. –data acquisition, calculation,
 Monitoring & displaying –charts & curves,
 Performing statistics on patient & control value –
flags.
 Analyzer computers have capacity to link to –
 lab. Info.System
 internet –companiesown manufacturing site –can
seereal time lab. data & can solve mechanical
problem in short duration.

AUTO
M
ATED SYSTEM D
ESI G
N
S
 Total Laboratory Automation -
 Japan,1980
 Integration of several instruments =processing
specimen management +transportationsystems
+analyzers +digital interpretation +dispatch of
results.
 creates inclusive, continuous networkmaking
each step in testingautomated.

AUTO
M
ATED SYSTEM D
ESI G
N
S
 Chemical, hematological, coagulation & immuno -
histochemical studies at the same time on asample.
 Advantage –reduction in –labeling errors,
turnaround times.

AUTO
M
ATED SYSTEM D
ESI G
N
S
 Disadvantages –significant financialinvestment
- Increased floor space.
- highly technicalpersonnel
- Infrastructure remodeling,
- personnel teambuilding,
- Software interfacing.

Automated blood grouping
 Groupometric:
 Matte in 1963
 It is an automated blood grouping machine
using multi-channel cuvettes for the reaction
mixtures
 Electro-mechanical units: for agglutination
reactions
 Electronic unit: for processing the results
of agglutination tests and print out of the
group.
 Autoanalyzer grouping machine with
continuous flow system and laser scanner.

The ORTHO AUTOVUE INNOVA System is an automated
imunohematology testing system used for blood typing,
antibody screening and compatibility testing using
ORTHOBIOVUE System cassettes. The ORTHO
AUTOVUE INNOVA System is a computersoftware driven,
fully automated system which provides automated liquid
pipetting,cassette handling, incubation, centrifugation,
reaction grading and interpretation.
The software used with the AUTOVUE INNOVA can be
interfaced with a laboratory information
system for test data transfer.

APHRESIS
 Apheresis is collection of anti-coagulated whole
blood from adonor, its separation into
components, retention of desired component
and return of remaining constituents backto the
donor with the help of automated cell separator
machines.

ADVANTAGES OF APHRESIS
 Reducedmultiple donorexposure
 Reduced risk ofalloimmunization
 Reduced incidence oftransfusion transmitted
diseases
 Full and effective transfusiondose
 Purer product:
 leucocyte reducedproducts
 High qualityproduct
 Fewerdonor reaction due to return of fluid

Typesof cellseparators
Intermittent flow cell separator (closedsystem)
Continuous flow cellseparator
Automated separation techniquesby
centrifugation
Cell separation by membranefiltration
Continuous magnetic cellseparator
(immunomagnetic)

 Automated separation techniqueby
centrifugation:
 Centrifugal force separates blood into
different component depending upon the
specific gravity.
 Blood is drawn from an a
u
t
o
m
a
t
i
cpump
Anticoagulant isadded tube blood is
pumped into roatating bowl,chamber in
which layering of components occursbased
on the density desired component
retained and rest returned to donor either
by continous flow or by intermittent flow.

 Separation by MembraneFiltration:
 Filtration of plasma through membrane which
allows collection of plasma from ahealthy
donor.
 Membranes are arranged ashollow fibres
which expelsthe cellular elements in the flow
of blood.
 Most commonlyused apheresis devices are:
Haemonetic corporation: Platelets,plasma,
leucocytes.
Baxter: Plasma, platelets, redcells,
leucocyte
Gambro: Plasma, platelets,leucocyte and
peripheral blood stemcells.

TECHNOLOGY FUCTIONN
HAEMONETIC Intermittent flow centrifuge separator.
Anticoagulant blood is pumped into rotating bowl
Incoming blood isseparated , red cells move to the
periphery and plasma to inside of rotating bowl and the white
cellsand plasma between red cellsand plasma
Using optical detectors and fluid surge elutriationprocess
desired component retained.
GAMBRO(Cobe) Continuous flow centrifuge cell separator where two arm
blood is drawn andreturned.
Here flat membrane is used to separate the cellsof blood from
plasma.
Allows lower WBCand RBCcontamination in platelets.
BAXTER Continuous flow technology.
CS3000 has two separation containers firstly for collection of
leucocyte reduced platelets and other for white cells (CS3000
plus).

COBE Spectra – Automated Apheresis

REFERENCES
 Transfusion Medicine-Technical Manual,2
edition 2003,WHO
 www.DiaMed.com
 AABB 20th Ed.

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