IVD for
Immunohematology
Dr. Cristina Boero
Bio Rad Laboratories - Immunohematology Division
R&D System Department
Cristina BOERO, PhD
• Biomedical Engineer from Politecnico di Torino (Italy)
• PhD in Microsystems and Microelectronics from EPFL
(Switzerland)
• May 2015
CGD, Immunohematology Division
Marketing
R&D System Department
Alain LOMMELÉ
System Departmentr Manager
Paul AERTS
Scientifc Affairs Manager
Olivier NOËL
System Engineer Manager
Cristina BOERO
System Engineer
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System
Engineers
Integration
and
Verification
Validation
Bio-Rad History
• Founded in 1952 by David and Alice Schwartz in Berkeley, CA
• The name “Bio-Rad” comes from biochemicals and radiochemicals
• In 2007 Bio Rad acquired
, leader in reagents and
instruments for blood typing and screening.
• Sales exceeded $2.1 billion in 2014
• Over 7,600 employees
• Balanced and complimentary business mix
Hercules, CA
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ID-Card by DiaMed
Sales by Market Segment
Industrial
3%
Hospital labs
34%
Biopharmaceutical
9%
Academic
23%
Reference labs
16%
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Transfusion labs
15%
Sales History
$2.1 billion
$1.9 billion
$1.8 billion
$1
billion
$900 million
$800 million
$700 million
$600 million
$500 million
$400 million
$300 million
$200 million
$100 million
1959
5
1964
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1969
1974
1979
1984
1989
1994
1999 2014
Clinical Diagnostics Products
AN INTEGRATED SYSTEMS APPROACH . . .
Diagnostic Tests
Quality Controls
Infectious Disease
AIDS/HIV
Blood Virus Detection
Bacteriology
Diabetes Monitoring
Genetic Disease Screening
Molecular Pathology
Autoimmune Testing
Blood Typing
Quality Controls
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Specialized
Testing
Instruments
Life Science Research Products
CIRCLE OF DISCOVERY . . .
Electrophoresis
Gene Transfer &
Modulation
Protein Expression
Protein Function
(identification &
interaction)
Food Safety
(food pathogen
testing)
Chromatography
Amplification
(PCR & qPCR)
Image Analysis
Digital Biology
(Droplet Digital PCR)
Cellular Analysis
(imaging & cell
sorting)
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Outline
• Introduction
- History of transfusion
- Human blood
• In-vitro diagnostics for immunohematology
- Transfusion and blood banks
- Principle of agglutination
- Analysis techniques in immunohematology
- Blood grouping and antibody identification
• IVD and automated instruments
- Automation in blood transfusion
- Example: IH-500
• Conclusion
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Introduction
History of transfusion
Human blood
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The Transfusion History
• Ancient Egyptian inscribed surgical
instruments on the wall of a temple (2nd c.
BC)
• Blood has been considered the «living
force» of the body
• Blood was considered one of the four
humors described by Hippocrates
• Blood letting was a common practice in
ancient time (until late 19th c.!!!)
http://collectmedicalantiques.com
/gallery/surgical-history
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http://www.history.com/news/history-lists/7-unusual-ancient-medical-techniques
The Transfusion History
Countess Elizabeth Bathory of Hungary took
daily baths in the blood of female virgins, killed by
her servants. She believed it would restore and
beautify the skin.
After 10 years, in 1610, the King ordered an
investigation, due to the lack of virgins in the area.
Soldiers found skeletons and a freshly killed
young female, drained of blood.
She was walled up in her bedchamber, where she
died 4 years later at age 54.
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The Transfusion History
• 1492, the first written mentioned transfusion for Pope
Innocentius VIII
• 1628, Discovering of the circulation of the blood due to
William Harvey
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The Transfusion History
• 1667, first blood transfusion experiments between
sheep and humans
• 1670, prohibition of the transfusion praxis due to high
incidence of mortality
Jean- Baptiste Denis, (1640–1704)
Montpelier, France. Physician to
Louis XIV.
1st Tx to 15 year old boy
Fever treated by blood letting (20!).
Given about 12 oz lamb‘s blood
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The Transfusion History
No more work on blood transfusion for 150 years…
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The Transfusion History
• 1818, James Blundell restarted experiments with blood
transfusion human to human (5 successful cases vs. 5
unsuccessful cases)
• 1850, Blood transfusion established in England due to
curative prescription of blood from newly executed
criminals with reduce of mortality to “just” 30%
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The ABO System
Karl Landsteiner
1868 - 1943
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• Born in Vienna in 1868
• Graduated from Vienna Medical
School in 1891
• He discovered the agglutination in
1900
• He defined the ABO blood group in
1901
• He got the Nobel prize for the
discovering of the ABO blood group in
1930
The ABO System
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The ABO System
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The ABO System
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The Rhesus System
Levine and Stetson - 1939
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The Rh(esus) System
Levine & Stetson
A mother ”Mary Seno” had a stillborn child
She needed a transfusion and was given blood from
her husband (both O group)
And suffered from a hemolytic transfusion reaction!
Her antibody reacted with ± 80% of human red cells
And was proven not be
•
•
•
•
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ABO
MN
P (now called P1PK)
The only systems known at the time
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Philip Levine,
1900-1987
35 Blood Group Systems
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Introduction
History of transfusion
Human blood
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Human Blood
Blood cells are made in the bone marrow before being
released into the circulation!
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Human Blood
Consists of different
parts or components
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Plasma 5050-55%
White cells <1<1-2%
Platelets <1%
Red cells 45%
Blood components: plasma
• Dissolved proteins (albumin, globulines, fibrinogen)
• Glucose
• Clotting factors
• Electrolytes
• Hormones
• Antibodies
www.the-scientist.com
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Blood components: Red Blood Cells
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In-vitro Diagnostics for
Immunohematology
Transfusion and blood banks
Principle of agglutination
Analysis techniques in Immunohematology
Blood grouping and antibody identification
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Why do we need a transfusion?
• Surgery
• Injuries
• Severe infections or liver disease
• Severe anemia
• Bleeding disorder (hemophilia or thrombocytopenia)
• Sickle disease
• …
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Blood donation
Registration
Donors receive basic eligibility (72 years old in Germany) and
donation information.
Health History
Answer a survey (private interview) about health history, drugs,
sexual activity, travels…
Check of temperature, pulse, blood pressure and hemoglobin
level (new capillary test)
The Donation
Clean an area on arm and insert needle
Classic donation takes about 8-15 minutes (500 ml of blood),
platelets, red cells or plasma up to 2 hours)
Sample tubes dedicated for virology and IH testing (in addition to
segments)
Refreshments
Snack and something to drink for 10-15 minutes.
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Identification and centrifugation of blood
bags
Blood bags are identified by barcodes in a
reception room
Centrifugation of blood bag to separate the
blood into Red Blood Cells (RBCs) and
plasma.
All components are still in the same bag but
separated in strips
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Separation of RBCs and Plasma
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Preparation of segments
RBCs
Plasma
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IH testing: sample preparation
Virological tests:
• Hepatitis B virus
• Hepatitis C virus
• HIV 1,2
• Syphilis
WHO recommandations
Immunohematological tests:
• ABO
• RhD
• CcEe
• Kell
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In-vitro Diagnostics for
Immunohematology
Transfusion and blood banks
Principle of agglutination
Analysis techniques in Immunohematology
Blood grouping and antibody identification
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Antigenes
• They may trigger an immuno-response
• They are mostly molecular connections of sugar, proteins,
lipid, as well as nucleoid acids
• The immune system usually does not react to selfantigens
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Antibodies
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•
An antibody also known as an immunoglobulin (Ig), is a
large, Y-shape protein produced by plasma cells
•
The antibody recognizes a unique molecule of the harmful
agent, called an antigen, via the variable region
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Antibodies
• “complete antibodies”
•
•
•
IgM-antibodies.
The big pentameric molecule is able to build a
bridge between 2 RBC’s by itself.
They react at 4 °C
• “incomplete antibodies”
•
•
•
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IgG-antibodies.
The smaller monomer antibodies are less able to
build a bridge between 2 RBC’s and need help
from a antibody against IgG.
They react at 37 °C
Agglutination principle
Complete antibodies
Incomplete antibodies
Spontaneous agglutination
Direct/Indirect antiglobulin
test
• Anti-A and Anti-B
• Anti-Lea
• Anti-M
• Anti-N
etc.
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• Anti-Rh
• Anti-Fya
• Anti-Jka
etc.
In-vitro Diagnostics for
Immunohematology
Transfusion and blood banks
Principle of agglutination
Analysis techniques in Immunohematology
Blood grouping and antibody identification
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Techniques in Immunohematology
There are 4 different techniques:
• Slide or tile method
• Tube method
• Microplate technique
• Gel card technology
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Slide or tile method
Pros:
• Cheap and fast
• Requiring few material and
space
• Slide and tile are re-usable after
decontamination
• No incubation and
centrifugation
• No need of further instruments
and electricity
Especially used in developing
countries
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Cons:
• Completely manual
• Only for ABO, Simonin, Rhesus
and phenotype tests
• High false negative
• Weak reaction are hard to
define
• When screening the D antigen,
a heating lamp is needed
(rhesuscope)
• No archive/history of the tests
performed (no stable reaction)
Tube method
Pros:
• Cheap and fast
• Tubes are re-usable after
decontamination
• Highly sensitive for reactions
involving IgM Abs (blood
grouping)
• For ABO, Simonin, Rhesus,
phenotype, Abs and Ags
screening
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Cons:
• Completely manual
• Tube consuming
• Need of 37°C and centrifuge for
certain tests (low throughput)
• Long incubation needed for
some tests (>30 mins)
• Low sensitivity for some tests
(D antigen)
• Risk of fault pos or neg due to
washing step
• No archive/history of the tests
performed
Microplate technique
Pros:
• High throughput (from 8 and 24
patients on the same MP)
• Usable on several automated
instruments
• Automated agitation and
centrifugation
• Possible visual or automated
reading
• Possibility to archive the results
• Not expensive
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Cons:
• Only for blood, Simonin,
Rhesus and phenotype tests
• Incubation needed for some
tests (10-15 mins)
• Risk of fault pos or neg due to
agitation
• Low sensitivity for weak D
• Results are not stable for longer
than 1 min
• If not automated, the handling
is difficult
Gel card technology
Pros:
• High throughput
• Both manual and automated
• All tests are covered
• High sensitivity
• Safe for the results and for the
user
• Automated reading
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Cons:
• Incubation needed for some
tests (10 mins)
• The centrifuge needs to be
adapted for cards
• Incubator is needed
In-vitro Diagnostics for
Immunohematology
Transfusion and blood banks
Principle of agglutination
Analysis techniques in Immunohematology
Blood grouping and antibody identification
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Blood grouping and antibody identification
Antigens identification
Antibodies identification
• ABO/RhD is very important in blood transfusion
• Antibody screening/identification is essential for pregnant
women and transfusion recipients
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Blood grouping
• ABO grouping
Forward grouping
(blood cell testing)
Reverse grouping
(sera testing)
• Standard phenotyping (anti-D, anti-C, anti-c, anti-E, anti-e anti-Kell)
• Extended phenotyping (MNS, P1, LU, etc. systems)
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Antibody screening
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Reagents – ID-System
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Example 1 – Crossmatch donor-receiver
A
(ABO1)
B
(ABO2)
AB
(ABO3)
D
(RH1)
CDE
(RH123)
Ctl
(Smurf)
(Brainy Smurf)
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(Hefty Smurf)
(Smurfette)
Example 2 – Hemolytic disease of the
newborn (HDN)
Blood grouping of the baby
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Antibody screening of the mother
Myths and Legends
Group A have the worst hangovers!
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Myths and Legends
More group A’s in the highest socioeconomic sector of society!
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Myths and Legends
Group B’s go to the toilet the most,
have more criminal tendencies and
are the most impulsive!
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Myths and Legends
Group A2 have higher IQ’s!
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Myths and Legends
Group O’s have the best
teeth....
but have weak characters
and little personality.
Mosquitoes prefer group O
blood!
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IVD and automated
instruments
Automation in Blood Transfusion
Example: IH-500
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How blood grouping is currently done?
Manually
Reagents
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Automated
Gel Cards
Samples
First step: pipetting
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Second and third steps
Incubation
Centrifugation
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Why automate?
•
•
•
•
•
Reduce errors in sample identification
Reduce errors in processing tests
Non-subjective interpretation
Prevent transcription errors
Improve objectivity, reproducibility, retrieval
and storage of results
• Reduce manual steps, facilitating improved
turnaround time
Adapated from:
Asian J Transfus Sci. 2012 Jul-Dec; 6(2): 140–144.
Why automate?
EFFICIENCY
ERRORS
PATIENT
CARE
• Cost
• Time
• Material
Technicon AutoAnalyzer I (1963)
IH-500
LEVEL OF AUTOMATION
TANGO infinityTM
Techno
TwinStation
Swing
Saxo
TwinSampler ID-Reader
Manual
workplace
Classic
ID-Gelstation
Lyra
MP-Reader
Banjo
ID-Reader
SAMPLES PER DAY
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IH-1000
HemOS™ SP II
ID-System
BIO-RAD Total Solution
SOFTWARE
REAGENTS
AUTOMATION
BIO-RAD
IHD
…….The most comprehensive solution for
Transfusion Medicine laboratories
IVD and automated
instruments
Automation in Blood Transfusion
Example: IH-500
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IH-500
Conclusion
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Summary
•
•
•
•
Blood Transfusion – Donor & Receiver
Techniques in Immunohematology
Blood grouping vs. antibody identification
Automation in blood transfusion
Prosense and Bio Rad: common interests
•
•
•
•
Antibody/Antigene-based detection
Optical biosensor
Qualitative rather then quantitative approach
Automation in blood transfusion
Acknowledgments
•
•
•
•
•
•
Alain Lommelé - System Department Manager
Paul Aerts – Scientific Affairs Manager
Olivier Noël – System Engineering Manager
Vincent Maisonniac – System Engineer
Paul Lamonby – International Product Manager
Wida Aziz – Validation Technician