WHAT IS PATHOGEN INACTIVATION?
● A process of killing micro-organisms in biological fluids including:
- Viruses
- Bacteria
- Parasites
● PI is a well-established approach to treat fractionated blood products (proteins) during manufacture.
● PI is thus currently being explored to increase the safety of plasma, platelets and blood components including
RBCs.
REDUCING THE RISK OF TRANSFUSION-TRANSMITTED INFECTIONS
Donor history
Donor examination
Donor testing
Diversion of initial aliquot
Leukoreduction
Post donation information Donor deferral registries
Limit donor exposure
ONGOING AND UNTESTED RISKS TO THE BLOOD SUPPLY
 Any agent known to cause disease in man and that has a viremic or bacteremic phase during its clinical course.
 Agents for which there are no routine screening tests in place include (partial list):

vCJD
HAV Foamy viruses

Malaria
HPV
HEV

HHV-8
Dengue Leishmania

Parvovirus
Rickettsia
SARS

Babesia
Chikungunya
etc.
PATHOGEN-INACTIVATED BLOODCOMPONENTS
 Goal: Eliminate transmission of viruses, bacteria and parasites (known and unknown)
 Secondary Specific Drivers:
Bacteria
Parasites
CMV
GvHD
Methods:
Well established/validated methods:
• Chemicals: Physical disruption
== > Solvent/ detergent technology
• Photoactive compounds: Genomic disruption
• Psoralen derivatives (amotosalen) • Riboflavin
Methylene blue
• Chemicals: Short-term activation
Genomic disruptions-303 (FRALE: Frangible Anchor Linker Extender)
• Direct radiation effect
Genomic disruption- I-JVC
Dual, dedicated viral reduction methods ( Combinations:)
Solvent-detergent
Pasteurisation pH 4 (IgG)
Caprylic acid (IgG)
To be implemented at large scale following relevant GMP
Types of ELISA
:Five Types of ELISA
Antiglobulin
Competitive
The best: Sandwich
the worst : Competitive (many false positives
the most specific: Combination
Variants of Elisa :
Microwells -- Beads
Viral Tx transmissible infections
HBV
HCV
Only DNA
Sandwich
Hepatitis A, E
Feaco-oral
Dry-heat treatment
Antibody Capture
Hepatitis G &TT virus
Not related to hepatitis
so no screening
HTLV 1,2
Human Herpes Virus 8
CMV
Epestien Barr virus
Endemic in
Kaposi-sarcoma in
Endemic in egypt Infectious
japan
immunocompromized
Cellular virus
mono-nucleosis
Parvovirous b19:
* in sickle cell and Thalassemia can cause = Aplastic Crisis
*severe fetal anemia fetal death or malformation
Nanofiltration
Combination assays
HIV 1, 2
West Nile virus
Bite of infected
mosquito
Evaluation and Use of Test Kits for Transfusion-Transmissible Infections
Definitions
Selection and evaluation
Validation
Control during routine use
1) Sensitivity
 The ability of an assay/reagent to detect very small amounts of analyte
 The ability of a test to detect positive cases (the absence of false negatives)
 Probability of an assay detecting all infected individuals
2) Specificity
 The degree of false reactivity associated with an assay/reagent
 The ability of the test to identify all negatives correctly: i.e. produces no false positives
Selection of Test Kits
 Directly contributes to the safety of the blood supply
 Must be of high quality, reliable and consistent
 Must do what is required of them
 Should be selected on the basis of laboratory/ quality requirements, not cost alone
 Cheap tests kits often actually cost a lot more because of poor specificity and failed test runs
3) Kit size
Number of tests per kit
Different sizes available
Other reagents in the kit: e.g. diluent
4) Shelf life
Overall shelf life of the kit and all
reagents in the kit
Life of reagent when delivered
5) Robustness during transportation
Time to ship from storage centre to
Storage/handling requirements
user (door to door)
during transport
6) What is the assay used for?
 Number of tests and frequency of testing
 How will it be used?
Manual or automated
Time between ordering and delivery
Actual conditions during transport
Methodology
7) Who will use it?
What constraints are there?
Resources
Methodology
What sensitivity?
What specificity?
National regulations
Testing strategy
What Determines Overall Performance?
 Specificity and sensitivity are key factors - BUT other factors should be considered: e.g.
Ease of use
Sample type and quantity
Sample/reagent addition checks
Available technology and methodology
Clear instructions
Competence of staff
Define specific requirements for the
test kit
Prepare a validation protocol for
laboratory assessment
Validate assay itself against known,
fully characterized material
Is the assay performing correctly?
According to manufacturer’s spcs
Evaluation and Final Selection
Collect all available relevant data
Validate most suitable selected test
kit
Validation
Review available data
**Evaluation by other laboratories
**List of test kits evaluated by WHO
Assess on paper against specific
requirements and list the most suitable
Review results
Select test kit
Equipment to be used, if relevant
Control During Routine Use
As expected following laboratory evaluation
Consistently
Reliably
Is the assay being used correctly?
Many problems are due to the user, NOT the manufacturer
Equipment must be properly maintained and calibrated
SOPs must be validated
staff must follow SOPs
Validation on receipt in the laboratory
* Shelf life
** Batch testing
Quality control in routine use
*For every batch of test
Storage conditions
* During use
** Stock
Role of the Quality Manager should ensure that
Evaluation is based on sound quality and scientific principles
SOPs are in place and are used
Staff are trained and certified as competent
Validation and re-validation are performed
Data are analysed and used to: **Improve quality **Identify problems
Best practice in Safe Injection
1. Elimination of Unnecessary Injection
Promoting Rational Prescribing
Educating the patients
2. Administer Injections Safely
== Make sure you are doing the ‘right’ things
Right Patient
Right Drug
Right formulation
Right dosage
Right time
Right route
3. Select safe medicines/blood component:
Proper handling of medicines/blood component
4. Use of sterile equipment
Use needle and syringe from sealed package
5. Avoid contamination
Wash hands Prepare on clean surface
Label clearly
Right injection equipment
Right storage
Observe proper storage conditions
Check expiry
Use syringes with re-use prevention features
Do not touch part of needle that will come in contact with patient’s tissue
6. Reconstitute drugs or vaccines safely
Use new sterile syringe and needle for
Use the correct diluents/water for
each reconstitution
injection
Reconstitute according to the
manufacturers’ specifications
7. Dispose of injection wastes and sharps properly
Immediate disposal of needle and syringe in puncture- and leak-proof container
8. Public health education
Anticoagulant &preservatives:
Whole blood volume collected into main bag is proportional to volume of Anticoagulant and preservatives used:
Ratio of Anticoagulant: Whole blood ( Which is a critical PROCESS) blood : anticoagulant = 6 : 1
With 70ml anticoagulant
With 63ml anticoagulant
Ideal Volume 500ml ± 50ml (450-550ml)
Ideal Volume450ml ± 45ml (405-495ml)
Ideal Weight 510gm - 620 gm
Ideal Weight 465gm - 560 gm
Anticoagulant
CPD / ACD → A=acid , C= citrate , D= Dextrose , P= phosphate===>
21 days expiry.
CPDA-1 →
A=acid , C= citrate , D= Dextrose , P= phosphate + A = adenosine ===> 35 days.
SAGM → S= saline , A= adenosine , G= glucose , M= mannitol =====>
42 days.
Citrate
Sodium
biphosphate
Dextrose
Adenine
Prevent clotting through chelating calcium , inhibiting calcium dependent steps of coagulation
cascade
Acts as buffer to control the decrease in pH expected from generation of lactic acid
Support ATP generation via glycolytic pathway
Acts as substrate for red cell synthesis of ATP resulting in improved viability
Types of Bags:
There are many types of blood bags to help in maintaining closed system throughout the separation procedures :
Single Double Triple Quadruple Pedi bags
Transfer bags with different capacities
Material Properties:
Pyrogen free
collapsible
colorless
Non toxic
Non fragile
No leakage
*Transparent and enables inspection of the bag content before, during collection and during blood transfusion
*Flexible enough to decrease eliminate resistance during filling and emptying of the blood bag
*To resist the extreme of temperatures [-80 to +50 °C]
*Does not cause any change in odour of any of its constituents
Tubing:
Leakage free
Characterized by flexibility and can be easily welded
collection and transfusion tubes should be 85 – 100
cm long
No cracks or distensions or kinks in the tube line.
Do not allow kinks
Have a stopper that can be broken to allow blood flow
Space between two successive tube numbers not more than 8 cm
Tubes must carry distinguished number, easily read and
unremovable
Needle:
Size 16-17 G
The design of the needle base enables proper fixation on
the donor arm
Covered with a cap that prevents leakage of anticoagulant
during storage
Exit Openings:
Compatible with transfusion set
Compatible with collecting tubes
Needle cover enables recapping and easily removed
Needle cover is sealed and the seal is destroyed when
removing the cover
Can be pierced and doesn't allow reclosure
Protection cap
Hanging position:
Must have an appropriate slit for hanging or placing it in the upright position
Sterilization:
Bag should be provided in a sterile state
Accessories:
Sampling port
Sampling bag
Needle Protector
Plastic clamps
What is NAT?
 Nucleic Acid Amplification Testing (NAT) :
 NAT is a molecular technology that focused on the detection of viral DNA or RNA of intended viruses.
 Highly sensitive and specific technique.
 Fully automated technique, either based on individual testing or pooling system.
Why NAT?
Highly sensitive & specific .
Targets specific viral nucleic acid sequences
Direct detection of low level of viral RNA or DNA.
Shortens the Window Period from infection to detection. Helps prevent transfusion transmitted disease.
Provides additional layer of safety to the blood supply.
Improves confidence in blood supply.
Window Period:
The most important factor for TTIs residual risk.
• The WP is defined as the time from infectivity to test reactivity.
• The chance of transmission is a function of both incidence and length of WP.
• Blood transfusion authorities and blood banks were concerned about the ability to close the gap of ‘window-period’
by additional steps to ensure quality and safety of blood and blood products.
NAT reduces the Window Period
Detection of HIV-1
HIV Ab from 21 days to 9 days
Detection of HCV
HCV Ab from 30-60 days to 7 days
HIV P24 Ag from 15 days to 9 days.
Detection of HBV
HBsAg from 44 days to 8 days
Screening Scenarios
 Why NAT with EIA and not NAT alone?
 NAT is complementary test to EIA screening and not supplementary.
 In some cases the viral load in the peripheral blood below the detection limit of the NAT assay due to wash of
the virus into hepatocytes or lymphocytes.
 The immunological markers will be the markers of infection detected in such cases.
 NAT testing will not replace the current serological tests in blood screening.
 So far no country has discontinued the serology screening after the implementation of NAT.
Residual Risk
 Residual risk =incidence rate X window period duration
 Incidence rate = seroconversions / Person / Years
 Sources of Residual Risk:
 Window period donations.
 Viral variants not detected by traditional serological tests.
 Immunosilent donors.
 Laboratory testing errors.
Techniques of NAT
: NAT utilizes either:
PCR: Polymerase chain reaction that permits the amplification of defined sequences of DNA, leading to
exponentially amplifying a target sequence. This significantly enhances the probability of detecting target gene
sequences in complex mixtures of DNA.
TMA : 'Transcription-mediated amplification" refers to nucleic acid amplification that uses an RNA polymerase to
produce multiple RNA transcripts from a nucleic acid template methods permitting the amplification of viral
sequences in vitro.
Steps of NAT
 Steps:
Target Capture and isolation
1) Samples are prepared for testing by lysing the viruses to release the genetic material – no pretreatment or
handling is required. Capture probes hybridize internal control (IC) and viral nucleic acids and bind them to magnetic
particles. Unbound material is washed away to remove non-specific material and to minimize potential inhibitors.
2) Amplification.
Transcription-mediated amplification (TMA) is used to amplify portions of the RNA and/or DNA. Reverse
transcriptase creates a DNA copy (cDNA) of the target nucleic acid. RNA polymerase initiates transcription,
synthesizing RNA. Some of the newly synthesized RNA amplification products reenter the TMA process and serve as
templates for new rounds of amplification. Potentially billions of copies are generated in less than one hour1.
3) Detection: Acridinium ester-labeled probes specifically hybridize to the amplification products.
Things to consider when planning to implement
1- NAT is technically demanding
2- Could interfere with timely release of critical blood components.
3- Would add to the cost of processing a unit of blood.
4- The retest algorithm should be well defined with NAT.
5- Turn around time for NAT results will be longer than any blood screening test currently in place
6- In case of Mini pool, the sample size should be considered regarding the sensitivity of the assay in addition to the
turn around time of the test.
7- Algorithm for resolving pools with reactive test results to determine individual donor source of a reactive pool.
EMERGENCY BLOOD TX
Acute blood loss can be:
*Visible such as that associated with open wounds.
*Invisible which may be associated with fracture femur or pelvis &uncontrolled GIT hemorrhage.
Symptoms of acute blood loss:
Symptoms appear after loss of 15 – 20% of blood volume (one liter in adults).
Hemorrhagic shock occurs with loss of sufficient quantities of blood (35 – 40%; 2 liters or more).
The goals for treatment of acute massive bleeding
1 Blood volume replacement to maintain tissue perfusion 2) Immediate intervention to stop bleeding from any site
3) Restoration of the oxygen carrying capacity of blood . 4 Correction&prevention of complications of massive Tx
Crystalloid
* Most common fluid used due to cheaper and available.
* Due to its low colloid oncotic pressure, only 20% remain within the circulation (IV space).
* Volume approximately 3 to 4 times of blood loss must be infused to maintain IV volume.
Colloid solution
Greater oncotic pressure and greater half life, so better
* Less used due to its cost and unavailability.
* Large dose can impair hemostasis.
* Which one is better has come into question.
2.Restoration of the oxygen carrying capacity ( RBCs transfusion)
Packed RBCs units are transfused to supply oxygen delivery to tissue ; whole blood may be used
The guidelines of RBCs transfusion: decision should be made on a case-by- case basis according to :
Ongoing blood loss Hb level Symptoms of impaired tissue oxygenation Signs of impending circulatory failure
* If whole blood is used the plasma contains active coagulation factors which may be of value.
The quality of RBCs for Tx is better to transfuse RBCs with storage time of less than 5-7 days because old RBCs :
a) Are deficient in 2,3 DPG. b) May adhere to the vascular endothelium secondary to the cytokines release
Hemoglobin level and the need for RBCs transfusion acute blood loss
*No transfusion when Hb is >10g%
*Transfuse RBCs when :
*Hb is 7 g% and Hct is 25%
* Massive uncontrolled bleeding what ever the Hb. level
*The dosing of RBCs transfusion is guided entirely by the extent of blood loss:
< 750ml : need 750-1500 ml crystalloid 1.5 -2 L: crystalloids and RBCs
>2 L : transfuse WB or PRBCs & saline.
Packed RBCs with saline transfusion is better than whole blood because:
Packed RBCs units may be transfused as type compatible for example :
2) Less anticoagulant is transfused.
3) Less products of the cellular elements ( cytokine , potassium, lactic acid….) as the are removed ..
4) The incidence of circulatory overload is much lower than whole blood
Disadvantages of Whole Blood Transfusion
1. When given alone to replace blood volume:
It increases the risk of disease transmission Should be of the same pt's group (limits use of type compatible RBCs
2. May induce circulatory over load.
3. For the blood bank :It will limit the preparation of other blood components (platelets & plasma).
Some times in emergency setting it may not be feasible to wait for completion of pre-transfusion testing (complete
blood grouping and cross-matching).
Similar or type-compatible blood group and even uncross-matched blood can be released in life-threatening emergency.
Regulations for the release of uncross-matched blood in urgent situation:
Still there is a considerable fear among doctors to use uncross-matched blood .
When the uncross-matched blood is requested urgently to a patient with massive uncontrolled bleeding, it is the
responsibility of both Physician and Blood bank personnel.
The physician must weight the hazard of giving uncross-matched blood against the risk of waiting for complete
cross-matched blood.
It is very important for the responsible physician to write down in the Pt’s records that the clinical situation was
sufficiently urgent.
This information may be useful for later transfusions to the same patient.
The blood bank personnel are responsible for the supplying :
* The safest available blood for the patient.
*
In the shortest time.
If the time is sufficient :
* Detect the patient’s blood grouping and transfuse uncross-matched RBCs units of a similar blood group
* This similar uncross-matched blood can be released with 99% of safety (the risk is 1 in 6000 units).
* In addition it will save the blood bank stock of O, Rh negative blood units for actual. Need.
*N.B. It is not allowed to trust any source for identification of the patient’s blood group (ID cards ,relatives ..)
for identification of the Pt’s blood group
If the patient's blood group is AB and. AB blood is not available at time in the blood bank give Compatible group
* It is very important not give whole blood ( A or B)to prevent reaction between the donor’s ABs & patient’s RBCs.
If the patient’s blood group cannot be done because:
* Transfuse 2 or more units of blood group O (Rh) negative packed red cells
N.B. : whole blood group O, Rh negative should not be used as it contains anti-A and Anti-B which cause hemolysis
3) If blood group O (Rh) negative blood is not available:
* Transfuse 2 units or more of blood group O (Rh) positive (especially if the patient is a male or post-menopausal)
* For women in the reproductive age it is better to give O Rh negative blood. However, in life threatening conditions
group O ;Rh positive blood may save the life of the woman because delaying transfusion may be more dangerous .
Follow up transfusion of un-crossmatched blood
a) The transfused blood type should be written in the Pt’s records ( it is of value if the Pt needs other transfusion) b) The
patient’s serum should be tested for :
1. Rh antibodies : In Rh negative patients transfused with Rh (+ve)
Premature Infant
90
2. Anti-A or Anti-B titer : In patients transfused with group {O} whole blood
Term Infant
80
If the Pt. needs blood ,transfuse the Pt ‘s group when the titer of Anti-Aor
Slim Male
75
Anti-B is not detectable
Obese Male
70
Slim Female
65
Obese Female
60
Massive Blood transfusion
It is generally defined as Tx of equivalent of one PT’s blood volume, or Tx of 10 units of blood within 24 hours.
OR , Replacement of more than 50% of circulating blood volume in less than 3 h
OR, Transfusion of >4 units of PRBCs in 1 h when on-going need is foreseeable
Transfusion at the rate of more than 150 ml/min or blood loss rate of 150 ml/min
Transfusion of more than 20 units of pRBCs in the course of hospital admission
The adverse effects of blood massive transfusion
Coagulopathy acidosis Hypothermia Hypocalcemia
Thrombocytopenia
The massive transfusion protocols ( MTPs)
The traditional protocol
Saline then PRBCs Then LABs FFp &PLTs if needed
The fixed ratio transfusion protocol
2:1:1 or 1:1:1 FFP:PLTS:pRBCs
TACO
TRALI
Hyperkalemia
Massive transfusion protocols are activated by a clinician in response to massive bleeding
YELLOW CODE
Generally this is activated after transfusion of 4-10 units.
Once the patient is in the protocol, the blood bank ensures rapid and timely delivery of all blood components together
to facilitate resuscitation.
This reduces dependency on laboratory testing during the acute resuscitation phase and decreases the need for
communication between the blood bank, laboratory and physician.
Limitations of massive transfusion protocols
Not standardized: The trigger for initiating the protocol as well as the optimum ratio of RBC: FFP: Platelets is
controversial. Therefore practice varies from centre to centre.
Wastage: If MTP is triggered for a non-massive blood loss situation, it may lead to wastage of blood products
.
Other haemostatic/blood replacement strategies
Activated factor VII:
manage uncontrolled bleeding is
unclear. However, it can be
considered as a rescue therapy in
patients with life-threatening
bleeding that is unresponsive to
standard haemostatic therapy.
recommended dose is 200 μg/kg
initially followed by repeat dose of
100 μg/kg at 1 h and 3 h
Antifibrinolytic agents:
Drugs like tranexemic acid may be
useful in bleeding complicated by
fibrinolysis such as cardiac surgery,
prostatectomy etc. Early
administration of tranexamic acid
in bleeding trauma patients has
been shown to significantly reduce
mortality.
Cell salvage:
Can be extremely useful in unanticipated
blood loss and in patients with rare blood
groups. This strategy is generally
reserved for massive blood loss in
operation theatres as asepsis can be
maintained easily. The relative contra
indications such as a possibility of
contamination with infected material and
malignant cells should be considered.
September 2019
1) Supportive management of leukemia.
2) Emergency blood transfusion.
3) Patient blood management , advantage and disadvantage
of RBCS transfusion.
4) Preparation, composition, and indications of FFP
therapeutic factors.
5) Non-ABO , RH blood group systems and their significance.
6) RBCS membrane diagram.
7) Quality control of platelets.
8) Complications of plasmapharesis .
9) AIHA difficulty in blood banks.
10) Indications of platelets transfusion, and indicator of
bleeding tendency.
11) HDFN.
12) Major ABO histocompitability prevention and treatment.
13) Pathogen inactivation of plasma.
14) Hazards waste and waste management.
15) Procedures of therapeutic plasma exchange.
16) Bacterial sepsis in blood transfusion , sources and
prevention.
17) Role of quality manager.
18) Factors and parameters affection RBCS and Ab production
and its significance.
19) Selective donor criteria.
20) Stages and enzymes of NAT.
October 2017
1) Quality elements .
2) HDN.
3) Differentiate between peripheral and bone marrow stem
cells.
4) Complications of plasmapharesis , describe one in details.
5) Types of leukemia , and how to diagnose.
6) Elisa types, and principles .
7) Factors affecting release and production of Ab ,and clinical
significance of each.
8) Types of autoimmune haemolytic anemia , and serological
findings of each.
9) Anticoagulant ratio to whole blood.
10) Diagram of red blood cell membrane.
11) Supportive management of leukemia.
12) Indications of therapeutic apharesis.
13) Mention viral transmitted infections.
14) Validation and evaluation of test kits.
15) Emergency blood transfusion.
16) QC of TTIs .
17) Pre-analytical errors in your lab.
18) Enumerate waste disposal steps.
19)Enumerate
methods of pathogen inactivation in plasma.
===========================================
Feb. 2019 1st.
1) Criteria of patient accepted in therapeutic unit.
2) External quality scheme.
3) Audit.
4) Types of Elisa.
5) Clinical uses of DAT.
6) Causes and prevention of HDN.
7) Types of blood bags and anti-coagualnts.
8) Screening assays. 10) Egyptian criteria of blood donor.
9) Categories of potential infection risk.
April 2019
1) Alloimmunization of blood transfusion.
2) Types of QC of TTIs.
3) Indications of TPE.
4) Mention viral TTIs.
5) Waste disposal.
6) Supportive treatment of leukemia.
7) Adverse reactions of donor.
8) Pre-transfusion compatibility testing .
9) Emergency blood release.
10) Define plasma , its derivative and its components.
11) Leukemia types and diagnosis.
12) How to secure national supply of plasma factors.
13) Platelets refractoriness causes and prevention and
treatment.
14) HCV , discuss mode of transmission.
15) Immune response to incompatible blood transfusion.
16) Types of ELISA.
17) Whats HDN , describe its 2 main types.
18) Define cross-matching , does ab screening replaces
cross-matching?
19) Donor deferral.
20) Appropriate use of FFP and cryopesipitate .
March 2017 1st
1) Emergency blood transfusion .
2) QC of packed RBCS and platelets.
3) Contingency plan , and hemovigilance.
4) Permanent donor deferral.
5) Different types of leukodepletion .
6) Anticoagulant ratio to whole blood.
7) Management of platelet refractoriness .
8) Elisa types and principles.
9) Methods of plasma inactivation.
10) Types of viral TTIs .
======================
Feb. 2018 1st
1) Mention serological methods of ag - ab detection, discuss
agglutination (principle, stages , reading and interpretation).
2) Role of hospital transfusion committee .
3) Types of RBCs.
4) QC of all blood components.
5) Crisis management for blood donation.
6) Discuss allergic blood reactions.
7) Discuss donor education program, motivation , recruitment
and counselling .
8) Patient management program , definition , goals and
position.
9) Coombs test direct and indirect (principle and uses).
10) Types of document in quality system.