blood banking india

1. UMBILICAL CORD BLOOD BANKING
(UCB)
Dr Atif Irfan Khan

2. OUTLINE
• Introduction
• Types of cord banks
• UCB collection
• UCB processing and storage
• Transplantation
• UCB banking in india

3. Stem cells
Umbilical cord stem cell Adult stem cell
HLA missmatch Higher tolerance Limited match
Viral contamination low higher
Proliferation capacity high limited
Risk of GVHD low higher
Collection procedure Safe and non invasive More invasive
supply abundant limited
Stem cells per unit less higher
INTRODUCTION

4. INTRODUCTION
Umbilical Cord Blood
• UCB was once considered a waste product and was discarded with the placenta after
delivery.
• With medical advancement now it is has been found to be a rich source of stem cells

5. INTRODUCTION
Historic of UCB
• 1970: First UCB transplant was performed in a 16 yr old with ALL
• 1988: First UCB bank was established in Paris France
• 1992: First public UCB bank was established in New York USA
• 1995: cord blood registry was established,
• Jeevan (also known as BeTheCure.in) first public cord blood bank set up in Tamil
Nadu India

6. UCB Banks types
Private bank Public bank
Donor Directed family or
autologous cord blood
Non-directed or
altruistic cord blood
Funding Family opting to store
cord
blood pays
Public funding not for
profit free of charge
when a unit is used
Aim Profit, aggressive
marketing
Creation of inventory
of cord units for
unrelated use
Beneficiary Available only to donor
of the cord, or a family
member
Available for matched
recipients nationally
and internationally

7. UCB collection
Donor screening
• Pregnant women
• Consent(before delivery): Collection, Processing, Testing, Storage, and Use
• History: Infectious diseases and Genetic disorders
• Testing (within 7 days before or after collection)
• TTI markers; HIV, hepatitis B, and hepatitis C, syphilis and cytomegalovirus
(CMV) antibodies, in some programs human T-lymphotropic virus (HTLV)-
I/II, malaria, Chagas disease, and West Nile virus

8. UCB collection
Collection
• Aseptic procedure
• Sterile collection bag
• Capacity: 200ml
• Anticoagulant: 35ml CPD, lyophilized heparin is also used
• Primary bag & Transfer bag- PVC+ plasticiser , Freezing bag- Ethyl venyl acetate
• Timing :
• In utero or Ex utero
• Volume Collected: 40-60 ml (within 2-5 min.)

9. UCB collection
IN-UTERO EX-UTERO
Performed by obstetrician or nurse/midwife UCB collection staff
place In OT/ delivery room special lab
Cost Cheaper Costly
Volume lesser higher
Personal No extra trained personal Trained personal
required
CD34 count Better Lesser than In-Utero

10. UCB collection
Processing
• Done within 48 hrs of collection
• Volume reduction: Final Volume should be smaller and Fixed
(a) smaller CBUs of equal, specified, size to allow for organized, easy identification within the
liquid nitrogen freezer facilitating individualized loading and retrieval operation;
(b) a consistent size allows for the use of the same amount of cryopreservative (DMSO) per unit,
for controlled freezing with a single cooling speed algorithm and for convenient automation of
freezing;
(c) a smaller DMSO volume, to limit DMSO infusion, particularly to pediatric recipients, as it may
cause adverse events including metabolic and hemodynamic stress

11. UCB Processing
1) Manual Method
• Standard blood bag centrifugation technique
• 6% HES is added to the anticoagulated UCB sample in a 1:5 ratio
• Centrifuged for 15 min at 4°C at 50 X g.
• The plasma with WBC fraction is transferred using a press.
• Second centrifugation 400 X g for 10 min at 4°C and the WBC-poor plasma
was transferred to a transfer bag., TNC- 87%, CD34- 90%
• DMSO is then added to the volume processed

12. UCB Processing
2) Leukocyte filtration and recovery method:
Reversible leukocyte adhesion on blood filters to easily trap and recovery
hematopoietic progenitors and reverse flushing with a protein-enriched saline
solution. TNC- 61%
3) Semi-automated methods:
• Top and Bottom method, similar to blood bags for blood component
separation
• TNC- variable in different studies from 61% to 92%
• Replaced by automated techniques now

13. UCB Processing
4) Automated methods: Sepax-
• consists of a centrifugal device a single-use
kit
• Uses cylindrical disposable spin around its
vertical axis
• TNC 76–87% and CD34+ cells 86%, with
36–45% hematocrit

14. UCB Processing
4) Automated methods: AutoXpress
• Plastic, battery-powered, microprocessor-controlled AXP
device, designed to fit standard cups of regular blood bank
centrifuges;
• A disposable bag set, comprising three connected bags
Can be used with or without HES
• TNC had high variability 76-95%, >98% CD34+ cells

15. UCB Processing
4) Automated methods: SynGenX-1000TM
• Disposable cartridge, has internal sections accessed through
separate tubing for red cells and buffy coat, and has a main
section for the incoming blood.
• The battery-driven microprocessor is guided by four optical
sensors that monitor the optical densities at strategic sites during
the centrifugation
• Is centrifuges in the standard bucket and blood bank centrifuge
• TNC- 87% and CD34+ cell 102%.

16. UCB Processing
PrepaCyte-CB
• A functionally closed sterile bag system composed of three integrally attached
processing and storage bags containing the PrepaCyte-CB separation solution
• Isolates nucleated cells and removes most red cells and nucleated red cells from the
final processed UCB unit.
• Carried out in standard blood bank centrifuge and extractors used

17. Conclusion- These results show that PrepaCyte-CB offers superior
separation of UCB when compared to Hetastarch.

18. UCB Processing
Processing- Quality Control Testing of UCB

19. UCB Processing
Processing- Quality Control Testing of UCB
• Done before cryopreservation

20. Cryopreservation and storage
• Cryopreservation:
• 10% DMSO
• Cryopreserved UCB are stored at ≤–150C at first
• Liquid nitrogen storage container at –196 C store upto 23 yrs
• Shipment: in frozen state after QC criteria and other test results are out.
• Thawing: in plastic zipper bag at 37 C waterbath
• Washing: with Equal volume of saline centrifuged at 400-600 × g for 15
minutes at 10 C and resuspended in appropriate volume of thaw solution

21. Transplantation of UCB
Infusion
• Transfused as soon as clinically possible
• Aseptic procedure
• The unit is infused by intravenous drip or syringe push directly into a central line without
a needle or a pump
• A bed side filter might be present if not filtered previously
• Patient’s vital signs is checked before, immediately after infusion, and 1 hour post
infusion

22. Indian perspective of UCB banking
• Parents opting for UCB banking in india is 2 per 1000 newborns which very low as
compared to US; 50 per 100 newborns
• 14 govt approved cord blood banks in india at present

23. Indian perspective of UCB banking
Regulatory bodies
• 3 major regulatory bodies, which are responsible for formulating policies in cord blood
banking sector
• Indian Council of Medical Research (ICMR)
• Department of Biotechnology (DBT)
• Drug Controller General of India (DCGI)

24. Prerequisites of UCB banking in india
Schedule F part XIID of Drug and cosmetic Rule ( 3rd amendment 2011)
A. GENERAL REQUIREMENTS-
1) Location surrounding and building
• Should comply with Factories Act, 1948
• Constructed in manner that functions are performed under hygienic and sterile conditions
• Adequately working space
2) Buildings and premises
• Avoid entry of rodents, walls and floors be smooth and free of cracks & Flooring shall
be unbroken and provided
• Processing and storage areas of products be separate

25. Prerequisites of UCB banking in india
3) Disposal of waste and infectious materials
• Facility should conform with requirements of the Pollution Control Board
• All bio-medical waste shall be dealt with Bio-medical Waste Management and
Handling Rules, 1996.
4) Health, clothing and Sanitation of personnel
• Employed personnel shall be free from infectious and contagious diseases and they
should be medically examined periodically at least once a year.
• Staff should ensure personal hygiene and a high level of personal hygiene.
• All personnel working in the Laboratory shall be protected against virus infections.

26. Prerequisites of UCB banking in india
5) Requirements for Processing, Testing and Storage Areas (all areas should be minimum
10.00 Sq. meters)
• Reception-
• Processing area-
• Clean and have an air handling System to provide a Class 10,000
environment, entry should be through air lock
• Room will house Class 100 biological safety cabinets
• Temp- of 20 ºC to 25ºC, Pressure +10-15 pascals, Relative humidity of 50-60%

27. Prerequisites of UCB banking in india
• Haematology and Serology Laboratory: 10.00 Sq. meters
• TTI lab: 10.00 Sq. meters
• Sterility Testing Laboratory: 10.00 Sq. meters
• HLA Typing Laboratory; Class 100,000 environment, air-conditioned with an area of at
least 10.00 Sq. meters
• Sterilization-cum-washing
• Records and Store Rooms: 10.00 Sq. meters each
• Cryogenic Storage room: 20.00 sq. meter, shall have temperature monitoring of
storage vessels, liquid nitrogen level in storage vessels and oxygen meter

28. Prerequisites of UCB banking in india
B. COLLECTION AND STORAGE OF PROCESSED UMBILICAL CORD BLOOD COMPONENT
1 ) Collection
• After Consent
• Under the supervision of the qualified Registered Medical Practitioner
2) Transportation-
• Transportation temperature : 18 to 28ºC
• The time period between collection and processing shall not exceed 72 hours.
3) Storage: at room temperature between 20 to 25ºC prior to processing

29. Prerequisites of UCB banking in india
C. PERSONNEL -
• Medical Director
• Whole time employ with MD in pathology/transfusion medicine/microbiology
and experience / training in cord blood processing and Cryogenic Storage
• Laboratory In-charge
• Post Graduate qualification in Physiology/Botany/Zoology/ Cell
Biology/Microbiology/Biochemistry/Life Sciences/Pharmacy with one year
working experience in pathological laboratory/ microbiology laboratory/testing
unit/training in cord blood processing cryogenic storage

30. Prerequisites of UCB banking in india
• Technical Supervisor (cord blood processing)
• Degree in Physiology/Botany/Zoology/Pharmacy/Cell Biology/Bio
Sciences/Microbiology/Biochemistry/M.L.T. with minimum of three years of
experience in the preparation of blood components and / or experience or
training in cord blood processing and Cryogenic Storage
• Diploma in M.L.T. with five years experience in the preparation of blood
components and experience or training in cord blood processing and Cryogenic
Storage shall be essential

31. Prerequisites of UCB banking in india
• Cord Blood Bank Technician
• Degree in Physiology/Botany/Zoology/Pharmacy/Cell Biology/Bio
Science/Microbiology /Biochemistry/M.L.T. with six months experience and or
training in cord blood processing and cryogenic storage; or
• Diploma in MLT with one year experience in the testing of blood and / or its
components and / or experience or training in cord blood processing and
Cryogenic Storage.

32. Prerequisites of UCB banking in india
E. QUALITY CONTROL
• Facilities shall be provided for Quality Control such as Hematological, Microbiological
and Instrumental testing
• Instruments which would be used to process test and store the UCB unit would be
validated before commissioning and calibrated from time to time
• Stem Cell Population, HLA typing and Genetic Disease Testing which may be
outsourced to a competent third party approved by the licensing authority.

33. Prerequisites of UCB banking in india
F. SCREENING TESTS
• Maternal blood sample shall be tested for, Hepatitis B, Hepatitis C, HIV 1 & 2,
Syphilis, Malaria, CMV, HTLV 2.
• UCB be tested for TNC, Total Mononuclear Cell Count, CD34+ enumeration, Cell
Viability, ABO Group and Rh Type, Sterility as regards Bacterial and Fungal
contamination status, HLA Matching (Only for allogenic Cord Blood Units)

34. Prerequisites of UCB banking in india
G. STORAGE
• UCB to be cryopreserved using a controlled rate freezing or equivalent validated
procedures.
• Storage temperature -196ºC and shall not be warmer than -150ºC.
H. REFERENCE SAMPLES
• Two reference samples prior to cryopreservation
• Stored at -196ºC and shall not be warmer than -150ºC
• One sample shall be stored at -76ºC

35. Prerequisites of UCB banking in India
I. LABELLING
• Initial label placed during collection shall specify
• Human Umbilical Cord Blood;
• Volume or weight of contents in the collection bag [UCB+ Anticoagulant];
• Mother‘s name;
• Place, Date and time of collection;
• Collected by;
• To be labeled in bold, ―ROOM TEMPERATURE ONLY– DO NOT REFRIGERATE, DO
NOT IRRADIATE
• Manufacturing license number.

36. Prerequisites of UCB banking in india
I. LABELLING
• Percentage of Cryoprotectant [DMSO] & any other additive / preserving
• Date of processing
• Issue label at the time of release of Cord Blood Unit
• Name of manufacturer & License number
• All details of the Cryogenic Storage Label
• The results of Total Nucleated Cells, Progenitor Cell percentage {CD34+), Viability;
• ABO Group, Rh Type & Results of TTI & HLA typing (allogenic)
• Statement ―leukoreduction filters should not be used; & Do not irradiate

37. Prerequisites of UCB banking in india
J. RECORDS OR DOCUMENTATION
• Client / donor enrolment / agreement record;
• Collection of unit and transportation record
• Master record of stored unit
• HLA Matching record
• Unit Release Register
• Stock Register for Collection Bag Cryoprotectant and Preservant, RBC Sedimentation
Enhancer
• Stock Register for Diagnostic Kits, Reagents and other consumables
• Record on feedback after use of cord blood / Adverse reaction record.
• Standard Operating Procedures

38. Prerequisites of UCB banking in india
K. CORD BLOOD RELEASE
• Written or electronic request from the transplant physician or designee for shipment
of the cord blood unit
• At the time of issue information include indications, contra-indications, caution,
instruction for handling and use of the cord blood unit including short-term storage
and preparation for transplantation

39. Ethical consideration
• UCB still an unproven treatment choice
• FDA considers it as investigational new drug
• Linking identity of donor to UCB
• Time taking money consuming
• Invading donor privacy
• Mobile population difficult to trace and increased chance of loss to follow up
• Autologous UCB is used up only by a minute fraction of the population
• Private sector mislead families with information and benefit of cord blood storage

40. Future prospects of UCB banking in India
• “hybrid model” where a vague division between public and private cord banks will exist
• Legislative measures have brought a proportion of cords in private banks for public access
in some places
• UCB in private banks would have an extremely low utility rate as the regenerative
medicine with potential uses in conditions like diabetes mellitus, etc. are still only
hypothetical.