Umbilical cord blood vs bone marrow vs peripheral stem cells
indications of stem cell transplant
Regulatory requirements for cord blood banking
Requirements for processing, testing and storage areas for UCB
Air-handling system
Personnel for Cord Blood Bank
Collection of processed UCB component
procedure- in utero ex utero
transportation
processing
freezing
storage
screening tests
quality control
advantages and disadvantages
labelling
tissue collection
live and deceased donors
The Embryology laboratory should been designed to provide an environment that is as close to optimum as possible for the growth of human embryos and to provide the best resulting pregnancy rates for patients undergoing IVF cycles.
Here, we discuss what is the components of IVF laboratory.
The Embryology laboratory should been designed to provide an environment that is as close to optimum as possible for the growth of human embryos and to provide the best resulting pregnancy rates for patients undergoing IVF cycles.
Here, we discuss what is the components of IVF laboratory.
There are three methods commonly used to initiate a culture from animals.
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2021 laboratory diagnosis of infectious diseases dr.ihsan alsaimarydr.Ihsan alsaimary
2021 laboratory diagnosis of infectious diseases
dr. ihsan alsaimary
university of basrah - college of medicine- DEPARTMENT OF MICROBIOLOGY
POBOX 696 ASHAR
BASRAH 42001
IRAQ
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Mastering Wealth: A Path to Financial FreedomFatimaMary4
### Understanding Wealth: A Comprehensive Guide
Wealth is a multifaceted concept that extends beyond mere financial assets. It encompasses a range of elements including money, investments, property, and other valuable resources. However, true wealth also includes non-material aspects such as health, relationships, and personal fulfillment. This guide delves into the various dimensions of wealth, exploring how it can be created, sustained, and enjoyed.
#### Defining Wealth
Traditionally, wealth is defined as the abundance of valuable resources or material possessions. It includes financial assets like cash, savings, stocks, bonds, and real estate. However, a broader understanding of wealth considers factors such as personal well-being, emotional health, social connections, and intellectual growth. This holistic view recognizes that true wealth is not solely about accumulating money but also about enhancing one's quality of life.
#### The Importance of Financial Wealth
Financial wealth remains a critical component of overall wealth. It provides security, freedom, and the ability to pursue opportunities. Key elements of financial wealth include:
1. **Savings**: Money set aside for future use. It is crucial for emergencies, large purchases, and financial goals.
2. **Investments**: Assets purchased with the expectation that they will generate income or appreciate over time. Common investments include stocks, bonds, mutual funds, real estate, and businesses.
3. **Income**: Regular earnings from work, investments, or other sources. Consistent income is essential for maintaining and growing wealth.
4. **Debt Management**: Effectively managing debt ensures that it does not erode financial wealth. This includes paying off high-interest debt and using credit wisely.
#### Creating Wealth
Creating wealth involves generating and accumulating financial and non-financial resources. The process can be broken down into several key strategies:
1. Education and Skill Development: Investing in education and skills enhances earning potential. Higher education, professional certifications, and continuous learning can lead to better job opportunities and higher salaries.
2. Entrepreneurship: Starting and running a successful business can be a significant source of wealth. Entrepreneurship requires innovation, risk-taking, and effective management.
3. Investing: Making smart investments is essential for wealth creation. This involves understanding different types of investments, assessing risks, and making informed decisions. Diversifying investments can reduce risk and increase potential returns.
4. Saving and Budgeting: Effective saving and budgeting help accumulate wealth over time. Setting financial goals, creating a budget, and sticking to it are foundational steps in wealth creation.
5. Real Estate: Investing in property can provide rental income and capital appreciation. Real estate is a tangible asset that can hedge against inflation
3. Umbilical cord blood
◦ Blood that remains in the placental blood vessels and in the attached umbilical
cord, which connects the unborn baby to the mother's womb
◦ UCB is collected from the placenta when the expectant mother is in the third
stage of labor (after delivery of baby) or after the delivery of the placenta.
◦ Rich in HSCs, which possess the properties of self-renewal as well as the
ability to differentiate into myeloid and lymphoid cell lineages
4. ◦ Rich in mesenchymal cells, are self-renewing and
minimally immunogenic, play a key role in immune
suppression in response to GVHD.
◦ 1988- First Cord blood used as an alternative
source of HSCs in Fanconi anemia
◦ The Indian rules define umbilical cord blood as “the
whole blood collected from placental and/or
Umbilical cord blood vessels after the umbilical
cord has been clamped”.
5. Characteristics Umbilical cord blood Bone marrow
Mobilised peripheral
blood stem cells
Ease of collection No safety risks for mother
or child
Needs to be
anesthetised,
Procedure in the OT,
takes several hours
Requires mobilization,
discomfort to the donor
Viral infections Rare common common
GVHD Uncommon and less
severe even if there is
GVHD
Common in
mismatched grafts
More risk of chronic
GVHD than that of
bone marrow
HLA mismatch HLA mismatch well
tolerated
Requires a perfect HLA
match
Requires a perfect HLA
match
6.
7.
8. Cord Blood Bank
◦ A place for carrying out operations of collection, processing, testing, banking,
selection, and release of cord blood units.
◦ The Drug Controller General of India (DGCI) established guidelines for cord blood
banking ,which became effective in 2012 and are designed to provide a framework
for facilities to obtain a license to manufacture and distribute cord blood in India.
9. Regulatory requirements for cord blood
banking:
◦ Under part XII D of the Drugs and Cosmetics Act, 1940 and Rules 1945 (amended up to
the 31st December 2016), the following are the requirements for cord blood banking-
a. Location and surroundings: no risk of contamination from the external environment,
no open sewage, drain, public lavatory, or any factory with excessive soot, smoke, etc.
b. Building and premises: Adequately provided with working space to allow logical
placement of equipment, material, and movement of personnel;
Designed/constructed/maintained to prevent insects, pests, and rodents entry.
10. ◦ Provided with the furniture in aseptic areas which is smooth, washable, and made of
stainless steel or any other appropriate non-shedding material other than wood.
◦ Provided with separate areas for processing and storage to prevent cross-contamination.
◦ Provided with defined environmental conditions for temperature, humidity, ventilation.
c. Disposal of waste and infectious materials: in conformity with Pollution Control
Board and the provisions Bio-Medical Waste Management (Amendment) Rules, 2018.
d. Health, clothing and sanitation of personnel: Medical examination at least once a
year and all persons shall wear clean body coverings appropriate for their duties before
entering the Processing Zone.
11. Requirements for processing, testing
and storage areas for UCB:
Separate areas for designated work purposes, namely: -
a. Cord blood reception: adequate registration, data entry, and generation of bar-
coded labels- An air-conditioned area of at least 10.00 sq. meters shall be provided.
b. Cord blood processing area: 20ºC to 25ºC with a positive differential pressure of
10-15 Pascal, relative humidity of 50-60%, minimum area shall be 10.00 sq. meters
c. Haematology and Serology Laboratory: for independent testing of UCB for
ABO grouping and Rh typing, total nucleated cell count, progenitor cell count, and
viability test (air-conditioned; at least 10.00 sq. meters area)
12. d. Transfusion transmissible infectious disease screening laboratory: HIV I
& II; Hepatitis B & C virus, syphilis, malaria, CMV, and HTLV
e. Sterility testing laboratory: The laboratory shall be used for performing
sterility tests on the UCB unit; adequate area for media preparation, sterilization,
incubation and decontamination.
f. HLA typing laboratory: HLA typing and genetic disease testing.
(All above- A.C and at least 10 sq. m)
g. Sterilization-cum-washing room
h. Records and store rooms
13. i. Cryogenic storage room: A minimum space of 20.00 sq. meters with
provision for temperature monitoring of storage vessels, liquid nitrogen
level in storage vessels, and oxygen meter.
The service space between each liquid nitrogen storage vessel, supply cylinders, and
connecting hose should be a minimum of 1.00 sq. meters (air-conditioned).
j. General storage area
14. Air-handling system
◦ Processing area shall have HVAC (heating, ventilation, and air conditioning)
system and be fitted with HEPA (High-Efficiency Particulate Air) filters having
Grade C (Class 10,000) environment. The entire processing shall be done
conforming to the Grade A (Class 100) Standard of air quality
15. Personnel for Cord Blood Bank:
Cord blood bank shall have the following categories of whole-time competent technical staff, namely: -
a. Medical director: possesses a post-graduate degree in MD [Pathology/Transfusion Medicine/Microbiology] and
has experience/training in cord blood processing and cryogenic storage.
b. Laboratory in-charge: post-graduate qualification in Physiology or Botany or Zoology or Cell Biology or
Microbiology or Biochemistry or Life Sciences or Graduate in Pharmacy and 1 yr experience.
c. Technical supervisor (cord blood processing): - The technical supervisor shall have a: Degree in Physiology or
Botany or Zoology, Pharmacy or Cell Biology or BioSciences or Microbiology or Biochemistry or M.L.T. with 3
years of experience/training in cord blood processing and Cryogenic Storage; or Diploma in Medical Laboratory
Technology (M.L.T.) with 5 years experience.
d. Cord blood bank technician(s): - A degree in Physiology or Botany or Zoology or Pharmacy or Cell Biology or
Bio Science or Microbiology or Biochemistry or Medical Laboratory Technology (M.L.T.) with 6 months’
experience or 1 year experience
16. UCB collectionprocedure
◦ Aseptically using 16 gauze needle, allowing placental blood to be removed by
gravity in a disposable PVC bag and containing an adequate quantity of sterile
pyrogen-free anticoagulant like CPD / CPD-A
◦ Collection may be in-utero (common) or ex-utero.
◦ Volume collection- 50 to 200 ml
17.
18.
19. In-utero
◦ Trained obstetricians after the delivery
of the infant (minimum of 34 weeks)
Advantages
◦ Avoids the possibility of failed
collection resulting from damage to
placenta during delivery
◦ Reduces delay in collection
◦ An increase in volume collected
◦ Reduces incidence of clotted collections
20. Ex-utero
◦ Trained UCB bank staff after the infant and placenta are delivered
◦ Following delivery, placenta is passed to UCB bank staff to harvest the blood
following cleaning the umbilical cord and aspiration of blood from placental vein
21. ◦ Higher UCB volume yields greater nucleated cells, CFUs and CD34+ cells on
engraftment, transplant-related events and survival.
◦ For double HPC(CB) transplants, each HPC(CB) unit has a minimum TNC count of
1.5×107/kg at cryopreservation.
◦ In double HPC(CB) transplants, pre cryopreservation CD34+ cell dose of ≥1×105/kg
used by several transplantation centres as the minimum cell doses for each unit
22. ◦ Collection within not greater than 5
minutes of placental delivery produced
higher volume and TNC count.
◦ Increased TNC count also seen in
Caucasian women, primi gravidae, female
newborns, and collection duration of more
than 5 minutes.
23. Transportation:
a. From the birthing centre to the designated laboratory.
b. The transportation procedure shall be validated to ensure the optimum survival
of the stem cells.
c. The transportation temperature should be between 18ºC to 28ºC.
d. The time period between collection and processing shall not exceed 72 hours.
Wada et al. served a 1% decrease in viability for every 4-h increase in transport
time for newly collected UCB units that were shipped at ambient temperature
24. Processing of UCB
◦ Centrifugation, sedimentation, for reducing red cell content, plasma volume
◦ Sedimenting agents such as hydroxyethyl starch (HES), gelatin and dextran
25. Freezing technique
◦ Dimethyl sulfoxide is used by most banks with cryogenic bags preferred over freezing vials
◦ DMSO should be added slowly (up to a final concentration of 10%; samples are kept at
4°C at all times) to prevent an osmotic shock to the cells.
◦ Slow cooling in a programmable controlled-rate freezing device at rate of 1 C/min will
result in adequate recovery of HPCs.
◦ After performing volume reduction, the amount of the final concentration of DMSO and
dextran in the bag was 10% and 15%, respectively
26. ◦ Both AABB and FACT-Netcord have defined
storage temperature limits for cryopreserved UCB to
be <−150°C.
◦ Once frozen, UCB units are typically transferred to
long-term storage into a monitored liquid nitrogen
container, either immersed in liquid (at −196°C) or in
vapor phase to minimize the potential for cross-
contamination
27. Storage:
a. Before processing, the UCB shall be stored at RT between 20ºC to 25ºC in the
reception area.
b. Samples pending tests for specific TTIs shall be stored in a segregated manner.
Note: Temperature range between 4ºC to 37°C, for the whole period of transit, may
be allowed beyond 18ºC to 28ºC in exceptional cases. The licensee shall adequately
explain the effects of deviation of transit temperature on the product in the client
education booklet.
28. Storage of UCB
◦ Cryopreserved using a controlled rate freezing,
the frozen storage shall be at -196ºC and shall
not be warmer than -150ºC.
◦ There will be no shelf life for this class of
product (ICMR 2023)
◦ UCB can be cryopreserved and stored for >15
years with efficient recovery of stem cells on
thawing
29. Screening tests
a. Maternal blood sample: Hepatitis B, Hepatitis C, HIV 1 & 2, Syphilis, Malaria,
CMV, HTLV.
b. The Umbilical cord blood sample: Total nucleated cell count, total
mononuclear cell count, progenitor cell (CD34+) enumeration, cell viability, ABO
group and Rh type, sterility test, HLA matching (for allogeneic cord blood units).
31. Reference samples:
a. At least two reference samples shall be collected from cord blood unit product
before cryopreservation and stored at minus 196ºC and shall not be warmer than
minus 150ºC.
b. At least one additional reference sample shall be stored at minus 76ºC or
colder for purposes other than viability analysis.
32. Labelling:
a. Initial label placed during collection shall specify:
i. Human Umbilical Cord Blood
ii. Approximate Volume or weight of contents in the
collection bag [UCB+ Anticoagulant]
iii. Mother’s name
iv. Date, time and place of collection
v. To be labelled in bold, —ROOM TEMPERATURE
ONLY– DO NOT REFRIGERATE, DO NOT
IRRADIATE
vi. Manufacturing license number
33. Label at the completion of processing and before issue
i. Name of product: Human Progenitor Cell [HPC] – Cord Blood
ii. Volume or weight of contents
iii. Percentage of cryoprotectant [DMSO]
iv. Percentage of any other additive
v. Date of collection [birth]
vi. Date of processing
vii. Name of manufacturer
viii. Manufacturing license number
ix. Storage temperature – not less than - 196ºC and shall not be warmer than minus 150ºC
x. Unique Traceability Number and/or bar code
34. Issue label at the time of the release of UCB unit
i. Name of manufacturer
ii. License number
iii. All details of the cryogenic storage label
iv. The results of Total Nucleated Cells, Progenitor Cell percentage [CD34+], Viability
v. Results of transfusion transmittable diseases testing on maternal blood
vi. ABO and Rh Group and HLA typing (allogeneic)
vii. Date of processing
viii. A statement indicating: - “Do not use leucoreduction filters” and “Do not irradiate”
ix. Name and address of receiving hospital
35. Standard Operating Procedures:
i. UCB collection
ii. Transportation of the collected UCB unit
iii. Processing of UCB unit
iv. Cryogenic storage of processed UCB unit
v. Testing of maternal blood for transfusion transmittable infections
vi. Testing of UCB for ABO Grouping and Rh Typing
vii. Testing of UCB unit for Total Nucleated Cell Count, Mononuclear Cell Count, Progenitor Cell
(CD34+)
enumeration, and viability
viii. Testing of UCB stem cell unit for sterility
ix. Disposal of biomedical waste
x. Dispensation of UCB unit
xi. Preventive maintenance protocol for all Instruments
xii. Acceptance / Rejection procedure of consumables
xiii. Environment monitoring of classified areas
xiv. Any other standard operative procedure as per requirements
36. Criteria for choosing UCB unit
◦ Should be HLA typed to high-resolution for HLA-A, -B, -C, and DRB1 loci
◦ In a double UCB transplant, both the UCBs should be at least 4/6 matched
to the patient using intermediate resolution (antigen level) for HLA loci A and
B and high resolution (allelic) typing for HLA DRB1
37. Thawing and washing of UCB
◦ Clean or sterile transparent bag (ie, a plastic zipper bag) and submerged in a 37 C
water bath of clean or sterile water or saline
◦ Thaw solution-10% dextran and a protein source, usually human serum albumin
in a final concentration of approximately 2.5% to 4.2% are used.
38. UCB release:
Cryopreserved UCB unit stored at -150ºC or colder shall be transported in a
liquid nitrogen cooled dry shipper that contains adequate absorbed liquid
nitrogen and has been validated to maintain the temperature below -150ºC for at
least 48 hours beyond the expected time of arrival at the receiving facility.
39. Advantages of UCB
◦ Ease of collection
◦ No risk for the mother or the child
◦ Less time needed for processing (more quickly available for use)
◦ More economical than bone marrow collection
◦ Decreased risk for transmission of TTI infection
◦ Lesser requirements for stringent HLA typing
◦ Relatively lesser possibility of GVHD
40.
41. Disadvantages:
◦ Slower degree of engraftment
◦ Limited cell dose in the inoculum making its use limited to children.
◦ Small volume of the Unit
◦ Additional cell doses unavailable
◦ Storage issues at ultra-low temperatures and related costs
◦ Poor immune reconstitution
42.
43. Public & Private UCBs
◦ India does not have any public UCB banks at present(paucity of public funded
UCB banks in India)- ICMR-2023
◦ The likelihood of the stored blood being used for HSCT is very small, probably
as low as 0.005 to 0.04% in the first 20 yrs of life (ICMR)
◦ Private banking is suggested in cases where there is a relative/sibling with a
condition or there is a family history of malignant or genetic conditions that can
be treated with HSC transplantation (Thalassemia)
◦ The chance of a cord blood being utilized is at least 100 times greater in a
public bank as compared to private bank
47. Tissue Typing
◦ Embryo created by IVF has the exact biological makeup to act as a future UCB
donor for an identified recipient, typically a sibling
◦ Basic procedure is performed by the removal of a single cell three to five days
after fertilization when the embryo is at the six-to-ten cell stage.
◦ Tissue can also be removed at further ages through surgical methods.
◦ In 1949, the U.S. Navy established the first tissue bank.
◦ The American association of Tissue Banks was established in 1976.
48.
49.
50.
51. Allografts and autografts
◦ Allografts are tissues transferred between individuals of the same species.
◦ They can be derived from a single tissue or multiple tissues acting as a functional unit.
◦ Autografts are tissues implanted into the individual from whom they were removed.
◦ For example, bone that has been surgically removed from a patient’s ilium can be
shaped to desired dimensions and implanted into the vertebral disk space of the same
patient.
52. Isografts and Xenografts
◦ Isografts, which are tissues transferred between genetically identical
individuals, such as identical twins, are uncommonly used.
◦ Xenografts are tissues transplanted from one species to another; a growing
number of medical products are being developed from highly processed
nonhuman animal tissues.
53.
54. Tissue collection
◦ Tissues are debrided of extraneous soft tissue and cut to specification.
◦ Different tissues are collected by different surgical procedures.
◦ In some cases, more than 100 grafts can be produced from a single donor.
55.
56. Tissue Processing
◦ After tissue has been procured from a donor, it is processed using aseptic
technique to prevent-contamination.
◦ Similar to good manufacturing practice, good tissue practice (GTP) requires
the facility to establish and maintain controls over temperature, humidity,
ventilation, and air filtration.
◦ Various solutions like ethylene oxide, antibiotics, alcohols, and surfactants,
may be used to reduce or eliminate bacterial contamination and remove
extraneous lipids and other biologic material.
57. Tissue preservation
◦ Several methods of tissue preservation are available for
long-term storage of human allografts, including
freezing and lyophilization (freeze drying).
◦ Tissue integrity can be enhanced through
cryopreservation, a process in which tissues are frozen
in a protective medium at a steady, controlled rate.
◦ The use of cryoprotectants, such as glycerol or
dimethyl sulfoxide, minimizes cell damage caused by
cell shrinkage and intracellular ice formation during
freezing.
66. Compatibility testing of allografts
◦ Although bone and soft tissue allografts may provoke a recipient immune
response, such responses are not usually clinically significant, presumably
due to a lack of residual cellular material in processed grafts.
◦ Therefore, it is not necessary to match most allografts to the recipient’s HLA
or ABO type.
◦ Possible exceptions include cryopreserved veins and arteries, for which some
clinicians request ABO compatibility, and frozen, unprocessed bone
allografts containing marrow or red cells.
67. ◦ Development of Rh(D), Fy(a), and Jk(b) antibodies in recipients following
transplantation of unprocessed bone allografts has been documented.
◦ If unprocessed bone is to be used for an Rh(D)-negative female of
childbearing potential, her future offspring may be at risk of hemolytic disease
of the fetus and newborn.
◦ Rh Immune Globulin prophylaxis should be considered if the Rh type of the
red cells or marrow contained in the allograft is positive or unknown.
68. REGULATIONS AND STANDARDS
◦ The Food and Drug Administration (FDA) regulates the activities of tissue
banks and tissue distribution intermediaries, which are establishments or
persons engaged in the recovery, screening, testing, labelling, processing,
storage, and/or distribution of human tissues for clinical use, under Title 21,
Parts 1270 and 1271 of the Code of Federal Regulations (CFR).
◦ These entities manufacture what the FDA classifies as human cells, tissues, and
cellular and tissue-based products (HCT/Ps).
69. The three rules of 21 CFR Part 1271 concern
1) registration of tissue bank establishments,
2) donor eligibility, and
3) GTP related to handling HCT/Ps.
70. HOSPITAL TISSUE SERVICES
◦ The AABB supports a tissue-dispensing service within the transfusion service,
which has expertise in providing human-derived products that are perishable,
potentially infectious, and sometimes in short supply and that require temperature-
controlled storage.
◦ The tasks of ordering, receiving, storing, distributing (issuing), tracking, and
tracing products as well as investigating adverse events, including complaints,
recalls, and look-back investigations, are activities that are common to the
transfusion service and tissue-dispensing service.
71.
72. Tissue Autograft
◦ Surgical reconstruction using the patient’s own tissues often has advantages
over the use of cadaveric tissue.
◦ Common autologous tissues include skull bone flaps (most common), ribs,
iliac crests, skin and endocrine tissue such as the pancreas and parathyroid.
◦ Autografts should not be collected from patients with systemic infections or if
the tissue is in close proximity to an infected area.
73. Autologous tissue collection and processing
1. Removal of tissue- by surgeon in aseptic manner
2. Culturing of tissue
3. Addition of storage solution
4. Storage- in sterile plastic bags or sterile Nalgene jars.
The Nalgene jars are useful because they are able to
withstand temperatures to –40°C for up to 5 years and
can be used with irradiation sterilization procedures.
5. Wrapping, labelling and preservation of container