Lab 12 Cord Blood by Ptcu8g

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									 Practical
Blood Bank


 Cord Blood
                  Umbilical cord
• In placental mammals, the umbilical cord (also called the
  birth cord or funiculus umbilicalis) is the connecting cord
  from the developing embryo or fetus to the placenta.
• During prenatal development, the umbilical cord is
  physiologically and genetically part of the fetus and (in
  humans) normally contains two arteries (the umbilical
  arteries) and one vein (the umbilical vein), buried within
  Wharton's jelly.
• The umbilical vein supplies the fetus with oxygenated,
  nutrient-rich blood from the placenta. Conversely, the fetal
  heart pumps deoxygenated, nutrient-depleted blood through
  the umbilical arteries back to the placenta.
                 Wharton's jelly
• Wharton's jelly is a gelatinous substance within the umbilical
  cord, largely made up of mucopolysaccharides (hyaluronic
  acid and chondroitin sulfate). It also contains some fibroblasts
  and macrophages.
• As a mucous tissue it protects and insulates umbilical blood
  vessels.
• Cells in Wharton's jelly express several stem cell genes,
  including telomerase. They can be extracted, cultured, and
  induced to differentiate into mature cell types such as
  neurons . Wharton's jelly is therefore a potential source of
  adult stem cells (also see the more common method of
  storing cord blood).
                  Cord blood testing
• Immediately after the birth of the baby, the umbilical cord is
  clamped and cut. If cord blood is to be drawn, another clamp
  is placed 8 to 10 inches away from the first, then the isolated
  section is cut and a blood sample is collected into a specimen
  tube.

• Cord blood testing is done to determine the following:
   –   Bilirubin levels
   –   Blood culture (if an infection is suspected)
   –   Blood gases, to evaluate the oxygen, carbon dioxide, and pH levels
   –   Blood sugar level
   –   Blood type and Rh
   –   Complete blood count (CBC)
          Abnormal Results Mean
• A low pH (less than 7.04 to 7.10) means there are higher
  levels of acids in the baby's blood. This might occur when the
  baby does not get enough oxygen during labor. One reason for
  this could be that the umbilical cord was compressed during
  labor or delivery.
• A blood culture that is positive for bacteria indicates
  septicemia.
• High levels of blood sugar (glucose) in the cord blood may be
  found if the mother has diabetes. The newborn will be
  watched for hypoglycemia (low blood sugar) after delivery.
• High levels of bilirubin could be due to:
   – HDN.
   – Infections that the baby gets before birth, including:
      •   Congenital CMV
      •   Congenital hepatitis
      •   Congenital rubella
      •   Congenital toxoplasmosis
   – Other possible causes include:
      •   Dubin-Johnson syndrome
      •   Jaundice in the mother
      •   Mother taking sulfa drugs during pregnancy
      •   Rh incompatibility
Collection of Cord Blood
• A cord sample (about 5 to 10 mls) should be taken from the
  babies of RhD negative women, women with known
  antibodies or in cases where there is insufficient
  documentation of maternal blood group or antibody status.
• The cord sample should be tested for blood group and direct
  antiglobulin test.
• Elution studies may be useful.
• Haemoglobin and bilirubin estimation should also be
  performed if DAT is positive.
• When the cord blood sample of the baby of an RhD negative
  woman is RhD positive, RhD immunoglobulin administration is
  indicated.
• When the cord blood is RhD negative, it is recommended that
  testing for the presence of the weak RhD antigen by the
  indirect antiglobulin test be performed. If positive, RhD
  immunoglobulin is indicated. If the direct antiglobulin test is
  positive, it may indicate fetomaternal incompatibility
• Antibody elution from the neonatal red cells can be
  performed to confirm the identity of the antibody coating the
  cord red blood cells.
• When haemolytic disease of the newborn is suspected, Hb,
  blood group, direct Coombs and bilirubin must be checked.
  Otherwise cord blood samples are not routinely tested though
  some collect the samples and store them for up to a week at
  4°C in case they should be needed.
• When the direct Coombs is positive, Rh typing must be
  confirmed and the coating antibody identified.
• The initial Rh typing will sometimes be found to be incorrect
  when the direct Coombs is positive, due to interference by the
  antibody.
                   Considerations
• Most hospitals routinely collect cord blood for testing at birth,
  since it is relatively convenient and the only time possible to
  collect such blood. Besides cord blood testing, cord blood can
  be used to treat certain types of bone marrow-related
  cancers. Some parents may choose to save (bank) their child's
  cord blood for this and other, future medical purposes.
• Cord blood banking for personal use is done by private
  companies, which charge for the service. Cord blood can also
  be donated to your local blood bank for use by others as
  needed to treat leukemia and other cancers.
             Cord Blood Banking
• Cord blood is rich in potentially life-saving stem cells. When
  they are transplanted, those stem cells are effective for the
  treatment of patients with diseases of the immune system
  (e.g., immunodeficiencies), blood (e.g., leukemia and sickle
  cell anemia) and selected metabolic disorders.

• The umbilical cord, placenta, and the blood they contain
  have no function after birth and they end up in waste. If
  exploited, this resource can be used for the good of the
  whole community.

• Stem cells in the cord blood similar to those found in bone
  marrow. This is why cord blood can be used for
  transplantation as an alternative to bone marrow.
• Stem-cell transplants have advantages over bone marrow
  transplants in the treatment, with a lower risk of tissue rejection
  (GvHD). The collection process is noninvasive, and involves a
  simple blood transfusion.

• As per the commitment of the Islamic University to serve the
  Palestinian community, this project is to set up the first National
  Umbilical Cord Blood Bank in Palestine.
 What are the advantages of cord blood?
• Cord blood offers a number of advantages to
  donors and transplant recipients.
  1. Cord blood collection is easy and poses no
     medical risk to the mother or newborn baby.
  2. Cord blood is collected in advance, tested and
     stored frozen, ready to use.
  3. Cord blood transplants do not require a perfect
     match.
  4. Cord blood transplants are associated with
     lower incidence of GvHD.
  5. Cord Blood Transplants are associated with
     lower risk of viral infections.
Are there any unfavorable aspects of cord blood
      transplants from unrelated donors?
 1. The main disadvantage of cord blood is that the
    volume collected is fixed and relatively small .
 2. A cord blood transplant also may give the
    patient one of the rare genetic diseases of the
    blood or immune system.
 3. The donor cells come from a newborn infant
    that will not be available for an additional
    donation of cells or bone marrow.
   Qualification of Cord Blood Units for the
NYBC's National Cord Blood Program Inventory.
•    A cord blood unit must meet all of the criteria (at a
     minimum) below to qualify for inclusion in the NCBP
     Search Inventory and be available for transplantation:
    1. The unit contains 1.1 billion total nucleated cells
       (TNC) or more before processing.
    2. The mother has given consent for donation
    3. The unit has been typed for HLA-A, -B and –DRB1 (A
       and B at low resolution and DRB1 at high resolution)
       and for the blood group antigens A, B and Rh:1 (D);
    4. The unit had acceptable results for CD34+ cells and
       CFU (colony forming units). Note: CFU evaluation has
       not been performed in all units;
5. There is no bacterial contamination of the unit;
6. The infant has no identified risks of genetic or
   infectious disease that might be transmitted in
   the cord blood;
7. The unit and the mother’s blood are negative
   for infectious disease markers;
8. The cord blood unit was processed and frozen
   following the NCBP protocol validated to
   maintain nucleated cell viability.
9. A sample of cord blood is available in a
   segment of the tubing integral to the bag
   containing the cord blood unit, for future
   confirmatory HLA typing.
        What is cord blood used for?
• The most common disease category has been
  leukemia. The next largest group is inherited
  diseases (of red blood cells, the immune system
  and certain metabolic abnormalities.)
• Patients with lymphoma, myelodysplasia and
  severe aplastic anemia have also been successfully
  transplanted with cord blood.
  How long do cord blood cells remain viable?

• It is not known how long cord blood can be stored
  in liquid nitrogen and its cells remain viable.
• NCBP's earliest units were stored in 1993. In checking
  the viability of cells in cord blood units that will not
  be used for transplantation, they did not detect any
  deterioration in the quality of the cells in cord blood
  units stored for up to 16 years.
• Units stored for up to 13 years have been used in
  transplants and the outcomes have been similar to
  those of newly collected units.
       Checking Donor's and Blood Tests?
• Normal procedure includes
   – Asking the mother and father for their health related history
   – Blood tests to rule out
       • HIV infections, other blood diseases human
         immunodeficiency virus (HIV, the cause of AIDS),
       • hepatitis B and C viruses (HBV and HCV),
       • human T-lymphotropic virus (HTVL),
       • syphilis,
       • West Nile Virus (WNV)
       • and Chagas disease (a disease common in South
         America).
       • test the baby's blood for Cytomegalovirus (CMV)
       • and for hemoglobin abnormalities such as sickle cell
         disease..
   – Signing of a consent form.
        Methodology
•   Collection
•   Processing
•   Storage
        How is cord blood collected?
• The cord blood collection process is simple, safe, and painless.
  It is usually completed in less than five minutes by your health
  care provider. Cord blood collection does not interfere with
  delivery and is possible with vaginal or cesarean deliveries.
  Your health care provider will use one of two options for cord
  blood collection: syringe method or bag method.
    – Syringe method: a syringe is used to draw blood from the
        umbilical cord shortly after the umbilical cord has been
        cut. The process is basically the same as drawing blood for
        a blood test.
    – Bag method: the umbilical cord is elevated to cause the
        blood to drain into a bag.
• The syringe or bag should be pre-labeled with a unique
  number that represents your baby. Cord blood may only be
  collected during the first 15 minutes following the birth, and
  should be processed by the laboratory within 48 hours.
• The Cord Blood Bank is responsible for collection of the blood.
• However, the related authorities (delivery doctors, nurses)
  must be aware of the mothers’ wish to donate or store Cord
  Blood after the birth.
• Minutes after a child’s birth and after the umbilical cord is cut,
  the placenta and its attached portion of the cord are
  delivered With maternal assent to collect the cord blood,
• staff move the placenta to an adjacent laboratory, where
  they collect the cord blood into a sterile plastic bag, much
  like those used for blood transfusion.
• Because they collect cord blood from the delivered placenta,
  no risk is incurred by mother or child.
• explains all aspects of the Program to the mother and requests
  her consent to donate the cord blood. Donation of the blood
  from a single placenta becomes a unique cord blood unit.
• The volume collected varies but usually ranges from 50 to 200 ml (about
  one-half to one cup).
                           Processing:
• to remove excess red blood cells (RBCs) and plasma and bring
  each collection down to a uniform single unit volume of 20 ml.
  1. Upon arrival in the Processing Laboratory, cord blood units
       are "logged-in" to the database and weighed to estimate the
       collected blood volume.
  2. The cord blood is transferred using a sterile-docking device,
       from the collection bag into a sterile, disposable processing
       bag set in preparation for its automated processing using the
       AXP (AutoXpress) platform (510 k-approved by FDA).
  3. Before and after processing, small samples are removed to
       count the total nucleated cells (TNC) and the number of
       nucleated cells that are positive for CD34. These samples are
       removed within plastic bubbles (pre-set in the tubing), sealed
       off from the processing bag set without exposing the
       products to possible infectious agents in the air of the
       laboratory.
4.   The bag set containing the cord blood unit is placed into the
     computerized AXP device, which is automatically centrifuged
     twice. The first centrifugation stratifies the cellular components
     [RBCs, white blood cells (WBC) and platelets] separated from
     the plasma. The second spin harvests the blood components
     into the three different bags of the AXP sterile bag set: RBC bag,
     freezing bag [containing the WBCs, predominantly the
     mononuclear cells (MNCs)] and plasma bag.
5.   The freezing bag contains the 20 ml final product (on average,
     more than 90% of the original MNCs and stem cells in some of
     the plasma, along with some red cells).
6.   Cold, sterile, Dimethylsulfoxide (DMSO, 5 ml of a 50% solution) is
     then added slowly. DMSO is a kind of "anti-freeze" or
     cryoprotectant that prevents ice crystals from forming inside of
     and destroying cells during freezing. It is also protective when the
     unit is eventually thawed.
                           Storage:
• The cord blood is frozen and stored in liquid nitrogen freezers
  designed especially for cord blood units.
• The BioArchive is a robotic freezer that controls and monitors
  the rate of freezing, then stores the unit in liquid nitrogen and
  provides quick access to stored units
• whenever needed, all under computer control and with
  complete documentation.
• When a transplant center requests a matched cord blood unit
  for a patient, the available inventory of cord blood units is
  “searched” for matches to the patient. The best matching unit
  (or units) can be selected, reported, confirmed and delivered
  rapidly upon request.
                    Freezing:
• Each cord blood unit is frozen (one at a time) in an
  individual “controlled-rate freezer” module within
  the BioArchive™ system. We use a freezing rate
  documented to maintain cell viability. A record of
  the freezing curve is printed and maintained in the
  BioArchive™ computer memory.
           Testing of cord blood units:
1. Typing for HLA-A, -B and -DRB1. Typing is performed at low
   intermediate resolution for HLA-A and -B and at high resolution
   (sequence based typing) for HLA-DRB1. In addition, before
   releasing a cord blood unit for transplantation, confirmatory HLA
   typing is performed on cells from a continuous segment for HLA-
   A, -B, -C at low intermediate resolution and HLA-DRB1 at high
   resolution.
2. Total nucleated cell enumeration is performed on all cord blood
   units before and after processing. Since mid-2000, cell counts
   have been done with an automated hematology analyzer
   (Sysmex XE-2100), that includes a count of nucleated red blood
   cells. The total nucleated cell count (TNC) of a unit includes all
   nucleated cells: white blood cells and nucleated red cells.
3.   CD34+ cell number has been assessed on all units since February
     2000. Currently, CD34+ cell count and viability are assessed in all
     cord blood units by flow cytometry before and after cord blood
     processing.
4.   Colony forming unit (CFU) assay., this assay is very reproducible
     and does not depend on the operator (laboratory technician).
     In addition, images of CFU from each cord blood unit are stored
     electronically and can be reviewed at later times.
5.   Bacterial culture (of the unit after processing) for both aerobic
     and anaerobic organisms. using the ESP Culture System (Versa
     TREK). The system screens a sample of the cord blood unit for
     bacterial infection, and if a positive signal is detected, the
     sample is sent to a Microbiology Reference Laboratory for
     bacterial identification and antibiotic sensitivity.
6.   Infectious disease testing of both the mother's blood and cord.
     Testing includes antibodies to HIV-1 and 2 (with a test that
     detects antibodies to HIV-O also), HTLV-1 and 2, HCV, HBV (anti-
     HBc), syphilis and hepatitis B virus surface antigen (HBsAg). Since
     2008, mother’s blood is also tested for antibodies to Chagas’
     disease.
7. Nucleic Acid Testing (NAT) for HIV, HCV and West Nile Virus
    are done on the maternal blood sample only
8. Cytomegalovirus (CMV) testing. Mother’s and cord blood
    samples are tested for total antibody to CMV, indicating past
    or recent exposure to the virus. In addition, perform CMV
    culture of the baby’s saliva. test the cord blood sample by
    PCR amplification of the viral DNA, in all cases that the
    mother’s antibody testing is positive for CMV.
9. ABO blood group and Rh type
10. Hemoglobinopathies screening. Sickle cell hemoglobin and
    hemoglobins C and E are detected by high performance
    liquid chromatography (HPLC)
11. Stored Samples. Aliquoted samples of plasma, viable white
    cells and DNA from the cord blood unit and from the
    mother's blood are kept frozen and are available for special
    testing in the future, as needed.
                  Finding a Match
• Several studies indicate that cord blood transplants do not
  need to be as perfectly HLA-matched to the recipient as
  bone marrow transplants do. However, patients have a better
  chance to survive and regain health if the cord blood graft is
  better matched (6/6 or 5/6) and provides cell doses of 20
  million nucleated cells per kg or more.
• Cord Blood Unit Match Reports: An individual Match Report gives
  detailed information about the cord blood unit, including
  number of total nucleated and CD34+ cells, ABO and Rh
  blood groups and results of other laboratory tests.
• The match referred to is required between the donor’s and
  recipient’s HLA antigens. The closer the match is, the better is
  the body’s chance of accepting the new stem cell.
Samples are saved and may be used for further
             testing in the future.


• Cord and mother’s blood samples are kept, frozen, and may
  be used in the future, even years after the baby’s birth. We
  must plan for the possibility that new tests for infectious or
  genetic diseases will be developed for which the cord blood
  must be tested in order to protect a transplant recipient from
  risk.

								
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