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Transfusion Medicine and Hematopoietic Transplantation

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Title: Transfusion Medicine and Hematopoietic Transplantation


1
Transfusion Medicine and Hematopoietic
Transplantation
  • Bill Zaloga, D.O.
  • MCG Transfusion Medicine
  • September 2005

2
Hematopoietic progenitor cells
  • Hematopoietic stem cells are self renewing and in
    sufficient numbers give rise to a complete
    sustained lymphohematopoietic graft.
    Hematopoietic progenitor cells are not self
    renewing and commited to give rise to a blood
    cell lineage. Both cell types are often referred
    to as hematopoietic progenitor cells (HPCs).
  • HPCs are given intravenously, migrate to the
    marrow, divide and mature.
  • Short-term repopulation (within about 2 weeks)
    potential is from lineage-committed HPCs, and
    long-term repopulating ability is necessary from
    the pluripotent HPCs for complete sustained
    engraftment.
  • Sources of progenitor cells include marrow,
    peripheral blood, umbilical cord blood, and fetal
    liver (experimental).
  • Historically marrow was the primary source of
    hematopoietic cells however, peripheral blood
    progenitor cell (PBPC, which contain stem cells
    and progenitor cells) transplants are more common
    for adult autologous transplantation.
  • HPCs can be collected with cytapheresis for bone
    marrow reconstitution. HPCs are then
    cryopreserved and, after treatment of the
    patient, infused.
  • Umbilical cord blood transplants show promise for
    pediatric patients and clinical studies are
    evaluating use in adults, safety and efficacy,
    and Human Leukocyte Antigen (HLA) mismatch extent
    that can produce a durable graft.
  • Allogeneic HPC transplants have the difficulty of
    finding an HLA match, rejection and graft-vs-host
    disease (GVHD), but the recipient may benefit
    from a graft versus tumor effect.
  • In the case of an identical twin donor the graft
    is called syngeneic.
  • Autologous grafts are technically not a
    transplant but a rescue with the patients own
    HPCs.

3
Hematopoietic progenitor cells
  • Increased age, underlying disease, and previous
    treatment may reduce HPC collection yield.
  • Once collected HPCs may have ex-vivo processing
    remove incompatible red cells or plasma, and/or
    cell selection (purging) such as classically
    selectively isolating CD34 cells, or antibody
    mediated lysis of malignant cells, or depletion
    of T cells which cause graft-vs-host disease.
  • Malignant diseases are the primary indications
    for HPCs although non-malignant disease have been
    treated.
  • Success of HPC transplantation depends on
    disease, condition of patient, and HLA match with
    overall survival 30-60 in otherwise fatal
    disease.

4
Hematopoietic progenitor cells
  • Regulations
  • In 1997 the US FDA made regulations for cellular
    and tissue based products that include PBPCs from
    placenta, umbilical cord and peripheral blood.
  • Adhere to current good tissue practice.
  • Proper handling, labelling, record keeping, and
    quality program maintenance.
  • Standards
  • AABB (American Association of Blood Banks) and
    Foundation for the Accreditation of Cellular
    Therapy (FACT) have similar published standards
    for HPCs.
  • AABB Standards for Hematopoietic Progenitor Cell
    and Cellular Product Services (addresses
    collection, processing, storage and distribution,
    while FACT standards also include clinical
    program issues).
  • National Marrow Donor Program (NMDP) has
    voluntary standards.
  • Institutional policies and protocols dictate
    specific contraindications for HPC
    transplantation.

5
Donor suitability
  • Autologous donor need history and physical to
    identify risks, other concerns are sensitivity of
    malignancy to myeloablative regimen and
    mobilization of sufficient cells to reconstitute
    marrow (need marrow biopsy after treatment).
  • Allogeneic donor Selection first based on HLA
    compatibility, other factors are CMV negativity
    (if recipient is CMV negative)
  • Sex mismatched individuals, parous female donors,
    and previously transfused donors report a higher
    risk of GVHD.
  • Allogeneic donors eligibility process includes
  • Health history,
  • Accurate, truthful information is essential to
    assess deferral.
  • Evaluation to meet minimum physiologic criteria
    is in AABB FACT standards for HPC services.
  • Donor blood tests samples must be tested 30 days
    before HPC collection,
  • ABO/Rh, antibody screen, HLA type, Hepatitis B
    C, HTLV, HIV, CMV, syphilis.
  • Augologous donors Hepatitis B C, HIV, HTLV.
  • Infectious disease tests routine donor blood
    tests regardless of HPC source since recipient is
    immunocompromised.
  • HIV positive donors are deferred.
  • Other positive disease markers do not rule out
    donors if informed consent is obtained from
    recipient and their physician the units are
    separately stored.
  • Cord blood is tested by testing the mothers
    blood within 48 hours cord blood collection.

6
HLA typing
  • Class I antigens HLA-A, B, and Cw
  • Class II antigens HLA-DR, DQ, DP
  • A and B traditionally tested serologically, but
    molecular typing is increasing.
  • Molecular typing provides greater resolution
    including allele subtypes identified as cross
    reactive groups by serology.
  • HLA-A, B, and DR disparities are important in
    graft failure, and HLA-C is being studied.
  • GVHD (graft-vs-host disease) is greater risk with
    Class II disparity than Class I.
  • Mismatching a single Class I or II allele has not
    affected survival, but mortality increases with
    more than one Class I or simultaneous Class I and
    II mismatches.
  • HLA-DRB1 and HLA-DQB1 allele disparity increases
    risk of GVHD.
  • GVHD is a greater risk for unrelated and related
    mismatched, than HLA-identical transplants.

7
GVHD
  • Immunocompetent donor T cells react against
    recipient tissues.
  • Nucleated cells have minor histocompatibility
    antigens not linked to the major
    histocompatibility complex antigens, so up to 6
    of HLA identically (6 antigen) matched HPC
    transplants fail and GVHD occurs in 20-60 of
    cases, despite immunosuppressive therapy for
    several months after transplant.
  • 2 forms, acute and chronic.
  • Acute form occurs a few to 100 days after
    transplant with skin, gastrointestinal tract and
    liver most affected.
  • Chronic form occurs in 30-40 of allogeneic
    transplants patients.
  • Chronic form occurs spontaneously months (after
    100 days) after transplant, or after acute form,
    with symptoms of acute form and often chronic
    autoimmune disorders.
  • Both forms predisposed to infections.
  • To reduce incidence T-cell depletion of
    transplant is being explored and
    immunosuppressive agents can be used
    prophylactically.
  • GVHD was found associated with decreased disease
    relapse (chronic GVHD) and improved survival in
    leukemia and a graft-vs-leukemia
    (GVL)/graft-vs-tumor effect is believed due to
    the graft attacking residual malignant cells.
  • Pediatric patients have a lower risk of
    susceptibility to GVHD.
  • Unrelated cord blood grafts and HLA-identical
    related cord blood grafts have a lower incidence
    than unrelated marrow grafts and HLA-identical
    related marrow grafts, respectively.

8
Donor lymphocyte infusion (DLI)
  • The GVL mechanism is not fully understood but
    donor cytotoxic T- cells are thought to react
    against specific recipient minor
    histocompatibility antigens.
  • DLI is attempted for leukemic relapse after
    allogeneic transplant to induce GVL.
  • Doctors attempt to maximize GVL while minimizing
    GVHD, but the optimal number of donor lymphocytes
    for GVL effect without significant GVHD is
    unknown and is a function of immunosuppression,
    myelosuppression, T-cell number transfused,
    T-cell phenotype, and timing of DLI.

9
Matched unrelated donations
  • 60-70 of candidates wont have a HLA identical
    sibling donor and a matched unrelated donation
    can be sought in several marrow donor databases
    worldwide.
  • 80 of searches usually find an HLA phenotype
    match with racial and ethnic minorities having a
    lower chance of success.
  • The median time from search start to transplant
    is 120 days.
  • The NMDP operates the National Bone Marrow Donor
    Registry under a contract from the US government
    and is the worlds largest unrelated donor blood
    stem cell registry.

10
ABO incompatibilities
  • Pluripotent and very early committed HPCs dont
    have ABH antigens, allowing successful
    engraftment regardless of ABO compatibility.
  • ABO incompatibility does not affect neutrophil or
    platelet engraftment, graft failure or rejection.
  • Delayed red cell engraftment and hemolysis does
    occurs.
  • A group O recipient may produce anti-A and anti-B
    for 3-4 months or longer.
  • Red cell engraftment may be delayed gt40 days, and
    red cells appear in circulation when antibody
    disappears.
  • With major ABO incompatibility (patient antigen-,
    donor antigen).
  • Donor red cell hemolysis in graft (immediate) and
    after engraftment (40-60 days).
  • Failure or delayed red cell engraftment.
  • Transfused red cells and plasma should be
    compatible with donor and recipient beginning at
    transplant conditioning regimen (until blood type
    is donors, then donor compatible).
  • Recipient antibody may be removed by
    plasmapheresis prior to HPC infusion.
  • With minor ABO incompatibilities (patient
    antigen, donor antigen-).
  • Patient red cell hemolysis from donors plasma
    antibody, and delayed antibody production from
    donor passenger lymphocytes (7-10 days).
  • Transient hemolysis may persist 2 weeks.
  • Transfused plasma should be compatible with donor
    and recipient, and red cell transfusions should
    be donor type beginning at transplant
    conditioning regimen (until blood type is donors,
    then donor compatible).
  • With Rh incompatibility.
  • Patient Rh- with anti-Rh and donor Rh, donor red
    cell hemolysis from engrafted HPCs.
  • Patient Rh and donor Rh- with anti-Rh, patient
    red cell hemolysis by donor anti-Rh after
    engraftment.
  • Despite an intensive pretransplant conditioning
    regimen (chemotherapy and radiation), some host
    HPCs may survive and coexist with the graft and
    is called hematopoietic chimerism which is being
    studied for improved immunocompetence and
    tolerance.

11
From AABB Technical Manual, 14 th Edition, 2002
12
Hematopoietic progenitor cells
  • Autologous marrow patient and marrow health
    limits use (ex. cant harvest marrow fibrosis,
    metastasis or necrosis).
  • PBPCs are a common option for these patients and
    in heavily treated patients.
  • Allogeneic marrow addresses problems with
    autologous transplant for malignant disease, and
    for patients incapable of supplying their own
    autologous normal HPCs,
  • but GVHD is a risk because of difficulty of
    having a good HLA match.

13
Marrow HPC collection
  • Same procedure for autologous or allogeneic.
  • Sterile procedure, with multiple needle
    aspirations done in OR from posterior iliac
    crests usually.
  • Prior radiation of marrow site may show
    hypocellularity there.
  • The harvest is mixed with anticoagulant (heparin
    or ACD), filtered, and put into a sterile blood
    bag.
  • Samples are removed for graft evaluation, quality
    assurance, and possible processing and
    cryopreservation.
  • Contains plasma, red cells, white cells,
    progenitor cells, platelets, and fat.
  • Target dose is 10-15 mL marrow/ kg of recipient,
    or a minimum, after processing, of 200,000,000
    nucleated cells/ kg of recipient.
  • NMDP limits maximum harvest to 1500 mL.
  • Extra marrow is necessary if alkylating agents
    were used before harvest since this decreases
    progenitor cell numbers, or post-harvest
    processing is done (increase up to double
    reinfusion target).
  • Blood transfusion may be necessary due to volume
    of marrow removed, and pre-/intra-procedure
    allogeneic units should be irradiated to avoid
    GVHD due to the harvest.

14
Peripheral blood HPCs
  • PBPC collection has no anesthesia risk, is less
    invasive, and has fewer tumor cells than marrow
    harvest.
  • Usually collected after recombinant hematopoietic
    growth factor, or chemotherapy mobilization.
  • Autologous PBPCs
  • Mobilization of hematopoietic cells from the
    marrow to the peripheral blood where they are
    collected by leukapheresis.
  • Mobilized autologous PBPCs vs autologous marrow
    harvest results show PBPCs have earlier
    engraftment, improved immune reconstitution,
    lower transplant morbidity, and similar incidence
    of GVHD.
  • Allogeneic PBPCs
  • Related transplant
  • Has mostly replaced marrow for related
    transplant.
  • Best results are complete HLA-matched related
    donor.
  • Best chance of six antigen HLA-match is from
    genetic siblings (25 chance complete match, 50
    chance of a haplotype match, and 25 chance of
    complete mismatch, but parents and children will
    also be at least a haplotype match).
  • Pediatric patients are more tolerant of partial
    HLA-mismatch.
  • Syngeneic grafts are genotypically identical with
    risk of GVHD reduced but no GVL effect.
  • Unrelated transplant
  • PBPCs have potential for better engraftment
    kinetics and enhanced GVL effect but studies are
    needed versus marrow.

15
Peripheral blood HPC collection
  • CD34 positive HPCs (lineage committed and
    pluripotent stem cells) are capable of trilineage
    hematopoietic reconstitution.
  • 1-3 of marrow and 0.01-0.1 of peripheral blood
    cells are CD34 positive.
  • Collection of increased numbers of leukocytes
    requires administration of drugs or adjuvants
    before leukapheresis.
  • Hematopoietic growth factors granulocyte-macropha
    ge colony-stimulating factor (GM-CSF, and
    granulocyte colony-stimulating factor (G-CSF).
  • Hematopoietic growth factors increase luekocyte
    yields and are well tolerated.
  • Hydroxyethyl Starch (HES) red cell
    aggregating/sedimenting agent minimizes
    inevitable red cells collected and enhances
    leukocyte collection (also causes volume
    expansion).
  • Chemotherapy and growth factors increase PBPC
    collection 50-200-fold, while each alone
    increases PBPCs by 10-30-fold.
  • Increased age, underlying disease, and previous
    treatment reduce collection yield.
  • May require increased number of collections,
    increased volume processed, or more growth
    factors.
  • Timing of collection length of time to
    engraftment correlates with CD34 cells in
    collection.
  • A peripheral blood CD34 cell count of
    10/microliter will yield at least 1,000,000 CD34
    cells/kg.
  • Clonogenic assays, WBC and mononuclear cells
    counts can determine time to initiate PBPC
    collection, but CD34 counts by flow cytometry
    provide a real time measure.
  • Usually G-CSF in normal donors mobilizes
    sufficient stem cells for allogeneic transplant.
  • Donors injected daily CD34 peripheral cell
    counts rising on day 3 and peaking after day 5-6.
  • Standard volume (8-9 liters) leukapheresis median
    yields WBC 32,400,000,000 mononuclear cells
    31,400,000,000 CD34 cells 330,000,000
    platelets 470,000,000,000 and red cells 7.6 mL.

16
Leukapheresis donor
  • The FDA has regulations, the AABB has voluntary
    standards, and the American Society for Apheresis
    has guidelines for apheresis donation.
  • Donor identification system relates donor to his
    components, samples, and records.
  • Requires informed consent.
  • Collection procedures shall not exacerbate a
    donors medical condition.
  • G-CSF complications in 90 bone pain, headache,
    body ache, fatigue, nausea.
  • G-CSF may cause false positive HCV antibody test.
  • Rarely splenic rupture, neutrophil skin
    infiltration, artery thrombosis, and anaphylaxis.
  • Cytopenias shall be tested for as appropriate
    before and after apheresis.
  • Normally volume replacement is not required.
  • During apheresis intravascular volume deficit
    shall be lt 10.5 mL/kg of donors weight.
  • Most donors will have a central venous
    double/triple lumen apheresis/dialysis catheter.
  • Peripheral venous access can also be used.
  • Most procedures of 2-3 blood volumes (standard
    volume 8-9 liters) requires 2-5 hours daily.
  • Large volume collections (at least 3 blood
    volumes, 15-20 liters) reduce number of
    procedures needed.
  • Large volume collections may recruit
    noncirculating PBPCs into the circulation.
  • Pediatric patients may need red cell prime of the
    apheresis machine.

17
Leukapheresis donor complications
  • Risks are similar for whole blood donation
    (nausea, vomiting, faintness, dizziness,
    hematoma, seizures, blood loss, air embolus,
    infection).
  • Anticoagulant is used to prevent donor blood
    clotting (MCG uses ACD-A).
  • Hypocalcemia due to citrate toxicity usually
    doesnt occur if donor has normal parathyroid and
    liver (metabolizes citrate) function.
  • Hypocalcemia is controlled by reducing the
    citrate, slowing the reinfusion rate, or
    administering calcium.
  • Vasovagal and hypovolemic reactions are rare, and
    serious reactions are less common than whole
    blood donation.
  • Hypovolemia/hypotension occur especially when
    extracorporeal volume gt 15 of total body volume.
  • Susceptible donors
  • children, elderly, neurology patients, anemic
    patients, and use of large extracorporeal volume
    intermittent flow devices. Also occurs with
    inadequate volume or protein replacement.
  • Antihypertensive medicines especially ACE
    inhibitors with albumin replacement cause
    hypovolemic reactions and flushing and medicine
    should be discontinued 72 hours prior.
  • Venous access complications 1 risk which
    include infection, hemorrhage, pneumothorax.
  • Catheter thrombosis is the most common
    complication.
  • Respiratory distress during or soon after can
    occur from pulmonary embolus, or ethylene oxide
    gas that sterilizes disposable plastic apheresis
    kits may cause a primarily ocular (periorbital
    and conjunctival edema, and tearing) allergic
    reaction in sensitized donors.
  • Fatalities during apheresis is rare with most due
    to cardiac dysrhythmias or acute pulmonary edema
    or ARDS during or soon after the procedure is
    begun when replacement fluids are infused.

18
Umbilical cord blood
  • Especially useful as a HPC source for children.
  • A smaller volume is adequate for successful
    engraftment.
  • Long-term frozen storage of a childs cord blood
    cells is being marketed.
  • The NMDP has a registry of cord blood banks.
  • Cord blood samples mothers serum is frozen for
    later testing before transplant.
  • HLA type, CD34, infectious disease, or other
    tests.
  • Informed consent required from mom ( family
    history to rule out genetic disease).
  • Drained by gravity from delivered/undelivered
    placenta (umbilical cord /- placental vessels)
    within 15 minutes of parturition.
  • Cord (umbilical vein area) is prepared as for
    blood donation to minimize bacteria.
  • A large bore needle connects the closed system to
    an anticoagulant blood bag.
  • typical volume 80-100 mL (range 40-240 mL).
  • Needs ABO/Rh type antibody screen from mom,
    infant or cord blood.
  • Cord blood white cells are HLA typed by ASHI or
    equivalent accredited lab.
  • ASHI is American Society for Histocompatibility
    and Immunogenetics.
  • Cord blood median nucleated cell count is
    1,200,000,000 with median CD34 cell count of
    3,100,000 (ex-vivo expansion of HPCs is being
    investigated).
  • Advantages more rapidly available, no donor
    risk, HPCs from under-represented populations,
    lower risk of viral infection and GVHD, and an
    increased capacity for proliferation and
    self-renewal compared with marrow.
  • Concerns ethics, informed consent, and ability
    to achieve long term engraftment in adult size
    patient from limited number of nucleated cells in
    cord blood.

19
HPC products
  • Can be categorized by degree of manipulation.
  • These should be infused or cryopreserved
    immediately after processing.
  • HPC, Marrow
  • Plasma depleted
  • Red cell depleted
  • Buffy coat preparation
  • Density separated
  • Cryopreserved
  • HPC, Apheresis
  • Plasma depleted
  • Red cell depleted
  • Buffy coat preparation
  • Density separated
  • Cryopreserved
  • HPC, Cord
  • Plasma depleted
  • Red cell depleted
  • Buffy coat preparation
  • Density separated

20
HPC graft processing
  • Allogeneic PBPCs are frequently infused
    unmodified.
  • Donor ABO incompatibility.
  • Red cells removed from graft.
  • Most commonly by sedimentation with hydroxyethyl
    starch (HES), or dextran in closed system.
  • HES causes rouleaux with red cells settling and
    nucleated cells remaining in plasma (gt 76
    nucleated cells retained, and lt1 residual red
    cells).
  • Cell washers and apheresis machines are also used
    with higher nucleated cell recovery and
    comparable red cell depletion.
  • Plasma reduced in graft to remove preformed
    antibody easily by centrifugation.
  • 75 of plasma volume removed, and gt70 initial
    nucleated cells retained.
  • Umbilical cord HPC banks usually immediately put
    graft in anticoagulant and cryopreserve, but try
    to reduce bulk through sedimentation and volume
    reduction.
  • Autologous or allogeneic marrow or PBPC banks may
    store hundreds of products, but cord banks will
    store thousands of products for long periods.

21
HPC graft processing marrow buffy coat
preparation
  • Plasma and red cells are removed
  • Density separation product is enriched in
    mononuclear cells and removes red cells,
    polymorphonuclear leukocytes, and pasma.
  • For autologous marrow harvest with subsequent
    myeloablative therapy you must process to
    facilitate cryopreservation.
  • Need to decrease marrow volume (can be up to 2000
    mL) so less dimethylsulfoxide (DMSO)
    cryopreservative toxicity is seen at infusion.
  • For allogeneic marrow harvest, red cells or
    plasma may need removal to reduce hemolytic
    reactions.
  • Current cryopreservation technique doesnt allow
    red cell or granulocyte preservation.
  • Lysed red cells may cause renal or hemolysis
    toxicity.
  • Lysed granulocyte products and platelets cause
    thawed product clumping and reduced HPC
    viability.
  • Want a buffy coat final product with depleted red
    cells and greatly reduced plasma.
  • By manual centrifugation
  • gt75 original nucleated cells recovered, but
    still a lot of red cells and granulocytes.
  • Density gradient separation with ficoll-hypaque
    (not approved for human use) eliminates red cells
    and granulocytes.
  • Open system and risk of bacterial contamination
  • By automated centrifugation (cell washers and
    apheresis).
  • Cell washers can produce gt80 original nucleated
    cells recovered, and volume decreased 80, but
    still a lot of red cells and granulocytes.
  • Apheresis instruments can recover gt94 of initial
    mononuclear cells and have 99 red cell reduction.

22
HPC graft processing cell selection
  • As cells mature CD34 antigen levels decrease.
  • Anti-CD34 antibody can select CD34 HPCs.
  • Flow cytometer can be used as a
    flourescence-activated cell sorter with physical
    separation droplet or fluid switch mechanisms
    that segregate individual cells.
  • Immunomagnetic separation An anti-CD34 antibody
    that has an attached magnetic bead is incubated
    with mononuclear cells and a magnet separates the
    appropriate cells.
  • Centrifugation can select CD34 HPCs.
  • A kit to seperate HPCs based on bouyancy is in
    clinical trials.
  • Counterflow centrifugal elutriation (CCE)
    seperates cells based on size and density.
  • Cells can be selected for specific patient
    applications.
  • Autologous tumor purging.
  • Minimal residual disease may cause disease
    relapse (PBPCs lt marrow HPCs).
  • Can detect in HPCs with monoclonal antibody to
    tumor and flow cytometry, or histochemistry.
  • Higher graft failure rate occurs in purging from
    damage to HPCs.
  • Pharmacologic high concentration chemotherapy in
    ex-vivo HPCs (not used currently).
  • Physical based on cell size and density, but
    doesnt reduce tumor enough.
  • Immunologic monoclonal antibodies select
    tumor-associated antigens with antibody bound
    toxin, complement or magnetic beads destroying or
    removing the tumor cells.
  • Allogeneic T-cell depletion (most common).
  • Want to maintain GVL effect while minimizing
    GVHD.
  • Higher graft failure rate in T-cell depleted
    grafts.
  • Research is ongoing for optimal T-cell depletion
    level.

23
HPC graft processing
  • Freezing.
  • Cryopreservation aims for minimal loss of cell
    viability and reconstitutive ability, due to
    intracellular ice crystal formation, (cell
    dehydration) increased external osmolarity, time
    for liquid to solid phase change, thermal shock,
    and post-transition freeze rate.
  • Ice crystals minimized by slow cooling, and DMSO
    which binds water.
  • Reduced cell dehydration (DMSO facilitates fluid
    flow across membranes).
  • Other effects are minimized by slow controlled
    rate of freezing.
  • Optimal freeze rate is 1-3 Celsius/minute, until
    90 to 100 Celsius, and computer controlled.
  • Non-controlled-rate freezing is described but not
    used widely with mechanical storage at 80
    Celsius.
  • Controlled-rate frozen HPCs are generally stored
    in liquid nitrogen.
  • HPCs in liquid phase of nitrogen, gt-180 Celsius
    (possibly more risk of cross-contamination vs.
    vapor phase, )
  • HPCs in vapor phase of nitrogen, -140 Celsius
    average (more temperature variation than liquid
    phase).

24
HPC graft processing
  • Storage.
  • Allogeneic products usually stored as liquid.
  • Collection generally timed to coincide with
    completion of recipients conditioning regimen.
  • Most HPCs remain viable for up to 3 days at 4
    Celsius or 22 Celsius in unmanipulated marrow,
    and for 24 hours at 4 Celsius in unmanipulated
    PBPCs.
  • Autologous products usually cryopreserved.
  • Due to long treatment times or long product
    collection times (PBPCs).
  • They can also be stored in case of relapse.
  • Grafts have succeeded after 11 years of
    crypreservation.
  • Infectious disease tests for donors should be
    complete 30 days before donation.
  • Each HPC product is also tested.
  • Regulations require alternative storage for
    untested or marker positive HPCs
  • Can store in vapor phase, or use a product
    overwrap.
  • Products need protection from rough handling,
    temperature pressure extremes, irradiation,
    breaking, and spilling.

25
HPC transplant hazards
  • Acute hemolytic reaction Usually ABO
    incompatibilities.
  • Delayed hemolytic reaction Usually ABO
    incompatibilities within days or weeks of
    infusion.
  • Alloimmunization to antigens of red or white
    cells, platelets or plasma.
  • Anaphylactic reaction Catastrophic symptoms and
    needs immediate treatment.
  • Anaphylactoid reaction Less severe than
    anaphylactic.
  • Allergic reaction Usually urticaria and no labs
    predict or prevent.
  • Febrile nonhemolytic reaction gt 1 Celsius
    elevation during or shortly after infusion.
  • Graft-vs-host disease
  • Graft failure
  • Infectious disease transmissionMay occur despite
    careful donor selection.
  • Circulatory overload Colloid and plasma
    containing product use should be a minimum.
  • Fat emboli From marrow graft.
  • Hypothermia But do not use a blood warmer with
    HPC products.
  • Nonimmunologic hemolysis Cell lysis from
    freeze/thaw, co-administered chemicals, bacterial
    toxins
  • Hypertension DMSO reaction which also is
    associated with bradycardia and tachycardia.
  • DMSO breakdown products excreted in lungs has
    garlic-like smell for 24-48 hours after infusion.
  • Flushing, rash, and chest tightness are related
    to histamine release by DMSO Prevented by
    premedication with anti-histamine.
  • Nausea/vomiting due to DMSO Prevented by
    premedication with anti-histamine or anti-emetic.

26
HPC thawing and infusion
  • Final identification is by infusing
    nurse/physician.
  • Central venous catheter infused by gravity drip,
    pump, or manual push (10-15 mL/minute minimizes
    clumps) /- inline standard blood filter with
    completion in lt 4 hours.
  • Do not infuse through leukoreduction or
    microaggregate filter, or irradiate.
  • The only infusion solution that may be used is
    normal saline if needed.
  • Infusion side effects of nausea, diarrhea,
    flushing, bradycardia, hypertenstion, abdominal
    pain warrant slowing infusion until they pass.
  • Red cell lysis occurs with freezing and
    hemoglobin clearance is assisted by hydrating the
    patient and alkylinating the urine (can see red
    urine).
  • Antihistamine premedication reduces hypotensive
    events.
  • If total infusion volume is gt 10 mL/kg or DMSO is
    gt 1 g/kg recipient weight, usually divide doses
    given in AM and PM or over two days.
  • The recipient is monitored with documentation
    before, during and after.
  • Exposure ex-vivo gt 1 hour of crypreservation
    concentrations of DMSO are toxic to PBPCs at
    22-37 Celsius.
  • To minimize thawed PBPCs DMSO exposure one bag at
    a time are thawed at the bedside.
  • Post-thaw lab samples from infusion bags are
    identical to that given to patient and more
    representative than pre-freeze samples.

27
HPC product evaluation
  • Cell counts determine total, mononuclear, and
    CD34 cell concentrations.
  • To determine dose of each product.
  • Since flow cytometry CD34 enumeration analysis
    methods differ between sites, correlation of
    their dose values are unreliable.
  • The International Society for Cellular Therapy
    has proposed guidelines for a standardized
    approach.
  • To determine number of collections to achieve
    engraftment.
  • To assess quality of processes and procedures.
  • Aerobic/Anaerobic bacteria and fungal cultures
    are done at least once during processing.
  • Sterility essential in immunosuppressed
    recipients, and in HPC products which have
    multiple manipulations.
  • Skin commensals are main isolates.
  • Since these are irreplaceable cells, a positive
    culture is evaluated by physician and not
    discarded.
  • The FACT and AABB require monitoring and
    documenting of days to engraftment of neutrophils
    and platelets.

28
Engraftment
  • The minimum number of HPCs for engraftment has
    not been definitely established, but experience
    and studies have provided guidance and
    institutional protocols may dictate minimum
    number collected and infused
  • Dose adequacy may be based on CD34 cell
    number/kg recipient body weight.
  • Minimum number of CD34 cells for autologous PBPC
    engraftment of neutrophils and platelets is about
    750,000-1,000,000/kg.
  • Minimum dose of CD34 cells for allogeneic PBPC
    transplants is about 2,000,000/kg.
  • Higher doses accelerate platelet engraftment, and
    reduce febrile periods and antibiotic use.
  • Mobilized autologous PBPCs vs autologous marrow
    harvest results show PBPCs have earlier
    engraftment with platelet reconstitution most
    affected
  • Chemotherapy G-CSF enhanced PBSC graft median
    time to 20,000/microliter platelet count is 10
    days vs 17 days for marrow graft.
  • Unrelated cord blood HPCs Higher risk of graft
    failure with weight gt 45 kg
  • Neutrophil median engraftment time
    (500/microliter) is 30 days
  • Platelets (50,000/microliter) is 56 days (longer
    than allogeneic marrow).
  • Related cord blood HPCs Time for neutrophil and
    platelet engraftment is longer than HLA-matched
    related marrow HPC graft.

29
Questions from Transfusion Medicine Self
Assessment and Review, proceed with slide show
for answers
  • Which cell type primarily mediates graft-vs-host
    disease (GVHD)?
  • A) B cells
  • B) T cells
  • C) Monocytes
  • D) Granulocytes
  • E) Progenitors

B
30
Question
  • Allogeneic major-mismatched marrow
    transplantation can expect delayed engraftment of
    which of the following?
  • A) Lymphocytes
  • B) Granulocytes
  • C) Platelets
  • D) Red cells
  • E) All of the above

D
31
Question
  • Adequacy of PBPC collection is best assessed by
    enumeration of cells bearing which antigen?
  • A) CD33
  • B) CD4
  • C) CD55
  • D) CD34
  • E) CD19

D
32
Question
  • Which of the following growth factors is used to
    mobilize progenitor cells for transplantation ?
  • A) G-CSF
  • B) Erythropoietin
  • C) IL-11
  • D) Thrombopoietin
  • E) Platelet-derived growth factor (PDGF)

A
33
Question
  • All of the following viral screen tests are
    required for autologous HPC donors except
  • A) anti-CMV
  • B) anti-HTLV
  • C) HBsAg
  • D) anti-HBC
  • E) anti-HIV

A
34
Question
  • The minimum number of CD34 cells in a PBPC
    collection for prompt engraftment is closest to
  • A) 200,000/kg
  • B) 2,000,000/kg
  • C) 20,000,000/kg
  • D) 200,000,000/kg
  • E) 2,000,000,000/kg

B
35
Question
  • A group B recipient receives a group A allogeneic
    HPC transplant. Which of the following would be
    the best choice for transfusion support during
    the transplant?
  • A) Group O red cells, group AB plasma/platelets.
  • B) Group O red cells, group A plasma/platelets.
  • C) Group B red cells, group A plasma/platelets.
  • D) Group B red cells, group AB plasma/platelets.
  • E) Group A red cells, group AB plasma/platelets.

A
36
Question
  • Which of the following is associated with minor
    ABO-mismatched (donor incompatible plasma)
    allogeneic HPC transplants?
  • A) Decreased risk of GVHD.
  • B) Hemolysis 7-10 days after transplantation.
  • C) Hemolysis 40-60 days after transplantation.
  • D) Delayed red cell engraftment.
  • E) Delayed granulocyte engraftment.

B
37
Question
  • In the setting of allogeneic HPC transplantation,
    HLA identical (6 antigen match) sibling donors
    are identified for what proportion of patients?
  • A) lt1
  • B) 5-10
  • C) 30-40
  • D) 60-70
  • E) gt90

C
38
Question
  • All of the following regarding autologous PBPC
    collection are true except
  • A) Requires mobilization with hematopoietic
    growth factors.
  • B) Results in more rapid platelet engraftment
    compared to autologous marrow.
  • C) Timing of collection can be optimally
    predicted using peripheral blood CD34 cell
    measurements.
  • D) Relatively contraindicated in patients who
    have received multiple courses of chemotherapy.
  • E) CD34 cell content in the graft correlates
    with length of time for engraftment.

D
39
References
  • AABB Technical Manual, 15 th Edition. Bethesda,
    MD AABB 2002 Chapter 25.
  • AABB, ARC, ABC Circular of Information for Use of
    Hematopoietic Progenitor Cell Products. Bethesda,
    MD AABB Jan 2000.
  • Helekar PS et al. Transfusion Medicine
    Self-Assessment and Review. Bethesda, MD AABB
    Press 1992 Chapter 7.
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