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.

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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.

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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.

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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.

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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.

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From AABB Technical Manual, 14 th Edition, 2002
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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.

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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.

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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.

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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.

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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.

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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.

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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.