Transfusion medicine

Transfusion therapy

Granulocyte use



Topic Completed: 19 July 2021

Minor changes: 19 July 2021

Copyright: 2020-2021, PathologyOutlines.com, Inc.

PubMed Search: Transfusion granulocyte use [TI] full text [SB]

Martin S. Ongkeko, M.D.
Kathleen (Cathy) Conry-Cantilena, M.D.
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Page views in 2021 to date: 156
Cite this page: Ongkeko MS, West KA, Conry-Cantilena K. Granulocyte use. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/transfusionmedgranulocyteuse.html. Accessed December 3rd, 2021.
Definition / general
  • Granulocytes are white blood cells with visible granules, the most abundant of which are neutrophils, with important roles in protecting against bacterial and fungal infections
  • Granulocyte transfusions are used for treatment of antimicrobial unresponsive bacterial and fungal infections in patients with severe neutropenia or defective granulocyte function (e.g. chronic granulomatous disease) when granulocyte recovery is anticipated
Essential features
  • Clinical indications for granulocyte transfusions (AABB: Circular of Information for the Use of Human Blood and Blood Components [Accessed 9 June 2021]):
    • Severe neutropenia with absolute neutrophil count (ANC) < 500/μL or impaired granulocyte function, such as chronic granulomatous disease
    • Documented evidence of fungal or bacterial infection that is unresponsive to appropriate antimicrobial therapy for at least 24 to 48 hours
    • To be used as a bridge with expectation of marrow recovery
  • Granulocyte product characteristics:
    • Each dose must contain at least 1.0 x 1010 granulocytes in at least 75% of units tested
    • Stored at 20 - 24 °C without agitation
    • Infused as soon as possible, with shelf life of 24 hours due to deterioration of function in storage
    • Irradiated to prevent transfusion associated graft versus host disease (TA-GVHD)
    • Crossmatch compatible with recipient's plasma due to 10 - 30 mL of red blood cells (RBCs) in the apheresis product
    • Emergency release: results of donor infectious disease testing are generally unavailable within 24 hours of collection
    • Cytomegalovirus (CMV) seronegative granulocytes must be provided for CMV seronegative recipients
    • Must not be leukoreduced but administered with a standard blood infusion set with a 170 to 260 micron filter
    • Granulocytes are not licensed blood products by the U.S. Food and Drug Administration (FDA)
Terminology
  • Apheresis granulocytes: prepared from stimulated, single donor leukapheresis collection, contain ≥ 1.0 x 1010 granulocytes, 10 - 30 mL of RBCs, ~1.0 x 1011 platelets and up to 200 mL of plasma; total volume of product approximately 300 mL (Cytotherapy 2017;19:1256)
  • Hydroxyethyl starch (HES): RBC sedimenting agent added during the leukapheresis collection to further separate RBCs from granulocytes
  • Whole blood derived buffy coat granulocytes: the source of granulocytes in the U.K., each component is 50 mL, with 1 - 2 x 109 leukocytes, hematocrit of 45%, 90 x 109 platelets and 9.5 g of hemoglobin; individual units must be pooled to provide an adequate patient dose (NHS Blood and Transplant: Clinical Guidelines for the Use of Granulocyte Transfusions [Accessed 9 June 2021])
Pathophysiology
  • Granulocytes migrate to sites of inflammation and infection to eliminate pathogens via phagocytosis and intracellular killing by generation of reactive oxygen species or by secretion of antimicrobial proteins to combat targets (Trends Immunol 2010;31:318)
  • Neutrophils can also kill microbes by releasing web-like chromatin structures called neutrophil extracellular traps (NETs) (Nat Rev Immunol 2018;18:134)
  • Infused granulocytes have been shown to migrate towards sites of superficial bacterial infections (Transfusion 2007;47:2185)
  • Infused granulocyte half life in the peripheral blood is short: 6 - 8 hours (Trends Immunol 2010;31:318)
  • Granulocytes are not recommended for prophylaxis during neutropenia in patients following chemotherapy or hematopoietic stem cell transplantation (Cytotherapy 2017;19:1256)
  • Evidence for survival benefit of granulocyte transfusions has not been firmly established but some studies, including a recent trial (Resolving Infection in Neutropenia with Granulocytes [RING] trial), suggest that larger doses of granulocytes may offer clinical benefit (Cytotherapy 2017;19:1256)
Blood donor screening
  • Community donors and directed (family) donors can be collected
  • Granulocyte donors must be healthy, meeting the same standards for donor screening (donor history questionnaire and physical exam) as blood donors
  • Additional considerations for blood donor screening are secondary to the adverse effects associated with the use of granulocyte colony stimulating factor (G-CSF), dexamethasone, HES and risks of apheresis procedures (see Apheresis granulocyte collection)
  • Granulocyte donor suitability criteria vary across different institutions but may include:
    • Adequate veins for peripheral vascular access
    • No history of hypersensitivity reactions to HES
    • No contraindications to steroid administration (hypertension, diabetes, gastrointestinal ulcers, cataract, active infections or other conditions adversely affected by steroids)
    • Assessment of renal function (creatinine, cystatin C, estimated glomerular filtration rate [eGFR]) due to use of HES
    • Eye exam: to screen for cataracts, especially for repeatedly stimulated donors
    • Assessment of pregnancy status
    • ABO / Rhesus (Rh) type
    • Human leukocyte antigen (HLA) type: if HLA matching is performed by the blood center
Blood donor testing
  • Similar to donor screening, donor infectious disease testing must abide by requirements outlined in Title 21 of the Code of Federal Regulations section 610.40 and 630.3(h), FDA Blood Guidances, standards of accreditation organizations such as American Association of Blood Banks (AABB) and state and local regulatory agencies (FDA: Code of Federal Regulations Title 21 [Accessed 21 June 2021], FDA: Blood Guidances [Accessed 21 June 2021])
  • Required allogeneic donor testing include HBV DNA, HBsAg, anti-HBc, anti-HCV, HCV RNA, anti-HIV 1 / 2, HIV 1 RNA, anti-HTLV I / II, WNV RNA and syphilis by a serologic test
  • CMV testing: CMV seronegative recipients must be administered granulocytes from CMV seronegative donors
  • HLA compatibility: further decreases the available donor pool
    • Patients with HLA antibodies should have HLA matched granulocytes or HLA cognate antigen type compatible with the patient's anti-HLA
    • Recipients planned to undergo hematopoietic stem cell transplantation should be provided with granulocytes lacking the prospective donor HLA antigens to avoid HLA alloimmunization and development of donor specific antibodies
  • Emergency release: granulocytes are transfused as soon as possible under emergency release or authorized exception because results of donor infectious disease testing are not available until 24 - 48 hours after sample collection (Acad Pathol 2020;7:2374289520909500)
    • Granulocyte donors may be selected from an established platelet donor pool whose donor infectious disease testing within the last 30 days is available
Donor selection / deferral
  • Donor suitability assessment: stimulation with G-CSF or dexamethasone, as well as addition of HES in the apheresis collection, require assessment for donor safety (Transfusion 2012;52:2646)
    • G-CSF adverse effects: bone pain, headache and fatigue; mild but treatable (J Clin Apher 2004;19:115)
    • Corticosteroid adverse effects: fluid accumulation, weight gain, insomnia, posterior subcapsular cataracts
      • Association of repeated corticosteroid stimulation with posterior subcapsular cataracts has been proposed but has not been established (Transfusion 2011;51:921)
    • HES adverse effects: minimal adverse effects; rare hypersensitivity and pruritic reactions (Transfusion 2015;55:911)
  • Long term followup of granulocyte donors repeatedly stimulated with G-CSF / dexamethasone appears to be safe (Transfusion 2009;49:513)
  • Logistics of donor selection, stimulation, testing and collection are cumbersome and may be inconvenient for the donor, adding to constraints in granulocyte availability
    • 2 - 3 donor visits / days are required for a collection procedure:
      1. Infectious disease testing
      2. Stimulation with steroid or G-CSF
      3. Day of granulocyte apheresis collection
Apheresis granulocyte collection
  • Collection of adequate numbers of granulocytes has historically been the limiting factor for the use of granulocytes because of both low yields and donor availability
  • Stimulation with corticosteroids with or without G-CSF has greatly increased the yield of granulocyte collections but with ensuing adverse effects on donors (Transfusion 2000;40:642, Transfusion 2001;41:1037)
    • Most blood centers in the U.S. provide steroid stimulated granulocytes
    • Corticosteroid only stimulation: 2 - 3 times absolute neutrophil (ANC) count increase with peak at 24 hours; yield: ~1 x 1010 granulocytes
    • G-CSF only stimulation: 7 - 10 times ANC increase with peak at 12 hours; yield: 2 - 6 x 1010 granulocytes
    • G-CSF + corticosteroid stimulation: 10 - 13.5 times ANC increase with peak at 12 hours; yield: 4.5 - 9 x 1010 granulocytes
  • Granulocyte donors can be community or family related (directed) donors
  • Apheresis collection utilizes HES to increase collection efficiency by separating the RBC from the granulocyte layer during centrifuge separation apheresis
    • Blood volume processing 7 - 10 L (procedure lasts for 2 - 3 hours)
  • Anticipated granulocyte increment in the neutropenic patient after receipt of G-CSF + corticosteroid stimulated apheresis granulocyte has been reported to be a median peak ANC increment of 1,000/μL measured at 12 - 24 hours postinfusion after an average granulocyte dose of 4 x 1010/L granulocytes (Bone Marrow Transplant 2015;50:846)
Laboratory
  • Storage condition: granulocytes are stored in 20 - 24 °C without agitation for a maximum of 24 hours
    • Must not be refrigerated
  • Irradiation: granulocytes must be irradiated to prevent transfusion associated graft versus host disease (TA-GVHD)
  • Compatibility testing: must be crossmatch compatible because of 10 - 30 mL RBCs in the granulocyte product
  • HLA compatibility: granulocyte transfusion should be avoided in patients with HLA antibodies; otherwise, the recipient should be HLA typed and the donor should be HLA matched or compatible with the patient's HLA antibodies, which may further decrease the available donor pool
  • Repeat screening for HLA, HPA (human platelet antigen) and neutrophil antibodies if refractoriness to platelet or granulocyte transfusions develop
  • Red cell incompatibility may be addressed by additional time on the bench for in vitro starch sedimentation, which removes 80 - 90% of RBC harvested during a granulocyte apheresis procedure; this may be necessary when (1) ABO major incompatibility exists and (2) recipient has clinically significant antibodies to RBC antigen(s) and an antigen negative donor cannot be recruited (Transfusion 2010;50:1203)
Administration
  • Must not be infused through a leukoreduction filter (only a standard blood administration set: 170 - 260 microns)
  • Potential adverse effects of granulocyte transfusions are similar to other blood products but more important associations include (Cytotherapy 2017;19:1256):
    • Fever and chills (5 - 10%)
    • Transfusion transmitted infections: especially CMV and leukocyte associated agents because leukoreduction cannot be performed
    • Pulmonary complications (10 - 18%): transfusion associated acute lung injury (TRALI), hypoxemia, hemoptysis
    • Alloimmunization to HLA and human neutrophil antigens (HNA): leading to reduced ANC increments to subsequent granulocyte transfusions and refractoriness to platelet transfusions
  • Contrary to previous reports, pulmonary complications due to the infusion of granulocytes with amphotericin B infusions have not been proven or replicated (Haematologica 1997;82:71)
  • Granulocyte transfusions are not widely utilized because of (Cytotherapy 2017;19:1256):f
    • Inconclusive clinical efficacy based on controlled clinical trials; most evidence is from case reports and case series
    • Challenges with collecting granulocytes by apheresis due to:
      1. Limited donor availability
      2. Low collection yield for an adequate dose
      3. Logistical difficulties with coordinating a multiday course of granulocyte transfusions
    • Improved treatment of neutropenic patients with antimicrobial agents and hematopoietic growth factors (G-CSF)
Case reports
  • 9 year old girl with leukocyte adhesion deficiency I (LAD I) who developed ecthyma gangrenosum treated with granulocyte transfusions (BMC Dermatol 2010;10:10)
  • 16 year old girl with severe aplastic anemia and invasive aspergillosis awaiting stem cell transplantation (Blood 2012;119:1353)
  • 35 year old woman with acute myeloid leukemia who developed TRALI after granulocyte transfusion (Transfusion 2003;43:1683)
Treatment
  • Infusion of allogeneic granulocytes in neutropenic patients follows the same approach as RBC and platelet transfusions but granulocytes are underused because of the challenges of collecting an adequate dose and the conflicting evidence for their clinical efficacy
  • Most published studies regarding efficacy of granulocyte transfusions are from case reports and case series
  • RING trial was designed to investigate the efficacy of granulocyte transfusions and it showed that there was no difference between the survival of patients receiving antimicrobial therapy and patients receiving antimicrobials and granulocyte transfusions; however, the study was underpowered due to low enrollment and a definitive conclusion could not be drawn
    • Post hoc secondary analysis suggested that patients who received higher doses have better outcomes than those who received a lower dose (Cytotherapy 2017;19:1256)
Videos

RBC sedimentation in granulocyte product

Sample assessment & plan
  • Assessment: 7 year old boy with severe aplastic anemia status post hematopoietic stem cell transplant (HSCT) with subsequent graft rejection / failure. He is now planned for second HSCT from a new donor and currently has febrile neutropenia with an ANC of < 200. He is diagnosed with fungal pneumonia based on radiology and identification of Aspergillus fumigatus in bronchoalveolar lavage. Despite broad spectrum antifungal treatment regimens, he remains febrile with rapid radiologic progression of peripheral right lower lung infiltrates on CT scan.
  • Plan: The patient is expected to undergo marrow reconstitution after a second HSCT as definitive therapy for severe aplastic anemia. Given the persistent febrile neutropenia and Aspergillus fungal pneumonia despite ongoing antifungal therapy, the use of granulocyte transfusions is warranted. The first granulocyte infusion will be available after approximately 4 - 7 days after donor recruitment with considerations given to HLA matching, CMV status and donor availability. Frequency and duration of granulocyte therapy will be determined by frequent reevaluation of the patient's clinical condition and will begin with 2 - 3 granulocyte transfusions/week during the neutropenic period
Board review style question #1
An 8 year old boy with acute lymphoblastic leukemia is admitted for induction chemotherapy. During his admission, he developed high grade fever (103.5 °F), hypoxia and hypotension. Workup showed blood culture positive for Pseudomonas aeruginosa susceptible to cefepime. After 3 days of antibiotic therapy, he continued to experience high grade fevers, positive serial blood cultures and acute kidney injury. His absolute neutrophil count is 0.2 x 103/μL. Other pertinent lab results are: blood type A positive with a negative antibody screen and anticytomegalovirus (anti-CMV) IgG and IgM are negative. The clinical care team consulted the transfusion medicine service to initiate a course of granulocyte transfusions. Which of the following products should be provided to the patient?

  1. ABO crossmatch compatible, irradiated, cytomegalovirus (CMV) seronegative granulocytes because the patient is CMV seronegative
  2. ABO crossmatch compatible, leukoreduced, irradiated, CMV seronegative granulocytes because the patient is CMV seronegative
  3. Irradiated granulocytes; no donor CMV testing is required because granulocytes are CMV safe
  4. Leukoreduced, irradiated, CMV seronegative granulocytes because the patient is CMV seronegative
  5. Leukoreduced, irradiated granulocytes; no donor CMV testing is required because granulocytes are CMV safe
Board review style answer #1
A. ABO crossmatch compatible, irradiated, cytomegalovirus (CMV) seronegative granulocytes because the patient is CMV seronegative. Granulocytes collected by leukapheresis contain 10 - 30 mL of red blood cells and must be crossmatch compatible to avoid hemolytic transfusion reactions. In instances when major ABO incompatibility exists, additional starch sedimentation can be performed to further decrease incompatible red blood cells. Irradiation must be performed because of lymphocytes in the product and the risk of transfusion associated graft versus host disease. It is apparent that leukoreduction cannot be performed in granulocyte products. The granulocyte product must be CMV matched with the recipient; therefore, a CMV seronegative product must be provided.

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Reference: Granulocyte use
Board review style question #2
What is true of granulocytes as a transfusion product?

  1. Can be stored after collection until infectious disease testing is reported
  2. Crossmatch is not necessary because it is released under emergent conditions
  3. Cytomegalovirus (CMV) status of the donor is not relevant because the product is irradiated
  4. Must be stored in a refrigerator to preserve phagocytic activity
  5. Must be stored without agitation with a maximum shelf life of 24 hours
Board review style answer #2
E. Must be stored without agitation with a maximum shelf life of 24 hours. Granulocytes are stored at room temperature (20 - 24 °C) without agitation. Infused granulocytes have a half life of 6 - 8 hours, must be infused under authorized medical exception and emergency release because infectious disease testing results are not available until 24 - 48 hours after collection. Because of the RBCs in granulocytes, the product must be crossmatched with the recipient's plasma. The only indication for irradiation is prevention of transfusion associated graft versus host disease (TA-GVHD), not CMV transmission. The granulocyte product must be CMV negative if the recipient is CMV negative due to the presence of lymphocytes in the product.

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Reference: Granulocyte use
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