Final Diagnosis --A Pediatric Male with Acute Myeloid Leukemia (AML) and Severe Neutropenia
PATIENT FOLLOW UP
The patient received multiple Rh-positive CMV negative, granulocyte transfusions on alternate days with improvement of the ANC. However the patient deteriorated clinically to have multiple organ failure syndrome with pancreatitis, cardiac dysfunction, liver and renal failure and eventually passed away.
History of granulocyte transfusions
Granulocyte transfusion (GTX) as replacement therapy is a logical therapeutic approach for neutropenia and has been available for last fifty years1, but has largely remained underutilized due to difficulties in collecting adequate numbers of viable and functional granulocytes. The normal human body produces 6x 1010 neutrophils /day2 but they have a short half-life of 6-7 hours and cannot be stored for more than a few hours; as they rapidly lose viability. Thus, granulocytes need to be collected and transfused on a daily basis. The collection methods significantly improved with the introduction of continuous flow apheresis blood collection devices in late 1960s that made collection of 1x 1010 neutrophils possible. These numbers improved further to 2-3 x 1010 when used in conjunction with stimulation of donors with corticosteroids and addition of Hydroxyethyl starch (HES) during apheresis. Although the earlier controlled trials for granulocyte transfusions in the 1970s and 1980s provided mixed results, overall the therapy was thought be moderately efficacious3 and granulocyte transfusions were rarely utilized between 1985 to 1995 due to some associated adverse events with GTX as well as the widespread use of antimicrobials for infection control. The evidence for the importance of higher granulocyte dose achieving favorable outcomes was seen even in the early uncontrolled trials and retrospective analysis of the early controlled trials4,5. The introduction of G-CSF, which leads to a rapid increase in the peripheral granulocyte counts in the range of 5 to 10 x 1010, renewed interest in this therapy. The granulocytes collected post G-CSF stimulation also appear to be of better quality.
Indications for granulocyte transfusions
- Severe neutropenia: ANC <0.5 x 109/L, due to congenital or acquired bone marrow failure syndromes.
- Receiving active treatment in an attempt to achieve disease remission.
- Proven or highly probable fungal or bacterial infection that is unresponsive to appropriate antimicrobial therapy.
- In whom neutrophil recovery is expected (ANC>0.5x109/l) in the near future and / or in whom definitive therapy of curative potential is planned.
Congenital disorder of neutrophil function:
- These patients receive GTX regardless of the neutrophil count.
- Patients with proven or highly probable fungal or bacterial infection unresponsive to antimicrobial therapy.
GTX should not be issued for therapeutic use in:
- Patients with bone marrow failure where neutrophil recovery is not anticipated to recur spontaneously and no further active treatment is planned.
- Sepsis in the absence of either neutropenia or known neutrophil dysfunction.
- Pyrexia of unknown origin.
The rationale for these criteria includes the difficulty in recruitment of donors, the huge burden on the donor, and other costs and risks associated with GTX.
Neutropenia from chemotherapy and hematopoietic cell transplantation (HCT) is the most common use of GTX6. G-CSF may be used to stimulate patients' bone marrow production of granulocytes, but response to G-CSF in this patient group is usually poor. The use of prophylactic GTX for infection prophylaxis remains controversial and is not currently recommended5.
Donor selection: Both community donors and directed donors can be used. Community donors are taken from an established donor pool and may have been recently tested for markers following a whole blood or platelet donation. Directed donors include family members and friends of the patient. Using only directed donors alone would delay the initiation of GTX7.
Donor qualifications: Granulocyte donors have to meet the minimum standards in donor history and physical examination set by the US Food and Drug Administration and possibly other country, state, and professional regulatory agencies. In addition to being qualified as a whole blood donor, granulocyte donors may have to meet the following requirements:
- Donors must be ABO and Rh matched to the recipient. This is a requirement as granulocyte concentrates are heavily mixed with red blood cells during apheresis.
- Negative for blood transfusion-associated infectious disease markers within 30 days of granulocyte donation.
- No history of allergies to steroids or starch.
- A history of hypertension, diabetes, gastrointestinal ulcers, glaucoma, tuberculosis, or any fungal infections may be a contraindication to steroid administration.
The inconvenience of multiple visits required for granulocyte donation adds to the donor burden and might be a limiting factor for the success of granulocyte transfusions.
Donor Stimulation: With the introduction of G-CSF and glucocorticoid-stimulated granulocyte collection, it is now possible to collect enough granulocytes from a single donor to produce a substantial increment in ANC in a neutropenic patient. There is variation in the dose of G-CSF used for donor stimulation across studies, ranging from 200 to 600 mcg. Use of lower G-CSF doses result in significant cost savings for the blood centers. The standard dose of dexamethasone used is 8 mg. Circulating neutrophil counts in G-CSF- and dexamethasone-stimulated donors are maximal at 12 hours after treatment8, so collections are planned accordingly. Granulocytes are collected with at least a 2-day interval between donations, with a maximum of 2 collections in a 7 day period and the total number of donations that are permissible for normal donors should not exceed 24 times in a rolling 12 month period.
Some of the side effects associated with G-CSF/dexamethasone administration include headache, arthralgia, bone pain, fatigue, and insomnia, generally are mild and self-limited 9-10. The circulating neutrophil counts return to normal a within a few days after the G-CSF administration is discontinued11.
Granulocyte Collection, Processing & Storage
Granulocytes can be harvested from both stimulated and unstimulated donors using apheresis. Citrate is commonly used as an anticoagulant. Hydroxyethyl starch (HES) is added to facilitate separation of granulocytes from RBCs, which improves granulocyte collection. The average granulocyte count per unit for stimulated donors is 6 x 1010 and for unstimulated donors is 1 x 1010.
1 apheresis granulocyte unit:
- Volume -250-350 ml
- RBCs (20-50 mL)
- PLTs (~1.0 x 1011)
- Plasma and anticoagulant.
Granulocyte concentrates should be irradiated to avoid the risk of transfusion-associated graft-versus-host disease especially in immune deficient patients. Approximately 25 Gy is used for irradiation, which renders lymphocytes within the granulocyte product incapable of dividing or attacking host tissues. Granulocytes have a short shelf life and can be stored for 24 hours at room temperature of 22+2ºC without agitation, but the recommendation is to use the granulocyte concentrates as soon as possible after collection.
Premedication is commonly used to minimize transfusion reactions associated with granulocyte transfusions. Granulocytes should be ABO/Rh/crossmatch compatible. When ABO-mismatched granulocytes need to be used in certain circumstances, RBC depletion techniques can be used to remove the unwanted RBCs from the granulocyte product12. Granulocytes are transfused slowly over a 2-4 hour period, using a standard blood administration set and a standard transfusion filter (pore size of 170 microns). Leukocyte reduction filters should never be used. The granulocyte dose for children is 10- 20ml/kg.
Recipient response post GTX is determined by monitoring post-transfusion ANC increments and changes in signs and symptoms of the underlying infection, using imaging studies and microbiological cultures.
Generally, accepted criteria for discontinuing GTX include the following:
- he clinical infection has been resolved based on clinical signs/symptoms, and laboratory/radiological test results.
- The patient's ANC is above 500 for three days without GTX, which is a sign of bone marrow recovery.
- The patient's clinical condition has worsened (i.e. poor response to GTX), and the treatment plan has changed to palliative care with patient and family consent. GTX is generally not considered as a part of palliative care.
Transfusion reaction rates are higher in GTX compared with RBC transfusion, with 25 to 50 percent having mild to moderate reactions13. The most frequently encountered reactions are fever and chills. Some of the other significant transfusion reactions associated with GTX are:
- Pulmonary adverse reactions - Moderate to severe reactions are more commonly associated with GTX .There is no increased risk of pulmonary complications with concomitant administration of Amphotericin B14.
- Transfusion-associated graft-versus-host disease (ta-GVHD) - are seen mostly in immunodeficient patients. Irradiation of the granulocyte concentrates is vital to eliminate this risk.
- HLA alloimmunization - Alloimmunization post GTX reduces in vivo granulocyte survival and can cause abnormal migration of neutrophils. In addition, refractoriness to platelet transfusion increases in patients receiving prophylactic GTX, due to the production of anti-HLA antibodies.
- Transfusion-transmitted infection - The risk of CMV may be greater in granulocyte concentrates, since CMV is harbored in peripheral blood leukocytes. Therefore CMV seronegative donors are selected as donors.
RING STUDY (RESOLVING INFECTION IN NEUTROPENIA WITH GRANULOCYTES)1
- The RING study was a multicenter randomized controlled clinical trial to test the efficacy of GTX from G-CSF/dexamethasone treated donors in neutropenic patients with infection. The criteria for eligibility were neutropenia (ANC<500/uL) due to aggressive chemotherapy or hematopoietic stem cell transplantation and proven/probable/presumed infection.
- Study design: Randomized two-arm, open label, phase III study.
- standard antimicrobial therapy
- standard antimicrobial therapy plus daily GTX from normal donors stimulated with subcutaneous G-CSF (480?g) and oral dexamethasone (8mg)
- Primary Endpoint: Meet two criteria
- Survival for 42 days after randomization
- Clinical response of the study-qualifying infection
- Evaluation of response - by an independent adjudication panel blinded to the subject's treatment arm
- Secondary Objectives:
- Primary outcome within each infection subgroup
- Frequency and nature of granulocyte transfusion reactions,
- Overall incidence of adverse effects
- Mortality through 3 months
- 114 subjects were enrolled and randomized; 58 to the control arm and 56 to the granulocyte transfusion arm. The study was terminated short of the desired enrollment (target sample size of 236) because of time limits on the funding of the TMH Clinical Trials Network.
The primary outcomes of the study were as follows:
- No significant difference was seen in the outcomes between the 2 treatment groups
- Success rates were 42% (20/48) and 43% (21/49) for the granulocyte and control groups, respectively (p> 0.99) on MITT analysis and 49% (17/35) and 41% (16/39), respectively, for subjects who adhered to their assigned treatments (PP analysis)(p=0.64)
- Primary outcome success was 59%, 15%, and 37% for the high dose, low dose, and control groups, respectively
- Subjects who received higher granulocyte dose fared better than those receiving lower dose, both in terms of primary outcome and survival (p<0.01 and p=0.02, respectively)
Limitations of the study
- Low accrual rate - only half the desired enrollment significantly decreased the power of the study to detect difference in treatments.
- The dose of granulocytes actually received by the subjects was not uniform in the study, with more than a quarter of the subjects receiving a mean dose less than the goal.
Conclusion of the Study
- Overall, no benefit of granulocyte transfusion therapy was observed, but the power of the study was reduced due to low accrual.
- Post-hoc secondary analysis suggested that patients receiving higher doses tended to have better outcomes than patients receiving lower doses.
- Granulocyte transfusions can be used in patients with severe neutropenia and infections not responding to routine treatment.
- High dose G-CSF/dexamethasone stimulated GTX give better patient outcomes and should be used.
- Price TH, Boeckh M, Harrison RW, McCullough J, Ness PM, Strauss RG, et al. Efficacy of transfusion with granulocytes from G-CSF/dexamethasone treated donors in neutropenic patients with infection. Blood. 2015;126(18):2153-61. doi: 10.1182/blood-2015-05-645986.
- Dancey JT, Deubelbeiss KA, Harker LA, Finch CA. Neutrophil kinetics in man. J Clin Invest 1976; 58:705.
- Strauss RG. Therapeutic granulocyte transfusions in 1993. Blood 1993; 81: 1675-78.
- Estcourt LJ, Stanworth SJ, Hopewell S, Doree C, Trivella M, Massey E. Granulocyte transfusions for treating infections in patients with neutropenia or neutrophil dysfunction.Cochrane Database of Systematic Reviews 2016;(4):CD005339.
- Estcourt LJ, Stanworth S, Doree C, Blanco P, Hopewell S, Trivella M, Massey E. Granulocyte transfusions for preventing infections in patients with neutropenia or neutrophil dysfunction. Cochrane Database of Systematic Reviews 2015;(6):CD005341.
- Goldfinger D, Qun Lu.Granulocyte transfusions.In: UpToDate, Post TW (Ed), UpToDate, Waltham, MA. (Accessed on March 20, 2017.)
- Hübel K, Carter RA, Liles WC, et al. Granulocyte transfusion therapy for infections in candidates and recipients of HPC transplantation: a comparative analysis of feasibility and outcome for community donors versus related donors. Transfusion 2002; 42:1414.
- Liles WC, Huang JE, Llewellyn C, et al. A comparative trial of granulocyte-colony-stimulating factor and dexamethasone, separately and in combination, for the mobilization of neutrophils in the peripheral blood of normal volunteers. Transfusion 1997; 37:182.
- Stroncek DF, Yau YY, Oblitas J, Leitman SF. Administration of G--CSF plus dexamethasone produces greater granulocyte concentrate yields while causing no more donor toxicity than G--CSF alone. Transfusion 2001; 41:1037.
- Heuft HG, Goudeva L, Sel S, Blasczyk R. Equivalent mobilization and collection of granulocytes for transfusion after administration of glycosylated G-CSF (3 microg/kg) plus dexamethasone versus glycosylated G-CSF (12 microg/kg) alone. Transfusion 2002; 42:928.
- Stroncek DF, Matthews CL, Follmann D, Leitman SF. Kinetics of G-CSF-induced granulocyte mobilization in healthy subjects: effects of route of administration and addition of dexamethasone. Transfusion 2002; 42:597.
- Wuest DL, Reich LM. Removal of ABO-incompatible red cells from lymphocytapheresis and granulocytapheresis components before transfusion. Transfusion 1997; 37:144.
- Petz, LD, Swisher, et al. Clinical Practice of Transfusion Medicine, 3rd ed, Churchill Livingstone, New York 1996. p.423.
- Catalano L, Fontana R, Scarpato N, et al. Combined treatment with amphotericin-B and granulocyte transfusion from G-CSF-stimulated donors in an aplastic patient with invasive aspergillosis undergoing bone marrow transplantation. Haematologica 1997; 82:71.
Shweta Bhavsar, MBBS, MD and Lirong Qu, MD, PhD