Use Of Granulocytes Research Articles

Granulocyte Transfusion Therapy as a Bridge to Hematopoietic Stem Cell Transplant in a Child with Aplastic Anemia and Refractory Perirectal Abscess

Granulocyte Transfusion Therapy as a Bridge to Hematopoietic Stem Cell Transplant in a Child with Aplastic Anemia and Refractory Perirectal Abscess

Successful Use of Granulocyte Transfusion Therapy As a Bridge to Hematopoietic Stem Cell Transplant in a Child with Aplastic Anemia and Refractory Perirectal Abscess

Background: Idiopathic aplastic anemia (IAA) is a rare, life-threatening acquired form of bone marrow failure characterized by multilineage cytopenias and hypocellular bone marrow. Profound neutropenia associated with very severe aplastic anemia (vsAA) is an important risk factor for development of life-threatening fungal and bacterial infections. Perirectal cellulitis and perianal abscess are one such infection and are associated with high rates of morbidity. The ability to eradicate infections in IAA is limited by the lack of imminent neutrophil recovery prior to treatment with either immune suppressive therapy (IST) or hematopoietic stem cell transplant (HSCT). We describe the successful use of granulocyte transfusion to treat antibiotic refractory proctitis with perianal abscess in a profoundly neutropenic child with vsAA. Case: A 7yoF presented with fever and pancytopenia in the setting of Human Metapneumovirus and Parainfluenza. Bone marrow biopsy showed hypocellularity (60-70%) with granulocytic and megakaryocytic hypoplasia, favoring viral suppression. Ultimately, she progressed to transfusion dependence. Repeat bone marrow aspirate and biopsy ultimately revealed bone marrow aplasia, DEB chromosomes, telomere lengths, and PNH clones were negative, confirming the diagnosis of vsAA. She was subsequently admitted for febrile neutropenia (F&N) and was found to have mild respiratory COVID-19, but began to complain of rectal pain that was initially attributed to her history of constipation. She defervesced on IV antibiotics (abx) and was discharged home with negative cultures. Within a week she represented with hematochezia and F&N and was found to have proctitis with perianal fistula on MRI (figure 1); GI PCR panel was + Enteroaggregative E. coli, no resistant organisms were identified. She clinically improved and discharged on IV piperacillin-tazobactam (pip-tazo). Despite 2 weeks of IV abx, she developed new fever and imaging confirmed persistent proctitis with peri-rectal abscess. As she had limited response to abx alone we trialed a short course of granulocytes with improvement in anorectal inflammation by CT. She was discharged with a plan to continue IV pip-tazo until achieving radiographic cure; however, interval imaging after an additional 2 weeks of abx showed rebound of her proctitis and she was again admitted with F&N and rising inflammatory markers. Her failure to eradicate her peri-rectal infection despite prolonged antibiotics (>4weeks), along with her previous response to a short course of granulocyte transfusion prompted us to prescribe a prolonged 10-day course of granulocytes with continued antibiosis. Her post-granulocyte CT showed resolution of her perirectal abscess and proctitis (figure 2). With resolution of her infection, she was able to proceed to HSCT which she completed without recurrence of her anorectal infection. Discussion: Conventional granulocyte transfusions are considered when patients have severe neutropenia (<500/uL), bacterial or fungal infection refractory to antimicrobials, and reasonable expectation of neutrophil recovery, but evidence for efficacy is limited. In our case, there was no expectation of imminent bone marrow recovery and our patient's infection limited her ability to proceed to IST or HSCT for treatment of her vsAA. Thus, in conjunction with infectious disease and transfusion medicine, and given her known response to granulocytes, we successfully re-attempted a prolonged, but limited, 10-day course of granulocyte transfusions and were able to eradicate a case of antibiotic-refractory proctitis with peri-anal fistula. We suggest an expanded application for the use of granulocytes in profoundly neutropenic patients without imminent recovery for severe, anti-microbial refractory infection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Use of Granulocyte Transfusions in Two National Cohorts and Association between Transfusion Dose and Patient Outcomes: The Best Collaborative Study

Granulocytes for transfusion (GTX) continue to be administered, mostly to treat refractory infection 1. Single center series continue to be reported, but evidence of GTX effectiveness remains uncertain. Recent attempts at randomized trials of GTX have not been completed to target although the largest trial, RING 2, identified potentially promising results in a sub-group analysis by dose.GTX can be produced by apheresis (from ‘stimulated’ donors), aGTX, or as a component pooled from whole blood (WB) donations. Logistic difficulties arise with aGTX, including recruitment of suitable donors willing to undergo cell mobilization and collection. In England GTX are now only supplied as pooled (10 WB donations/pool, mean volume single pooled unit 207 ml, mean granulocyte yield/unit 0.9 x10 10, typical adult dose 2 pools, available 6 days per week). In France, production moved from aGTX to pooled in 2020 (20 WB donations/pool, mean volume single pooled component 428 ml, mean granulocyte yield/unit 1.8 x10 10, typical adult dose 1 pool, available 6 days per week). There have been no recent published national data on the clinical use of pooled granulocytes, or comparisons between large datasets.The aim of this study was to 1) describe the use of GTX, including pooled granulocytes, in two national cohorts of recipients; 2) estimate the effect of dose on patient mortality using statistical methods which could be applied to a larger dataset in the future.A pre-piloted data collection form (DCF) was used to collect prospective audit data on all GTX given to patients in participating hospitals in England from March 2017 -Sept 2020 in England. The PROspective Granulocyte usage and outcomEs Survey (ProGrES) was deemed a national registry/audit with no individual patient consent required for anonymized data. Information on patient characteristics and outcomes was collected at the time of the request for GTX, following completion of GTX and at 28 days and 6 months follow up. DCFs were adapted for international use, and French data were collected from Jan 2018 to Dec 2020.Descriptive analyses were performed to summarize GTX dose, patient characteristics, and outcomes. Using the English data, we investigated the association between GTX dose and mortality within 28 days of final GTX using logistic regression models with and without adjustment for possible confounders (age, infection source, use of renal therapy) and using the target trial framework. Ethical approval was obtained from London School of Hygiene and Tropical Medicine for analysis of English data.Results are presented on 224 (England, pooled) and 139 (France) patients who received GTX. In France, 95 and 44 patients received aGTX and pooled GTX, respectively. Table 1 shows patient features and outcomes. The most frequent underlying diagnosis was acute myeloid leukemia in all groups, and the most common indication was treatment of refractory infection. Median number of transfusions was similar in all groups (5.0, 5.3, 6.0). The mean dose/kg/transfusion was highest in the aGTX group (0.55 x10 9/kg), followed by French pooled (0.40) and English pooled (0.2). Death within 28 days of last GTX varied from 20.2% (French apheresis) to 32.5% (French pooled).Additional analyses were performed on the English data; for death within 28 days, a 0.1 x10 9 increase in GTX dose was associated with a 22% reduction (95% CI -45% to +0%) in odds of death within 28 days of referral; odds ratio 0.78 (95% CI 0.57 to 1.00). After adjustment for potential confounders, the odds ratio was attenuated to 0.92 (95% CI 0.67, 1.23).In summary, GTX continue to be requested by clinicians in patients with poor outcomes, despite an uncertain evidence-base. Regression analysis found dose was associated with lower odds of 28-day mortality. This was not significant after confounder adjustment, but this proof-of-principle analysis was limited by the sample size. A potential dose effect was also seen in the RING study. Our analysis using a target trial approach provides an approach for estimating GTX effectiveness in a larger cohort, which is required given the well-recognized challenges of undertaking randomized trials. The analysis provides a range of sample size calculations for testing an association between dose and outcomes for a defined effect size in a larger international cohort, supported by the BEST Collaborative 3.

Use of granulocyte transfusions among haematology units in England and North Wales.

To establish the current use of granulocyte transfusions in haematology patients and explore interest in further research. Granulocytes may be used for the treatment of severe infection in neutropenic patients or for primary or secondary prophylaxis. Clinical utility of granulocyte transfusions is unclear, and recent studies have demonstrated equivocal outcomes. Pooled granulocytes are the main granulocyte product used in England and Wales, but there are no data on the patterns of use and little consensus on accepted indications. A survey was distributed to UK hospitals delivering intensive chemotherapy. Clinical scenarios were posed, with further questions on clinician experience of using granulocytes, availability of the product, barriers to use and interest in further research. The response rate was 57%; 34·9% of all responses were from allogeneic stem cell transplant centres. Paediatric centres comprised 9·5% respondents, and 19% centres had access to apheresis granulocytes. Of respondents, 58·7% had used granulocytes in the last 3 years, 89·2% of whom used granulocytes to treat refractory infection. There was little consensus on use of granulocytes in the given clinical scenarios even when patients clearly met national guideline criteria. Paediatric centres were overall more likely to recommend granulocyte use. The most frequently identified barrier to use of granulocytes was lack of evidence of effect. Of the respondents, 75% indicated a willingness to participate in further research. There remains a lack of consistency about use of granulocytes, which is unsurprising given the lack of clinical data to support their efficacy. We did, however, demonstrate a willingness to participate in further research.

Changing Trends in the Use of Granulocyte Transfusions in Neutropenic Children with Sepsis in India.

We present our data on granulocyte transfusions in children undergoing treatment for cancer and HSCT at our centre and their changing indications. In this retrospective observational analysis of children who received granulocytes from 2007 to 2015, we divided children receiving granulocytes into two groups-the first from January 2007 till December 2013 and the second from January 2014 till December 2015. This division is based on the change in our policy to use granulocytes within 48h of septicemia as the incidence of drug resistant bacterial strains had increased at our centre. Data on 72 children with febrile neutropenia treated with 230 granulocyte infusions was analyzed. From 2007 to 2013 (n=48/72), 27/48 (56%) had culture proven sepsis of which 14 (51%) were carbapenem resistant gram negative bacilli. 11 of the 27 children survived the crisis (41%). We then changed our policy to transfuse granulocytes early during sepsis. From 2014 to 2015 (n=24/72) 22 patients had culture proven sepsis (91%) of which 20 had carbapenem resistant gram negative bacilli. 12/22 (54%) with culture proven sepsis survived the episode. The survival rate improved from 41% in first group to 54% after early intervention with granulocytes (P value 0.0347). Despite the increased incidence of resistant bacteria during the period of 2014-2015, early use of granulocytes improved survival rate from 41 to 54%. This intervention cannot be taken in isolation and needs to be offered early in parallel with appropriate antibiotics.

Granulocyte Transfusions for Children with Infection and Neutropenia or Granulocyte Dysfunction

Transfusions of granulocytes can be used as an adjunct therapy to antimicrobials in patients with infection and neutropenia or granulocyte dysfunction. However, there is a lack of strong clinical evidence to support the use of this treatment strategy, particularly in children. We retrospectively reviewed the medical records of children who received granulocytes at our institution from April 2009 to October 2012, with emphasis on primary indication for the transfusion and clinical outcome in terms of infection. The patients had granulocyte dysfunction or severe neutropenia, defined as absolute neutrophil count (ANC) < 500 cells/mm3 due to chemotherapy or hematopoietic stem cell transplant (HSCT), and reasonable hope for bone marrow recovery or engraftment. Eighteen children received granulocytes during 20 distinct episodes: 62% (n = 13) for acute infection, 29% (n = 5) for unresolved chronic infection during the time of HSCT, and 9% (n = 2) for other clinical conditions such as typhilitis and appendectomy. Overall, 92% (n = 12) of the episodes of acute infection had complete or partial resolution, as determined by review of vital signs, physical exam findings and discontinuation of antimicrobials. A substantial number (46%) of children who received granulocytes for acute infection developed respiratory adverse events, but all of these recovered. We conclude that granulocyte transfusions continue to be primarily used in neutropenic patients with acute infections, and that its use in this group of patients is reasonable. However, a prospective randomized clinical trial is needed to evaluate safety and whether the use of granulocytes is superior to antimicrobial-only therapy.

Use of granulocytes concentrates from a single high yield apheresis to support more than one patient.

6622 Background: Allogeneic granulocyte transfusion therapy is sometimes the only therapeutic option in immunocompromised neutropenic leukemic/HSCT patients with life threatening bacterial and fungal infections. Collection of larger cell doses of granulocyte concentrates (GC) by automated apheresis following G-CSF/steroids administration has renewed interest and its use has grown steadily. Methods: Donors were recruited from family/friends of the patients and informed consent was obtained. GCs were collected via donor stimulation with a single subcutaneous dose of G-CSF (600 mcg). First time donors received an oral dose of dexamethasone (8 mg). GCs were harvested by Hetastarch-assisted leukapheresis 12 hours later via peripheral venous access with the continuous flow cell separator Spectra (COBE Caridian BCT, Lakewood, CO). Results: GCs were collected from 93 donors (67M: 26F; age median 40 (19-73 years) and 1 ½ x blood volume processed median 7048 (3212-8085 mL). The pre/post wbc were median 8.1 (4.1-22.8)/ 42.7 (22.4-77.5). The volume collected was based on the post G-CSF wbc count (&gt; 35 K/UL - 800 mL; &lt;35 K/UL between 600-700 mL). The original yield (10 x10e) was median 9.7 (5.1-17.2 units), volume median 718 (538-860 mL) and WBC median 132.7 (84.2 -241.6 K/UL per bag). The Split GCs were median 4.48 (2.70-7.72 units), with neutrophils median 87% (54-97% per bag). GCs were transfused to 51 patients (31M: 20F; age median 58 [19-83 years]) who received median 3.5 (1-18 split units), containing wbcs median 10734.69 [5778-42 -162698.09 (x10^3/uL)/unit) and achieved a WBC increment median 0.2 (0.0-8.3K/UL). Conclusions: The GCs were split due to the primary care team's reluctance to transfuse the entire volume of the product due to volume restrictions or for some other medical reason. The same rationale to split platelets from high yield single apheresis was used to split our GC products. We found that GCs from each donor was utilized maximally and also brought partial relief to other patients who lacked donors. We would like to make the case that when adequate numbers of GCs are collected it may be feasible to split the unit. This may offer a glimmer of hope to other patients in need of GCs who lack a donor network support.

Safety Evaluation for a Cell‐based Immune Support System in an Ex Vivo Rat Model of Gram‐negative Sepsis

Granulocyte dysfunction is a central component of immunodeficiency in septic patients. Granulocyte transfusions appear to be pathophysiologically useful; however, they cause unwanted side-effects in the lungs and other organs. This study evaluates the safety of an extracorporeal immune support system with granulocytic cells in a rat model of Gram-negative sepsis. Three groups of male CD rats received either saline (control group, I), a dose of Escherichia coli O7:K1 lethal to 90% of the animals (LD90) (septic group, II), or an LD90 dose of E. coli that was incubated with the human promyelocytic leukemia cell line (HL-60) (differentiated into the granulocytic direction) for 20 min prior to infusion (second septic group, III). The animals were observed for seven days. Pre-treatment with HL-60 cells resulted in no adverse effects in the group III animals. Significantly lower bacterial counts and endotoxin levels in the plasma were detected after 24 h as compared to group II (P < 0.05). Group III animals had better weight gain and more stable hemodynamics than group II animals (P < 0.01). Seven day survival was 0/8 in group II, 6/8 in group III, and 8/9 in group I (log-rank test: II-III: P < 0.001). The data suggest that extracorporeal use of granulocytes allows the therapeutic use of these cells while avoiding unwanted effects resulting from direct contact to internal organs.

T-lymphocyte function from peripheral blood stem-cell donors is inhibited by activated granulocytes.

PBSC transplant provides 10 times more T cells than BMT However, the incidence and severity of acute GvHD is similar among recipients of both types of transplants. Studies in mouse models suggest that the similar clinical outcome in BMT and PBSCT is due to differences in the lymphokine profiles. PBMC, PBMC from G-CSF mobilized donors (G-PBMC)and BM mononuclear cells (BM-MC) were analyzed by flow cytometry and ELISA to detect gamma-IFN and IL-4 production. Hematoxylin and eosin staining was used to identify morphology and annexin/propidium-iodide was used for apoptosis assays. We show decreased production of gamma-interferon (85%) and IL-4 (60%) in G-PBMC when compared with either PBMC or BM-MCT cells on ex vivo assays. Surprisingly, 85% of fresh G-PBMC is composed of low-density granulocytes (LDG), which undergo apoptosis after 48 h in culture. At this same time, gamma-IFN production from G-PBMC T cell was reverted. In vitro, G-CSF converts granulocytes into LDGs, able to inhibit T-cell function by H2O2 production, and not through immune-deviation towards a Th2-type phenotype. We show that the estimated numbers of Th1 and Th2 cells infused in BMT and PBSCT do not differ significantly. These findings are discussed with reference to the relatively low incidence of acute GvHD in PBSCT shown in the literature. We suggest that these results might depend on the high number of granulocytes and progenitors infused. The potential use of granulocytes as immunosupressive short-term therapy is now being investigated by our group using a mouse experimental model.

Analysis of granulocyte-reactive antibodies using an immunoassay based upon monoclonal-antibody-specific immobilization of granulocyte antigens.

To detect human granulocyte-reactive antibodies, a glycoprotein-specific enzyme immunoassay for platelet antibodies was adapted for the use of granulocytes as target cells. Peripheral blood granulocytes were simultaneously incubated with a monoclonal antibody (mAb) and the serum to be investigated. After solubilization, aliquots of the cell lysate were transferred to plastic tubes coated with goat anti-mouse antibodies. Following immobilization of the trimolecular (mAb-glycoprotein-human antibody) complex it was detected by addition of enzyme-labelled goat anti-human antibodies using a luminescence technique. This assay allowed identification of different granulocyte-reactive antibodies present in the same sample without the need for complicated absorption studies. Alloantibodies against HLA and the granulocyte-specific NA antigens as well as isoantibodies against the Fc-gamma-receptor III (FcRIII) were detectable using mAb-specific immobilization of granulocyte antigens (MAIGA). Binding of autoantibodies to the FcRIII and to the CD 11b/CD18 complex could be shown.

Comparison of in-111 Granulocytes and Tc-99m Albumin Colloid for Bone Marrow Scintigraphy by the Use of Quantitative SPECT Imaging

A comparison of In-111 oxinate labeled autologous granulocytes and Tc-99m albumin colloid for bone marrow scintigraphy is reported. The aim of this report was to determine if the intense uptake in the liver and spleen with nano-sized colloids, which hampers the evaluation of the skeletal parts surrounding the liver, is reduced by the use of radiolabeled granulocytes. This study is based on a retrospective analysis of 19 abdominal tomographic examinations with In-111 granulocytes performed to detect septic foci. After correction for attenuation and scattering of photons was performed, the uptake in the bone marrow of the lumbar spine was seen to be related to the liver, spleen, and tissue background activity. The results in this study were compared with corresponding data from 20 normal liver/spleen tomographic examinations performed with Tc-99m nanocolloid, which is routinely used for bone marrow scintigraphy. The bone marrow/liver activity ratio for granulocytes varied, but it exceeded the corresponding mean ratio for colloid in all examinations. The mean values for granulocyte uptake 3 and 20 hours after injection was, respectively, about 6 and almost 10 times higher than were those for colloid. The activity ratios between bone marrow and spleen as well as between bone marrow and tissue background was not improved and or may even have been reduced by the use of granulocytes. It is suggested that granulocytes labeled in vitro by Tc-99m hexamethylpropylene amineoxime (HMPAO) or in vivo by monoclonal antigranulocyte antibodies may provide techniques for improved bone marrow imaging.

Prophylaxis and Treatment of Infection of the Immunocompromised Host by Granulocyte Transfusions

Granulocyte transfusions are expensive and involve healthy donors in a procedure which is time-consuming, uncomfortable and not without risk. It is therefore important to consider carefully the circumstances which justify their use. The indiscriminate transfusion of inadequate quantities of granulocytes from random donors into sensitized recipients should be discouraged. Severely neutropenic patients with bacterial infection persisting in the face of maximum antibiotic therapy are clearly appropriate recipients of granulocyte transfusion therapy. Well-designed programmes of prophylactic granulocyte transfusion can reduce the acquisition of bacterial infection in granulocytopenic patients. However, there are very few clinical situations in which their use is justified. A wider use of granulocytes must await better techniques of granulocyte procurement and donor selection.

Granulocyte Transfusion—A Review

Advances in component transfusion therapy over the last two decades have improved the management of hematologic diseases. In patients undergoing chemotherapy for leukemia, hemorrhagic problems have been minimized with platelet transfusions. However, infectious complications due to severe granulocytopenia have surfaced as the leading cause of death in these patients. This finding stimulated vigorous research in the use of granulocytes for transfusion, resulting in progress in granulocyte collection, short-term liquid storage and clinical use. The effectiveness of granulocyte concentrates in the management of infections in severely neutropenic patients (fewer than 500 neutrophils per μl]) with documented bacterial sepsis has been amply demonstrated. Serious untoward reactions to granulocyte transfusions have been relatively rare. Some areas of granulocyte transfusion that require further investigation are 1) more precise definition of criteria for the selection of patients most likely to benefit; 2) improvements in efficiency of collection and preservation; and 3) better understanding of immunologic factors affecting efficacy of therapy.

Hematology: An Annotated Bibliography of Recent Literature

Hematology: An Annotated Bibliography of Recent Literature