Transient Neutropenia Research Articles

Granulocyte collections: comparison of two apheresis systems

Donor granulocyte concentrates are routinely administered to patients with granulocyte function defects or transient neutropenia and (risk of) bacterial or fungal exacerbations, despite lack of definitive clinical proof for patient-relevant outcome improvement. Granulocytes are collected by apheresis from healthy donors treated with granulocyte-colony-stimulating factor and/or steroids for neutrophil mobilization the evening before apheresis, as well as with hydroxyethyl starch during apheresis, to enhance sedimentation of red blood cells (RBCs) and thus to facilitate accessibility of neutrophils for collection. Granulocyte apheresis procedures are performed with standard apheresis equipment, including with the frequently used apheresis system for peripheral blood "stem cell" collection, COBE Spectra MNC (Terumo BCT), using the same tubing set as for MNC collection, but a different software protocol, PMN. An automated apheresis system for granulocyte collection, Spectra Optia IDL (Terumo BCT), became available in October 2011. Since then, 70 granulocyte apheresis procedures have been performed at our site, 35 each with the new and old systems. Apheresis procedures were well tolerated throughout. The target dose of 1 × 10(10) neutrophils was achieved in all but one collection with Spectra Optia IDL. Spectra Optia IDL collections were approximately 20% more efficient. Products contained more nontarget white blood cells (mononuclear cells), but fewer RBCs and platelets. Although less blood had to be processed with Spectra Optia IDL to achieve the same granulocyte dose, clinically relevant differences between the two apheresis devices were not apparent. Both apheresis systems are similarly capable of generating granulocyte concentrates.

Transient neutropenia in children with febrile illness and associated infectious agents: 2 years’ follow-up

The aim of the study was to identify the relationship of acquired neutropenia with childhood infections and to assess its clinical course, complications, and outcome. Children admitted to two pediatric wards over a 4-year period with febrile neutropenia were prospectively investigated for underlying infections with inflammatory markers, cultures of body fluids, and serological tests. The study included 161 previously healthy children with febrile neutropenia/leukopenia aged (mean ± SD) 3.02 ± 3.86 years (range, 0.1-14). One hundred and thirty-six out of 161 patients (84.5 %) had transient neutropenia (TN), while in 25 patients, neutropenia was chronic (CN) and persisted for ≥180 days. An infectious agent was isolated in 98/161 (60.9 %) cases, in 68.4 % patients with TN, and in 20 % of those with CN (p = 0.001). Among the patients with CN, seven had positive antineutrophil antibodies (autoimmune neutropenia) and four were eventually diagnosed with hematological malignancy. In all age groups, TN was of short duration (<1 month), of mild to moderate severity, and was predominantly associated with viral infections. Two years after diagnosis, 143/161 children (88.8 %) were available for follow-up. One hundred and thirty-seven of 143 (95.8 %) had recovered completely, while the rest remained neutropenic. The latter patients had a benign course despite severe neutropenia. In conclusion, febrile neutropenia during childhood is usually transient, often following viral and common bacterial infections, without serious complications and in the majority of cases it resolves spontaneously. However, in a considerable percentage of patients, neutropenia is discovered incidentally during the course of an infection on the ground of an underlying hematological disease.

Trabectedin as single agent in relapsed advanced ovarian cancer: results from a retrospective pooled analysis of three phase II trials

Three phase II studies evaluated trabectedin monotherapy as second-/third-line therapy in patients with refractory/recurrent ovarian cancer (ROC). Three different schedules were investigated: 3-h infusion every 3 weeks (3-h_q3w), 24-h infusion q3w (24-h_q3w), and 3-h weekly infusion for 3 weeks of a 4-week cycle. This retrospective pooled analysis evaluated the efficacy and the safety profile of trabectedin according to each administered regimen. Data from 295 patients were used to compare weekly versus q3w schedules, and 3-h versus 24-h infusion given q3w. Both q3w regimens showed higher overall response rate (36 vs. 16 %; p = 0.0001), disease control rate (66 vs. 46 %; p = 0.0007), and longer median progression-free survival (5.6 vs. 2.8 months; p < 0.0001) than the weekly schedule. Comparable activity was observed for the 3- and 24-h infusions q3w. Common adverse events were nausea, fatigue, vomiting, transient neutropenia, and transaminase increases. A better safety profile regarding neutropenia, fatigue, and vomiting was seen for the 3-h_q3w regimen as compared to the 24-h_q3w one. Trabectedin given as a single agent q3w as 3-h infusion is the schedule of choice for the treatment of ROC, and its efficacy and safety profile favorably compares with other active salvage treatments.

Incidence of Late Chronic Anemia in Newly Diagnosed Patients with Chronic Myelogenous Leukemia Responsive to Imatinib

Abstract 3769Anemia has been reported in about 5–8% of newly diagnosed patients with Chronic Myelogenous Leukemia (CML) treated with imatinib in the first 6 months of therapy, and has been generally regarded as transient like neutropenia and thrombocytopenia: in patients responsive to imatinib, however, it is still unclear if the prolonged treatment could induce the appearance of a late chronic anemia. To highlight this issue, we revised 104 CML patients (M/F 54/50, median age at diagnosis 58.0 years, interquartile range 43.3 – 70.2) treated at our Institution with 1st line imatinib therapy for at least 36 months and in stable complete cytogenetic response (CCyR). At the 36th month of imatinib treatment, a late chronic anemia (defined as Hb levels < 11 g/dl for > 6 months) was present in 16/104 patients (15.4%): the anemia was severe (Hb < 10 g/dl) in 10 patients (9.6%) and moderate/mild (Hb ≥ 10 < 11 g/dl) in 6 patients (5.8%). Clinical features at diagnosis of patients who had late chronic anemia at the 36th month compared to the remaining 88 patients without late anemia are shown in the Table:Hb < 11 g/dl at the 36th monthHb ≥ 11 g/dl at the 36th monthpM/F6/1048/400.209Median age (yrs) (IR)68.0 (58.9 – 75.4)56.6 (43.1 – 69.1)0.016Sokal Score: Low3440.002Int839High55Median Hb (g/dl) (IR)11.6 (10.4 – 13.3)12.7 (11.2 – 13.8)0.098Median WBC (× 109/l) (IR)79.0 (33.2 – 136.6)60.5 (31.8 – 112.4)0.423Median PLTS (× 109/l) (IR)624 (299 – 835)412 (296 – 557)0.122All patients with late chronic anemia had a low reticulocyte count and 7/16 a condition of iron deficiency with reduced serum ferritin but no clinical and instrumental sign of chronic blood loss: in this latter group, oral iron supplementation was always ineffective. Four out 16 patients (25%) needed 1 or more red cell transfusions during the follow-up. At landmark analysis from the 36th month of imatinib treatment, there was no significant difference in the cumulative 4-year overall survival for patients with and for those without late chronic anemia (80%, 95% CI 44.8 – 100%, vs 94%, 95%CI 85.8 – 100%, respectively; p=0.068). In conclusion, the occurrence of a late chronic anemia during long-lasting treatment with imatinib has been observed in > 15% of our responsive patients and was severe in about 10% of cases: its occurrence seems more common in elderly patients with higher Sokal score at diagnosis. Late chronic anemia does not seem to affect OS, but the real impact should be evaluated on a large cohort of patients. Disclosures:Tafuri:Sigma Tau Pharmaceuticals: Research Funding.

Pharmacokinetic Exploration of Potential for Bendamustine to Affect Rituximab Concentrations and of Cytochrome P450 1A2 Inhibitors to Affect Bendamustine Concentrations in Patients with Non-Hodgkin and Mantle Cell Lymphoma

AbstractAbstract 4919 BackgroundBendamustine, a unique alkylating agent that leads to cancer cell death via several pathways, and rituximab, an anti-CD20 monoclonal antibody, are effective as combination therapy for non-Hodgkin's lymphoma (NHL) and mantle cell lymphoma (MCL). Because bendamustine and rituximab are molecularly unique, drug-drug interactions may be unlikely. Bendamustine is primarily metabolized via hydrolysis, also reducing the likelihood of drug-drug interactions. A recently reported analysis confirmed that rituximab does not affect systemic exposure to bendamustine, but the potential impact of bendamustine on rituximab has not been reported. Moreover, because bendamustine is a CYP1A2 substrate, the potential may exist for drug-drug interactions with CYP1A2 inhibitors and inducers. This analysis graphically explored the potential for drug-drug interactions by examining (1) the effect of bendamustine on rituximab pharmacokinetics (PK) and (2) bendamustine metabolism in the presence of cytochrome P450 (CYP) 1A2 inhibitors. MethodsData from a phase 3 study of combination bendamustine-rituximab in patients with advanced indolent NHL or MCL were assessed to evaluate the potential of bendamustine to affect systemic rituximab exposure. Rituximab 375 mg/m2 was administered on day 1 followed by bendamustine 90 mg/m2 30-min infusion on days 1 and 2 of 6 to 8 consecutive 28-day cycles. Serum rituximab concentrations at end of rituximab infusion (ie, rituximab monotherapy), 24 hours and 7 days postinfusion (ie, after bendamustine exposure) were measured. Data were compared with published literature on PK of rituximab administered without bendamustine (data on methods used in the published reports were limited). To evaluate effect of CYP1A2 inhibitors on bendamustine PK, graphical displays of observed bendamustine concentrations versus time since previous dose stratified by presence or absence of CYP1A2 inhibitors were generated. ResultsThe full analysis population included 49 patients (median age, 63. 1 years; range 37–84). To analyze the effect of bendamustine on rituximab PK, 77 rituximab PK sample records from a subset of 19 patients were available. Minimum, median, and maximum observed serum rituximab concentrations in the absence (at end of rituximab infusion) and presence (at 24 hours and 7 days after rituximab infusion) of bendamustine are shown in Figure 1. The slope of rituximab concentrations from end of infusion to 24 hours and 7 days was similar to that seen in rituximab studies without bendamustine. In this study, rituximab concentrations with and without bendamustine were consistently lower than those in the literature (24% at end of infusion, 30% at 24 hours, and 53% at 7 days).To analyze the effect of CYP1A2 inhibitors on bendamustine PK, 141 samples from 29/49 (59%) patients were taken in the presence of CYP1A2 inhibitors, and 102 samples from the remaining 20/49 patients were CYP1A2 inhibitor-free. Bendamustine concentrations were similar in observed time plots stratified by presence or absence of CYP1A2 inhibitors (Figure 2).Serious adverse events considered related to bendamustine plus rituximab were neutropenia, transient acantholytic dermatosis, bacteremia, pyrexia, and febrile neutropenia, which is consistent with the serious adverse event profile for this combination in larger studies. ConclusionsThis graphical exploration suggests that no drug-drug interactions were demonstrated between bendamustine and rituximab or CYP1A2 inhibitors. Just as rituximab does not affect bendamustine PK, bendamustine did not appear to affect rituximab PK over time. Differences in observed rituximab concentration could be due to potential differences in assay methods/sensitivity or the length of rituximab infusion. Analysis of graphical displays of bendamustine concentrations in the presence and absence of CYP1A2 inhibitors suggests that CYP1A2 inhibitors do not increase systemic exposure to bendamustine, a result that is consistent with the near instantaneous hydrolytic metabolism of bendamustine. These results suggest that CYP1A2 is not a substantial metabolic pathway for bendamustine; however, whether CYP1A2 inducers have clinically relevant drug-drug interactions was not analyzed in this study. Based on results from this study, the likelihood of drug-drug interactions with bendamustine appears to be low.Support: Teva Pharmaceutical Industries Ltd. [Display omitted] [Display omitted] Disclosures:Darwish:Teva Pharmaceuticals: Employment. Bond:Teva Pharmaceuticals: Employment. Burke:Spectrum Pharmaceuticals: Consultancy. Hellriegel:Teva Pharmaceutical Industries Ltd: Employment. Robertson:Teva Pharmaceutical Industries Ltd: Employment. Phillips:Cognigen Corporation, which received research funding from Teva Pharmaceuticals: Employment, Research Funding. Grasela:Cognigen Corporation, which received research funding from Teva Pharmaceuticals: Employment, Research Funding.

Ecthyma Gangrenosum in a Previously Healthy Infant

Ecthyma Gangrenosum in a Previously Healthy Infant

Cytarabine and clofarabine after high‐dose cytarabine in relapsed or refractory AML patients

Clofarabine has been shown to be effective in AML patients, either as single agent or, mainly, in association with intermediate dose cytarabine. Based on these reports, we conducted a preliminary study combining clofarabine and intermediate dose cytarabine in AML patients who relapsed or failed to respond to at least two induction therapies. We treated 47 patients affected by relapsed/refractory AML with a regimen including clofarabine at 22.5 mg/m(2) daily on days 1-5, followed after 3 hr by cytarabine at 1 g/m(2) daily on days 1-5. Ten patients received a further consolidation cycle with clofarabine at 22.5 mg/m(2) and cytarabine at 1 g/m(2) day 1-4. Among the 47 patients, 24/47 (51%) achieved a complete remission, 5/47 (10.5%) a partial response, 10/47 (21%) had a resistant disease, and 6/47 (13%) died of complications during the aplastic phase. The most frequent nonhematologic adverse events were vomiting, diarrhea, transient liver toxicity, febrile neutropenia, and infections microbiologically documented. Among the 24 patients who obtained a CR 13 underwent allogeneic bone marrow transplantation. In 14 patients, complete remission duration was shorter than 12 months, whereas 10 patients experienced longer complete remission duration. These very preliminary results suggest that clofarabine-cytarabine regimen is effective in this particularly poor prognosis category of patients, representing a potential "bridge" toward bone marrow transplant procedures. Safety data were consistent with previously reported salvage therapies. Further studies and a longer follow up are warranted.

Infectious Etiologies of Transient Neutropenia in Previously Healthy Children

Healthy children presenting with neutropenia are often hospitalized and treated empirically with antibiotics without an evidence of infection. The objective of this study was to investigate the infectious causes of isolated transient neutropenia in otherwise previously healthy children. A 2-year prospective study was conducted at a tertiary hospital in Kuwait. All previously healthy children (aged 1 month to 12 years) hospitalized with isolated neutropenia defined as absolute neutrophil count (ANC) ≤ 1.5 × 10/L were enrolled in the study. Investigations to identify the infectious causes included blood and urine culture for bacteria whereas for viruses, serology for Epstein-Barr virus, cytomegalovirus, adenovirus, parvovirus and polymerase chain reaction for human herpes virus 6 and enterovirus were performed. Fifty-five children were enrolled during the study. Children less than 2 years of age constituted 73% of the sample. There were 2 peaks of presentation: March-May (33%) and September-November (38%). Associated features were congested throat (56%), runny nose (53%) and cervical lymphadenopathy (20%). The median ANC on admission was 0.6 × 10/L. Associated infections were documented in 55% of enrolled children and were as follows: human herpesvirus 6, 30%; enterovirus, 23%; influenza A H1N1, 13%; parvovirus, 10%; Epstein-Barr virus, 10%; urinary tract infection, (Eshcherichia coli) 7%; and adenovirus, 7%. No serious bacterial infection was identified, and the mean time for recovery of the ANC was 16.7 ± 15 days. Neutropenia in previously healthy children in Kuwait is caused by demonstrable infections in 55% of cases. Majority of children will recover their ANC completely within 1 month without significant infectious complications.

Multicenter phase II study of trabectedin in patients with metastatic castration-resistant prostate cancer

This multicenter phase II trial evaluated the efficacy and safety of trabectedin in metastatic castration-resistant prostate cancer (CRPC). Two schedules were evaluated in three cohorts: weekly as 3-h i.v. infusion at 0.58 mg/m(2) for 3 out of 4 weeks (Cohort A, n = 33), and every 3 weeks (q3wk) as 24-h infusion at 1.5 mg/m(2) (Cohort B1, n = 5) and 1.2 mg/m(2) (Cohort B2, n = 20). The primary end point was prostate-specific antigen (PSA) response; secondary end points included safety, tolerability and time to progression (TTP). Trabectedin resulted in PSA declines ≥ 50% in 12.5% (Cohort A) and 10.5% (Cohort B2) of patients. Among men pretreated with taxane-based chemotherapy, PSA response was 13.6% (Cohort A) and 15.4% (Cohort B2). PSA responses lasted 4.1-8.6 months, and median TTP was 1.5 months (Cohort A) and 1.9 months (Cohort B2). The dose of 1.5 mg/m(2) (approved for soft tissue sarcoma) given as 24-h infusion q3wk was not tolerable in these patients. At 1.2 mg/m(2) q3wk and 0.58 mg/m(2) weekly, the most common adverse events were nausea, fatigue and transient neutropenia and transaminase increase. Two different trabectedin schedules showed modest activity in metastatic CRPC. Further studies may require identification of predictive factors of response in prostate cancer.

The effect of combined therapy with deferoxamine and deferiprone on serum ferritin level of beta-thalassemic patients

ObjectiveTo determine the effect of combined therapy with deferoxamine and deferiprone on serum ferritin level of beta-thalassemic patients.Method and materialsThis controlled clinical trial was conducted on 26 major beta-thalassemic patients. Twelve patients in case group received deferiprone 75 mg/kg/day three times a day (TDS) orally with deferoxamine 30–50 mg/kg subcutaneously every other day. Fourteen patients in control group received deferoxamine 30–50 mg/kg/day subcutaneously for 6–12 hours/day and 5–6 days per week. Serum ferritin level was measured at the beginning and at the end of the third and the sixth months of study. Side effects of combined therapy were assessed. Data were analyzed by SPSS software v: 16.0 and t-Student and compared t-tests.ResultsThe mean of serum ferritin level in case group significantly decreased from 7539.8 ± 3434.9 µg/l at the beginning of study to 4848.7 ± 2706.2 µg/l (P < 0.001) and to 4338.3 ± 2308.8 µg/l (P < 0.001) at the end of the third and the sixth months of study, respectively. The mean of serum ferritin level in control group insignificantly increased from 5668 ± 3613.8 to 6210.8 ± 3940.9 µg/l and to 5742 ± 3205.9 µg/l at the end of the third and the sixth months of study, respectively. The common side effects of combined therapy were nausea (8.3%) and arthropathy (8.3%). A mild transient neutropenia and liver enzymes elevation happened in four patients of case group.ConclusionCombined therapy with deferoxamine and deferiprone significantly decreases serum ferritin level. It has little side effect and is suggested for major beta-thalassemic patients as a suitable therapy.

Anti-TNF therapy induced immune neutropenia in Crohns disease- report of 2 cases and review of literature

Transient neutropenia is reported in some patients on biologic therapy. We report two cases of severe neutropenia in patients with Crohn`s disease following treatment with anti-TNF therapy. In both cases neutrophil specific granulocyte autoantibodies were detected during period of neutropenia and disappeared on cessation of anti-TNF therapy. These may indicate that anti-TNF agents may produce autoimmune agranulocytosis by triggering production granulocyte autoantibodies. The long term management strategy for patients with anti-TNF therapy induced autoimmune neutropenia is uncertain.

Fractionated 90y Ibritumomab Tiuxetan (Zevalin™) Radioimmunotherapy As An Initial Therapy of Follicular Lymphoma – First Results From a Phase II Study in Patients Requiring Treatment According to GELF/BNLI Criteria

Abstract 102 Background:Radioimmunotherapy (RIT) has proven to be highly active in relapsed follicular lymphoma (FL) and the best single agent efficacy results in frontline therapy of FL were obtained with Iodine-131 Tositumomab (Bexxar™) (Kaminski et al NEJM 2005); albeit half of the patients had low tumor burden. In patients with higher tumor burden, using more than one fraction of RIT increases the overall radiation dose over that of a single fraction of treatment, thereby potentially improving both the response rates and survival (Illidge et al, Blood 2009). Methods:We conducted an international, multicenter phase 2 trial to evaluate the efficacy and toxicity of Fractionated 90Y Ibritumomab tiuxetan (Zevalin™) RIT as an initial therapy of Follicular Lymphoma. Eligible patients had untreated follicular lymphoma (grade 1, 2, or 3a) and at least one criterion of high tumour burden - one lymphoma lesion greater than 7 cm or three separate nodes of 3 cm or more; symptomatic splenic enlargement; raised serum concentrations of either lactate dehydrogenase or β2-microglobulin; compressive syndrome; or the presence of B symptoms. Treatment consisted of two doses of 90Y-ibritumomab tiuxetan (11.1 MBq/kg) given 8–12 weeks apart. Patients with greater than 20% bone marrow involvement (BM) with lymphoma received 4 weekly infusions Rituximab (375 mg/m2) and proceeded to fractionated RIT only if a repeat BM biopsy demonstrated clearing of lymphoma with less than or equal to 20% involvement. The primary endpoint was end of treatment response (EOR) of the intent-to-treat (ITT) population according to IWC 1999, assessed 12 weeks after last 90Y Ibritumomab tiuxetan infusion (21 weeks after treatment start). Secondary objectives were safety and progression free survival (PFS). Results:74 patients with a median age of 61 years (28−80), including 58 (78%) with stage III–IV stage, 23 (31%) intermediate risk FLIPI 2 and 34 (46%) with high risk FLIPI 3–5; were included between June 2007 and June 2010 in 7 centres. Thirteen (18%) patients with >20% BM involvement required rituximab pre-treatment, 2/74 did not qualify for RIT, meaning 72 received the first 90Y Ibritumomab tiuxetan infusion and 55 (76%) completed the full treatment schedule. The 2nd infusion of RIT was withheld secondary to hematologic toxicity with 1st infusion (n=12, 17%) or human anti murine antibodies positive testing (n = 4; 5.6%) or other (n = 1, 1.4%). Two out of 72 patients did not have recorded response data and the EOR was 95.7% (67/70) with CR/CRu of 57.1% (40/71). Six patients subsequently improved response making an ORR of 97.1% (68/70) (95% CI 90.0% – 99.7%), and CR/CRu of 64.3%% (45/70) (95% CI 51.9% – 75.4%). For the subset of 17 patients who only received a single 90Y Ibritumomab tiuxetan infusion, ORR (CR/CRu) was 100% (76,5%). At a median follow-up of 1.52 years (range 0.13 – 3.69 years) the PFS is 67 %, 20 patients have progressed and 12 of these have required further treatment (8 chemotherapy, 2 radiotherapy, 2 other). Updated data with median follow-up of more than 2 years will be presented.Ten patients experienced at least one SAE during the treatment period, with 3 related to study treatment (one case of rigors associated with the first infusion of rituximab and 2 cases of neutropenic sepsis both associated with the second RIT dose. The most common toxicity was hematologic: after the first 90Y Ibritumomab tiuxetan dose, related G3-4 hematological AEs were transient neutropenia (20.8%, 18 days median duration) and thrombocytopenia (20.8%; 20 days median duration). After the second 90Y Ibritumomab tiuxetan dose, related G3-4 hematological AEs increased to 36.4% for neutropenia (31days median duration), 14% for anemia (8/55 required transfusion) and 56.4% for thrombocytopenia (40 days median duration). There has been one case of MDS diagnosed 26 months after treatment and one death due to metastatic breast cancer diagnosed 9 months post last dose of 90Y Ibritumomab tiuxetan Conclusion:Fractionated RIT using 90Y-ibritumomab tiuxetan is an effective frontline treatment of advanced-stage follicular lymphoma in patients with high tumor burden requiring treatment and delivers high response rates. The treatment was well tolerated by patients with few infectious episodes and AE's and manageable hematologic toxicity. Disclosures:Illidge:Bayer Schering Pharma: Honoraria, Research Funding, Speakers Bureau. Morschhauser:Roche: Consultancy, Honoraria.

Leukocyte Integrin Activation and Alterations in Parathyroid Hormone Levels Following G-CSF Administration

Abstract 2164Following administration of granulocyte colony-stimulating factor (G-CSF) there is a marked but transient drop in circulating neutrophils. To determine the contribution of integrin activation to these events, a monoclonal antibody (mAb 24) that recognizes the activated α-subunit cation binding domain of the integrin family of receptors was used to evaluate human leukocytes following G-CSF administration. A single 480 mcg dose of human recombinant G-CSF (Neupogen, Amgen) was administered in the upper arm of six healthy human subjects (3 male, 3 female). EDTA and heparinized blood samples were collected prior to and at 15, 30, 60, 90, 120 and 240 minutes after injection. Mean absolute neutrophil count decreased from 2,557 ± 654/μl at baseline to 227 ± 193 (p<0.001) at 30 and 45 minutes. Flow cytometric analysis revealed a dramatic increase in binding of mAb24, which recognizes the occupied divalent cation binding site of the activated integrin α subunit, to neutrophils but not other circulating nucleated cells within 15 to 30 minutes after G-CSF administration, concurrent with a dramatic decrease in circulating neutrophil numbers. This activation, like the drop in neutrophil count, was transient, with a return to baseline status by 60 minutes. Intact parathyroid hormone (iPTH) levels were also evaluated. Within 30–90 minutes following G-CSF injection there was a significant drop in iPTH from a baseline of 52.5 ± 31.1 to a nadir value of 30.6 ± 20.9 pg/ml (p=0.01). A rebound in iPTH levels to 63.4 ± 27.0 pg/ml (p<0.001) occurred at 90 to 240 minutes. There were no significant changes in ionized calcium or magnesium levels. These results demonstrate that the integrin on circulating neutrophils is transiently activated following G-CSF administration and that this activation may mediate the transient neutropenia seen in this setting. In addition, alterations in PTH levels occur following G-CSF administration. As it is known that iPTH influences the hematopoietic stem cell (HSC) niche, fluctuations in iPTH levels following G-CSF administration may play a role in HSC expansion. Disclosures:No relevant conflicts of interest to declare.

The fundamentals of neutrophil antigen and antibody investigations

Background The neutrophil population makes up the largest proportion (40–70%) of white blood cells and is crucial to our innate immune response. An unexpectedly significant proportion of neutrophils (∼28%) is located in the pulmonary circulation. This is an immune system front line wherein neutrophils guard an important interface between the external environment and the body. Neutrophils migrate to sites of infection and/or inflammation, where they phagocytose and destroy foreign microorganisms and mediate inflammation and eventual healing. Therefore, a decrease in neutrophil numbers often results in increased susceptibility to infection. Sustained or transient immune neutropenia occurs when neutrophil‐reactive alloantibodies or autoantibodies destroy neutrophils, or significantly compromise their function. Neutrophil‐reactive antibodies are capable of activating the neutrophil’s microbicidal arsenal with resultant damage to the surrounding tissue.Antigens and Antibodies There are currently eight well characterized human neutrophil antigens (HNA): HNA‐1a, HNA‐1b, HNA‐1c, HNA‐2a, HNA‐3a, HNA‐3b, HNA‐4a and HNA‐5a. Only HNA‐1a, HNA‐1b and HNA‐1c are specific to neutrophils, whilst the others are also expressed on other cell types. It is important to remember that neutrophils also express HLA Class I antigens, and that there is some evidence that adequately activated neutrophils may express HLA Class II antigens. This provides an interface between the innate and adaptive immune response. Therefore, both HNA and HLA Class I and II antibodies are capable of reacting with neutrophils in vivo and in vitro.Routine Antigen and Antibody Investigation Antigen genotyping protocols are now available for HNA‐1a, HNA‐1b, HNA‐1c, HNA‐3a, HNA‐3b, HNA‐4a and HNA‐5a. The serological examination of neutrophils is reliant on harvesting a representative, pure population of neutrophils, which is difficult because neutrophils are short lived (6–8 h) and fragile. The International Granulocyte Immunobiology Workshop recommends the use of both the granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT), complemented by the monoclonal antibody immobilization of granulocyte antigen (MAIGA) assay. The GIFT uses paraformaldehyde‐fixed neutrophils and detects the binding of antibodies to surface HNA and HLA antigens. Serological reaction in the GAT is reliant on viable neutrophils. It demonstrates the ability of antibodies to sensitize neutrophils and stimulate them to undergo chemotaxis towards other sensitized neutrophils, thus forming microscopic agglutinates. The MAIGA is useful in confirming antibody specificities as it employs monoclonal antibodies to known HNA epitopes. To screen for neutrophil‐reactive antibodies, serum or plasma is exposed to a screening panel of neutrophils in the GIFT and GAT. To differentiate between HNA and HLA antibodies, the simple strategy of using platelet pools to absorb out HLA antibodies can be used. Specificity is determined using the GIFT and GAT, and a phenotyped and genotyped neutrophil panel and confirmed with the MAIGA.Application The combination of the GIFT, GAT and MAIGA was originally developed for the reliable investigation of immune neutropenias. However, in the last decade these techniques have become fundamental to the investigation of immune mediated TRALI where neutrophils are a key target and effector cell.

Optimizing Patient Safety During Hemodialysis

PUBLICATION OF THE LANDMARK REPORT TO ERR IS Human: Building a Safer Health System by the Institute of Medicine galvanized the health care system to focus relentlessly on improving patient safety. These efforts have many forms, often leveraging current health information technology to collect and analyze information about the characteristics of avoidable complications. Many efforts focus on known weaknesses in the health care delivery process, such as discontinuity of care, lack of integrated accountability among clinicians for patient outcomes, and communication errors. Failure mode analysis, root cause analysis, and examination of “near misses” are often tools used for improving patient safety. However, observers agree that there remain fundamental problems with patient safety in an increasingly complex medical environment. The use of hemodialysis for the treatment of irreversible kidney failure represents an important opportunity to improve patient safety. Hemodialysis has gradually been transformed from an extraordinary to a commonplace medical procedure during the past 50 years. Today, more than 50 million hemodialysis treatments are performed annually in the United States alone. Moreover, as dialysis therapy has matured, an increasingly elderly, frail population with extensive comorbidities is now being treated with high attendant risks for procedure-related complications. Meanwhile, technical improvements in nearly all aspects of the hemodialysis procedure have greatly increased overall safety. Today, there is better standardization of equipment, more use of automated controls and real-time monitors built into dialysis equipment for adjusting critical components of the dialysis procedure, and use of higher purity of water in dialysate than in the past. However, many gaps remain in the safety net around this complex medical procedure and current approaches for improving safety in the dialysis population are often suboptimal. Published studies that systematically evaluate the frequency and type of adverse events and medical errors occurring in dialysis units are rare. Many patient safety problems in dialysis units are “known knowns,” including medication errors, risks for intradialytic hypotension, and complications related to vascular access. These problems can be addressed using strategies already proven to have benefit in other clinical settings, such as medication reconciliation, and targeted interventions for prevention of falls. Other safety problems, such as clusters of unusual infections, fit readily into failure mode analysis models. The greatest opportunities and challenges, however, probably involve identifying “unknown unknowns” that affect patient safety. Two salient studies, separated by more than 40 years and each published in JAMA, illustrate the inherent value of alert clinicians and researchers working together to illuminate “unknown unknowns” in dialysis care. In 1968, during the early days of dialysis therapy, clinicians made the serendipitous observation that transient neutropenia occurred 2 to 15 minutes after starting dialysis. The neutropenia was followed by the appearance of circulating immature neutrophils and by the end of dialysis the white blood cell count was normal. The occurrence of severe transient neutropenia during dialysis was then confirmed in 33 consecutive patients undergoing dialysis treatments. The clinicians became investigators and demonstrated that this was a systemic phenomenon by drawing blood samples from the antecubital vein of the contralateral arm (ie, venipuncture in the arm away from the dialysis access site) and further noted that the neutropenia developed when only a small fraction of the blood volume had passed through the dialyzer. Subsequent research clarified that when blood came into contact with dialysis membranes manufactured out of unmodified cellulose, commonly used in that era, vigorous activation of the alternate complement pathway ensued. The transient neutropenia was due to profound complementmediated neutrophil activation, which caused aggregation of neutrophils in tissue beds such as the lungs. When later work established that there were important clinical consequences from repetitive neutrophil activation with each dialysis treatment, new more biocompatible dialysis membranes were developed, and eventually unmodified cellulosic dialyzers were removed from the market.

Leukocyte integrin activation mediates transient neutropenia after G-CSF administration

AbstractAfter administration of granulocyte colony-stimulating factor (G-CSF), there is a marked, albeit transient, drop in circulating neutrophils. To determine the role of leukocyte integrins in this disappearance, a dog having canine leukocyte adhesion deficiency (CLAD) or CLAD dogs who had undergone gene correction either by matched littermate allogeneic transplant or autologous gene therapy were evaluated. Shortly after G-CSF administration, a dramatic, yet transient, neutropenia was observed in the control littermates. This neutropenia was not as marked in the CLAD dogs. In all instances, it was CD18+ neutrophils that preferentially egressed from the circulation. The association of CD18 with this rapid loss suggested leukocyte integrin activation after G-CSF administration. To determine the activation status of the integrin, a monoclonal antibody recognizing the activated α-subunit cation binding domain (mAb24) was used to evaluate human leukocytes after G-CSF administration. Mirroring the dramatic decrease in circulating neutrophil numbers, there was a dramatic and specific increase in the activation of the α-subunit after G-CSF expression on polymorphonuclear leukocytes. This activation, like the drop in neutrophil count, was transient. These results demonstrate that the leukocyte integrin on circulating neutrophils is transiently activated after G-CSF administration and mediates the transient neutropenia observed after G-CSF administration.

Diagnosis of Autoimmune Neutropenia by Neutrophil-bound IgG and IgM Antibodies

Autoimmune neutropenia (AIN) in infancy is caused by antineutrophil (granulocyte-specific) autoantibodies. These antibodies are rarely found in circulation because their serum levels are extremely low. We hypothesized that a direct granulocyte immunofluorescence test (D-GIFT) that enables us to detect neutrophil-bound autoantibodies consisting of both immunoglobulin (Ig) G and IgM has better diagnostic value than the detection of circulating autoantibodies. Whole blood (100 μL) was obtained from 50 infants with AIN, 12 infants with transient neutropenia, and 37 control infants. D-GIFT was performed using both fluorescein isothiocyanate-conjugated antihuman IgG Fc portion monoclonal antibodies and fluorescein isothiocyanate antihuman IgM monoclonal antibodies. Results were assessed as relative fluorescence intensity (RFI). The RFIs of antineutrophil IgG-bound and antineutrophil IgM-bound cells in patients with AIN were significantly higher than those in patients with transient neutropenia and in controls. Positive results, as assessed by RFI scores of more than 1.81 in either antineutrophil IgG-bound or antineutrophil IgM-bound cells, showed the sensitivity and specificity of D-GIFT, and the areas under the receiver operating characteristic curve (0.98, 0.98, and 0.997, respectively) in the diagnosis of AIN. D-GIFT detecting both neutrophil-bound IgG autoantibodies and IgM autoantibodies has discriminatory power for identifying patients with AIN and, therefore, can be a useful diagnostic test.

A single‐arm, open‐label study of alemtuzumab in treatment‐refractory patients with multiple sclerosis

Alemtuzumab (CD52-specific humanized monoclonal antibody) was found to be an effective therapy for treatment-naive patients with relapsing-remitting multiple sclerosis. Evaluate alemtuzumab's effects in patients with treatment-refractory relapsing-remitting multiple sclerosis. Forty-five relapsing-remitting multiple sclerosis patients who experienced ≥2 relapses during 2 years prior to the study entry whilst receiving interferon therapy were administered 24 mg i.v. alemtuzumab/day for 5 days at baseline and 3 days 12 months later. Patients received premedication with 1 g i.v. methylprednisolone on days 1-3 at both times. After 2-year follow-up, the annualized relapse rate was reduced by 94% compared to pre-treatment levels, from 1.6 (2 years prior to treatment) to 0.17 for the 2 years following (P<0.0001). Moreover, 86% of patients showed stable or improved scores on the Expanded Disability Status Scale, and only 1 experienced an increase in disability lasting ≥6 months. The majority (70-88%) showed stable or improved leg, arm and cognitive function as measured by the Multiple Sclerosis Functional Composite. Serious adverse events observed in single patients were transient neutropenia and pneumonia, pulmonary emboli and deep vein thrombosis. Five patients developed clinical thyroid disorders but no opportunistic infections or cases of immune thrombocytopenic purpura were observed. Alemtuzumab effectively reduced relapse rates and improved clinical scores in patients with active relapsing-remitting multiple sclerosis not controlled by interferon therapy.

Ecthyma Gangrenosum Secondary toStaphylococcus aureusin an Infant with Transient Neutropenia

Ecthyma gangrenosum is classically a cutaneous manifestation of a pseudomonal septicemia that presents in a patient with an immunodeficiency or hematologic malignancy. We describe a previously healthy 8-month-old girl who developed transient neutropenia and characteristic ecthyma gangrenosum lesions secondary to methicillin-resistant Staphylococcus aureus. This unique presentation of methicillin-resistant Staphylococcus aureus ecthyma gangrenosum emphasizes the importance of broad empiric coverage and early culturing for microorganism and susceptibilities in any patient presenting with ecthyma gangrenosum.

Safety (toxicity), pharmacokinetics, immunogenicity, and impact on elements of the normal immune system of recombinant human IL-15 in rhesus macaques

AbstractIL-15 uses the heterotrimeric receptor IL-2/IL-15Rβ and the γ chain shared with IL-2 and the cytokine-specific IL-15Rα. Although IL-15 shares actions with IL-2 that include activation of natural killer (NK) and CD8 T cells, IL-15 is not associated with capillary leak syndrome, activation-induced cell death, or with a major effect on the number of functional regulatory T cells. To prepare for human trials to determine whether IL-15 is superior to IL-2 in cancer therapy, recombinant human IL-15 (rhIL-15) was produced under current good manufacturing practices. A safety study in rhesus macaques was performed in 4 groups of 6 animals each that received vehicle diluent control or rhIL-15 at 10, 20, or 50 μg/kg/d IV for 12 days. The major toxicity was grade 3/4 transient neutropenia. Bone marrow examinations demonstrated increased marrow cellularity, including cells of the neutrophil series. Furthermore, neutrophils were observed in sinusoids of enlarged livers and spleens, suggesting that IL-15 mediated neutrophil redistribution from the circulation to tissues. The observation that IL-15 administration was associated with increased numbers of circulating NK and CD8 central and effector-memory T cells, in conjunction with efficacy studies in murine tumor models, supports the use of multiple daily infusions of rhIL-15 in patients with metastatic malignancies.