A comparison of methods for detecting leukocyte antibodies in autoimmune neutropenia

A six-month-old girl and an 18-month-old boy with autoimmune neutropenia due to anti-NA1 are described. The antibodies were detected by granulocyte microagglutination, and their disappearance in the girl coincided with a return of a normal neutrophil count. The autoantibodies in both patients also reacted in the granulocyte cytotoxicity (GC) assay, and in one patient, in the staphylococcal protein A (SPA) assay. However, neither the GC nor the SPA assays showed the anti-NA1 specificity found by agglutination, and the presence of GC and SPA antibodies did not coincide with neutropenia. These three leukocyte antibody techniques may detect different antibodies and have different clinical significances. This report provides additional evidence of the existence of autoimmune neutropenia and indicates that the clinical role of neutrophil antibodies detected by different serologic techniques is not yet established. Antibodies detected by granulocyte agglutination were clinically significant in these two patients with autoimmune neutropenia, while the results of testing with GC and SPA were not.     

Granulocyte immunology.

Antibodies directed against antigens on the granulocyte (neutrophil) membrane can cause a variety of disorders including neonatal immune neutropenia, immune neutropenia after bone marrow transplantation, autoimmune neutropenia, and drug-induced immune neutropenia. Since granulocyte alloantibodies can lead to severe pulmonary transfusion reactions (TRALI), febrile transfusion reactions and refractoriness to granulocyte transfusions, they also play an important part in blood transfusion. The implicated human neutrophil alloantigens (HNA) have been renamed in the recently introduced HNA nomenclature which is based on the antigen's glycoprotein location. The Fc gamma Receptor IIIb (CD16, HNA-1) and the NB1 glycoprotein (CD177, HNA-2) represent the major immunogenic molecules of the neutrophil membrane. They bear the clinically most important antigens HNA-1a,-1b,-1c (NA1, NA2, SH) and HNA-2a (NB1), respectively. For the detection of granulocyte antibodies, a combination of immunofluorescence and agglutination tests together with a panel of freshly isolated, typed test neutrophils has been shown to represent the best means of detection. The introduction of the glycoprotein-specific assay "MAIGA" has improved alloantibody identification considerably. To facilitate and improve neutrophil typing, PCR-SSP techniques have been established for HNA-1a,-1b, and -1c genotyping.     

Identification of a new white cell antigen

BACKGROUND: Antibodies to white cell antigens can cause alloimmune neonatal neutropenia, autoimmune neutropenia, and transfusion reactions. CASE REPORT: A full-term male infant developed a skin infection and was found to be neutropenic on his fourth day of life. He had a transient increase in his neutrophil count after treatment with intravenous immunoglobulin, but his neutrophil count was not consistently normal until he was 6 weeks old. Serum from the baby's mother reacted in a granulocyte immunofluorescence assay but not in a granulocyte agglutination assay. The mother's serum was tested in the granulocyte immunofluorescence assay against neutrophils from 103 healthy, unrelated people, and it reacted with cells from 66 percent of those people. The expression of SL correlated weakly with the expression of NA1 (r = 0.23; p = 0.02) and 5a (r = 0.20; p = 0.05) antigens. SL antigen expression on neutrophils was not associated with the expression of NA2, NB1, NB2, NC1, 5b, 9a, or Mart. The expression of SL on neutrophils from members of an extended family was analyzed, and the antigen was found to be inherited in an autosomal-dominant manner. Anti-SL also reacted with T-lymphocytes in a flow cytometry assay but did not react with red cells or platelets. No lymphocytotoxic antibodies were detected in the mother's sera. The anti-SL was tested against neutrophils in an immunoprecipitation and immunoblotting assay, but no molecules were identified. The neutrophil-specific antigens NA are located on Fc gamma receptor III (CD16). To determine if the SL antigen was also located on Fc gamma receptor III, anti-SL was also tested in a monoclonal antibody immobilization of granulocyte antigens assay. Anti-SL did not react with molecules recognized by CD16 monoclonal antibodies. CONCLUSION: A new white cell antigen SL, with a frequency of 66 percent, was identified on neutrophils and T- lymphocytes as a result of the evaluation of a case of neonatal alloimmune neutropenia. The molecule bearing the SL antigen was not identified in immunoblotting, immunoprecipitation, or monoclonal antibody immobilization of granulocyte antigens assays.     

Neutrophil alloantigens

Antibodies to neutrophil antigens can cause neonatal alloimmune neutropenia, autoimmune neutropenia, febrile transfusion reactions, and transfusion-related acute lung injury. Several neutrophil antigen systems have been described serologically, but only the human neutrophil antigen-1 (HNA-1) or NA and HNA-2 or NB systems have been well characterized biochemically and molecularly. HNA-1 antigens are located on FcgammaRIIIb, CD16. HNA-2 antigens are located on 58- to 64-Kd glycoprotein, CD177, and are encoded by a gene on chromosome 19 that belongs to the Ly-6 family. The function of the CD177 is not known, but the CD177 gene is highly homologous to a gene overexpressed in neutrophils from patients with polycythemia rubra vera called PRV-1. New polymorphisms in these antigen systems are still being described, but the complete understanding of these neutrophil antigen systems has been slow because of the complexity of these genes.     

Antibodies to granulocytes in patients infected with human immunodeficiency virus.

Individuals who are infected with human immunodeficiency virus (HIV) are known to have a high incidence of autoantibodies. In this study, serum samples from 100 individuals with HIV infection were tested for granulocyte antibodies (red cell antibodies, lymphocytotoxic antibodies, circulating immune complexes, and serum immunoglobulin G levels) by granulocyte agglutination (GA) and granulocyte immunofluorescence (GIF) assays. Granulocyte antibodies were detected in 66% of serum samples by GIF and in 21% of serum samples by GA. None of the positive sera reacted with granulocyte antigens of known specificity. Antibodies that reacted with red cell antigens other than ABO were detected in only three serum samples, but lymphocytotoxic antibodies were detected in 62% of patients. All serum samples were tested by immunoblotting with granulocyte plasma membranes. Only two samples were found to be positive; one sample reacted with a 58 kd protein and one reacted with a 55 kd protein, but neither serum sample immunoprecipitated any protein from granulocytes that were labeled at the cell surface with iodine 125. Since immune complexes that are bound to granulocyte membranes can be detected by GIF, circulating immune complex levels were measured in all 100 samples. Immune complexes were increased in GIF-reactive serum samples compared with GIF-nonreactive serum samples (23.3 +/- 19.5 micrograms Eq/ml [mean +/- SD] vs 9.6 +/- 8.1 micrograms Eq/ml, p less than 0.001) but not in GA-reactive serum samples compared with GA-nonreactive sera (24.4 +/- 21.3 micrograms Eq/ml versus 16.9 +/- 16.0 micrograms Eq/ml, p = 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Granulocyte antigens and antibodies

Granulocyte-specific antigens defined by human alloantisera are clinically important in neonatal neutropenia, autoimmune neutropenia, transfusion reactions, drug-induced immune neutropenia, and poor response to granulocyte transfusions. Many different antigens have been defined by human alloantisera using different assays. Only antigens of the N system of Lalezari are commonly studied today. The composition and location of these antigens within the granulocyte membrane are not known, but work is now proceeding on those issues. It also appears that these antigens may have some structural or functional role in the granulocyte. Although many myeloid MoAbs have been developed, there are very few reports to establish whether these identify structures that are related to the N series of granulocyte antigens. However, these MoAbs can serve as excellent reagents for better understanding the granulocyte membrane. It appears that studies during the next several years will provide exciting information about the structure-function relationships in the granulocyte membrane.     

Primary autoimmune neutropenia in children in Taiwan

BACKGROUND: Autoimmune neutropenia in children is caused by granulocyte-specific autoantibodies. These antibodies react to the patient's own neutrophils but disappear when the neutropenia spontaneously remits. This study reviewed our experience with autoimmune neutropenia in children and investigated possible associations with HLA-DR and HLA-DQ alleles.
STUDY DESIGN AND METHODS: From 1993 to 2006, our laboratory received 155 blood samples from children with neutropenia. Of these samples, 55 had granulocyte-specific autoantibodies on the indirect granulocyte immunofluorescence test. As the children had no other disorders associated with neutropenia, they were diagnosed with primary autoimmune neutropenia. HLA-DRB1 and -DQB1 allele typing was performed in 31 cases, and the results were compared with those of 190 normal healthy unrelated Taiwanese controls.
RESULTS: The mean ages of onset and resolution of neutropenia were 9.8 months (median, 9.0 months; range, 4-28 months) and 22.5 months (median, 20.0 months; range, 13-44 months), respectively. The male-to-female ratio was 1.2:1. The mean absolute neutrophil count was 190 per µL (standard deviation, 213/µL). Most patients (74%) had antibodies against HNA-1a. Autoimmune neutropenia in children in Taiwan was significantly associated with HLA-DQB1*0503 (odds ratio, 6.48; p = 0.0002; p_c = 0.003) allele.
CONCLUSION: In Taiwan, autoimmune neutropenia in children is associated with HLA-DQB1*0503. The autoantibody in autoimmune neutropenia is most commonly anti-HNA-1a.     

Invasive aspergillosis in neutropenic patients: rapid neutrophil recovery is a risk factor for severe pulmonary complications

BACKGROUND: In invasive aspergillosis, the duration of neutropenia is an accepted risk factor, and recovery from neutropenia is generally associated with a favourable outcome. However, the rapidity of granulocyte recovery may rarely be associated with adverse sequelae. The purpose of this study was to define the relationship between neutrophil (polymorphonuclear, PMN) recovery after chemotherapy-induced bone marrow aplasia and the occurrence of severe pulmonary complications (haemoptysis, pneumothorax and death) in patients with haematological malignancies who developed invasive fungal pneumonias. METHODS: Twenty consecutive patients were retrospectively studied; eight of them had developed pulmonary events between 5 and 11 days after neutrophil recovery that followed deep neutropenia (PMN < 100 microL-1). RESULTS: Five patients had haemoptysis (one of these also had pneumothorax) and three had pneumothorax. According to the multiplicative logistic model, the odds of occurrence of a pulmonary event increased significantly with increasing PMN count on the fifth day (P < 0.001). Five of the eight patients who had pulmonary complications died. Also, the risk of death was larger in the presence of rapid neutrophil recovery, although the difference was not statistically significant (P = 0.111). Analysis of clinical and laboratory data showed that the risk of pulmonary complications significantly increased when the neutrophil concentration was > 4500 microL-1 on day 5 after deep granulocyte neutropenia (PMN < 100 microL-1). There was no correlation between pulmonary complications, dosage of amphotericin B and deaths. CONCLUSION: The occurrence of life-threatening complications in patients with invasive fungal pneumonia is closely related to rapid PMN recovery.     

Monoclonal antibodies reacting with placental protein 5: use in radioimmunoassay, Western blot analysis, and immunohistochemistry

Monoclonal antibodies were raised reacting with placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor. Immunization was carried out with an antigen purified from late pregnancy placenta tissues. After fusion with myeloma cells, clones producing antibodies reacting with PP5 were isolated. Antibodies produced by two of the established hybridoma clones were characterized. The Ka of the antibodies was 0.22 x 10(9) L/mol and 0.3 x 10(8) L/mol. in Western blot analysis, both monoclonal antibodies reacted with the purified antigen that had a relative molecular weight (Mr) of 30 kd, but minor components of Mr 27 kd, 56 kd, and 62 kd were also identified. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions, the purified protein yielded three polypeptides (Mrs of 16.4 kd, 16.8 kd, and 18.3 kd) that did not react with the monoclonal antibodies in Western blot analysis. By immunoperoxidase staining with monoclonal and polyclonal antibodies, PP5 was localized to the syncytiotrophoblast, cytotrophoblast, and endothelium of early and late pregnancy placenta tissues, whereas various other tissues were PP5-negative. In immunofluorescence staining, isolated endothelial cells were stained with both monoclonal antibodies. Endothelial cells in monolayer culture released into the medium a substance that is immunologically similar to purified PP5.     

Severe congenital neutropenia: clinical effects and neutrophil function during treatment with granulocyte colony-stimulating factor.

We studied neutrophil function and clinical responses in seven patients with severe congenital neutropenia (SCN) after they received treatment with recombinant human granulocyte colony stimulating factor (rhG-CSF). Two subpopulations of patients with SCN were defined by their pattern of absolute neutrophil response, superoxide production, and cytochrome b559 levels. One group had an oscillating absolute neutrophil count and reduced ability to produce superoxide and cytochrome b559 (n = 4), and the second group had a relatively constant absolute neutrophil count response with normal superoxide and cytochrome levels (n = 3). Neutrophils from both groups had decreased surface expression of FcRIII and abnormal upregulation of the C3bi receptor (CR3). All patient neutrophils, however, had normal contents of the primary granule constituent, beta-glucuronidase, and the specific granule constituent, vitamin B 12 binding protein. The clinical response to rhG-CSF was evident by marked improvement in the degree of periodontitis and reduction in the number of oral ulcers in both groups of patients. Although neutrophil function is not completely normal in patients with SCN, it is likely that enough redundancy exists in neutrophil bactericidal capacity to promote normal host response to inflammation.     

Neonatal neutropenia and bacterial sepsis associated with placental transfer of maternal neutrophil-specific autoantibodies

BACKGROUND: Passively acquired neonatal neutropenia is an infrequently reported complication of maternal autoimmune neutropenia (AIN). Two affected siblings are described. The firstborn developed Citrobacter meningitis and was permanently disabled. The second was success-fully managed with pre- and postnatal injections of recombinant human granulocyte colony-stimulating factor (rHuG-CSF). STUDY DESIGN AND METHODS: Neutrophil-specific antibodies were evaluated by flow cytometry (FC), monoclonal antibody immobilization of granulocyte antigens, and granulocyte agglutination assays. RESULTS: A neutrophil-reactive antibody was detected by FC in samples of the mother's serum spanning a 4-year time frame. This antibody reacted with neutrophils from the mother, father, and their first infant and with 18 of 20 target neutrophils tested. In serologic studies, it was shown that the antibody was not specific for the commonly recognized neutrophil-specific alloantigens HNA-1a (NA1), HNA-1b (NA2), HNA-1c (SH), HNA-2a (NB1), or HNA-3a (5b). CONCLUSION: Severe neonatal neutropenia in the two siblings appears to have been caused by placental transfer of a maternal neutrophil-reactive autoantibody of undetermined specificity. Neutrophil counts should be evaluated in infants born to mothers with chronic neutropenia of possible autoimmune origin so that neutropenic infants can be carefully monitored and antibiotics and/or rHuG-CSF administered if indicated.     

Antibodies, clinical and hematologic findings in immunoneutropenias

Sera were analyzed from patients who were suspected to have antibodies to neutrophils. The analysis comprised five methods which avoid heterologous antibodies to human immunoglobulin. These methods were the granulocyte agglutination test (GAT), the granulocyte cytotoxicity test (GCT), the monocyte cytotoxicity test (MCT), the lymphocyte cytotoxicity test (LCT) and immune phagocytosis inhibition test (IPI). Each serum was tested with cells from five healthy donors, at least, and some with cells from relatives. After exclusion of sera containing multiple antibodies and HLA-antibodies with positive LCT- and IPI-tests, the GAT- or GCT-reactive antibodies were significantly (p = 0.00005) more frequent among patients with neutrophil counts less than or equal to 1.0 X 10(9)/l (30%; n = 117) than among patients with neutrophil counts greater than 1.0 X 10(9)/l (9%; n = 111). Within the group of neutropenic patients (less than or equal to 1.0 X 10(9)/l) these antibodies were significantly (p = 0.001) more frequent in patients without (48%; n = 46) than with reduction of the granulopoiesis (13%; n = 31). This typical feature of an immune cytopenia also could be shown with GAT-reactive antibodies alone. From all five antibody tests the diagnostic criteria of the autoimmune neutropenia (AINP) and neonatal alloimmune neutropenia (NIN) were infered. 25 patients with clearly defined AINP presented with significantly more infectious complication than 23 patients with only assumed AINP (48% versus 13%). Further, the clinical findings of eight patients with NIN were described. --Antibodies only reactive in the GAT were detected in healthy individuals (1.3%; n = 75) and patients without indication for AINP or NIN (8%; n = 111), also.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged neutropenia resulting from antibodies to neutrophil-specific antigen NB1 following marrow transplantation

Marrow graft failure is a significant cause of morbidity following bone marrow transplantation. A case is reported of marrow graft failure due to neutrophil antibodies. A 13-year-old girl with a large granular lymphocytosis and chronic neutropenia was treated with granulocyte transfusions prior to undergoing a transplant with bone marrow from a partially matched, unrelated donor. Following the transplant, a bone marrow biopsy showed engraftment of donor myeloid cells, but the recipient remained neutropenic. Testing of the serum for neutrophil antibodies found that the recipient's serum had a high-titer neutrophil antibody. Immunoprecipitation studies using the marrow recipient's serum and 125I surface-labeled neutrophils showed that the antibody reacted to the neutrophil-specific antigen NB1. Phenotyping of neutrophils from the marrow donor found that they expressed NB1 antigen, and, in a crossmatch assay, the recipient's serum reacted with donor neutrophils. Despite treatment with granulocyte-macrophage– colony-stimulating factor, the marrow transplant recipient remained neutropenic and died of polymicrobial sepsis and aspergillosis 38 days after the transplant. The presence of high-titer antibodies to neutrophil-specific antigen NB1 in this patient following transplant likely prevented the recovery of her peripheral blood neutrophils and contributed to her death.     

Managing passively acquired autoimmune neonatal neutropenia: a case study

Pregnant women with autoimmune neutropenia (AIN) and circulating neutrophil-specific autoantibodies can deliver neutropenic neonates at risk of sepsis. We report the case of a woman who had two such pregnancies. The woman had been on prophylactic granulocyte colony-stimulating factor (G-CSF) treatment, but this was ceased prior to conception in both pregnancies. In the first pregnancy, there was no monitoring or interventions, and the neonate was neutropenic and required intensive care treatment. In the second pregnancy, the maternal neutrophil autoantibody level was monitored, and G-CSF treatment was introduced in the third trimester. The second infant had no neutropenia at delivery and an excellent Apgar score. We discuss the management strategy in the second pregnancy that included monitoring of serial titres of the maternal autoantibody and the introduction of G-CSF in the third trimester, which may have contributed to a more favourable clinical outcome. This may assist other clinicians faced with similar dilemmas in the future.     

Effects of recombinant human granulocyte colony-stimulating factor on neutrophil function in vitro and in vivo following chemotherapy and autologous bone marrow transplantation.

Recombinant granulocyte colony-stimulating factor (rG-CSF) primed the ability of human neutrophils to generate increased levels of reactive oxidants in response to fMet-Leu-Phe, and also resulted in an increased rate of protein biosynthesis which was similar to that induced by granulocyte-macrophage colony-stimulating factor. However, rG-CSF reduced the chemotactic activity of neutrophils in response to endotoxin and did not result in an enhanced rate of killing of Staphylococcus aureus. rG-CSF was administered to patients after high dose chemotherapy and autologous bone marrow transplantation for the treatment of either Hodgkin's disease or multiple myeloma. This cytokine decreased the period of neutropenia following such treatment. Neutrophil function in two patients, measured seven days after the final administration of rG-CSF, was severely impaired as indicated by a greatly decreased ability to generate reactive oxidants. However, seven days later (i.e. 14 days post-therapy), the functional activity of the neutrophils from these patients had returned to normal. These data indicate that assays of neutrophil function together with morphological assessment of neutrophil numbers and maturity should be performed in order to evaluate the immune status of patients undergoing such therapy.     

Characterization of the neutrophil molecules identified by quinine- dependent antibodies from two patients

BACKGROUND: Two patients with episodic pancytopenia and renal failure associated with quinine (Qn) ingestion were previously found to have Qn- dependent antibodies that reacted with red cells, platelets, and neutrophils. The purpose of these studies was to characterize the neutrophil antigens recognized by Qn-dependent antibodies from these two patients. STUDY DESIGN AND METHODS: The neutrophil molecules recognized by the Qn-dependent antibodies in the sera from the two patients were analyzed by immunoprecipitation using 125I-labeled neutrophils. Neutrophils from 13 different donors were tested. RESULTS: The Qn-dependent antibodies from Patient 1 immunoprecipitated a 60-kDa molecule on neutrophils from seven donors and an 85-kDa molecule on neutrophils from three donors. The Qn-dependent antibodies from Patient 2 reacted with a 32-kDa molecule on neutrophils from 5 donors, a 60-kDa molecule on neutrophils from 9 donors, and an 85-kDa molecule on neutrophils from 10 donors. Neutrophil-specific antigen NB1 is also located on a 60-kDa glycoprotein (GP). While the antibody in serum from Patient 1 did not show specificity for NB1, the antibody from Patient 2 detected the 60-kDa molecule on NB1-positive neutrophils from 9 of 11 donors tested and did not detect the 60-kDa molecule on NB1-negative neutrophils from 2 donors. In a monoclonal antibody immobilization of granulocyte antigens assay, the Qn-dependent antibody from both patients reacted with the 60-kDa molecule carrying NB1. The Qn- dependent antibody from a third patient, Patient 3, was previously found to react with an 85-kDa GP and the 60-kDa NB1 GP. To determine if the Qn-dependent antibodies from Patients 2 and 3 recognized the same 85-kDa GP, neutrophils were treated with serum from Patient 3 plus Qn to remove the 85-kDa GP. Then, serum from Patient 2 plus Qn no longer immunoprecipitated the 85-kDa GP. CONCLUSION: The antigens recognized by Qn-dependent neutrophil antibodies were located on molecules of 85, 60, and 32 kDa. Qn-dependent antibodies from two patients reacted with the same 85-kDa GP and those from three patients reacted with the same 60-kDa GP. The 60-kDa molecule recognized by the Qn-dependent antibodies carried the NB1 antigen.     

A prospective study of neutropenia induced by high doses of beta-lactam antibiotics

A prospective study on the effect of beta-lactam antibiotics on granulopoiesis was carried out in 29 consecutive patients with bacterial endocarditis. Fourteen patients received a high dose of benzylpenicillin, up to 18 g/day, but in only three of them could the treatment be fulfilled as planned, for a mean time of 25 days. In 11 benzylpenicillin treated patients treatment had to be discontinued because of fever, rash or neutropenia. Neutropenia appeared in seven patients after 14-24 (mean 22) days. No superinfection occurred during the neutropenic phase which lasted 2-12 days. Patients with neutropenia differed significantly from others in having a lowered pretreatment neutrophil count (3.2 vs 10.4). In 15 patients treated with other beta-lactams, three cases of fever and rash and one case of neutropenia were seen in patients treated with cloxacillin 12 g daily. It was concluded that a daily dose of 18 g of benzylpenicillin is too high for longer treatment periods and that patients with initial low counts of neutrophils have an increased risk of developing neutropenia.     

流式细胞术检测抗中性粒细胞抗体方法的建立及临床初步应用

目的建立适用于临床的流式细胞术（FCM）检测抗中性粒细胞抗体（ANA）的方法,并作临床初步应用。方法采用FCM,以前向散射和侧向散射参数区分全血标本中的淋巴细胞和粒细胞,设定粒细胞门,用CD16b-藻红蛋白（PE）和CD177-别藻蓝蛋白（APC）单克隆抗体分别检测中性粒细胞中表面表达CD16b和CD177抗原细胞的百分率。用所建方法检测健康对照32名,确定参考范围,以此作为判断受检者血液中抗CD16b或CD177抗体存在与否的依据。检测外周血白细胞（WBC）总数〈4.0×109/L且中性粒细胞绝对计数（ANC）〈2.8×109/L的59例患者的CD16b和CD177百分率,并对其中中性粒细胞表达CD16b和/或CD177百分率低于参考范围（即判为相应抗体阳性）的患者给予激素治疗,观察疗效。结果健康人群中性粒细胞中表达CD16b和CD177的百分率分别为98.36%±1.53%和74.95%±11.07%,据此分别确定了健康人群中性粒细胞表达CD16b和CD177的参考范围分别为CD16b〉95.36%、CD177〉53.25%（即〉x珋-1.96s）。59例患者中有23例（39.0%）ANA阳性,其中ANC〈2.0×109/L患者的ANA阳性率（50.0%）远高于ANC〉2.0×109/L患者（27.6%）。ANA阳性患者经激素治疗后,WBC和ANC上升。结论 FCM检测抗中性粒细胞CD16b和CD177抗体是自身免疫性中性粒细胞减少症（AIN）实验简便、快捷的诊断方法,有助于AIN的诊断、鉴别诊断和疗效判断。     

Survival among children and adults treated with granulocyte transfusions: Twenty years’ experience at a Brazilian blood center

BackgroundIt remains controversial whether granulocyte transfusions as a supportive treatment improve survival in patients with febrile neutropenia or granulocyte dysfunctions. We describe survival rates subsequent to granulocyte transfusions in pediatric and adults patients treated at a major blood center in Brazil.Material and methodsWe retrospectively reviewed the clinical charts of pediatric and adult patients treated with granulocyte transfusions at our institution from January 2000 to October 2019. We assessed demographic characteristics, clinical features, indications for transfusion, units transfused, dose of granulocytes administered and survival rates 30 and 100 days after the initial transfusion.ResultsWe identified 64 pediatric and 67 adult patients treated with 262 granulocyte transfusions. An optimal dose (> 0.6 × 10⁹ granulocytes per kilogram per transfused unit) was available for transfusion in 80.4 % of pediatric patients but in only 19.6 % of adults (p = 0.017). Thirty days after their first granulocyte transfusion, 38 (59.4 %) pediatric and 61 (91 %) adult patients had died. Patients receiving the optimal dose of granulocytes had better survival outcomes, but even among this sub-group, adults were more likely to die than were children either at 30 days (OR = 8.67, 95 %CI 2.69–34.9) or 100 days (OR = 6.27, 95 %CI 1.86–25.9) after their initial granulocyte transfusion.ConclusionSurvival rates following granulocyte transfusion varied by the dose transfused and were higher in children than in adults.     

Exacerbation by granulocyte colony-stimulating factor of prior acute lung injury: implication of neutrophils

OBJECTIVE: Granulocyte colony-stimulating factor is widely prescribed to hasten recovery from cancer chemotherapy-induced neutropenia and has been reported to induce pulmonary toxicity. However, circumstances and mechanisms of this toxicity remain poorly known. DESIGN: To reproduce a routine situation in cancer patients receiving chemotherapy, we investigated the mechanisms underlying granulocyte colony-stimulating factor-induced exacerbation of alpha-naphthylthiourea-related pulmonary edema. SETTING: Laboratory research unit. SUBJECTS: Male specific-pathogen-free Sprague-Dawley rats. INTERVENTIONS: The effects of granulocyte colony-stimulating factor given alone or after alpha-naphthylthiourea used to induce acute lung injury were investigated. MEASUREMENTS AND MAIN RESULTS: Lung injury was assessed based on neutrophil sequestration (myeloperoxidase activity in lung tissue) and influx into alveolar spaces (bronchoalveolar lavage fluid cell quantification) and on edema formation (wet/dry lung weight ratio) and alveolar protein concentration into bronchoalveolar lavage fluid. Tumor necrosis factor-alpha and interleukin-1beta were measured in serum, lung homogenates, and isolated alveolar macrophage supernatants. In control rats, granulocyte colony-stimulating factor (25 microg/kg) significantly elevated circulating neutrophil counts without producing alveolar recruitment or pulmonary edema. alpha-Naphthylthiourea significantly increased the wet/dry lung weight ratio (4.68 +/- 0.04 vs. 4.38 +/- 0.07 in controls, p=.04) and induced alveolar protein leakage. Adding granulocyte colony-stimulating factor to alpha-naphthylthiourea exacerbated pulmonary edema, causing neutrophil sequestration in pulmonary vessels, significantly increasing lung myeloperoxidase activity (12.7 +/- 2.0 mOD/min/g vs. 1.1 +/- 0.4 mOD/min/g with alpha-naphthylthiourea alone; p<.0001), and increasing proinflammatory cytokine secretion. alpha-Naphthylthiourea-related pulmonary edema was not exacerbated by granulocyte colony-stimulating factor during cyclophosphamide-induced neutropenia or after lidocaine, which antagonizes neutrophil adhesion to endothelial cells. Tumor necrosis factor-alpha and interleukin-1beta concentrations in alveolar macrophage supernatants and lung homogenates were significantly higher with alpha-naphthylthiourea + granulocyte colony-stimulating factor than with either agent alone, and anti-tumor necrosis factor-alpha antibodies abolished granulocyte colony-stimulating factor-related exacerbation of alpha-naphthylthiourea-induced pulmonary edema. In rats with cyclophosphamide-induced neutropenia, tumor necrosis factor-alpha concentrations in alveolar macrophage supernatants and lung homogenates were significantly decreased compared with rats without neutropenia. CONCLUSION: Granulocyte colony-stimulating factor-related pulmonary toxicity may involve migration of neutrophils to vascular spaces, adhesion of neutrophils to previously injured endothelial cells, and potentiation of proinflammatory cytokine expression.