Indirect Granulocyte Immunofluorescence Test Research Articles

The Cs a and Cs b Red Cell Antigens of the Cost Blood Group Collection Correspond to the HNA-3a and HNA-3b Neutrophil Antigens: Unexpected Twins with Implications for Sickle Cell Anemia

Recent genome-wide association and murine studies identified the human neutrophil antigen 3a/b polymorphism (HNA-3a/b) in SLC44A2 (rs2288904-G/A) as a risk factor in venous thromboembolism (VTE). We tested whether this polymorphism associates with clinical features in SCA patients in whose VTE and stroke are frequently encountered complications. Incidentally, we discovered that rare subjects with the homozygous HNA-3b/b genotype (genotype frequency < 2%) also carry the uncommon Cs(a-) red cell phenotype. Despite its discovery more than 50 years ago, the molecular basis of this red cell phenotype and the presumptive protein carrier for the antigens of the COST blood group collection (Cs a and Cs b) has remained unknown. Inspired by this fortuitous discovery, we hypothesized that rs2288904 and COST antigens are closely associated. Thus, we genotyped this SNP in a cohort of 35 Cs(a-) subjects and found that all of them showed a HNA-3b/b genotype, indicating that the high-frequency allele of rs2288904 (HNA-3a; 461G) encoding Arg154 might encode the Cs a antigen (COST1). Interestingly, anti-Cs a does not react with SLC44A2 null RBCs (exceptional VER- blood type) and SLC44A2 knockout K562 cell line, confirming that the COST antigens are carried on this protein. In addition, using a panel of K562 cells expressing HNA-3a or 3b antigens developed by CRISPR/Cas9 genome editing, we confirmed that anti-Cs a recognizes the HNA-3a antigen. Overall, our results confirmed that HNA-3 and COST antigens have the same molecular basis. As a result, the Cs a and Cs b red cell antigens can now be included in the CTL2 blood group system, encoded by the SLC44A2 gene. This new finding could be of medical and biological value because anti-HNA-3a may be responsible for severe cases of neonatal neutropenia and transfusion-related acute lung injury (TRALI). Current testing for HNA-3a antibodies includes the granulocyte agglutination test and granulocyteindirect immunofluorescence test, as recommended by the ISBT Working Party on Granulocyte Immunobiology. These classical methods, however, are cumbersome because they require fresh granulocytes that have to be prepared daily and used immediately. Consequently, screening for HNA-3a antibodies is restricted to a few expert laboratories. Therefore, we propose to use the simple indirect antiglobulin red cell agglutination test (IAT) and Cs(a-) RBCs to screen for this antibody in the plasma of donors and patients. Finally, given that the starting point for this project was the study of a potential association of the HNA-3 polymorphism with clinical and biological features in SCA patients, we found that the HNA-3b allele was significantly associated with a higher monocyte and platelet count (p<0.02) and the occurrence of leg ulcer (p=0.01), suggesting an important role of the SLC44A2 protein in the SCD physiopathology.

Work‐up in the case of granulocyte antibodies

Granulocyte allo‐ and autoantibodies have been implicated in a variety of clinical conditions such as primary and secondary autoimmune neutropenia (AIN), alloimmune neutropenia (NAIN), transfusion‐related acute lung injury (TRALI) as well as febrile and severe pulmonary transfusion reactions. The classic granulocyte agglutination test (GAT) in combination with the granulocyte indirect immunofluorescence test (GIFT) can detect nearly all relevant antibodies. HNA‐1, ‐2, 4, 5 and HLA class I antibodies are clearly detectable in the GIFT while HNA‐3 antibodies strongly agglutinate neutrophils in the GAT. The glycoprotein‐specific and highly sensitive monoclonal antibody‐specific immobilization of granulocyte antigens (MAIGA) test detects all human neutrophil antigen (HNA) antibodies except for HNA‐3 but is time‐consuming and requires highly skilled personnel. For TRALI diagnostics, laboratory testing is completed by the indirect lymphocyte immunofluorescence test (LIFT), and HLA class I and II ELISAs or fluorescent bead‐based assays (Luminex). The latter also enable faster and more automated HNA antibody detection but to date not all specificities, especially HNA‐3, can be reliably detected so that still the classical tests have to complete the methodological spectrum. HNA allelotyping by PCR methods is the first choice for all HNA except for HNA‐2 because the molecular reason for the HNA‐2null phenotype is not completely understood. Establishing a PCR‐SSP reaction for the main CD177*787A>T polymorphism comprises the risk to miss other causes so that HNA‐2 is still typed serologically. Granulocyte serology even today is widely based on a variety of manual methods, requires experienced laboratory staff and profound knowledge of granulocyte immunobiology.

Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN.

Alloimmunization against platelets, granulocytes and erythrocytes in multi-transfused patients in Iranian population

BackgroundThis study aims at alloantibody screening, determination of the types of these antibodies in multiple-transfused patients with chronic hematologic diseases. Patients and MethodsThis descriptive study was performed on 240 patients with chronic hematological diseases referred to public hospitals in Iran. Single blood sample was taken and tested for the presence of antibodies. In case of a positive antibody screening, antibody identification was performed using granulocyte agglutination test (GAT), granulocyte indirect immunofluorescence test (GIIFT), platelet indirect immunofluorescence test (PIIFT), monoclonal antibody-specific immobilization of platelet antigen (MAIPA) and panel cells. ResultsOut of 240 patients, 105 patients (43.75 %) had been alloimmunized. The incidence of alloantibodies against red blood cells (RBCs) in positive alloantibodies patients were 84.76% (89/105). The most common alloantibody was against antigens of the kell (anti-K) and Rh (anti-E) and (anti D) systems (46.66%, 18.09% and 11.43% respectively). The overall incidence of anti- human leukocyte antigen (HLA) antibodies were 65.7% (69/105). Polymorphonuclear (PMN)-specific antibodies were found in 6.66% (7/105). Also from 105 patients, 14 patients had alloantibodies against platelet. DiscussionIn general, it is recommend that to decrease the rate of alloantibody synthesis, the packed cells should be cross matched for minor blood groups especially for Rh (E) and kell. In addition, the use of leukodepleted blood products can decrease the frequency of alloimmunization against platelet (PLT), PMN and HLA antigens.

Chronic Idiopathic Neutropenia In Adults: Clinical Features In a 4-Year Prospective Study

Chronic idiopathic neutropenia (CIN) is a rare acquired hematological condition, defined by an absolute neutrophil count (ANCs) lower than 1.8 x103/µL in white and 1.5 x103/µL in black people for more than 3 months, either in the absence or in the presence of anti-neutrophils antibodies (autoimmune forms). CIN is usually diagnosed after the exclusion of congenital and secondary forms. The former are usually marked by frequent and severe infections, that occur early in life, and by and increased risk of evolution to acute myeloid leukemia or myelodisplastic syndromes. In this prospective study we followed up 56 patients with CIN (21 males and 35 females, median age 55 years, range 25-86 years) for a median time of 48 months from January 2009 (10 patients had a previous follow-up of 8 years and 2 of 10 years), focusing on 1) severity of neutropenia, 2) ANCs variations (by general estimating equations GEE models), 3) positivity for anti-neutrophil antibodies (by direct and indirect granulocyte immunofluorescence test), 4) bone marrow features, 5) incidence of infectious episodes, and 6) evolution to definite clonal hematologic diseases (hairy cell leukemia HCL, chronic expansion of NK cells and myelodisplastic syndrome MDS). The mean ANCs were stably under the normal range (1.5-6.5 x103/µL) at all the time points considered; by GEE analysis, a great inter-subject variability was observed during the follow-up (p=0.012), whereas no significant intra-subject variations were found. Considering the severity of neutropenia, 21 patients (47%) showed neutrophils lower than 1x103/µL at enrollment (median 0.49 x103/µL, range 0.1-0.969 x103/µL), and 8 cases <0.5 x103/µL. The mean ANCs observed during the follow up were significantly lower in males than in females (p=0.023) and in cases with mild splenomegaly, although not significantly (11 cases, 20%, mean maximal diameter 11,4 cm by ultrasonography), independently from gender (multivariate analysis). Anti-neutrophil antibodies were detected in 19/56 patients (34%), and mean ANCs values over the follow up were significantly lower in positive versus negative cases (p=0.027). Lymphocyte values greater than the upper normal value of our series (3.4 x103/µL) were observed in 5/56 patients (9%). By peripheral blood immunophenotyping (N=23), 13 (56.5%) patients displayed absolute NK+ cells greater than 0.2 x103/mL (normal NK cut-off value), but all under below 2 x103/µL. Bone marrow evaluation was performed in 27 patients: median cellularity was 35% (range 13-75), and 10/27 (37%) displayed a value lower than 25% (threshold for hypocellularity in aplastic anemia); 19/27 (70%) showed some dysplastic cells, even if less than 10% dysplastic cells and without coexistent MDS-related karyotype; cytogenetic was normal in 24 cases (89%), while 3 males, all older than 60 years, displayed a 45, X0 karyotype (7, 6 and 3 metaphases respectively. Finally, 10 patients (18%) showed monocytosis, and 6 (10%) a MGUS. An infection needing oral antibiotic or antiviral therapy occurred in 13 patients (25%) (2 pneumonias, 7 upper respiratory tract, 3 Herpes Zoster Virus and 1 urinary tract infections), without relationship with the patient's mean ANCs value, the nadir of ANC value, nor with the presence of anti-neutrophil antibodies. During the follow-up, because of suspected progression/evolution, 14 patients were re-evaluated by bone marrow biopsy or peripheral immune-phenotyping: 4 cases were diagnosed with chronic NK expansion, 4 with HCL, and 2 with MDS, one refractory cytopenia with unilineage dysplasia (RCUD) and one with multilineage dysplasia (RCMD). No association was found between evolution and ANCs, both as values at enrolment and mean counts over the follow up, nor with gender, presence of anti-neutrophils antibodies, monocytosis, splenomegaly, electrophoresis abnormalities and infections. All 4 patients, who developed an NK-expansion, showed peripheral lymphocytes >3.4 x103/µL at enrolment (>5x103/µL in only 1 case), and 3 cases displayed increased NK cells at peripheral immune-phenotyping (p= 0.018). In conclusion, CIN in adults is a benign disease, with an infectious rate not superior to that of the general population and a great variability in ANCs values. During this prospective observation, 10 CIN patients evolved, reaching the criteria for clonal hematological diseases, suggesting that this condition deserves clinical follow up. Disclosures:No relevant conflicts of interest to declare.

A Novel Method Using Extracted Human Neutrophil Antigens From HNA Gene-Transfected Cell Lines for Detection of Antibodies Against Human Neutrophil Antigens

Abstract 3272Antibodies against Human Neutrophil Antigen (anti-HNA antibodies) have been implicated in immune neutropenia and transfusion-related acute lung injury. Those have been usually analyzed by Granulocyte indirect immunofluorescence test by flow-cytometry (GIFT-FCM), which requires fresh blood for panel neutrophils that cannot be preserved more than a few hours. Furthermore, specificity for anti-HNA antibodies cannot be easily identified by GIFT-FCM because of several antigens on membrane of panel neutrophil. In this study, we have developed a novel method designated as KY-Luminex Method, which was modified from Monoclonal antibody-specific immobilization of granulocyte antigens.For KY-Luminex Method, extracted human neutrophil antigens (eHNAs) were established from gene-transfected cell lines (KY-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b) that express HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b, respectively. These eHNAs were immobilized by monoclonal antibodies on microspheres (eHNA-microspheres). Sera were tested to the eHNA-microspheres in wells of a 96-well microplate, respectively. The reactivities of the sera were analyzed by Luminex100.From May 2010 to March 2012, sera were obtained from 101 patients with autoimmune neutropenia in children. The age distribution was from 2 months to 72 months. Sixty-three patients were females and 38 were males.As shown in Table, the sera from 74 patients showed positive reaction both in standard GIFT-FCM and KY-Luminex method. The sera from 11 patients showed positive in GIFT-FCM, and negative in KY-Luminex. These sera finally proved to have antibodies against antigens other than neutrophil such as anti-HLA antibody. The sera from 13 patients showed negative in GIFT-FCM, and positive in KY-Luminex. The sera from 3 patients showed negative both in GIFT-FCM and KY-Luminex. Sera with positive reaction against each panel neutrophil in GIFT-FCM have tendency to react against multiple antigens in KY-luminex method. Especially, majoryty of analyzed sera had antibodies against HNA-4a, 4b, 5a and 5b. No antibodies against each antigens were detected in sera from normal controls by KY-luminex method.These results indicate that KY-luminex method has superior to GIFT-FCM in both sensitivity and specificity of anti-HNA antibodies. Furthermore, eHNA-microspheres are stable at 4 centigrade for more than a half year, and many samples such as 96 samples could be analyzes at once in simple and easy method.TableRelationship of reactivities of patient's sera between GIFT-FCM and KY-luminex method. Data represent the number in 101 patientsGIFT-FCMKY-Luminex methodpositivenegativepositive7411negative133 Disclosures:No relevant conflicts of interest to declare.

Autoimmune neutropenia due to antineutrophil antibodies in a patient with primary sclerosing cholangitis.

Autoimmune neutropenia (AIN) is defined as a decrease in the circulating absolute neutrophil count (ANC) to less than 1500/μl caused by serum antineutrophil antibodies. Secondary AIN is associated with various autoimmune diseases. Herein we present the case of a patient with primary sclerosing cholangitis (PSC) who developed secondary AIN. A 19-year-old man was admitted due to liver injury, and a diagnosis of PSC was established by cholangiogram and liver biopsy. Severe neutropenia, with the ANC down to 130/μl, developed during his hospital course. No medications had been given at that time and bone marrow aspiration revealed no abnormality. Therefore we suspected secondary AIN as a causative etiology and examined whether antineutrophil antibodies were detectable in the patient's sera by flow cytometric analysis of the granulocyte indirect immunofluorescence test. We found that antineutrophil antibody was strongly positive on admission, and the titer decreased along with recovery from neutropenia. This is the first reported case of a patient with PSC who developed AIN, with detection of serum antineutrophil antibodies.

Primary autoimmune neutropenia in children in Taiwan

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. 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. 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 microL (standard deviation, 213/microL). 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. In Taiwan, autoimmune neutropenia in children is associated with HLA-DQB1*0503. The autoantibody in autoimmune neutropenia is most commonly anti-HNA-1a.

Clinical Characteristics in Neonates with Alloimmune Neutropenia: Significance of the Detection of Antineutrophil Antibodies

Neonate alloimmune neutropenia (NAIN) is caused by the transplacental transfer of maternal alloantibodies directed against antigens on the infant's neutrophils. To date, there are scant studies about its clinical characteristics and characterization of antineutrophil antibodies though some case reports are found. In this study we analyzed 11 cases with NAIN from January 2005 to December 2007. The diagnosis of NAIN was confirmed by the transient neutropenia less than 500/μl of absolute neutrophil count (ANC), the detection of maternal antineutrophil antibody, the incompatibility of neutrophil antigens between parents, and their mothers without autoimmune diseases. Antineutrophil antibodies were detected by granulocyte indirect immunofluorescence test using flow cytometry. To quantify the strength of the antibodies, the ratio of the mean fluorescence channel of each sample to that of control serum was expressed as relative fluorescence intensity according to the method reported previously (Blood 99: 3468, 2002). The median age at diagnosis in NAIN patients was 8 days after birth ranged from 0 to 30 days. The average of ANC at the presentation was 170/μl ranged from 0 to 500/μl. All antineutrophil antibodies detected in sera of both neonates and their mothers were against HNA-1 antigens. The alloantibody against HNA-1a was found in 2 cases, that against HNA-1b was in 6 cases, and that against FcγR IIIb was found in 3 cases. The fact that the frequencies of homozygote of HNA-1a and HNA-1b in Japanese population were approximately 50% and 12%, respectively may reflect the frequency of alloantibody specificity in NAIN. During the neutropenic period, 7 cases with NAIN showed mild to moderate infections associated with neutropenia, such as pyrexia and pyodermia. In contrast, 4 of 11 cases with NAIN did not have any infectious episodes in their clinical course. In all patients, the spontaneous recovery of neutropenia with the disappearance of alloantibody was observed within 6 months (median 85 days ranged from 3 weeks to 6 months). The duration until spontaneous resolution of neutropenia was dependent on the strength of alloantibody found in sera of the mothers and neonates. Two patients with significantly high strength of alloantibodies had the relatively long duration to restore the neutropenia(4 months and 6 months). In conclusion, the specificity of antineutrophil antibodies in patients with alloimmune neutropenia is dependent on the frequencies of neutrophil antigens in Japanese population. The quantification of alloantibodies in neonates and their mothers may be useful in considering the clinical course of neutropenia in neonates.

Auto-immune neutropenia associated with metformin in a patient with splenic marginal zone lymphoma

A 75-year-old man with splenic marginal zone lymphoma had been treated by fludarabine. The 3 first courses were very well tolerated, with no cytopenia. To treat diabetes mellitus, metformin was added at day 6 after the beginning of the fourth course of fludarabine treatment. He had previously been briefly treated with metformin a few months before. Four days later, at day 10, haemogram revealed severe neutropenia (neutrophils: 0.7 g/l). Neutrophil count continued to decrease reaching 0.283 g/l at day 17. A bone marrow aspirate was performed and showed very active granulopoiesis with maturation arrest at the promyelocyte stage. Metformin was discontinued on day 22 (16 days after reintroduction) and neutropenia promptly reversed during next week with 1.9 g/l (day 29). Direct and indirect granulocyte immunofluorescence test were used to detect anti-neutrophil autoantibodies. The antibodies were related to metformin and were detectable by granulocyte agglutination test and the monoclonal antibody-specific immobilization of granulocyte antigens assay. A possible explanation is the fludarabine-metformin combination, since fludarabine could deplete regulator T-lymphocytes and lead to the emergence of metformin-induced anti-neutrophil antibodies.

Free Versus Neutrophil-Bound Antibodies in 100 Consecutive Italian Children with Chronic Neutropenia.

Chronic neutropenia (CN) is defined as a blood absolute neutrophil count (NC) below 1500/ml, lasting for at least 6 months. We report the results of serological screening for anti-neutrophil antibodies, performed in our laboratory during 2002–2005, in 100 consecutive children affected from CN and followed in 4 Italian pediatric centers.The patients, 65 males (m) and 35 females (f), had a median age of 18 months (range: 1–180). Both IgG bound to patient neutrophils (CD16+ cells) and the patient serum reactivity against healthy donor granulocytes (Direct and Indirect Granulocyte-Immunofluorescence Test, D-GIFT and I-GIFT) were assessed by flow-cytometry. The results are shown in the table, grouped according to the pattern of reactivity observed with D- and I-GIFT:A NC above 1500/ μl on the day of serological screening was found in 5.5%, 22.4%, 0% and 35% of patients in groups 1, 2, 3 and 4, respectively. Secondary CN, usually in association with other autoimmune disorders or malignant diseases, was reported in 5.5%, 10.3%, 0% and 50% of patients in groups 1, 2, 3 and 4, respectively. Infections were present in 72%, 79%, 25% and 8% of patients in groups 1, 2, 3 and 4, respectively. In group 2, 50 of the 58 patients (86%), who didn't show anti-neutrophil antibodies in their sera, presented neutropenia with concomitant infections. In these patients only D-GIFT was able to detect neutrophil antibodies.Patients testing positive for I-GIFT had more severe neutropenia. Patients negative by both techniques were mostly found to have non-immunomediated neutropenia at clinical follow up. Although many laboratories perform only an indirect test to detect antineutrophil antibodies, our results suggest that direct test is more sensitive in identifying immunomediated chronic neutropenia.tablegroupGIFTno. of patientsm/fNC (cells/μl)*WBC (x102/μl)*1D+ I+1815/3757 ± 5207.58 ± 3.12D+ I−5836/22968 ± 8187.29 ± 5.33D– I+43/1525 ± 3696.71 ± 2.34D- I−2011/91693 ± 15108.28 ± 3.2* Mean ± SD

Rituximab in the Treatment of Secondary Autoimmune Neutropenia.

Autoimmune cyclical neutropenia is a rare syndrome with periodically severe but temporary neutropenia with occurrence of 3–4 weeks, but not rarely in 2 or 5 week intervals. Antibodies with specificity for the neutrophils has been demonstrated in a number of different neutropenic disorders and is often associated with systemic autoimmune disease. Since the role of monoclonal antibodies in the treatment of autoimmune disorders is not clearly optimized, we evaluated the effectiveness of Rituximab in the treatment of immune neutropenia.We present two cases (male 66 years and 42 years female) with immune neutropenia, associated with systemic autoimmune disease, Chron's disease in male patient and rheumatoid arthritis in female patient. Bone marrow biopsy revealed reduction of granulopoesis and absence of neutrophils in the peripheral smear-agranulocytosis. Immunological investigations revealed increased polyclonal IgG, positive indirect granulocyte immunofluorescence test. Both patients had a clinical course with continuously intermittent neutropenia every 2 weeks, presented with recurrent febrile episodes and serious lower respiratory tract infections. Treatment with G-CSF was immediately started in both patients to reach neutrophil recovery, but after one year of periodically persistent neutropenia it was decided to start treatment with Rituximab, an infusion with standard single agent 375 mg/m2 weekly for 4 weeks. After 4 infusions neutrophils reached normal levels and the treatment continued with every 2 weeks infusion of monoclonal antibody in duration of 2 months and now once a month as maintenance treatment. The patients are one year and six months after beginning treatment with monoclonal antibodies, in stable clinical and laboratory condition. We can conclude that B cells play a central role in the autoimmune process and Rituximab treatment has been used in patients with autoimmune neutropenia. Further randomized studies will define optimal role of Rituximab in the treatment of autoimmune diseases.

Alloimmune neonatal neutropenia and thrombocytopenia associated with maternal anti HNA‐1a, HPA‐3b and HLA antibodies

The incidence of alloimmune neonatal neutropenia combined with neonatal alloimmune thrombocytopenia is very low. We report a case of a neonate who suffered severe neutropenia and thombocytopenia with widespread petechial spots. The presence of alloantibodies in mother's and patient's sera was analyzed by lymphocytotoxicity test, agglutination test, granulocyte indirect immunofluorescence test, platelet immunofluorescence test (PIFT) and solid phase enzyme-linked immunosorbent assay. Human neutrophil antigens (HNA) and human platelet antigen (HPA) genotypes were tested by polymerase chain reaction analyses. The mother's and patient's sera reacted with neutrophils and lymphocytes of the father. PIFT revealed the presence of IgG anti-platelet antibodies in the patient's serum but the test was negative in the maternal serum. Analyses of HNA-1 and HPA genotypes of the family revealed maternal-neonatal HNA-1a and HPA-3b mismatch. The study of the mother's and patient's sera showed the presence of anti HNA1a, HPA-3b and HLA antibodies specific for HLA-A3 and HLA-B38 antigens. These results suggest that the transplacental passage of maternal HNA-1a, HPA-3b and HLA alloantibodies caused neutropenia and thrombocytopenia in this patient.

Primary autoimmune neutropenia in children: a study of neutrophil antibodies and clinical course

Primary autoimmune neutropenia (AIN) in children is characterized by severe neutropenia, but mild bacterial infections and a spontaneous resolution. Neutrophil autoantibodies are involved in the disease. The precise relationship between the specificity and level of reactivity of the antibodies with the absolute neutrophil count and frequency of infections is not known. To obtain a better insight into this relationship, we performed a follow-up study in 15 patients with primary AIN. In addition, we performed two different neutrophil antibody tests to evaluate their sensitivity and specificity. Blood samples from 15 children were tested for neutrophil antibodies, at different time-points during the disease, by using the indirect granulocyte immunofluorescence test (GIFT) and the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay. Clinical data related to the occurrence of bacterial infections and treatment, and neutrophil counts were collected. Early in the disease, antibodies with pan-FcRIIIb specificity were detected, and HNA-1a or HNA-1b specificity of the antibodies developed over time. The sensitivity and specificity of neutrophil antibody detection tests were higher in the GIFT than in the MAIGA assay. Variables predicting time of recovery from neutropenia were not found. Prophylactic antibiotics led to the almost complete disappearance of infections. In patients with primary neutropenia, neutrophil antibody specificity changes over time. Prophylactic antibiotics do benefit the patients.

Differential diagnosis and clinical course of autoimmune neutropenia in infancy: comparison with congenital neutropenia

Aim: Autoimmune neutropenia in infancy (AIN) is caused by granulocyte‐specific autoantibodies. Clinical presentation and diagnosis have not been well studied, resulting in cumbersome diagnostic investigations and unnecessary treatment such as granulocyte colony‐stimulating factor (G‐CSF) therapy. Methods: Clinical, laboratory and immunological data of 18 infants with AIN were evaluated. Granulocyte‐specific autoantibodies were detected by the direct granulocyte immuno‐fluorescence test (D‐GIFT), indirect granulocyte immunofluorescence test (I‐GIFT) and immuno‐blotting. Results: The average age of onset and resolution of neutropenia in AIN was 7.4 ± 3.4 mo (mean ± SD) and 20.4 ± 4.9 mo, respectively. Sixteen of the 18 patients presented with mild infectious symptoms; the other 2 patients were detected by chance and presented with no infectious symptoms. D‐GIFT was positive in all patients, and I‐GIFT was positive in 17 of these 18 patients. Most patients showed preferential binding to neutrophils from NA(1 + 2−)‐phenotyped donors by I‐GIFT and immunoblotting. An antibiotic (sulfamethoxazole‐trimethoprim) was given to 15 patients for prophylaxis. G‐CSF was given to only one infectious patient. Conclusion: A combination of diagnostic tests for the detection of granulocyte‐specific autoantibodies was useful in diagnosing AIN, thus avoiding unnecessary investigations. Continuous treatment with G‐CSF was not necessary for prophylaxis, even if neutrophil counts were extremely low.

FcγRIII b and FcγRIIa polymorphism may affect the production of specific NA1 autoantibody and clinical course of autoimmune neutropenia of infancy

We studied 18 children with autoimmune neutropenia (AIN) to evaluate whether there was a possible relationship between the specificity of granulocyte autoantibodies (anti-NA1,2) and the phenotype of the NA system. Direct granulocyte immunofluorescence test (D-GIFT) was positive in all patients, and indirect granulocyte immunofluorescence test (I-GIFT) was positive in 17 of these 18 patients, respectively. Fourteen of 18 patients showed preferential binding to neutrophils from NA(1+2−) phenotyped donors. Immunoblotting with anti-FcγRIIImAb showed that IgG prepared from 7 of 12 patients precipitated both FcγRIIIb from NA1 and NA2 neutrophil lysate, whereas the other 5 precipitated only NA1. Patients’ IgG did not react with purified FcγRIIa. FcγRIIIb genotype were NA(1+2−) in 15 of 18 patients and NA(1+2+) in the other 3. FcγRIIa type of all patients were (H+R−). These distributions were significantly different from those of healthy Japanese blood donors ( n = 608). The genotype of FcγRIIIb and FcγRIIa may affect the production of neutrophil specific auto-antibodies in AIN of infancy and influence its clinical course.

Neutrophil Antibody Specificity in Different Types of Childhood Autoimmune Neutropenia

Autoimmune neutropenia (AIN) in children can be divided into 2 forms. In primary AIN, neutropenia is the sole abnormality, and although neutrophil counts are generally below 500 μL−1, mild bacterial infections occur. Primary AIN is mostly seen in young children and shows a self-limited course. AIN occurring in association with autoimmune diseases (secondary AIN) often shows more severe infectious complications. We analyzed clinical and serological data from 28 pediatric patients with AIN to evaluate whether there is a possible relationship between specificity of the neutrophil autoantibodies and the clinical course of the disease. Specificity of the circulating antibodies was determined with the indirect granulocyte immunofluorescence test (GIFT) and a panel of phenotyped donor neutrophils. The samples were further analyzed in the monoclonal antibody immobilization of granulocyte antigens assay (MAIGA) for neutrophil antigen (NA)1, NA2, CD11a, and CD11b specificity. With the indirect GIFT, an antibody specificity was deduced in 26 of the 28 analyzed samples. In all but 3 sera from patients with primary AIN, NA1-(76%) or NA2-(10%) specific antibodies were detected with the indirect GIFT. In 2 samples, the reactivity in the indirect GIFT was too weak to draw conclusions, but the MAIGA showed NA1 and/or NA2 specificity of the antibodies. One serum, from a patient with primary AIN with a persistent neutropenia for more than 6 years, contained NA1, possibly pan-FcγRIIIb, and CD11a antibodies. In 4 sera from patients with primary AIN, weak antibodies with CD11a or CD11b specificity were detected with the MAIGA. Sera from 7 patients with secondary AIN contained in all cases antibodies with pan-FcγRIIIb specificity, as deduced from the indirect GIFT results and absorbance/elution experiments performed with 2 sera. The MAIGA confirmed this for only 1 of the 5 tested sera. Furthermore, CD11a antibodies were detected in 1 of the 5 tested sera. In conclusion, our results indicate that primary AIN is usually associated with NA-specific antibodies, whereas secondary AIN seems to be associated with pan-FcγRIIIb antibodies. Thus, characterization of the antibodies in sera from children with AIN discriminates patients with primary AIN from those with secondary AIN.

Neutrophil Antibody Specificity in Different Types of Childhood Autoimmune Neutropenia

AbstractAutoimmune neutropenia (AIN) in children can be divided into 2 forms. In primary AIN, neutropenia is the sole abnormality, and although neutrophil counts are generally below 500 μL−1, mild bacterial infections occur. Primary AIN is mostly seen in young children and shows a self-limited course. AIN occurring in association with autoimmune diseases (secondary AIN) often shows more severe infectious complications. We analyzed clinical and serological data from 28 pediatric patients with AIN to evaluate whether there is a possible relationship between specificity of the neutrophil autoantibodies and the clinical course of the disease. Specificity of the circulating antibodies was determined with the indirect granulocyte immunofluorescence test (GIFT) and a panel of phenotyped donor neutrophils. The samples were further analyzed in the monoclonal antibody immobilization of granulocyte antigens assay (MAIGA) for neutrophil antigen (NA)1, NA2, CD11a, and CD11b specificity. With the indirect GIFT, an antibody specificity was deduced in 26 of the 28 analyzed samples. In all but 3 sera from patients with primary AIN, NA1-(76%) or NA2-(10%) specific antibodies were detected with the indirect GIFT. In 2 samples, the reactivity in the indirect GIFT was too weak to draw conclusions, but the MAIGA showed NA1 and/or NA2 specificity of the antibodies. One serum, from a patient with primary AIN with a persistent neutropenia for more than 6 years, contained NA1, possibly pan-FcγRIIIb, and CD11a antibodies. In 4 sera from patients with primary AIN, weak antibodies with CD11a or CD11b specificity were detected with the MAIGA. Sera from 7 patients with secondary AIN contained in all cases antibodies with pan-FcγRIIIb specificity, as deduced from the indirect GIFT results and absorbance/elution experiments performed with 2 sera. The MAIGA confirmed this for only 1 of the 5 tested sera. Furthermore, CD11a antibodies were detected in 1 of the 5 tested sera. In conclusion, our results indicate that primary AIN is usually associated with NA-specific antibodies, whereas secondary AIN seems to be associated with pan-FcγRIIIb antibodies. Thus, characterization of the antibodies in sera from children with AIN discriminates patients with primary AIN from those with secondary AIN.

Frequency of neutrophil-specific antigens among Koreans using the granulocyte indirect immunofluorescence test (GIFT)

NA1, NA2, NB1, NB2, NC1, and NE1 are a group of antigens specifically expressed on neutrophils. Antibodies against neutrophil-specific antigens are involved in alloimmune neonatal neutropenia (ANN), autoimmune neutropenia (AIN), and transfusion-related acute lung injury (TRALI). We investigated the frequencies of NA1, NA2, NB1, and Mart antigens in 105 healthy Korean blood donors (65 males, 40 females) by the granulocyte indirect immunofluorescence test (GIFT) employing flow cytometry. Antigen and gene frequencies were: NA1, 0.78 and 0.53, respectively; NA2, 0.75 and 0.50, respectively; and NB1, 0.86 and 0.62, respectively. The Mart antigen was positive in all 105 subjects included in this study. The proportions of NB1-positive neutrophils among NB1-positive individuals were variable with a mean value of 72.0 ± 20.2% (range, 27–100%). Differences in the frequency of the NA1 antigen have been reported between Asian and Caucasian populations. Our data support this variation. In addition, it was found that the frequency of the NA1 antigen in Koreans is significantly lower than in Japanese and Chinese populations (p &lt; 0.05). Immunohematology 1997;13:15–16.

Clinical significance of white cell antibodies in febrile nonhemolytic transfusion reactions

Febrile nonhemolytic transfusion reactions (FNHTRs) are associated with white cell (WBC) antibodies. The purposes of this study were to determine the frequency of WBC antibodies, to associate the severity of reactions with antibody specificity, and to distinguish FNHTRs from infection and postoperative fever. By using the granulocyte indirect immunofluorescence test in conjunction with lymphocytotoxicity testing, it was found that 70 percent of FNHTRs in 24 patients involved WBC antibodies. The remaining 30 percent of apparent FNHTRs were associated with infections and postoperative fever. Granulocyte-specific antibodies were as prevalent as HLA antibodies and were associated with the severest reactions. Because FNHTRs occur with granulocyte-specific antibodies, HLA antibodies, and possible monocyte-specific antibodies (untested in this and other studies), these reactions could be grouped together as WBC-associated reactions.