DISCUSSION
The true incidence of AIN amongst those diagnosed as CBN remains unknown as anti-neutrophil antibody testing is not routinely available amongst laboratories and cell-based antibody detection remains a technically demanding and time-consuming process. In the past, there has been conflicting opinions on the utility of antibody testing among CBN patients to specifically identify AIN individuals, given the overall benign and self-limiting clinical course of these individuals with only a small proportion of patients with significant infections.(4, 5, 9) Previously, some have commented that anti- neutrophil antibody detection did not correlate with the severity of neutropenia nor does it play any role in predicting prognosis in AIN (6, 10), and therefore routine testing is not recommended.(7, 8) In practice, anti-neutrophil antibodies are often difficult to detect as they often present in low titers and bind to their targets with poor avidity.(10) The detection rate varied amongst study groups ranging from 10 -98% (1, 4-6, 11), though all using different combination of testing methods. We report on a cohort of 81 Chinese children with presumed CBN, using the combination of cell-based and bead-based method, anti-neutrophil antibody was detected and therefore subclassified as AIN in 30.9% of the neutropenic cohort. Compared by an earlier study done by our group in 2004 reviewing the characteristics among Chinese children with CBN, using only cell-based method anti-neutrophil antibody was found in 21% of the group.(5) This demonstrates that using multiple methods of testing increases the detection rate of anti-neutrophil antibody, this was also recommended by previous studies.(10, 12) Our overall detection rate for AIN is lower than recent studies that evaluated detection rate of LABScreen MULTI, which quoted up to 98 % detection of HNA-1a, HNA-1b and HNA 2 antibodies, note that this study used pre-selected plasma donors previously identified to have antibodies with GIFT, GAT and MAIGA. In the same context, within our cohort, amongst 25 individuals identified to be antibody positive by GIFT/GAT, the LABScreen MULTI correctly identified 23/25 individuals, which demonstrates a similar specificity of 92%.
When comparing the two cell-based detection methods and bead-based methods, the combination of GIFT and GAT remains the gold standard in HNA antibody detection, however these tests are not suitable for high- throughput of samples.(11) It is known that GAT is very specific for detecting HNA antibodies, especially against HNA-3a due to their unique aggregation pattern. LABScreen MULTI, whilst easy to handle and suitable for high-throughput screening, remains costly and not ideal for detecting HNA-3a antibodies.(13) In this cohort, LABScreen MULTI detected antibodies where GIFT and GAT were negative in 13.6% (11/81) of the tested sample, whilst it was unable to detect 2.5% (2/81) of antibodies which were picked up subsequently by GAT and identified as against HNA-3a. These LABScreen MULTI figures differed from those reported by Schulze et al.(11) and Schonbacher et al.(13) who reported a false negative of 5.5 % and 5.6 % respectively and false positive result in 10% and 18% respectively. The contrasting numbers could be the result of those studies using donor sera pre-tested with GIFT/GAT, whilst in reality the antibody titers among patients with CBN could vary largely. Based on these findings, LABScreen MULTI should at least be applied in combination with the GAT to ensure reliable detection of anti HNA-3a and other more time-consuming methods such as GIFT, MAIGA should be implemented together to increase accuracy.
Previous groups including Madyastha et al.(14) and Boxer et al(6) were not able to identify clinical characteristics that correlated with the presence of anti-neutrophil antibodies. Among our children, we were able to establish with statistical significance that the presence of antibodies was associated with older age of onset, more severe neutropenia on presentation, lower median neutrophil count and more likely to have invasive infection with abscess being the top cause. Furthermore, AIN individuals will recover slower and later compared to their antibody negative counterparts. This plays significant role in the counselling of parents of newly diagnosed with AIN on the clinical course and prognosis on their disease course, these children are often subject to regular blood taking and frequent hospital visits, with these findings, we are more confident in reassuring them and managing their expectations.
We also identified that amongst the initial presentations for neutropenia, the top reason being infection accounted for 48.1% (66.7%, 26/39 patients with self-limiting viral infections), this was followed closely by infants coming in for investigation for prolonged jaundice exceeding the first two weeks of life (33.3%). In our hospital’s neonatology unit, neonates screened to have jaundice beyond first two weeks of life in the community are called back for blood screening to rule out conditions that could predispose individuals to prolonged jaundice, the complete blood profile is often ordered along with the liver function. Among these 27 infants, further investigations for the child and parents for maternal alloantibodies in the setting of neonatal alloimmune neutropenia (NAIN), nor HNA genotyping were done, and could therefore not be excluded in this cohort. Given that these infants were not reported to experience invasive infection during the study period, and the overall low incidence of NAIN which often are asymptomatic,(15) further extensive work up was not done. The anti-neutrophil antibody detection rate was only 0.1% amongst these infants, which was lower that the literature reported 0.35-1.1%. Another postulated explanation could be that since these infants are mostly breastfed, antibodies could deliver through their mother’s breastmilk and therefore the pathophysiology may slightly differ then those of classical CBN, AIN or NAIN. The inclusion of this group of children may contribute to overall lower age of onset of neutropenia within this cohort compared to the usual 5-15months(1, 4) and may also contribute to the lower antibody detection rate. If excluded, our overall antibody detection rate would increase to 40.7 % (22/54). Given the opportunity, it may be useful to look into similar aged infants and testing their parents to determine whether they’re truly cases of NAIN and comparing their clinical course with those with classical CBN and AIN.
In summary, following this cohort of Chinese children, we can conclude that the presence of anti-neutrophil antibodies in AIN has implications on the severity of neutropenia, infective complications and also prognosis. This serves as an important tool for clinicians to counsel parents and guides clinician’s follow up approach in patients with AIN. We have included a suggested diagnostic pathway for children with isolated neutropenia for guidance. (Supplementary Figure S1) What’s more, we have demonstrated that the combined use of cell-based and bead-based detection methods increases the detection rate. Whilst the classical cell-based methods are labor intensive and arduous, it should be implemented together with bead-based methods to increase its diagnostic accuracy.