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.