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\begin{document}

\title{Significance of anti-neutrophil antibody in chronic benign neutropenia
in Chinese children}



\author[1]{Shau yin Ha}%
\author[1]{Sophie Lai}%
\author[1]{Wing Shan See}%
\author[2]{Jaime Duque}%
\author[3]{Daniel Ka Leung Cheuk}%
\author[4]{Jenny  Ho}%
\author[4]{Stephen Cheung }%
\author[4]{Patrick Chu}%
\author[2]{Pamela PW Lee}%
\author[4]{Janette SY Kwok}%
\affil[1]{Queen Mary Hospital}%
\affil[2]{The University of Hong Kong}%
\affil[3]{Hong Kong Children's Hospital}%
\affil[4]{Division of Transplantation and Immunogenetics, Department of Pathology \& Clinical Biochemistry, Queen Mary Hospital}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
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\selectlanguage{english}
\begin{abstract}
Background: Chronic benign neutropenia (CBN) and autoimmune neutropenia
(AIN) are notoriously difficult to differentiate in children owing to
their indistinguishable clinical course and varying availability and
accuracy in the methods of anti-neutrophil antibody detection. This
study aims to investigate whether the presence of anti-neutrophil
antibody has implications on the disease course in Chinese children with
AIN, as well as evaluating the various methods including LABScreen
MULTI, granulocyte agglutination test (GAT) and granulocyte
immunofluorescence test (GIFT) in anti-neutrophil antibody detection.
Procedure: Chinese children under 18 years of age with neutropenia [?] 1.5
x 109/L lasting 6 months or more in our single center were recruited
into the study between 2016 to 2018. Patients with secondary causes of
neutropenia were excluded. Blood for anti-neutrophil antibody and
genotyping was taken once at the time of recruitment and subsequently
when neutropenia recovered to [?] 1.5 x 109/L. A combination of two
in-house methods including GIFT, GAT and commercial kit LABScreenTM
multipanel were used for the detection of antibodies. The age of onset,
age of recovery, duration of neutropenia, gender, serial neutrophil
counts, incidence of invasive infection, use of G-CSF were examined.
Results: Using combined testing methods, anti-neutrophil antibody was
detected in 30.8 \% of patients and positivity was associated with more
severe neutropenia, higher likelihood of infection and slower and later
recovery compared to those without antibodies. Conclusions: The presence
of anti-neutrophil antibody was useful in predicting the clinical course
of patients with AIN. The use of combined testing methods increased the
detection rate.%
\end{abstract}%



\sloppy


\textbf{Significance of anti-neutrophil antibody in chronic benign
neutropenia in Chinese children}

Sophie HY Lai,\textsuperscript{1*} Queenie WS See\textsuperscript{1},
Jaime S Rosa Duque, MD\textsuperscript{1,2} Daniel KL
Cheuk\textsuperscript{1,3}, Dr Jenny CY Ho\textsuperscript{4}, Dr
Stephen KF Cheung\textsuperscript{4}, Mr Patrick Chu\textsuperscript{4},
Pamela Lee\textsuperscript{1,2,3,} Dr Janette SY
Kwok\textsuperscript{4}, Professor SY Ha\textsuperscript{1,2,3\#}

\textsuperscript{1}Department of Paediatrics and Adolescent Medicine,
Queen Mary Hospital, Hong Kong,

\textsuperscript{2}Li Ka Shing Faculty of Medicine, The University of
Hong Kong, Hong Kong.

\textsuperscript{3}Department of Paediatrics, Hong Kong Children's
Hospital, Hong Kong

\textsuperscript{4}Division of Transplantation and Immunogenetics,
Department of Pathology, Queen Mary Hospital, Hong Kong.

\textsuperscript{\#}Correspondence to:

Prof Shau-Yin Ha, Consultant\textbf{,} Honorary
Professor\textbf{,}Department of Paediatrics and Adolescent
Medicine\textbf{,} LKS Faculty of Medicine, The University of Hong
Kong\textbf{.} Hong Kong Children's Hospital, Hong Kong\textbf{.} Email:
syha@hku.hk Tel: (852) 5741-3204 Fax: (852) 3512-7598 Address: Doctor's
Office, 9/F, Tower B, Hong Kong Children's Hospital, 1 Shing Cheong
Road, Kowloon Bay, Kowloon, Hong Kong

\textbf{Text word count:} 2944

\textbf{Abstract Word Count:} 247

\textbf{Brief running title:} Utility of anti-neutrophil antibody in
neutropenia

\textbf{Key words:} immune cytopenia, autoimmune neutropenia, chronic
benign, neutropenia, paediatric haematology/oncology, anti-neutrophil
antibody

\textbf{References:} 15

\textbf{Table(s):} 1 table

\textbf{Figure (s):} 2 figures, 1 supplementary figure

*, denotes first author

names in any order they wish when citing this work

\#, denotes corresponding author

Abbreviations

Abbreviations

AIN

Autoimmune neutropenia

CBN

Chronic benign neutropenia

CIN

Chronic idiopathic neutropenia

GAT

Granulocyte agglutination test

GIFT

Granulocyte immunofluorescence test

G-CSF

Granulocyte Colony Stimulating Factor

HNA

Human neutrophil alloantigen

MAIGA

Monoclonal antibody--specific immobilization of granulocyte antigens.

NAIN

Neonatal alloimmune neutropenia

\textbf{Background:}

Chronic benign neutropenia (CBN) and autoimmune neutropenia (AIN) are
notoriously difficult to differentiate in children owing to their
indistinguishable clinical course and varying availability and accuracy
in the methods of anti-neutrophil antibody detection. This study aims to
investigate whether the presence of anti-neutrophil antibody has
implications on the disease course in Chinese children with AIN, as well
as evaluating the various methods including LABScreen MULTI, granulocyte
agglutination test (GAT) and granulocyte immunofluorescence test (GIFT)
in anti-neutrophil antibody detection.

\textbf{Procedure:}

Chinese children under 18 years of age with neutropenia [?] 1.5 x
10\textsuperscript{9}/L lasting 6 months or more in our single center
were recruited into the study between 2016 to 2018. Patients with
secondary causes of neutropenia were excluded. Blood for anti-neutrophil
antibody and genotyping was taken once at the time of recruitment and
subsequently when neutropenia recovered to [?] 1.5 x
10\textsuperscript{9}/L. A combination of two in-house methods including
GIFT, GAT and commercial kit LABScreenTM multipanel were used for the
detection of antibodies. The age of onset, age of recovery, duration of
neutropenia, gender, serial neutrophil counts, incidence of invasive
infection, use of G-CSF were examined.

\textbf{Results:}

Using combined testing methods, anti-neutrophil antibody was detected in
30.8 \% of patients and positivity was associated with more severe
neutropenia, higher likelihood of infection and slower and later
recovery compared to those without antibodies.

\textbf{Conclusions:}

The presence of anti-neutrophil antibody was useful in predicting the
clinical course of patients with AIN. The use of combined testing
methods increased the detection rate.

\textbf{INTRODUCTION}

Chronic benign neutropenia (CBN) of infancy and childhood is
characterized by a self-limiting low neutrophil count below 1.5 x
10\textsuperscript{9}/L lasting for at least 6 months.(1) The condition
encompasses both autoimmune neutropenia (AIN) and chronic idiopathic
neutropenia (CIN). There continues to be a confusing overlap in the
nomenclature and classification among the immune neutropenia syndromes
despite sharing a similar benign clinical course, differing only in the
presence or absence of anti-neutrophil antibodies.(2) The difficulty in
differentiating the two lies with the variable accuracy and availability
of the numerous testing modalities available for antibody detection (3)
namely classical tests such as granulocyte agglutination tests (GAT),
immunofluorescence tests (GIFT), monoclonal antibody-specific
immobilization of granulocyte antigens (MAIGA) and newer bead-based
assay such as LABScreen MULTI (One Lamda, Inc). The detection rate of
anti-neutrophil antibodies in patients with CBN could be as high as
98-100\% when several testing methods are used. (1, 4) According to an
earlier cohort study by our group, Chinese children with AIN share
similar clinical characteristics with patients of other western series,
including the age of onset (9 months) and male to female ratio (4:6).(1,
4, 5) Among the 24 children studied, they all experienced a relatively
benign course regardless of their remission status.(5) Whether
anti-neutrophil antibody testing plays a role in establishing a
diagnosis, predicting outcome or have implications on follow up or
management remains controversial, with various studies demonstrating
conflicting evidence.(1, 6-8) This prospective study aims to determine
the prevalence of anti-neutrophil antibody among a larger group of
Chinese children using multiple modalities of testing and whether the
presence or absence of antibodies has implications on the clinical
characteristics and prognosis of these patients.

\textbf{METHODS}

This is a prospective cohort study conducted at a single center in the
Department of Paediatrics and Adolescent Medicine, Queen Mary Hospital,
Hong Kong. Prior approval with the hospital Clinical Research Ethics and
Institution Review Board was received HKU/HKWC IRB UW 15-379. Chinese
children who were under 18 years old of age and found to have
neutropenia, defined as absolute neutrophil count (ANC) [?] 1.5 x
10\textsuperscript{9}/L lasting 6 months or more, were recruited into
the study between 2016 to 2018. Information sheets are given and
explained and informed consent was obtained. We excluded all patients
with other causes of neutropenia for example: congenital neutropenia,
cyclic neutropenia, neutropenia `secondary' to allo-immunity, or other
immune or systemic diseases, because the etiologies, clinical
manifestations, natural courses, and prognosis of these disease entities
were different from those of CBN. Blood for anti-neutrophil antibody and
genotyping was taken once at the time of recruitment and subsequently
again when neutropenia recovered, which was defined as ANC [?] 1.5 x
10\textsuperscript{9}/L for more than 3 months. These patients were
followed up every 3 months with regular blood count monitoring for the
duration they are neutropenic, anti-neutrophil antibody was repeated
when the neutrophil count recovered. The cohort was followed up between
the period of April 2016 until April 2021. A initial screening with
LABScreen MULTI(One Lambda, Canoga Park, Ca, USA) was first performed
and positive samples were then further tested by a combination of two
in-house methods including GIFT, GAT for the detection of
anti-neutrophil antibodies. GIFT detects antibodies anchored on the
patient's neutrophils using fluorescent labelled anti human IgG and can
be detected by flow cytometry or by fluorescence microscopy. Whilst GAT
involves incubating patient's serum with neutrophils followed by
microscopic evaluation for leucoagglutination. LABScreen MULTI is a
multiplex commercial assay which allows for the simultaneous detection
of human leucocyte antigen (HLA) and human neutrophil antigen (HNA)
antibodies, where patient's serum is mixed with microbeads coated with
HNA and HLA antigens, bound antibodies were detected with
R-phycoerythrin (PE)- conjugated anti-Human IGG. Florescence was
detected with a Luminex flow analyzer and evaluated with HLA
Fusion\textsuperscript{TM} software (One Lamda).

A total of 100 participants were identified during the study period of
April 2016 until April 2021. One was excluded as he defaulted further
blood taking after the initial recruitment and testing, 6 were excluded
as non-ethnic Chinese and 12 excluded for other causes of neutropenia
found later on. Secondary causes for exclusion included: severe aplastic
anaemia, severe congenital neutropenia, Schwachman- Diamond Syndrome,
X-linked chronic granulomatous disease, acute lymphoblastic leukaemia.
81 participants with chronic benign neutropenia were qualified for the
study. The age of onset, age of recovery, duration of neutropenia,
gender, serial neutrophil counts, incidence of invasive infection, use
of Granulocyte- colony stimulator factory (G-CSF) were examined.
Invasive infection was defined arbitrarily as infection requiring
hospitalization plus either surgical intervention or intravenous
therapy.

The Kaplan-Meier method was used to estimate median overall recovery
time for the neutropenia patients. Two-sided 95\% confidence intervals
(CIs) for the median survival time were calculated by the
Brookmeyer-Crowley method. Cox regression analysis was used to determine
the relationship of the anti-neutrophil antibody status with the
recovery ratio, adjusting for the follow up time and their age of onset.
Demographic information and results are presented as group medians,
absolute counts and 95th interpercentile ranges or percentages. Stepwise
multivariate logistic regression with the forward selection method was
used to detect independent associations with the outcomes of interest,
and P \textless{} 0.05 was considered significant. Statistical
comparisons were completed using SPSS (IBM, Armonk, NY, USA) and JMP
(SAS Institute, Cary, NC, USA)

\textbf{RESULTS}

The baseline characteristics of the 81 participants were similar in the
gender distribution (Female=37 (45.7\%), the median age of onset of
neutropenia was 3.7 months (1.1- 48)). The majority of patients
presented with infection (n=39, 48.1\%), predominantly viral infection
(n= 26, 66.7\%), whilst invasive infection in the form of abscess made
up 15.4\%. No patients from this cohort developed bacteremia, invasive
respiratory, central nervous system, urinary tract or bone infection at
presentation. The second most common presentation was incidental finding
from blood taking in neonates with prolonged neonatal jaundice (n= 27,
33.3\%), the rest made up by incidental finding during investigation for
other conditions including failure to thrive, premature thelarche,
congenital cataract and per rectal bleeding. Among these 27 infants
picked up by neonatal jaundice screening, only 3/27 had detectable
anti-neutrophil antibodies, 2/27 had persistent neutropenia during the
period of follow up whilst the rest recovered, their median time to
recover was 1.61 years which was shorter than the 2.65 years of the
whole cohort. In the 81 participants with presumed CBN, anti-neutrophil
antibodies were detected in 30.9\% (n=25) of individuals. Antibodies
against HNA-1a was detected in 21 individuals, whilst 4 were against
HNA-3a. When comparing those with anti-neutrophil antibodies versus
without (Table 1), AIN patients were significantly older in age of onset
(median, 6.7 versus 2.1 months, p=0.0029), more likely to have severe
neutropenia on presentation (neutrophil count \textless{}0.5 x
10\textsuperscript{9}/L, p=0.0437), more individuals with profound
neutropenia (neutrophil count \textless{}1 x 10\textsuperscript{9}/L,
p=0.0152) with the lowest neutrophil count also found to be lower (0.1 x
10\textsuperscript{9}/L versus 0.4 x 10\textsuperscript{9}/L, p=0.0005)
and more likely to have invasive infection (n= 6, 24\% versus n=4,
7.1\%, p=0.041). The majority of invasive infection occurred in the
first year of diagnosis. Abscess was the most common presentation and
involvement varied from skin, submandibular, perianal and in the finger.
Methicillin-sensitive Staphylococcal aureus was the most commonly
cultured bacteria.

The time for 50\% recovery probability for this cohort of neutropenia
patients was 2.34 years from onset (95\% C.I. 1.98 to 2.71). (Fig. 1A)
Furthermore, anti-neutrophil antibody positive individuals were found to
recover slower and later, based on the reverse Kaplan-Meier curve, it
can be deduced that at 5 years follow up, 87.9\% of anti-neutrophil
antibody negative individuals recovered versus 69\% of antineutrophil
antibody positive individuals (p=0.035). (Fig. 1B) Cox regression
analysis found that the anti-neutrophil antibody status was
independently associated with neutropenia recovery, where those with
antibody-negative status had a 1.87 times higher chance of neutropenia
recovery compared to antibody-positive individuals irrespective of
follow-up time and age of onset.

Amongst the 81 samples processed, other than for 11 samples, the rest of
the results were concordant amongst the three tests used. Among the
discrepant samples, LABScreen MULTI detected anti-neutrophil antibodies
in 9 samples, which was not detected by GIFT and GAT. Whilst for 2
samples, LABScreen MULTI was negative and GIFT or GAT detected
anti-neutrophil antibodies against HNA-3a antigen.

\textbf{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.

\textbf{CONFLICTS OF INTEREST STATEMENT}

Sophie HY Lai declares no conflicts of interest (Conflicts of interest:
none).

Queenie WS declares no conflicts of interest (Conflicts of interest:
none).

Jaime S Rosa Duque declares no conflicts of interest (Conflicts of
interest: none).

Daniel KL Cheuk declares no conflicts of interest (Conflicts of
interest: none).

Jenny CY Ho declares no conflicts of interest (Conflicts of interest:
none).

Stephen KF Cheung declares no conflicts of interest (Conflicts of
interest: none).

Patrick Chu declares no conflicts of interest (Conflicts of interest:
none).

Pamela Lee declares no conflicts of interest (Conflicts of interest:
none).

Janette SY Kwok declares no conflicts of interest (Conflicts of
interest: none).

Shau-Yin Ha declares no conflicts of interest (Conflicts of interest:
none).

\textbf{Acknowledgements:}

Transplantation and Immunogenetics Division, Queen Mary Hospital, Hong
Kong Wilfred Wong contributed with data analysis

Naomi WT Wong contributed with data analysis

\textbf{ETHICAL APPROVAL}

Consent has been obtained and no approval was required for this case
report.

\textbf{FUNDING SOURCES}

Donation fund to Haematology and Oncology team, Queen Mary Hospital,
Hong Kong

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\textbf{LEGENDS}

Figure 1A. This reverse Kaplan-Meier curve demonstrates a recovery rate
of 50\% at 2.34 years follow up with 95\% C.I. 1.98 -- 2.71. Censored
individuals refer to those inviduals who had not recovered at the time
of analysis.

Figure 1B. The two reverse Kaplan-Meier curve of anti-neutrophil
antibody positive and negative patients are compared and demonstrates
that antibody positive patients recovered slower and later compared to
their antibody negative counterparts.

\textbf{Hosted file}

\verb`Table 1.docx`  available at \url{https://authorea.com/users/359987/articles/565159-significance-of-anti-neutrophil-antibody-in-chronic-benign-neutropenia-in-chinese-children}

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