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
109/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 109/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 FusionTM 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 < 0.05 was considered significant. Statistical
comparisons were completed using SPSS (IBM, Armonk, NY, USA) and JMP
(SAS Institute, Cary, NC, USA)