Methods:
We performed a prospective observational study after obtaining an
institutional ethical clearance (INT/IEC/2017/000299). Informed written
consent and wherever applicable, assent was taken from parents and
patients. Children who presented to our centre and diagnosed as HUS were
screened. Infection associated and secondary causes of HUS (HIV,
malignancy, cobalamin deficiency, drug induced, autoimmune diseases)
were ruled out. All those with aHUS were enrolled in the study.
Relevant history was documented. Examination was done at enrolment and
blood pressure was staged according to AAP guidelines.[2]
Investigations including complete blood count (SYSMEX XN-1000; Japan)
and renal function was performed at three points of care namely at
admission, minimum value reached and maximum value achieved during
hospital stay. We categorized anemia according to WHO classification.
[3] Children who had platelet count less than 150 x
109cells/L were considered to have thrombocytopenia,
which was categorized as mild (100-150x109/L),
moderate (50-100x109/L) and severe
(<50x109/L). Fragmented red blood cells or
schistocytes were documented according to the standards of ICSH
recommendations by an expert Hematopathologist. [4] Lactate
dehydrogenase (LDH) level was also recorded, >500 U/L
indicative of hemolysis. AKI was staged according to KDIGO guidelines.
[5] Estimated glomerular filtration rate (eGFR) was calculated using
bedside modified Schwartz formula which utilizes length/ height of child
in centimetre and serum creatinine values in milligram per decilitre.
[6] Urine routine microscopy was performed to look for microscopic
hematuria. Proteinuria was analysed as negative, non-nephrotic and
nephrotic range.
Human immunodeficiency virus (HIV) serology, anti-nuclear antibodies
(ANA), and blood culture were done to rule out secondary causes like
infection triggered or autoimmune etiologies. Stool culture and STEC-PCR
was performed if there was a prodrome of diarrhea to ensure that STEC
HUS was not included.
All children with aHUS were worked up for underlying complement defects
(i). Complement C3 and C4 was performed by end-point nephelometry.
Interpretation was according to age-wise normative data which were
available from our immunology laboratory.
(ii). Anti-FH antibody (IgG) testing was done by ELISA method using
VIDITEST ELISA kit (Intra-Assay: CV < 6%, Inter-Assay
<10%; Detection limit- 0.6 AU/ml). 26 AU/ml was taken as the
cut off.
(iii).Factor B, H and I were also done by ELISA according to the
manufacturers (SinoGeneclon Ltd.) described method.
(vi).CD46 {membrane co-factor protein (MCP)} was estimated by flow
cytometry. The median fluorescent index was assessed by flow-cytometry
in all the leucocytes (neutrophils, monocytes and lymphocytes). It was
measured in both stimulated and unstimulated phase. Later δ MFI and
staining index were calculated for standardization.
For analysis of complement parameters by flow-cytometry, 40 healthy
children were also analysed and reference range was derived from the
mean of δMFI expression of those 40 healthy controls.
Statistical analysis: Data was analyzed using SPSS Version 23
(Chicago, IL). Standard statistical tests were used for analysis. All
quantitative data were analyzed for their normal distribution using
tests of normality (Kolmogrov test). Descriptive statistics was used for
baseline comparison. For normally distributed data (platelet count at
admission, urea at admission, C3, C4) independent t-test was used for
comparison of means. For skewed data (age, creatinine) non-parametric
test was used for comparison of medians. For continuous independent
variables, unpaired t test was performed while for categorical
variables, Chi-square tests or Fisher’s Exact tests (if cell frequency
< 5), was used.