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.