Chitotriosidase determination in plasma and in dried blood spots: a comparison using two different substrates in a microplate assay.
BACKGROUND
Chitotriosidase (CT) is a macrophage glycosylhydrolase referred as a biochemical marker on diagnosis and prognosis for Gaucher Disease (GD). The aim of the study was to validate a microplate assay for plasma and dried blood spots on filter paper (DBS) for CT activity determination and to compare CT activity using 4-methylumbelliferyl-b-D-N,N',N''-triacetyl-chitotrioside (4MU-C3) and 4-methylumbelliferyl-deoxychitobiose (4MU-dC2) as substrates.


METHODS
Heparinized blood was collected from 12 healthy volunteers (HV) and 14 treated GD patients. Total blood was used to prepare DBS and plasma was obtained by centrifugation. CT activity measurements were done on tube and microplate by a fluorimetric assay using plasma and punches of the DBS samples.


RESULTS
The use of 4MU-C3 presented a good correlation among plasma and DBS when analyzing the groups altogether (Pearson=0.76), but the correlation was weaker when we isolated GD patients (Pearson=0.67). The use of 4MU-dC2 increased the correlation among the samples for this group of individuals (Pearson=0.89).


CONCLUSIONS
The possibility of DBS assay facilitates sample transport and storage and CT activity determination on a microplate assay improves laboratory routine. The use of 4MU-dC2 may be more appropriate for GD patients diagnosis on DBS.
Rodrigues
Mayra D B
MD
Department of Pediatrics, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil.
de Oliveira
Allan C
AC
Müller
Karen B
KB
Martins
Ana Maria
AM
D'Almeida
Vânia
V
D000293:Adolescent; D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods*; D016022:Case-Control Studies; D002648:Child; D005260:Female; D005776:Gaucher Disease / Q000097:blood / Q000175:diagnosis / Q000628:therapy; D006596:Hexosaminidases / Q000097:blood* / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
D014312:Trisaccharides; D006596:Hexosaminidases; C086400:chitotriosidase