Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

Transcription

1 Brazilian Journal of Medical and Biological Research (2) 33: Influence of sample handling on the measurement of lysosomal enzymes ISSN 1-879X 13 Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes M. Burin 1, C. Dutra-Filho 2, J. Brum 3, T. Mauricio 4, M. Amorim 5 and R. Giugliani 1 1 Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre and 2 Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil 3 Hospital Sarah Kubischeck, Brasília, DF, Brasil 4 Serviço de Genética Médica, Universidade Federal da Paraíba, João Pessoa, PB, Brasil 5 Departamento de Genética, Universidade Federal do Pará, Belém, PA, Brasil Correspondence M. Burin Serviço de Genética Médica Hospital de Clínicas de Porto Alegre Ramiro Barcelos, Porto Alegre, RS Brasil Fax: mburin@hcpa.ufrgs.br Research supported by CNPq. Received June 5, 1998 Accepted May 3, 2 Abstract Key words Lysosomal storage diseases Lysosomal enzymes Inborn errors of metabolism Reference laboratories Storage of blood samples Sample handling Introduction Braz J Med Biol Res 33(9) 2

2 14 M. Burin et al. Material and Methods Protocol and sample preparation Braz J Med Biol Res 33(9) 2

3 Influence of sample handling on the measurement of lysosomal enzymes 15 Enzyme assays Statistical analysis Braz J Med Biol Res 33(9) 2

4 16 M. Burin et al. Figure 1 - Comparison of leukocyte (A) and plasma (B) activities of different lysosomal enzymes when blood collection was performed with a plastic syringe (syr) or with a vacuum glass tube (vac). Values are the mean ± SEM and are reported as nmol h -1 mg protein -1 and nmol h -1 ml -1 for leukocytes and plasma, respectively. asa, Arylsulfatase A; B-gal, ß-galactosidase; hex (x.1), hexosaminidase; %hex A, hexosaminidase A percentage; B-glu, ß-glucuronidase. Enzyme activities A B asa B-gal hex (x.1) %hex A Leukocytes syr B-glu hex (x.1) %hex A Plasma vac Table 1 - Distribution of plasma and leukocytes from 6 individuals divided into two aliquots according to mode of transportation and time of processing. P<.5 compared to 24 h (chi-square test). Results Number of samples isolated x samples not isolated (in parentheses) Plasma Air company 45 (1) 1 () 5 () (refrigerated) Regular mail 35 () 1 () 15 (1) (room temperature) Leukocytes Air company 45 (1) 1 () 5 () (refrigerated) Regular mail 35 () 1 () 15 (1) (room temperature) Braz J Med Biol Res 33(9) 2

5 Influence of sample handling on the measurement of lysosomal enzymes Arylsulfatase A (leukocytes) 6/8 h 3/32 h 54/56 h ß-Galactosidase (leukocytes) ß-Glucuronidase (plasma) 6/8 h 3/32 h 54/56 h Hexosaminidase (plasma) Figure 2 - Effect of different heparin concentrations and storage times on the activity of lysosomal enzymes. Values are the mean ± SEM and are reported as nmol h -1 mg protein -1 and nmol h -1 ml -1 for leukocytes and plasma, respectively. Hexosaminidase A (Hex A) is reported as % hexosaminidase. P<.5 compared to 6/8 h at the same heparin concentration. P<.5 compared to the heparinized syringe group at the same storage time Enzyme activities /8 h 3/32 h 54/56 h Hexosaminidase (leukocytes) /8 h 3/32 h 54/56 h % Hex A (plasma) /8 h 3/32 h 54/56 h 6/8 h 3/32 h 54/56 h 12 % Hex A (leukocytes) % Heparin 5% Heparin Heparinized syringe 6/8 h 3/32 h 54/56 h Storage time Braz J Med Biol Res 33(9) 2

6 18 M. Burin et al. Figure 3 - Effect of different means of transportation and shipping times on the activity of lysosomal enzymes. Values are the mean ± SEM and are reported as nmol h -1 mg protein -1 and nmol h -1 ml -1 for leukocytes and plasma, respectively. Hexosaminidase A (Hex A) is reported as % hexosaminidase. P<.5 compared to 24 h considering the same means of transportation. P<.5 compared to samples shipped by air considering the same shipping time Arylsulfatase A (leukocytes) ß-Galactosidase (leukocytes) ß-Glucuronidase (plasma) Hexosaminidase (plasma) Enzyme activities Hexosaminidase (leukocytes) % Hex A (plasma) % Hex A (leukocytes) Room temperature (mail) Refrigerated (air company) Mailing time Braz J Med Biol Res 33(9) 2

7 Influence of sample handling on the measurement of lysosomal enzymes Arylsulfatase A (leukocytes) ß-Glucuronidase (plasma) h Figure 4 - Effect of different temperatures and times of storage. Values are the mean ± SEM and are reported as nmol h -1 mg protein -1 and nmol h -1 ml -1 for leukocytes and plasma, respectively. Hexosaminidase A (Hex A) is reported as % hexosaminidase. P<.5 compared to 24 h. P<.5 compared to the refrigerated group at the same storage time. 3 ß-Galactosidase (leukocytes) 18 Hexosaminidase (plasma) Enzyme activities 3 Hexosaminidase (leukocytes) 1 % Hex A (plasma) 96 h h 1 % Hex A (leukocytes) Refrigerated Room temperature 2 Storage time Braz J Med Biol Res 33(9) 2

8 11 M. Burin et al. Figure 5 - Effect of different anticoagulants for samples processed at different times after collection. Values are means ± SEM and are reported as nmol h -1 mg protein -1 and nmol h -1 ml -1 for leukocytes and plasma, respectively. Hexosaminidase A (Hex A) is reported as % hexosaminidase. P<.5 compared to the heparinized group at the same storage time. ACD, Acid citrate-dextrose Arylsulfatase A (leukocytes) ß-Galactosidase (leukocytes) ß-Glucuronidase (plasma) Hexosaminidase (plasma) Enzyme activities 3 24 Hexosaminidase (leukocytes) 1 8 % Hex A (plasma) % Hex A (leukocytes) Heparinized syringe ACD 2 Storage time Braz J Med Biol Res 33(9) 2

9 Influence of sample handling on the measurement of lysosomal enzymes 111 Discussion Table 2 - Distribution of leukocyte samples according to time between collection and processing and storage temperature. P<.5 compared to 24 h (chi-square test). RT, Room temperature. Storage condition Number of samples isolated x samples not isolated (in parentheses) 96 h RT 1 () 8 (2) 4 (6) (1) 4 o C 1 () 1 () 1 () 1 () Braz J Med Biol Res 33(9) 2

10 112 M. Burin et al. Braz J Med Biol Res 33(9) 2

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