Brazilian Journal of Medical and Biological Research (2) 33: 13-113 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, 235 935-3 Porto Alegre, RS Brasil Fax: 55-51-316-81 E-mail: 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
14 M. Burin et al. Material and Methods Protocol and sample preparation Braz J Med Biol Res 33(9) 2
Influence of sample handling on the measurement of lysosomal enzymes 15 Enzyme assays Statistical analysis Braz J Med Biol Res 33(9) 2
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 3 25 24 18 12 6 asa B-gal hex (x.1) %hex A 2 15 1 5 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
Influence of sample handling on the measurement of lysosomal enzymes 17 4 32 24 16 8 4 Arylsulfatase A (leukocytes) 6/8 h 3/32 h 54/56 h ß-Galactosidase (leukocytes) 5 4 3 2 1 32 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. 32 256 24 192 16 8 128 64 Enzyme activities 4 32 6/8 h 3/32 h 54/56 h Hexosaminidase (leukocytes) 12 96 6/8 h 3/32 h 54/56 h % Hex A (plasma) 24 72 16 48 8 24 6/8 h 3/32 h 54/56 h 6/8 h 3/32 h 54/56 h 12 % Hex A (leukocytes) 96 72 48 24 1% Heparin 5% Heparin Heparinized syringe 6/8 h 3/32 h 54/56 h Storage time Braz J Med Biol Res 33(9) 2
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. 2 1 3 2 Arylsulfatase A (leukocytes) ß-Galactosidase (leukocytes) 12 96 72 48 24 2 16 12 ß-Glucuronidase (plasma) Hexosaminidase (plasma) 1 8 4 Enzyme activities 3 2 1 Hexosaminidase (leukocytes) 12 96 72 48 % Hex A (plasma) 24 9 8 7 6 5 4 3 2 1 % Hex A (leukocytes) Room temperature (mail) Refrigerated (air company) Mailing time Braz J Med Biol Res 33(9) 2
Influence of sample handling on the measurement of lysosomal enzymes 19 3 2 1 Arylsulfatase A (leukocytes) ß-Glucuronidase (plasma) 15 12 9 6 3 96 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) 144 2 1 18 72 36 Enzyme activities 3 Hexosaminidase (leukocytes) 1 % Hex A (plasma) 96 h 2 1 8 6 4 2 96 h 1 % Hex A (leukocytes) 8 6 4 Refrigerated Room temperature 2 Storage time Braz J Med Biol Res 33(9) 2
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. 25 2 15 1 5 4 Arylsulfatase A (leukocytes) ß-Galactosidase (leukocytes) 18 144 18 72 36 18 ß-Glucuronidase (plasma) Hexosaminidase (plasma) 32 24 144 18 16 72 8 36 Enzyme activities 3 24 Hexosaminidase (leukocytes) 1 8 % Hex A (plasma) 18 6 12 4 6 2 1 % Hex A (leukocytes) 8 6 4 Heparinized syringe ACD 2 Storage time Braz J Med Biol Res 33(9) 2
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
112 M. Burin et al. Braz J Med Biol Res 33(9) 2
Influence of sample handling on the measurement of lysosomal enzymes 113 References 1. Tager JM, Jonsson LM, Aerts JG, Oude EJ, Schram AW, Erickson AH & Barranger JA (1984). Metabolic consequences of genetic defects in lysosomes. Biochemical Society Transactions, 12: 92-95. 2. Lowden JA (1982). Enzymological diagnosis of lysosomal storage diseases. In: Willey AM, Carter TP, Kelly S & Porter IH (Editors), Clinical Genetics - Problems in Diagnosis and Counseling. Academic Press, New York. 3. Wenger DA & Williams C (1991). Screening for lysosomal disorders. In: Hommes FA (Editor), Techniques in Diagnostic Human Biochemical Genetics - A Laboratory Manual. Wiley-Liss Inc., New York. 4. Landels EC, Ellis IH, Bobrow M & Fensom AH (1991). Tay-Sachs disease heterozygote detection: use of centrifugal analyser for automation of hexosaminidase assays with two different artificial substrates. Journal of Medical Genetics, 28: 11-19. 5. Bailey DN & Bove JR (1975). Chemical and hematological changes in stored CPD blood. Transfusion, 15: 244-249. 6. Sasakawa S & Tokunaga E (1976). Physical and chemical changes of ACD preserved blood: a comparison of blood in glass bottles and plastic bags. Vox Sanguinis, 31: 199-21. 7. Lombardo A, Goi G, Guagnellini E, Fabi A, Sciorelli G, Burlina AB & Tettamanti G (1984). Behaviour of several enzymes of lysosomal origin in human plasma during whole blood storage. Clinica Chimica Acta, 143: 343-353. 8. Skoog WA & Beck WS (1956). Studies on the fibrogen, dextran and phytohemagglutinin methods of isolating leukocytes. Blood, 11: 436-454. 9. Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193: 265-275. 1. Suzuki K (1977). Globoid cell leukodystrophy (Krabbe disease) and GM1-gangliosidosis. In: Glew RH & Peters SP (Editors), Practical Enzymology of Sphingolipidoses. A.R. Liss, New York. 11. Singer JD, Cotlier E & Krimmer R (1973). Hexosaminidase A in tears and saliva for rapid identification of Tay-Sachs diseases and its carriers. Lancet, 2: 116-119. 12. Beaudet AL, Diferrante NM, Ferry GD, Nichols BL & Mullins CE (1975). Variation in the phenotypic expression of ß-glucuronidase deficiency. Pediatrics, 86: 388-394. 13. Lee-Vaupel M & Conzelmann E (1987). A simple chromogenic assay for arylsulfatase A. Clinica Chimica Acta, 164: 171-18. 14. Kolodny EH & Mumford RA (1976). Human leukocyte acid hydrolases: characterization of eleven lysosomal enzymes and study of reaction conditions for their automated analysis. Clinica Chimica Acta, 7: 247-257. 15. Avila JL & Convit J (1975). Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro. Biochemical Journal, 152: 57-64. 16. Avila JL & Convit J (1976). Physicochemical characteristics of the glycosaminoglycan lysosomal enzyme interaction in vitro. Biochemical Journal, 16: 129-136. 17. Mikulíková D & Trnavský K (1982). Influence of a glycosaminoglycan polysulfate (arteparon) on lysosomal enzyme release from human polymorphonuclear leukocytes. Zeitschrift für Rheumatologie, 41: 5-53. 18. Lund-Hansen T, Hoyer PE & Andersen H (1984). A quantitative cytochemical assay of ß-galactosidase in single cultured human skin fibroblasts. Histochemistry, 81: 321-33. 19. Singer HS, Nankervis GA & Schafer IA (1972). Leukocyte beta-galactosidase activity in the diagnosis of generalized GM1 gangliosidosis. Pediatrics, 49: 352-361. 2. Raghavan S, Gajewski A & Kolodny EH (1977). GM1-Ganglioside ß-galactosidase in leukocytes and cultured fibroblasts. Clinica Chimica Acta, 81: 47-56. 21. Mutoh T, Kiuchi K, Sobue I & Naoi M (1984). Application of a GM1 ganglioside ß-galactosidase microassay method to diagnosis of GM1 gangliosidosis. Clinica Chimica Acta, 14: 223-23. 22. Triebling A, Dlugosz J, Brzozowski J, Lukaszewicz W & Gabryelewicz A (1979). Heparin effect on the activity of certain lysosomal hydrolases in dog pancreas (in vivo investigations). Acta Physiologica Polonica, 3: 527-532. 23. Branum E, Cummins L, Bartilson M, Hopper M, Pruett S & O Brien JF (1988). Effect of two anticoagulants on leukocyte yield and function, and on lysosomal enzyme activity. Clinical Chemistry, 34: 11-113. 24. Schechter DC & Swan H (1962). Biochemical alterations of preserved blood. Archives of Surgery, 84: 269-276. 25. Moore SB, Beckala H, DeGoey S & Leavelle D (1981). A report on the use of ACD (solution B) as whole blood transport medium for recovery of lymphocytes for HLA typing. In: Zachary AA & Braun WE (Editors), Laboratory Manual, American Association for Clinical Histocompatibility Testing. American Association for Clinical Histocompatibility Testing, New York. 26. Walker RH (199). Blood and blood components: preparation, storage, and shipment. In: Walker RH (Editor), Technical Manual. American Association of Blood Banks, Arlington. 27. Draper RK, Fiskum GM & Edmond J (1976). Purification, molecular weight, amino acid and subunit composition of arylsulfatase A from human liver. Archives of Biochemistry and Biophysics, 177: 525-538. 28. Lombardo A, Caimi L, Marchesini S, Goi GC & Tettamanti G (198). Enzymes of lysosomal origin in human plasma and serum: assay conditions and parameters influencing the assay. Clinica Chimica Acta, 18: 337-346. Braz J Med Biol Res 33(9) 2