FAMILY HEART STUDY LABORATORY TESTS

 

  1. Albumin, Serum
  2.  

    Albumin was measured by a thin film adaptation of a bromcresol green colorimetric procedure using the Vitros analyzer (Johnson & Johnson Clinical Diagnostics, Inc. Rochester NY 14650).

     

    Corcoran RM, Durnan SM. Albumin determination by a modified bromocresol green method. Clin Chem 1977; 23(4): 765.

     

  3. Angiotensin converting enzyme genotyping
  4.  

    (Performed by Dr. Mark Leppert’s Laboratory)

     

  5. Angiotensinogen Genotyping
  6. a. Angiotensinogen M235T Genotyping

     

    Angiotensinogen genotyping of the M235T polymorphism was performed using the method of Russ et al., 1993. In this method, DNA is amplified using primers designed to insert a restriction endonuclease site into the product. The product is digested with Sau96, followed by electrophoresis of the digested DNA on agarose gel and evaluation based on ethidium bromide staining of the separated products.

     

    Russ AP, Maerz W, Ruzicka V, Stein U, and Werner G. Rapid detection of the hypertension-associated Met235-Thr allele of the human angiotensinogen gene. Human Molecular Genetics 1993;2:609-610.

     

    b. Angiotensinogen -6 Genotyping

     

    (Performed by Dr. Mark Leppert’s Laboratory.)

     

  7. Apolipoprotein AI Quantitation, EDTA plasma
  8.  

    Apolipoprotein A1 quantitation is performed using the Beckman Array (Beckman, Brea, CA 92621-6209) kinetic rate nephelometric method which is designed to measure the rate of increase in intensity of light scatter from particles suspended in solution as a results of complexes formed during an antigen-antibody reaction.

     

    Stein EA. Lipids, lipoproteins, and apolipoproteins. In: Tietz NW, ed. Fundamentals of Clinical Chemistry 3rd ed. Philadelphia: WB Saunders, 1987: 478-479.

     

  9. Apolipoprotein B Quantitation, EDTA plasma
  10.  

    Apolipoprotein B quantitation was performed using the Beckman Array (Beckman, Brea, CA 92621-6209) kinetic rate nephelometric method which is designed to measure the rate of increase in intensity of light scatter from complexes, suspended in solution, formed as a result of an antigen-antibody reaction.

     

    Stein EA. Lipids, lipoproteins, and apolipoproteins. In: Tietz NW, ed. Fundamentals of Clinical Chemistry 3rd ed. Philadelphia: WB Saunders, 1987: 478-479.

     

  11. Apolipoprotein E Genotyping
  12.  

    Apolipoprotein E (apo E) genotyping is performed using PCR to amplify a 267 bp fragment from exon 3 of the apo E gene (Reymer et al., 1995). The PCR product is digested with the Hha I restriction endonuclease (an isoschizomer of Cfo I) which results in a specific banding pattern for the three isoforms of the apo E protein when separated by polyacrylamide gel electrophoresis and silver stained.

     

    Reymer WA, Groenemeyer BE, Burg R, Kastelein J. Apolipoprotein E genotyping on agarose gels. Clin Chem 1995; 41: 1046-1047.

     

  13. Calcium, Total, Serum
  14.  

    Total serum calcium was measured by a thin film adaptation of a Arsenazo III dye-binding, colorimetric method using the Vitros analyzer (Johnson & Johnson Clinical Diagnostics, Inc. Rochester NY 14650).

     

    Leary NO, Pembroke A, Duggan PF. Single stable reagent (Arsenazo III) for optically robust measurement of calcium in serum and plasma. Clin Chem 1992; 38(6): 904-908.

     

  15. Cholesterol, Total, EDTA plasma
  16.  

    Cholesterol was performed using a commercial cholesterol oxidase method on a Roche COBAS FARA centrifugal analyzer (Boehringer Mannheim Diagnostics , Indianapolis, IN 46250). In this method (Allain et al., 1974), cholesterol esters are hydrolyzed to free cholesterol by cholesterol esterase. Free cholesterol is then oxidized by cholesterol oxidase producing hydrogen peroxide which when combined with 4-aminophenazone and phenol forms a red chromophore. Formation of this chromophore is measured at 520 nm at 37 C and is directly proportional to the cholesterol concentration of the sample.

     

    Allain CC, Poon LS, Chan C, Richmond W, and Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20(4): 470-475.

     

  17. Creatinine, Serum
  18.  

    Creatinine was measured by a thin film adaptation of the amidohydrolase enzymatic method using the Vitros analyzer (Johnson & Johnson Clinical Diagnostics, Inc. Rochester NY 14650).

     

    Mauck JC, Mauck L, Novros J, Norton GE. Development of a single slide Kodak Ektachem thin-film assay for serum and urine creatinine. Clin Chem 1986; 32(6): 1197.

     

  19. C-Reactive Protein, Serum
  20.  

    C-Reactive Protein (CRP) was analyzed on serum by an ELISA method calibrated with WHO reference material. The intra- and interassay CV's for this method were 3.0% and 6.0% respectively.

     

    Macy EM, Hayes TE, Tracy RP. Variability in the measurement of C-reactive

    protein in healthy subjects: implications for reference intervals and epidemiological applications. Clin Chem 1997;43(1):52-8.

     

  21. Cystathionine Beta-Synthase (CBS) Genotyping
  22.  

    The method of Tsai et al., 1996 which detects both the T833C and the G919A mutation was used for genotyping CBS. Exon 8 of the CBS gene is amplified and the PCR product is digested with Bsr I to detect the T833C mutation and with Alu I to detect the G919A mutation. The digested product is separated on a 12% polyacrylamide gel and silver stained.

     

    Tsai MY, Bignell M, Schwichtenberg K, Hanson, N. High prevalence of a mutation in the cystathionine-b-synthase gene. Am J Hum Genet 1996; 59: 1262-1267.

     

  23. Factor V (Leiden) Genotyping
  24.  

    Factor V Leiden genotyping was performed using a modification of the method of Bertina et al. in which a 192 base pair PCR product of exon 10 of the factor V gene was digested with Mnl I restriction endonuclease. The digest was analyzed on a silver stained polyacrylamide gel. PCR product from the wildtype gene has an endonuclease recognition site resulting in 92 bp and 36 bp fragments while PCR product from the Leiden gene does nothave this recognition site resulting in a 128 bp fragment. (There is another cut site in this PCR product that gives 64 bp product regardless of the presence or absence of the Leiden mutation.)

     

    Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, deRonde H et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-67.

     

  25. Factor VII, Citrate plasma
  26.  

    Factor VII activity was measured using a functional assay on the MLA 700 analyzer (Medical Laboratory Automation, Inc., Pleasantville, NY 10570-9977). In this assay, dilutions of the sample or standard plasma are added to a substrate which is plasma deficient in factor VII with normal activity for the other factors. Clotting times are determined by adding thromboplastin C.

     

    Edson JR, Krivit W, White JG. Kaolin partial thromboplastin time: high levels of procoagulants producing short clotting times masking deficiencies of other procoagulants or low concentrates of anticoagulants. J Lab Clin Med 1967; 70(1): 463-470.

     

  27. Fibrinogen, Citrate plasma
  28.  

    Fibrinogen was determined by the method of Clauss et al., 1957 adapted to the MLA 800 analyzer (Medical Laboratory Automation, Inc., Pleasantville, NY 10570-9977) in which excess bovine thrombin is added to dilutions of plasma converting the fibrinogen to a fibrin clot. The rate of clotting time is proportional to the amount of fibrinogen in the sample.

     

    Clauss A. Gerinnungphysiologische schnellmethode zur bestimmung des fibrinogens. Acta Haemat 1957; 17: 237-246.

     

     

  29. Folate, Serum
  30.  

    Folate was analyzed on the Access® automated immunoassay analyzer from Beckman (Chaska, MN 55318) using a competitve-binding receptor assay. After treatment of serum to release folate from endogenous binding proteins, sample folate competes with folic acid-alkaline phosphatase conjugate for binding sites on a limited amount of folate binding protein. Resulting complexes bind to a solid-phase via mouse anti-folate binding protein and are measured by a luminometer when reacted with the chemiluminescent substrate Lumi-Phos 530Ò.

     

    Fairbanks VF, Klee GG. Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry 3rd ed. Philadelphia: WB Saunders, 1999: 1696-1697.

     

  31. Glucose, Serum
  32.  

    Glucose was measured by a thin film adaptation of a glucose oxidase enzymatic, spectrophotometric procedure using the Vitros analyzer (Johnson & Johnson Clinical Diagnostics, Inc. Rochester NY 14650).

     

    Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen receptor. Ann Clin Biochem 1969; 6: 24.

     

  33. HDL-Cholesterol, EDTA plasma
  34.  

    HDL-cholesterol quantification was performed with the above described cholesterol method after precipitation of non-HDL-cholesterol with magnesium/dextran (Warnick et al. 1982).

     

    Warnick GR, Benderson J, Albers JJ. Dextrab sulfate-Mg 2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 1982; 28(6): 1379-1388.

     

  35. Homocysteine, Total, EDTA plasma
  36.  

    Total Homocysteine was measured by an HPLC method with a fluorescent detector (Araki et al., 1991). In this method, tri-n-butylphosphine is used to liberate thiol compounds from plasma proteins and reduce the disulfide bonds in homocystine and cystine to homocysteine and cysteine, respectively. After deproteinization with trichloroacetic acid and derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate, samples are separated on a Supelcosil LC-18-DB column and read by fluorometric detection.

     

    Araki A, Sako H. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1991; 565:441-446.

     

  37. Insulin, Serum
  38.  

    Insulin was performed by the coated-tube RIA method distributed by Diagnostic Products Corporation (Los Angeles, CA 90045). In this solid-phase RIA, insulin specific antibody is immobilized on the wall of a polypropylene tube. Radiolabeled antigen and antigen from the sample compete for a limited number of antibody binding sites. The amount of labeled antigen bound to antibody is inversely proportional to the amount of insulin in the sample.

     

    Yalow RS, Berson SA. Immunoassay of plasma insulin. Meth Biochem Anal 1964; 12: 69

     

     

  39. LDL-Cholesterol, Calculated
  40.  

    For samples with triglyceride levels less than 400 mg/dL, LDL-cholesterol was calculated using the Friedwald formula (Friedwald et al., 1972).

     

    Friedwald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma without the use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499.

     

  41. LDL-Cholesterol Quantitation by Ultracentrifugation
  42.  

    LDL-cholesterol quantitation was performed on EDTA plasma by ultracentrifugation (David et al., 1986) using a Beckman TLA 100 table top ultracentrifuge with a Beckman TLA 100.3 fixed angle rotor (Beckman, Brea, CA 92621-6209). In this method, 1.0 mL of EDTA plasma is sandwiched between two layers each containing 1.0 mL of 0.150 mol/L sodium chloride (d=1.006 g/mL). After centrifugation for 2.5 hours at 100,000 rpm, the top half of the tube which contains VLDL and any chylomicrons is removed and the lower half contents are washed into a volumetric flask which is then adjusted to 2.0 mL total volume. The LDL-cholesterol is calculated as the difference between the measured cholesterol in the volume-adjusted infranate times two minus the HDL-cholesterol measured in the original plasma.

     

    David JA, Naito NK. Separation of lipoprotein (Lp) fraction by the Beckman TL-100 table-top ultracentrifuge (UC). Clin Chem 1986; 32: 1094.

     

  43. Lewis Blood Group Typing
  44.  

    Lewis blood group phenotyping was performed by a hemagglutination method using commercially available monoclonal antibodies (Gamma Biologicals Inc. Houston, TX 77092).

     

    Mourant AE. A "new" blood group antigen of frequent occurrence. Nature 1946; 158: 237.

     

  45. Lipoprotein (a) , EDTA plasma
  46.  

    Lipoprotein (a) was performed using a commercial immunoprecipitin assay (DiaSorin, Stillwater, MN 55082-0285) on the COBAS FARA centrifugal analyzer (Boehringer Mannheim Corp., Indianapolis, IN 46250-0457). In this analysis, goat antibody monospecific for lipoprotein (a) is mixed with the sample. Insoluble antigen-antibody complexes are formed producing turbidity and increased light scatter.

     

    Rifai N, Bachorik PS, Albers JJ. Lipids, lipoproteins, and apolipoproteins. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry 3rd ed. Philadelphia: WB Saunders, 1999: 855.

     

  47. Magnesium, Serum
  48.  

    Magnesium was measured by a thin film adaptation of a formazan dye-binding, colorimetric method using the Vitros analyzer (Johnson & Johnson Clinical Diagnostics, Inc. Rochester NY 14650).

     

    Smith-Lewis MJ, Babb BE, Hilborn DA, Mauck JC, Norkus NS, Toner JL, Weaver MS. Thin-film colorimetric assay for magnesium. Clin Chem 1986; 32(6): 1200.

     

  49. Methylene Tetrahydrofolate Reductase (MTHFR) Genotyping
  50.  

    MTHFR genotyping was performed using the method of Frosst et al., 1995 which detects the C677T mutation. Exon 4 of the MTHFR gene is amplified and the PCR product is digested with Hinf I. The mutation creates a recognition sequence for Hinf I which digests the normal 198 bp product into a 175 bp and 23 bp fragments. The digested product is separated on a 12% polyacrylamide gel and stained with ethidium bromide.

     

    Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet 1995; 10: 111-113.

     

  51. Methionine Synthase Genotyping
  52.  

    (Performed by Dr. Rima Rozen’s Laboratory)

     

  53. Plasminogen Activator Inhibitor 1 (PAI-1) Quantitation, Citrate plasma
  54.  

    In this ELISA assay (Declerck et al., 1988), PAI-1 forms a sandwich by binding to both a mouse monoclonal anti-human PAI-1 antibody which is coated to microwells and to a second monoclonal anti-PAI-1 antibody coupled with peroxidase. The amount of bound peroxidase coupled antibody, which is determined by measuring the cleavage of ortho-phenylenediamine substrate, is directly related to the amount of PAI-1 present in the sample.

     

    Declerck PJ, Alessim C, Verstreken M, et al. Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay. Blood 1988; 71(1): 220-225.

     

  55. Sodium and Potassium, Serum
  56.  

    Analysis of serum sodium and potassium was performed by a thin film adaptation of an ion selective electrode method using the Vitros analyzer (Johnson & Johnson Clinical Diagnostics, Inc. Rochester NY 14650).

     

    Tietz NW, Pruden EL, Siggaard-Andersen O. Electrolytes, blood gases and acid-base balance. In: Tietz NW, ed. Fundamentals of Clinical Chemistry 3rd ed. Philadelphia: WB Saunders, 1987: 614-620.

     

  57. Triglyceride, EDTA plasma
  58.  

    Triglyceride was measured using Triglyceride GB reagent on the Roche COBAS FARA centrifugal analyzer (Boehringer Mannheim Corp., Indianapolis, IN 46250-0457). In this method, free glycerol is eliminated in an initial blank reaction that differs from the final reaction only in the omission of lipase and 4-aminophenazone. The initial reaction is followed by enzymatic hydrolysis of triglycerides with lipase and determination of the liberated glycerol by an enzymatic, colorimetric reaction of peroxide and 4-aminophenazone to form color that is directly proportional to the amount of triglycerides present in the sample.

     

    McGowan MW, Artiss JD, Strandbergh DR, Zork B. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 1983; 29(3): 538-542.

     

  59. Uric Acid, Serum
  60.  

    Uric acid was measured by a thin film adaptation of a uricase enzymatic method using the Vitros analyzer (Johnson & Johnson Clinical Diagnostics, Inc. Rochester NY 14650).

     

    Rock RC, Walker WG, Jennings CD. Nitrogen metabolites and renal function. In: Tietz NW, ed. Fundamentals of Clinical Chemistry 3rd ed. Philadelphia: WB Saunders, 1987: 684-686.

     

     

  61. Vitamin B6, EDTA plasma
  62.  

    Vitamin B6 analysis was performed using a commercial radioenzymatic assay (American Laboratory Products Co., Windham, NH 03087) which is a modification of the method described by Shin et al., 1981. In this assay, tridiated tryrosin is decarboxylated to tridiated tyramine by the vitamin B6 dependent enzyme, tryrosine apodecarboxylase. The amount of tridiated tyramine formed in this reaction is directly related to the amount of vitamin B6 present in the sample.

     

    Shin-Buehring Y, et al. A new enzymatic method for pyridoxyl-5’-phosphate determination. J Inter Metab Dis 1981;4:123.

     

  63. Vitamin B12
  64.  

    Vitamin B12 was analyzed on the Access® automated immunoassay analyzer from Beckman (Chaska, MN 55318) using a competitive-binding immunoenzymatic assay. After treatment of serum with alkaline potassium cyanide and dithiothreitol to convert all forms of vitamin B12 to the cyanocobalamin form, vitamin B12 in the sample binds to the intrinsic factor alkaline phosphatase conjugate, preventing the conjugate from binding to the solid phase anti-intrinsic factor. Separation and washing removes materials not bound to the solid phase, and the light generated by the reaction with the chemiluminescent substrate Lumi-Phos 530Ò as measured by a luminometer is inversely proportional to the concentration of vitamin B12 in the sample.

     

    Fairbanks VF, Klee GG. Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry 3rd ed. Philadelphia: WB Saunders, 1999: 1696-1697.

     

  65. VLDL-Cholesterol to Total Triglyceride Ratio

 

VLDL-cholesterol was performed on EDTA plasma by ultracentrifugation (David et al., 1986) using a Beckman TLA 100 table top ultracentrifuge with a Beckman TLA 100.3 fixed angle rotor (Beckman, Brea, CA 92621-6209). In this method, 1.0 mL of EDTA plasma is sandwiched between two layers each containing 1.0 mL of 0.150 mol/L sodium chloride (d=1.006 g/mL). After centrifugation for 2.5 hours at 100,000 rpm, the top half of the tube which contains VLDL and any chylomicrons is removed. Using the standard cholesterol method, cholesterol is then measured in this fraction and the ratio to the total triglycerides calculated.

 

David JA, Naito NK. Separation of lipoprotein (Lp) fraction by the Beckman TL-100 table-top ultracentrifuge (UC). Clin Chem 1986; 32: 1094.