- Email: [email protected]
Chemiluminescence screening assays for erythrocytes and leukocytes in urine
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[28] Chemiluminescence Screening Assays for Erythrocytes and Leukocytes in Urine By VALERIE
J. BUSH, BRENDA J. HALLAWAY, THOMAS A . EBERT,
DAVID M. WILSON, and DENNIS J. O'KANE Introduction The use of chemiluminescence as a detection system within laboratory medicine, especially applied toward immunoassays, provides the advantages of high specificity and sensitivity, rapid analysis, low cost, and high stability. I Only recently, however, has chemiluminescence become practical as a screening system in urinalysis.2 Current, clinical methods used to detect erythrocytes and leukocytes in urine include manual microscopic analysis, dipsticks, and automated imaging. 3-5 These procedures are time-consuming, expensive, prone to error, and cannot exclude normal samples efficiently from analysis. Applying chemiluminescence screening systems for erythrocytes and leukocytes in urine has the potential to reduce the number of normal urine samples requiring additional diagnostic testing and may facilitate reprioritizing laboratory resources through the use of automation. The routine application of automation to urinalysis, however, is compromised by cell sedimentation in the sample vessels over time, giving rise to heterogeneous sampling artifacts, as well as by variation in urine composition, patient to patient. This impacts the establishment of stringent guidelines for the validation of method performance and clinical utility. Additional difficulties for the validation of chemiluminescence method performance in urines include stability of the urine matrix, quantification and uniformity of controls and standards, effective cell lysis, and the effects of hyperosmolality on cellular components in the assays.6-8 1 H. A. H. Rongen, R. M. W. Hoetelmans, A. Bult, and W. P. Van Bennekom, Z Pharm. Biom. Anal, 12, 433 (1994). 2 B. J. Hallaway, M. E. Copeman, B. S. Stevens, T.S. Larson, D. M. Wilson, and D. J. O'Kane, in "Bioluminescence and Chemiluminescence: Molecular Reporting with Photons" (J. W. Hastings, L. J. Kricka and P. E. Stanley, eds.), p. 517. Wiley, Chichester, 1997. 3 D. J. Holland, K. J. Bliss, C. D. Allen, and G. L. Gilbert, Pathology 27, 91 (1995). 4 R. C. Bartlett, D. A. Zern, I. Ratkiewicz, and J. Z. Tetreault, Arch. Pathol. Lab. Med. 118, 1096 (1994). 5 M. MeGinley, L. L. Wong, J. H. McBride, and D. O. Rodgerson, J. Clin. Lab. Anal, 6, 359 (1992). 6 S. Kubo, T. Matsumoto, M. Sakumoto, O. Mochida, Y. Abe, and J. Kumazawa, Renal Failure 20, 75 (1998).
METHODS IN ENZYMOLOGY,VOL. 305
Copyright© 2000by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/00$30.00
[28]
ERYTHROCYTES AND LEUKOCYTES IN URINE
403
Screening for Erythrocytes in Urine
Introduction The presence of red blood cells (hematuria) and/or hemoglobin (hemoglobinuria) in urine can indicate significant health problems and patient morbidity. Hematuria results from any disease or trauma to the kidney and/or urinary tract and is also seen with the presence of calculi, neoplasms, and infections or with the use of certain nephrotoxic drugs. Hemoglobinuria is a less common clinical finding and results from either acquired hemolytic disease or genetic defects. However, certain conditions (pH, specific gravity, or osmolality) within the urine result in the lysis of erythrocytes leading to hemoglobinuria. Lysis of erythrocytes in the urine can occur in the bladder or in the specimen container after the urine is voided. A sensitive, screening chemiluminescence (CL) assay has been developed for determining hemoglobinuria/hematuria. The pseudoperoxidase activity of hemoglobin reacts with a cyclic hydrazide substrate in the presence of hydrogen peroxide to form a superoxide radical that proceeds spontaneously to form a product with the emission of light (Fig. 1).
Materials and Methods Assay Reagents Required Cetyltrimethylammonium chloride (CTAC), 25% solution (Aldrich Chemicals, Milwaukee, WI) Dimethyl sulfoxide (DMSO): minimum 99.5% (GC) grade Sodium acetate: anhydrous, ACS reagent Sodium chloride Acetic acid: glacial, +99% purity Nitric acid, 70%, ACS reagent Hydrogen peroxide, ACS reagent, assay, 30.0% Hemoglobin (Hb), human, purified, crystallized, dialyzed, and lyophilized (Sigma Chemicals, St. Louis, MO)
Assay Solutions Required 7-Dimethylaminonaphthalene-l,2-dicarbonic acid hydrazide (7-DNH) (Assay Designs, Inc., Ann Arbor, MI): Dissolve 5.0 mg 7-DNH in
7 M. Qian, J. W. Eaton, and S. P. Wolff, Biochem. J. 326, 159 (1997). 8 T. Matsumoto, K. Takahashi, Y. Mizunoe, N. Ogata, M. Tanaka, J. Kumazawa, M. Nozaki, and P. van der Auwera, Eur. Urol. 20, 232 (1991).
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LUMINESCENCE-BASED ASSAYSin Vitro
[28]
(H3C)2N
CTAC
RBCs
(Lysis)
~
Hb
+ H202
+
o
O-I o
OH OH
+ N2
+ CL Emission
o FIo. 1. Assay of erythrocytes in urine with 7-DNH. Erythrocytes are first lysed with detergent, liberating hemoglobin. Hemoglobin pseudoperoxidase activity catalyzes the oxidation of 7-DNH, resulting in CL emission.
5.0 ml of DMSO and vortex. Dilute to 200/zg/ml by adding 20 ml of DMSO and vortex. Perform a spectrographic analysis, comparing absorbance at 324 and 414 nm, to any previous 7-DNH stocks. If the new and previous stock have < 10% coefficient of variation (CV), the new stock is acceptable for routine assay use. Aliquot 1.0 ml 7-DNH stocks and wrap containers or tubes with aluminum foil to block light. Store at - 2 0 °. The 7-DNH stock substrate can be frozen and thawed repeatedly without detrimental effects. 7-DNH substrate buffer: Dissolve 4.1 g of sodium acetate and 292.2 g NaC1 in 1.0 liter of distilled H20 to make a 50 mM sodium acetate and 5 M NaCI solution. Adjust to pH 5.5 with 1 to 2 drops of glacial acetic acid. The substrate buffer will be used to dilute the 7-DNH to 68 ng/ml, or approximately 1:3000. Diluting 3.4/xl of 7-DNH stock substrate into 10.0 ml of substrate buffer is sufficient for 34 tubes. Add 34/zl CTAC to the 7-DNH substrate solution to permit prelysis of erythrocytes. 1% nitric acid: In a 15-ml polypropylene tube, dilute 100/zl of nitric acid into 10.0 ml of distilled H20 for a 1.0% solution. Store at room temperature for up to 6 months, then discard. Chemiluminescence trigger solution: Dilute 166 ml of 30.0% H202 with 834 ml of distilled H20 (5% H202). Adjust the pH with 1% nitric
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acid to 3.25 ± 0.02, using a pipettor if necessary. Transfer 5.0% H202 to the large trigger reservoir and place in the luminometer. One liter is sufficient for 3250 injections. Store at room temperature for 2 weeks, then discard. Negative urine matrix. The requirements for selecting suitable urine samples for use as a negative urine matrix are that the samples are negative by microscopy for erythrocytes, leukocytes, casts, and crystals and are negative for free hemoglobin and leukocyte esterase by dipstick analyses. Also, the urine samples must have less than 15 mg/dl glucose, preferably 1-5 mg/dl; that the samples have less than 8 mg/dl of protein, preferably 1-5 mg/dl; that the samples have a normal pH in the range of 5.0 to 7.0; and that the samples have a normal osmolality in the range of 300-800 mOsm/kg. Combine selected urine samples to form at least a 2.0 liter pool and recheck the parameters. Prepare 10- to 12-ml aliquots of the pooled negative urine matrix and store at - 2 0 ° for up to 6 months, then discard. Thaw a single aliquot for each assay run, preparing other assay reagents during this time. Four urine blanks are assayed at the beginning of each run. Hb standard dilution buffer: 0.15 M sodium chloride (NaC1). Dissolve 87.6 mg into 10.0 ml of distilled H20. Vortex and adjust pH to 5.0. Store at room temperature for up to 3 months, then discard. Hb standards: Dissolve 1.0 mg of Hb into 1.0 ml of 0.15 M sodium chloride, pH 5.0. Vortex and incubate for 15 min. Vortex again. Wrap tube or container with aluminum foil to block light and store Hb stock standard for up to 1 week, then discard. Prepare a new set of Hb standards for each assay run using the negative urine matrix. Five standards, at concentrations of 32, 160, and 800 ng/ml and 4 and 20/~g/ml, are assayed at the end of each run and are prepared from the stock Hb standard of 1.0 mg/ml as a fivefold, serial dilution, using the negative urine matrix. Instrumentation. The assay is performed using a CLS-ID chemiluminescence detection system (Nichols Institute Diagnostics, San Juan Capistrano, CA), which includes a four probe Tecan diluter and a luminometer with injectors for assay solutions and triggers. All assay procedural steps are programmed using the CLS-ID system. Assay Procedures. Urine samples are prelysed by the addition of Triton X-100 to 0.01% final concentration. Ten microliters of prelysed urine is added to 290/~1 of the 7-DNH substrate solution in a 12 x 75-mm polystyrene tube. Three hundred microliters of 5% H202 is added by the luminometer and chemiluminescence emission is detected. CL decision levels must be determined with each luminometer. Under the conditions of the
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assay used in these studies, samples emitting ---2000 relative light units (RLU) are reported as normal. Samples emitting >2000 RLU are considered positive for erythrocytes and are considered potentially abnormal. Positive samples are examined further by manual microscopic cell analysis or by hemocytometer counting. Results and Discussion Kinetics of CL Emission. The kinetics of CL was determined by monitoring the signal generated from the substrate over a 20-sec time period added to filtered urine in the presence and absence of Hb (background RLU). Background CL emission was low with the 7-DNH substrate in contrast to other cyclic hydrazide derivatives investigated. The low background was in part attributed to the low pH of the assay (pH 5.5). Maximum background emission occurred within the first second after the addition of HEO2 followed by a rapid decline to the level of instrument noise. The addition of hemoglobin to the filtered urine altered the course of CL such that the emission peaked at 2 sec with a more gradual decay. A high signalto-noise ratio was obtained by integrating photons emitted in the first 5 sec. Interferences. Uric acid, ascorbic acid, and bilirubin do not affect assay performance. Myoglobin (Mb) at >5.0/zg/ml and myeloperoxidase at >2.5 /zg/ml provided positive interferences that are detected by the substrate. High levels of bacteria do not affect the CL emission. Method Comparison. A patient study was performed on urine samples from 500 patients (246 females and 254 males) and compared to values obtained by automated imaging. The sensitivity and specificity of the assay were 95 and 95.5% (>3 RBC/high power microscopic field) using a CL emission cutoff of >2000 RLU. The normal reference range for urinary Hb is <150 ng/ml (calculated from the number of erythrocytes per high power field and assuming 1 RBC -- 30 pg of Hb). Urines emitting CL >2000 RLU (or >150 ng/ml Hb) are reflexed for automated or manual microscopic analysis. The false negative rate was low (3/246 females). The CL screen, manual microscopic analysis, and dipstick were negative for these patients. The apparent false-positive rate was 12/246 females and 8/254 males. Screening for Leukocytes in Urine High levels of leukocytes in urine may be an indication of urinary tract infection (UTI), cystitis, nephritis, various autoimmune diseases, and physiological responses to drugs. Leukocytes, including monocytes and
[28]
ERYTHROCYTES AND LEUKOCYTESIN URINE F
F
407 O
H
C F 0
MPO
I O~C C
H
3
0
0
~ H
H202
~
C
H202
I O~C
H
3
0
HO ~
I CH3
I CH3
Acridan
Acridinium
"-
I CH3
N-Methyl-Acridone
+ CL Glow Emission
FtG. 2. Assay of leukocytes in urine with an acridan substrate. Leukocytes are first prelysed with detergent, liberating myeloperoxidase. Myeloperoxidase catalyzes the dehydrogenation of the acridan to form an acridinium species that decomposes spontaneously with CL emission.
granulocytes, express myeloperoxidase (MPO) activity. MPO has been assayed with luminol derivatives, 9,1° but luminol also reacts with other heme proteins found in urine, such as hemoglobin (Hb) and myoglobin (Mb). ill2 A rapid, inexpensive CL screening procedure has been developed using the peroxidase triggering of acridinium ester 13 by MPO present in leukocytes2 (Fig. 2).
Materials and Methods Assay Reagent Solutions Required Dimethyl sulfoxide: minimum 99.5% (GC) grade Triton X-100 stock solution: 1% by volume Methimazole stock solution (optional): dissolve 11.4 mg methimazole (Sigma Chemicals) in 10.0 ml of DMSO and store in 0.5-ml aliquots at 0.01 M. Dilute to 5.0 mM for working stock. Acridan substrate (PS-1, solutions A and B, Lumigen Inc., Southfield, MI): prior to use add 0.01% Triton (v/v) to solution A. Use this 9 G. Briheim, O. Stendahl, and C. Dahlgren, Infect. Immun. 45, 1 (1984). 10p. Roschger, W. Graninger, and H. Klima, Biochem. Biophys. Res. Commun. 123, 1047 (1984). 11T. Olsson, K. Bergstrom, and A. Thore, Clin. Chim. Acta 122, 125 (1982). 12T. Olsson, K. Bergstrom, and A. Thore, Clin. Chim. Acta 138, 31 (1984). 13H. Akhavan-Tafti, K. Sugioka, Z. Arghavani, R. DeSiva, R. S.Handley, Y, Sugioka, R. A. Eickholt, M. P. Perkins, and A. P. Schaap, Clin. Chem. 41, 1368 (1995).
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solution to dilute solution B 1:100 for immediate use. Optional add methimazole t.o 50/zM. This additive selectively inhibits the activities of most peroxidase activities, but is a poor inhibitor of MPO. Negative urine matrix: prepared as described earlier for screening for erythrocytes. Peroxidase controls: Soybean peroxidase (SBP) (Pierce Chemicals, Rockford, IL): dissolve 1.0 mg into 0.911 ml of negative urine matrix for a concentration of 1.1 mg/ml. Vortex, invert, and wrap tube with aluminum foil or place control in a light-sensitive container to block light. Incubate for 15 min at ambient temperature and vortex again. Store stock concentration of 1.1 mg/ml at 4° for 4 weeks. Prepare a new set of controls for each assay run, using the corresponding urine matrix. Three controls at concentrations of 22, 55, and 110 ng/ ml are assayed at the end of each run and are prepared from a stock SBP of 1.1 mg/ml and the negative urine matrix. Soybean peroxidase is utilized instead of MPO because of its greater stability in urine. Instrumentation. The assay is performed using a CLS-ID chemiluminescence detection system (Nichols Institute Diagnostics), which includes a four probe Tecan diluter and a luminometer with injectors for assay solutions and triggers. All assay procedural steps are programmed using the CLS-ID system. Assay Procedure. Urine samples are prelysed with 0.01% Triton X-100 and diluted 1 : 8 (v/v) with distilled H20. Ten microliters of dilute urine is added to 50/xl of PS-1 substrate and incubated at ambient temperature for 10 rain. MPO activity is detected by counting the CL emissions for 1 sec. CL decision levels must be determined for each luminometer and each assay condition. CL emission <1500 R L U under the conditions of assay used for these studies are considered normal (---5 leukocytes per high power microscope field). Samples with CL emission >1500 R L U are considered abnormal and are considered as potentially positive for leukocytes. Potentially positive samples are examined further by hemacytometer counting, or by a manual microscopic analysis, to determine the number of leukocytes per milliliter. Results and Discussion
Luminol derivatives react with a wide variety of heme proteins. This lack of specificity limits its application in urinalysis where Hb and Mb may be present and have peroxidase activities. The reactivity of PS-1 with heme proteins was assessed to determine if this substrate had a more restricted
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ERYTHROCYTES AND LEUKOCYTES IN URINE
409
specificity. Several peroxidase activities are detected with the PS-1 substrate, including eosinophil peroxidase (EPO), MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). The addition of 50/~M methimazole, an inhibitor of EPO, TM reduces activities of EPO, TPO, and LPO by >90%, whereas MPO is inhibited by only 50%. The addition of methimazole to PS-1 assay allows MPO activity to be detected selectively. Alternatively, omitting methimazole permits the detection of eosinophils in urine but with an increased background CL. Prelysing Cells. A major advantage of this assay is the use of automated pipettors and sample identification. However, while the primary sample tube is in queue for sampling and dilution, cells in the urine are settling due to gravity, causing decreased signal levels and potentially resulting in false-negative results. The samples must be prelysed to overcome this difficulty. Several lysis reagents were investigated, including saponin, mellitin, Tween 20, CHAPS, and Triton X-100. Triton X-100 proved to be the most useful in that it lysed leukocytes and erythrocytes, did not interfere in the leukocyte assay, and did not interfere in the erythrocyte assay, permitting one prelysed sample to be used in both assays. Osmolality. Hyperosmolal urine samples (>600 mOsm) do not give as high a CL signal as lower osmolal samples with the same number of leukocytes present. Hyperosmolal urine samples inhibit MPO activity.8 Consequently, the effect of urine osmolality on the acridan CL assay was investigated. Filtered urine samples with osmolalities ranging from 260 to 1053 mOsm are spiked with the same amount of MPO. The quenching effect of hyperosmolal urines is minimized at an eightfold dilution with water. 2 Interferences. Uric acid, ascorbic acid, bilirubin, and trolox (watersoluble vitamin E analog) do not affect assay performance. Hemoglobin at >10/~g/ml and myoglobin at >2/~g/ml are detected by the PS-1 substrate. Samples populated grossly with bacteria caused slight inhibition of the MPO activity. Method Comparison. A patient study was performed on samples from 99 males and 89 females to compare cell quantification by automated imaging with MPO CL emission with the PS-1 substrate. 2 Arbitrary set points to define abnormal urines were established from literature values as > 10,000 WBC/ml for males and >16,000 WBC/ml for females. Assay sensitivity and specificity are 92 and 86% (->5 WBC/high power microscopic field). The specificity of the assay is lower because of the apparent false-positive results (11/89 females and 12/99 males). Apparent false-negative rates were 3/99 (males) and 2/89 (females). The number of samples submitted 14 A. Taurog and M. L. Dorris, Arch. Biochem. Biophys. 296, 239 (1992).
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for WBC quantification by manual microscopic analysis was reduced by 70%. Conclusions Erythrocyte and leukocyte prescreening assay sensitivities for both assays are equivalent to those obtained by either manual microscopic analysis or automated imaging. Only 30% of urine samples need to be analyzed by quantitative methods for the presence of erythrocytes and leukocytes. The proportion of false-negative samples is small. Rapid, inexpensive prescreening for erythrocytes and leukocytes allows for high throughput of samples and fast turnaround times. This also allows focusing laboratory resources on the analysis of abnormal samples.
[29] Immunoassay Protocol for Quantitation of Protein Kinase Activities B y JENNIFER M O S I E R , CORINNE E . M . O L E S E N , JOHN C . V O Y T A ,
and
I R E N A BRONSTEIN
Introduction Phosphorylation and dephosphorylation play a critical role in many cellular signal transduction processes, including control of cell cycle, response to growth factors, apoptosis, cellular transformation by oncogenes, and infection. >3 Protein kinases, which catalyze phosphoryl transfer from ATP to a protein or peptide substrate, are critical regulatory enzymes in many pathways and are the focus of biomedical and pharmaceutical research. These enzymes are important therapeutic targets for cancer, immune response disorders, infectious disease, and cardiovascular disease. 4 Protein kinase assays typically measure phosphorylation of a peptide or protein substrate. Traditionally, kinase assays are performed with radioactively labeled ATP by the measurement of 32p incorporation into the 1 T. Hunter, Cell 80, 225 (1995). 2 j. Cleaveland, P. Kiener, D. Hammond, and B. Schacter, Anal, Biochem. 190, 249 (1990). 3 G. Rijksen, B. van Oirschot, and G. Staal, Anal, Biochem. 182, 98 (1989). 4 j. G. Foulkes, M. Chow, C. Gorka, A. R. Frackelton, Jr., and D. Baltimore, J. Biol. Chem. 260, 8070 (1985).
METHODS IN ENZYMOLOGY,VOL. 305
Copyright © 2000 by AcademicPress All rightsof reproductionin any form reserved. 0076-6879100 $30.00
LUMINESCENCE-BASED ASSAYSill Vitro
[28]
[28] Chemiluminescence Screening Assays for Erythrocytes and Leukocytes in Urine By VALERIE
J. BUSH, BRENDA J. HALLAWAY, THOMAS A . EBERT,
DAVID M. WILSON, and DENNIS J. O'KANE Introduction The use of chemiluminescence as a detection system within laboratory medicine, especially applied toward immunoassays, provides the advantages of high specificity and sensitivity, rapid analysis, low cost, and high stability. I Only recently, however, has chemiluminescence become practical as a screening system in urinalysis.2 Current, clinical methods used to detect erythrocytes and leukocytes in urine include manual microscopic analysis, dipsticks, and automated imaging. 3-5 These procedures are time-consuming, expensive, prone to error, and cannot exclude normal samples efficiently from analysis. Applying chemiluminescence screening systems for erythrocytes and leukocytes in urine has the potential to reduce the number of normal urine samples requiring additional diagnostic testing and may facilitate reprioritizing laboratory resources through the use of automation. The routine application of automation to urinalysis, however, is compromised by cell sedimentation in the sample vessels over time, giving rise to heterogeneous sampling artifacts, as well as by variation in urine composition, patient to patient. This impacts the establishment of stringent guidelines for the validation of method performance and clinical utility. Additional difficulties for the validation of chemiluminescence method performance in urines include stability of the urine matrix, quantification and uniformity of controls and standards, effective cell lysis, and the effects of hyperosmolality on cellular components in the assays.6-8 1 H. A. H. Rongen, R. M. W. Hoetelmans, A. Bult, and W. P. Van Bennekom, Z Pharm. Biom. Anal, 12, 433 (1994). 2 B. J. Hallaway, M. E. Copeman, B. S. Stevens, T.S. Larson, D. M. Wilson, and D. J. O'Kane, in "Bioluminescence and Chemiluminescence: Molecular Reporting with Photons" (J. W. Hastings, L. J. Kricka and P. E. Stanley, eds.), p. 517. Wiley, Chichester, 1997. 3 D. J. Holland, K. J. Bliss, C. D. Allen, and G. L. Gilbert, Pathology 27, 91 (1995). 4 R. C. Bartlett, D. A. Zern, I. Ratkiewicz, and J. Z. Tetreault, Arch. Pathol. Lab. Med. 118, 1096 (1994). 5 M. MeGinley, L. L. Wong, J. H. McBride, and D. O. Rodgerson, J. Clin. Lab. Anal, 6, 359 (1992). 6 S. Kubo, T. Matsumoto, M. Sakumoto, O. Mochida, Y. Abe, and J. Kumazawa, Renal Failure 20, 75 (1998).
METHODS IN ENZYMOLOGY,VOL. 305
Copyright© 2000by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/00$30.00
[28]
ERYTHROCYTES AND LEUKOCYTES IN URINE
403
Screening for Erythrocytes in Urine
Introduction The presence of red blood cells (hematuria) and/or hemoglobin (hemoglobinuria) in urine can indicate significant health problems and patient morbidity. Hematuria results from any disease or trauma to the kidney and/or urinary tract and is also seen with the presence of calculi, neoplasms, and infections or with the use of certain nephrotoxic drugs. Hemoglobinuria is a less common clinical finding and results from either acquired hemolytic disease or genetic defects. However, certain conditions (pH, specific gravity, or osmolality) within the urine result in the lysis of erythrocytes leading to hemoglobinuria. Lysis of erythrocytes in the urine can occur in the bladder or in the specimen container after the urine is voided. A sensitive, screening chemiluminescence (CL) assay has been developed for determining hemoglobinuria/hematuria. The pseudoperoxidase activity of hemoglobin reacts with a cyclic hydrazide substrate in the presence of hydrogen peroxide to form a superoxide radical that proceeds spontaneously to form a product with the emission of light (Fig. 1).
Materials and Methods Assay Reagents Required Cetyltrimethylammonium chloride (CTAC), 25% solution (Aldrich Chemicals, Milwaukee, WI) Dimethyl sulfoxide (DMSO): minimum 99.5% (GC) grade Sodium acetate: anhydrous, ACS reagent Sodium chloride Acetic acid: glacial, +99% purity Nitric acid, 70%, ACS reagent Hydrogen peroxide, ACS reagent, assay, 30.0% Hemoglobin (Hb), human, purified, crystallized, dialyzed, and lyophilized (Sigma Chemicals, St. Louis, MO)
Assay Solutions Required 7-Dimethylaminonaphthalene-l,2-dicarbonic acid hydrazide (7-DNH) (Assay Designs, Inc., Ann Arbor, MI): Dissolve 5.0 mg 7-DNH in
7 M. Qian, J. W. Eaton, and S. P. Wolff, Biochem. J. 326, 159 (1997). 8 T. Matsumoto, K. Takahashi, Y. Mizunoe, N. Ogata, M. Tanaka, J. Kumazawa, M. Nozaki, and P. van der Auwera, Eur. Urol. 20, 232 (1991).
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LUMINESCENCE-BASED ASSAYSin Vitro
[28]
(H3C)2N
CTAC
RBCs
(Lysis)
~
Hb
+ H202
+
o
O-I o
OH OH
+ N2
+ CL Emission
o FIo. 1. Assay of erythrocytes in urine with 7-DNH. Erythrocytes are first lysed with detergent, liberating hemoglobin. Hemoglobin pseudoperoxidase activity catalyzes the oxidation of 7-DNH, resulting in CL emission.
5.0 ml of DMSO and vortex. Dilute to 200/zg/ml by adding 20 ml of DMSO and vortex. Perform a spectrographic analysis, comparing absorbance at 324 and 414 nm, to any previous 7-DNH stocks. If the new and previous stock have < 10% coefficient of variation (CV), the new stock is acceptable for routine assay use. Aliquot 1.0 ml 7-DNH stocks and wrap containers or tubes with aluminum foil to block light. Store at - 2 0 °. The 7-DNH stock substrate can be frozen and thawed repeatedly without detrimental effects. 7-DNH substrate buffer: Dissolve 4.1 g of sodium acetate and 292.2 g NaC1 in 1.0 liter of distilled H20 to make a 50 mM sodium acetate and 5 M NaCI solution. Adjust to pH 5.5 with 1 to 2 drops of glacial acetic acid. The substrate buffer will be used to dilute the 7-DNH to 68 ng/ml, or approximately 1:3000. Diluting 3.4/xl of 7-DNH stock substrate into 10.0 ml of substrate buffer is sufficient for 34 tubes. Add 34/zl CTAC to the 7-DNH substrate solution to permit prelysis of erythrocytes. 1% nitric acid: In a 15-ml polypropylene tube, dilute 100/zl of nitric acid into 10.0 ml of distilled H20 for a 1.0% solution. Store at room temperature for up to 6 months, then discard. Chemiluminescence trigger solution: Dilute 166 ml of 30.0% H202 with 834 ml of distilled H20 (5% H202). Adjust the pH with 1% nitric
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acid to 3.25 ± 0.02, using a pipettor if necessary. Transfer 5.0% H202 to the large trigger reservoir and place in the luminometer. One liter is sufficient for 3250 injections. Store at room temperature for 2 weeks, then discard. Negative urine matrix. The requirements for selecting suitable urine samples for use as a negative urine matrix are that the samples are negative by microscopy for erythrocytes, leukocytes, casts, and crystals and are negative for free hemoglobin and leukocyte esterase by dipstick analyses. Also, the urine samples must have less than 15 mg/dl glucose, preferably 1-5 mg/dl; that the samples have less than 8 mg/dl of protein, preferably 1-5 mg/dl; that the samples have a normal pH in the range of 5.0 to 7.0; and that the samples have a normal osmolality in the range of 300-800 mOsm/kg. Combine selected urine samples to form at least a 2.0 liter pool and recheck the parameters. Prepare 10- to 12-ml aliquots of the pooled negative urine matrix and store at - 2 0 ° for up to 6 months, then discard. Thaw a single aliquot for each assay run, preparing other assay reagents during this time. Four urine blanks are assayed at the beginning of each run. Hb standard dilution buffer: 0.15 M sodium chloride (NaC1). Dissolve 87.6 mg into 10.0 ml of distilled H20. Vortex and adjust pH to 5.0. Store at room temperature for up to 3 months, then discard. Hb standards: Dissolve 1.0 mg of Hb into 1.0 ml of 0.15 M sodium chloride, pH 5.0. Vortex and incubate for 15 min. Vortex again. Wrap tube or container with aluminum foil to block light and store Hb stock standard for up to 1 week, then discard. Prepare a new set of Hb standards for each assay run using the negative urine matrix. Five standards, at concentrations of 32, 160, and 800 ng/ml and 4 and 20/~g/ml, are assayed at the end of each run and are prepared from the stock Hb standard of 1.0 mg/ml as a fivefold, serial dilution, using the negative urine matrix. Instrumentation. The assay is performed using a CLS-ID chemiluminescence detection system (Nichols Institute Diagnostics, San Juan Capistrano, CA), which includes a four probe Tecan diluter and a luminometer with injectors for assay solutions and triggers. All assay procedural steps are programmed using the CLS-ID system. Assay Procedures. Urine samples are prelysed by the addition of Triton X-100 to 0.01% final concentration. Ten microliters of prelysed urine is added to 290/~1 of the 7-DNH substrate solution in a 12 x 75-mm polystyrene tube. Three hundred microliters of 5% H202 is added by the luminometer and chemiluminescence emission is detected. CL decision levels must be determined with each luminometer. Under the conditions of the
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LUMINESCENCE-BASED ASSAYSin Vitro
[28]
assay used in these studies, samples emitting ---2000 relative light units (RLU) are reported as normal. Samples emitting >2000 RLU are considered positive for erythrocytes and are considered potentially abnormal. Positive samples are examined further by manual microscopic cell analysis or by hemocytometer counting. Results and Discussion Kinetics of CL Emission. The kinetics of CL was determined by monitoring the signal generated from the substrate over a 20-sec time period added to filtered urine in the presence and absence of Hb (background RLU). Background CL emission was low with the 7-DNH substrate in contrast to other cyclic hydrazide derivatives investigated. The low background was in part attributed to the low pH of the assay (pH 5.5). Maximum background emission occurred within the first second after the addition of HEO2 followed by a rapid decline to the level of instrument noise. The addition of hemoglobin to the filtered urine altered the course of CL such that the emission peaked at 2 sec with a more gradual decay. A high signalto-noise ratio was obtained by integrating photons emitted in the first 5 sec. Interferences. Uric acid, ascorbic acid, and bilirubin do not affect assay performance. Myoglobin (Mb) at >5.0/zg/ml and myeloperoxidase at >2.5 /zg/ml provided positive interferences that are detected by the substrate. High levels of bacteria do not affect the CL emission. Method Comparison. A patient study was performed on urine samples from 500 patients (246 females and 254 males) and compared to values obtained by automated imaging. The sensitivity and specificity of the assay were 95 and 95.5% (>3 RBC/high power microscopic field) using a CL emission cutoff of >2000 RLU. The normal reference range for urinary Hb is <150 ng/ml (calculated from the number of erythrocytes per high power field and assuming 1 RBC -- 30 pg of Hb). Urines emitting CL >2000 RLU (or >150 ng/ml Hb) are reflexed for automated or manual microscopic analysis. The false negative rate was low (3/246 females). The CL screen, manual microscopic analysis, and dipstick were negative for these patients. The apparent false-positive rate was 12/246 females and 8/254 males. Screening for Leukocytes in Urine High levels of leukocytes in urine may be an indication of urinary tract infection (UTI), cystitis, nephritis, various autoimmune diseases, and physiological responses to drugs. Leukocytes, including monocytes and
[28]
ERYTHROCYTES AND LEUKOCYTESIN URINE F
F
407 O
H
C F 0
MPO
I O~C C
H
3
0
0
~ H
H202
~
C
H202
I O~C
H
3
0
HO ~
I CH3
I CH3
Acridan
Acridinium
"-
I CH3
N-Methyl-Acridone
+ CL Glow Emission
FtG. 2. Assay of leukocytes in urine with an acridan substrate. Leukocytes are first prelysed with detergent, liberating myeloperoxidase. Myeloperoxidase catalyzes the dehydrogenation of the acridan to form an acridinium species that decomposes spontaneously with CL emission.
granulocytes, express myeloperoxidase (MPO) activity. MPO has been assayed with luminol derivatives, 9,1° but luminol also reacts with other heme proteins found in urine, such as hemoglobin (Hb) and myoglobin (Mb). ill2 A rapid, inexpensive CL screening procedure has been developed using the peroxidase triggering of acridinium ester 13 by MPO present in leukocytes2 (Fig. 2).
Materials and Methods Assay Reagent Solutions Required Dimethyl sulfoxide: minimum 99.5% (GC) grade Triton X-100 stock solution: 1% by volume Methimazole stock solution (optional): dissolve 11.4 mg methimazole (Sigma Chemicals) in 10.0 ml of DMSO and store in 0.5-ml aliquots at 0.01 M. Dilute to 5.0 mM for working stock. Acridan substrate (PS-1, solutions A and B, Lumigen Inc., Southfield, MI): prior to use add 0.01% Triton (v/v) to solution A. Use this 9 G. Briheim, O. Stendahl, and C. Dahlgren, Infect. Immun. 45, 1 (1984). 10p. Roschger, W. Graninger, and H. Klima, Biochem. Biophys. Res. Commun. 123, 1047 (1984). 11T. Olsson, K. Bergstrom, and A. Thore, Clin. Chim. Acta 122, 125 (1982). 12T. Olsson, K. Bergstrom, and A. Thore, Clin. Chim. Acta 138, 31 (1984). 13H. Akhavan-Tafti, K. Sugioka, Z. Arghavani, R. DeSiva, R. S.Handley, Y, Sugioka, R. A. Eickholt, M. P. Perkins, and A. P. Schaap, Clin. Chem. 41, 1368 (1995).
408
LUMINESCENCE-BASED ASSAYSin Vitro
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solution to dilute solution B 1:100 for immediate use. Optional add methimazole t.o 50/zM. This additive selectively inhibits the activities of most peroxidase activities, but is a poor inhibitor of MPO. Negative urine matrix: prepared as described earlier for screening for erythrocytes. Peroxidase controls: Soybean peroxidase (SBP) (Pierce Chemicals, Rockford, IL): dissolve 1.0 mg into 0.911 ml of negative urine matrix for a concentration of 1.1 mg/ml. Vortex, invert, and wrap tube with aluminum foil or place control in a light-sensitive container to block light. Incubate for 15 min at ambient temperature and vortex again. Store stock concentration of 1.1 mg/ml at 4° for 4 weeks. Prepare a new set of controls for each assay run, using the corresponding urine matrix. Three controls at concentrations of 22, 55, and 110 ng/ ml are assayed at the end of each run and are prepared from a stock SBP of 1.1 mg/ml and the negative urine matrix. Soybean peroxidase is utilized instead of MPO because of its greater stability in urine. Instrumentation. The assay is performed using a CLS-ID chemiluminescence detection system (Nichols Institute Diagnostics), which includes a four probe Tecan diluter and a luminometer with injectors for assay solutions and triggers. All assay procedural steps are programmed using the CLS-ID system. Assay Procedure. Urine samples are prelysed with 0.01% Triton X-100 and diluted 1 : 8 (v/v) with distilled H20. Ten microliters of dilute urine is added to 50/xl of PS-1 substrate and incubated at ambient temperature for 10 rain. MPO activity is detected by counting the CL emissions for 1 sec. CL decision levels must be determined for each luminometer and each assay condition. CL emission <1500 R L U under the conditions of assay used for these studies are considered normal (---5 leukocytes per high power microscope field). Samples with CL emission >1500 R L U are considered abnormal and are considered as potentially positive for leukocytes. Potentially positive samples are examined further by hemacytometer counting, or by a manual microscopic analysis, to determine the number of leukocytes per milliliter. Results and Discussion
Luminol derivatives react with a wide variety of heme proteins. This lack of specificity limits its application in urinalysis where Hb and Mb may be present and have peroxidase activities. The reactivity of PS-1 with heme proteins was assessed to determine if this substrate had a more restricted
[28]
ERYTHROCYTES AND LEUKOCYTES IN URINE
409
specificity. Several peroxidase activities are detected with the PS-1 substrate, including eosinophil peroxidase (EPO), MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). The addition of 50/~M methimazole, an inhibitor of EPO, TM reduces activities of EPO, TPO, and LPO by >90%, whereas MPO is inhibited by only 50%. The addition of methimazole to PS-1 assay allows MPO activity to be detected selectively. Alternatively, omitting methimazole permits the detection of eosinophils in urine but with an increased background CL. Prelysing Cells. A major advantage of this assay is the use of automated pipettors and sample identification. However, while the primary sample tube is in queue for sampling and dilution, cells in the urine are settling due to gravity, causing decreased signal levels and potentially resulting in false-negative results. The samples must be prelysed to overcome this difficulty. Several lysis reagents were investigated, including saponin, mellitin, Tween 20, CHAPS, and Triton X-100. Triton X-100 proved to be the most useful in that it lysed leukocytes and erythrocytes, did not interfere in the leukocyte assay, and did not interfere in the erythrocyte assay, permitting one prelysed sample to be used in both assays. Osmolality. Hyperosmolal urine samples (>600 mOsm) do not give as high a CL signal as lower osmolal samples with the same number of leukocytes present. Hyperosmolal urine samples inhibit MPO activity.8 Consequently, the effect of urine osmolality on the acridan CL assay was investigated. Filtered urine samples with osmolalities ranging from 260 to 1053 mOsm are spiked with the same amount of MPO. The quenching effect of hyperosmolal urines is minimized at an eightfold dilution with water. 2 Interferences. Uric acid, ascorbic acid, bilirubin, and trolox (watersoluble vitamin E analog) do not affect assay performance. Hemoglobin at >10/~g/ml and myoglobin at >2/~g/ml are detected by the PS-1 substrate. Samples populated grossly with bacteria caused slight inhibition of the MPO activity. Method Comparison. A patient study was performed on samples from 99 males and 89 females to compare cell quantification by automated imaging with MPO CL emission with the PS-1 substrate. 2 Arbitrary set points to define abnormal urines were established from literature values as > 10,000 WBC/ml for males and >16,000 WBC/ml for females. Assay sensitivity and specificity are 92 and 86% (->5 WBC/high power microscopic field). The specificity of the assay is lower because of the apparent false-positive results (11/89 females and 12/99 males). Apparent false-negative rates were 3/99 (males) and 2/89 (females). The number of samples submitted 14 A. Taurog and M. L. Dorris, Arch. Biochem. Biophys. 296, 239 (1992).
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LUMINESCENCE-BASED ASSAYSin Vitro
[29] •
for WBC quantification by manual microscopic analysis was reduced by 70%. Conclusions Erythrocyte and leukocyte prescreening assay sensitivities for both assays are equivalent to those obtained by either manual microscopic analysis or automated imaging. Only 30% of urine samples need to be analyzed by quantitative methods for the presence of erythrocytes and leukocytes. The proportion of false-negative samples is small. Rapid, inexpensive prescreening for erythrocytes and leukocytes allows for high throughput of samples and fast turnaround times. This also allows focusing laboratory resources on the analysis of abnormal samples.
[29] Immunoassay Protocol for Quantitation of Protein Kinase Activities B y JENNIFER M O S I E R , CORINNE E . M . O L E S E N , JOHN C . V O Y T A ,
and
I R E N A BRONSTEIN
Introduction Phosphorylation and dephosphorylation play a critical role in many cellular signal transduction processes, including control of cell cycle, response to growth factors, apoptosis, cellular transformation by oncogenes, and infection. >3 Protein kinases, which catalyze phosphoryl transfer from ATP to a protein or peptide substrate, are critical regulatory enzymes in many pathways and are the focus of biomedical and pharmaceutical research. These enzymes are important therapeutic targets for cancer, immune response disorders, infectious disease, and cardiovascular disease. 4 Protein kinase assays typically measure phosphorylation of a peptide or protein substrate. Traditionally, kinase assays are performed with radioactively labeled ATP by the measurement of 32p incorporation into the 1 T. Hunter, Cell 80, 225 (1995). 2 j. Cleaveland, P. Kiener, D. Hammond, and B. Schacter, Anal, Biochem. 190, 249 (1990). 3 G. Rijksen, B. van Oirschot, and G. Staal, Anal, Biochem. 182, 98 (1989). 4 j. G. Foulkes, M. Chow, C. Gorka, A. R. Frackelton, Jr., and D. Baltimore, J. Biol. Chem. 260, 8070 (1985).
METHODS IN ENZYMOLOGY,VOL. 305
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