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Evaluation of Micro-Bumintest reagent tablets for screening of microalbuminuria
Diabetes Research and Clinical Practice, 9 (1990)
137-142
137
Elsevier DIABET 00390
Evaluation of Micro-Bumintest reagent tablets for screening of microalbuminuria Joseph Tai and Wah Jun Tze Departments
of Pathology and Pediatrics, University of Britih
Columbia, Vancouver, BC. Canada
(Received 28 June 1989) (Revision received 21 November 1989) (Accepted 28 December 1989)
Summary The presence of significant microalbuminuria is an important predictor of early diabetic nephropathy. Currently methods available to detect microalbuminuria are not suitable for routine screening. This study assessed the performance of Micro-Bumintest reagent tablets (Ames Laboratory) as a visual screening test for qualitative measurement of urinary albumin. 150 urine specimens from type 1 diabetic patients with a disease duration of < 1-19 years were assayed over 15 runs using quantitative albumin (radioimmunoassay) and total protein assays. In parallel, 600 determinants were made with the tablets. Each sample was assessed by two readers using a color chart with patterns illustrating typical negative and positive color reactions with a grading from 1 to 7. Urinary protein was determined with an Ames Clinitek 10 urine chemistry analyzer and the Multistix 10 SG reagent strips. The results revealed that as the albumin concentration increased, the percentage of specimens detected visually as presumptive positive (grading = 3) reactions and positive reactions (grading > 3) increased. A positive Micro-Bumintest reaction was obtained with an albumin concentration of > 40 pg/ml. Presumptive positive reactions occur more than 50% of the time at protein concentrations > 14 mg/dl. The tablet test is more sensitive than the Clinitek lO/Multistix 10 SG strip system. These data show that the Micro-Bumintest reagent tablets are a sensitive and convenient screening test for detection of microalbuminuria. Key words: Microalbuminuria;
Protein;
Children
Introduction It has been shown that a urine albumin concentration between 15 and 200 pg/ml, termed Address for correspondence: Dr. W.J. Tze, Dept. of Paediatrics, University of British Columbia, 4480 Oak Street, Room lA46, Vancouver, B.C., Canada V6H 3V4. 0168-8227/90/$03.50
0 1990 Elsevier Science Publishers
microalbuminuria, is an early sign of presymptomatic diabetic nephropathy [ 1,2]. Two studies showed that about 80% of adult insulindependent diabetic patients with 24-h urinary albumin excretion rates over 50 mg/24 h will develop clinical macroproteinuria and some degree of renal failure over the next 6-24 years and only 4% with albumin excretion rates below
B.V. (Biomedical
Division)
138
50 mg/24 h develop macroproteinuria [ 3,4]. In patients with overt diabetic nephropathy, improved metabolic control had no effect on diabetic nephropathy [ 51. However, recent studies suggest that early aggressive therapy of the diabetes and any associated raised blood pressure will arrest the progression or even reverse the incipient diabetic nephropathy [ 61. Therefore, the detection of microalbuminuria is important in the clinicalmanagement ofdiabetes mellitus. Methods for quantitative measurement of urinary albumin are available, but they are all rather expensive or require further experimentation in individual laboratories and results are not obtained immediately [7,8]. The Albustix test can only detect albumin concentrations above approximately 150-200 pg/ml and is not sensitive enough for the detection of microalbuminuria. A quick and sensitive screening method would therefore be helpful. The purpose of the study was to test the performance of the Ames Micro-Bumintest Tablet relative to radioimmunoassay and Multistix 10 SG test strips for detecting microalbuminuria. Materials and methods 150 urine specimens from IDDM patients with a disease duration of < 1-19 years were tested over 15 runs, using quantitative albumin (double antibody radioimmunoassay kit KHAD, Diagnostic Products Corp., Los Angeles, CA) and total protein (Bio-Rad Protein Assay Kit II, Mississauga, Ontario) assays, Ames Multistix 10 SG urinalysis reagent strips with a Clinitek Instrument and two lots of Micro-Bumintest reagent tablets (Ames Division, Miles Laboratories Ltd., Rexdale, Ontario). Specimen collection and preparation
24-h urine samples were collected without addition of preservative and the specimens were stored frozen at - 20°C until use. Specimens were removed from the freezer and stored overnight in a refrigerator to thaw. Three ml of each sample was centrifuged at 1500 x g for 30 min to
remove any precipitation. The supernatant was decanted into fresh tubes, mixed by inversion, and used for the Micro-Bumintest and the comparative assays. The urine specimens were tested in duplicates for albumin by radioimmunoassay and for total protein according to the manufacturer’s instructions. A standard curve was prepared each day using human serum albumin (CAP, UPRM, 2.0 g/l) diluted in 0.9% saline. To confirm assay performance of the albumin radioimmunoassay and total protein assay, control charts were established before clinical testing by testing in duplicate control samples over 20 individual runs. The coefficient of variation of radioimmunoassay for 20 runs was 4.4 7.. The mean and established control limits were established and were adhered to during the study. Micro-Bumintest
Two Micro-Bumintest readers assessed each specimen run and both readers passed a short test for defective red-green color vision before the beginning of the tablet evaluation. In addition, each reader completed a 5-run familiarization study using Ames supplied urine controls before testing patient specimens. The testings were performed under laboratory lighting and each specimen was tested with two lots of Micro-Bumintest tablets for a total of four results. For testing, a tablet was placed on a clean paper towel. Using a disposable dropper, about 30 ~1 (1 drop) of urine was added to the centre of the tablet which was allowed to be absorbed. With a clean dropper, 1 drop of distilled water was added on the tablet. A second drop was added after the water had been absorbed. After the absorbance of the second drop the readers visually compared the reacted tablet color to the three reference color patterns (levels 2, 4 and 6) and recorded the number (l-7) that most closely matched the color intensity. A negative reaction with Micro-Bumintest reagent tablets is one in which there is no defined bluish-green spot left on the tablet. A positive reaction is defined as any trace of a bluish-green spot or ring that is seen on the tablet surface after the water has been
139
absorbed. Micro-Bumintest results reported as 1 or 2 represent a negative reaction, a result reported as 3 shows a presumptive positive reaction, and results reported as 4 to 7 are positive reactions with increasing color intensity. The urine specimens were also tested with the Ames Multistix 10 SG reagent strip to measure the presence or absence of analytes and/or metabolites. Also, a specific gravity measurement with a clinical refractometer was obtained.
Results 150 urine specimens were tested resulting in 600 Micro-Bumintest determinations. The albumin and total protein assay mean results are compared to Micro-Bumintest visual results in Tables 1 and 2, respectively. Micro-Bumintest results are compared to Multistix 10 SG reagent strip results in Table 3. Table 1 illustrates the sensitivity of MicroBumintest for albumin as determined relative to the results of the albumin radioimmunoassay. As the albumin concentration increased, the percentage of specimens detected visually as presumptive positive reactions and positive reactions increased. A consistent positive Micro-Bumintest reaction was obtained with an albumin concentration of greater than 40 pg/ml, with observer concordance of 95 %. Table 2 shows the sensitiviTABLE
1
Comparison of urinary albumin concentration by radioimmunoassay and Micro-Bumintest frequency distribution Albumin @g/ml)
Micro-Bumintest <3
0 - 20 20.1- 40 40.1- 60 60.1- 80 80.1-100 > 100
158 (50.0) 10 (9.3) 0 (0) 0 (0) 0 (0) 0 (0)
a Percent is shown in brackets.
frequency” 3
144 (45.6) 71 (65.7) 6 (10.7) 1 (3.6) 0 (0) 0 (0)
>3 14 (4.4) 27 (25) 50 (89.3) 27 (96.4) 16 (100) 76 (100)
TABLE
2
Comparison of urinary total protein Micro-Bumintest frequency distribution Protein (mg/dl)
o- 1.0 l.l- 3.0 3.1- 4.0 4.1- 5.0 5.1- 6.0 61.- 7.0 7.1- 8.0 8.1- 9.0 9.1-10.0 10.1-11.0 11.1-12.0 12.1-13.0 13.1-14.0 14.1-15.0 15.1-16.0 16.1-17.0 17.1-18.0 >18
concentration
Micro-Bumintest
frequency”
<3
3
12 (100) 19 (67.9) 46 (82.1) 26 (46.4) 30 (48.4) 5 (25.0) 10 (26.3) 10 (20.8) 2 (7.1) 2 (4.5) 2 (10.0) 0 0 0 0 0 0 0
(0) (0) (0) (0) (0) (0) (0)
0 (0)
and
23
0 (0)
6 (21.4)
3 (10.7)
10 (17.9) 19 (33.9) 31 (50.0) 11 (55.0) 22 (57.9) 27 (56.3) 17 (60.7) 23 (52.3) 5 (25.0) 14 (38.9) 10 (50.0) 3 (18.8) 2 (25.0) 0 (0)
0 (0) 11 (19.7) 1 (1.6) 4 (20.0) 6 (15.8) 11 (22.9) 9 (32.2) 19 (43.2) 13 (65.0) 22 (61.1) 10 (50.0) 13 (81.2) 6 (75.0) 12 (100)
4 (100) 0 (0)
0 (0) 92 (100)
a Percent is shown in brackets.
ty of Micro-Bumintest for total protein in urine relative to the comparative total protein assay. Micro-Bumintest visual presumptive positive reactions occur more than 50% of the time in the protein range of 6-13 mg/dl and positive reactions occur more than 50% of the time at protein concentrations 2 14 mg/dl. Table 3 shows the findings of Multistix teststrips and Micro-Bumintest tablets classified by the albumin concentration as determined by radioimmunoassay. The urine samples are classified into four groups according to albumin concentrations (Group 1: O-20 pg/ml, n = 79; Group 2: 20.1-40 pg/ml, n = 27 ; Group 3 : 40.1-100 pg/ml, n = 25; Group 4: > 100 pg/ml, n = 19). As the urinary albumin concentration increased the percentage of Micro-Bumintest results giving presumptive positive or positive reactions also increased. Therefore for all urine samples with albumin concentration of > 40.1 pg/ml, 96.0% (169/ 176)
140 TABLE 3 Comparison
of Micro-Bumintest Number
Albumin cont.
tablet, Multistix test strip readings and urinary albumin by radioimmunoassay Mean”
Range
S.G.”
Glucose”
PH”
Micro-Bumintest <3/nb
O-20
79
20.1-40
27
40.1-100
25
> 100
19
5.92 4.55 32.15 5.35 61.26 14.16 383.51 244.65
a Mean f SD. b Number of observations
O.l18.9 20.339.65 42.1591.50 lOO.i999.0
+ & f f
TABLE 4 Comparison of Clinitek 10 protein results with albumin concentration by radioimmunoassay Clintek reading”
@g/ml) Neg.
Trace
0 - 20 20.1- 40
67 (84.8) 12 (44.4)
40.1-100 > 100
11 (44.0) 0 (0)
+ + f i
6.63 1.12 6.48 0.89 6.24 0.71 6.08 0.56
+ k f k
689.24 420.67 522.20 460.84 526.92 420.77 305.56 424.92
f k f f
> 3/n
158/316 141316 144/3 16 lo/108 27,‘108 711108 o/100 93/100 7/100 O/76 76176 O/76
per total number of observations.
of the Micro-Bumintest readings were > 3 (positive). With urine albumin concentration of > 100 pg/ml, 100% gave readings of > 3 (positive). Table 3 also shows that the specific gravity, pH and ketone content of urine samples did not influence the outcome of the Micro-Bumintest results. The urine glucose content of Group 1 samples was significantly higher than that of Group 4 samples (P = 0.0019) but not different from the rest. Table 4 shows that when the results of protein determination by Multistix strips were correlated with albumin concentration as determined by radioimmunoassay the Multistix did not detect microalbumin in 1 l/25 cases or 44% of the Group
Albumin
1.028 0.007 1.027 0.007 1.022 0.008 1.021 0.009
3/n
100
2300
8 (10.1) 4 (5.1) 11 (40.7) 3 (11.1)
0 (0) 0 (0)
0 (0) 1 (3.7)
14 (56.0) 0 (0) 4 (21.0) 4 (21.0)
0 (0) 9 (47.4)
0 (0) 2 (10.5)
a Percent is shown in brackets.
30
3 urine samples with 40.1-100 pg/ml range.
albumin
in
the
Discussion Microalbuminuria, a marker for predicting overt clinical nephropathy is of clinical importance in and insulin-dependent both maturity-onset diabetes mellitus [l-4,9]. Unfortunately, lack of a simple procedure to detect albumin in urine at concentrations not detectable by conventional dipstick methods has limited this assessment to a few research laboratories. The aim of the present study was to test the performance of Micro-Bumintest. In this study Micro-Bumintest detected all samples with albumin concentration > 40 pg/ml as positive with 96% sensitivity (n = 44). The remaining samples were detected as presumptive positive. Observer concordance (n = 2) for a positive test with Micro-Bumintest is 95 %. It is more sensitive than the dipstick method which has a sensitivity of 75% (33/44 determinations). In the 20.1-40 pg/ml range the test is less certain, with 65.7 and 25% of the results giving presumptive positive and positive readings, respectively. At the O-20 pg/ml level, 45.6% of urine samples gave reactions at presumptive positive level and 4.4% false-positive reactions.
141
The rather high incidence of presumptive positive results could be due to the color characteristics of the tables at low urinary protein concentrations. This is compounded by the fact that the color pattern for level 3 is absent from the color pattern chart. The Micro-Bumintest reaction detects urinary albumin at concentrations 2 40 pg/ml and urinary total protein concentrations of 2 14 mg/dl. The metabolic condition of the diabetic patient did not affect the outcome of the Micro-Bumintest results. However, how the sample is prepared is important, since frozen-thawed samples which were not centrifuged or filtered with filter paper to remove precipitates consistently gave false positive readings, despite low albumin and protein concentrations as determined by the comparative assays. Despite the minor problems, MicroBumintest could be used to identify individuals with microalbuminuria and clinical albuminuria, but further quantitative assessment would be necessary in positive cases to allow close monitoring of these patients. Also the fact that day-to-day variation in urinary albumin excretion is around 40-50x would suggest that patients with urine samples giving presumptive positive tablet reactions should be investigated within reasonably short intervals of 3-6 months [lo]. If a second reading is positive then a quantitative measurement should be made by radioimmunoassay or other quantitative procedure. A group of 15 diabetic patients with urinary albumin concentrations of > 20.1 pg/ml was followed for a year. Urinary albumin concentration was measured on 21 separate occasions and Micro-Bumintest assessments also performed. The urine concentration fluctuated in these patients but remained above 20.1 pug/ml in all cases, with 12 samples in the 20.1-40 pg/ml range and 18 samples >40.1 pg/ml. One sample in the tirst group gave a negative reading and 6 and 5 samples gave presumptive positive and positive readings, respectively. One sample in the second group gave a presumptive positive reading and the remainder gave positive readings. This follow-up observation confirmed that Micro-Bumintest as-
sessment is quite reproducible. The present investigation agrees with the earlier studies of Schmitz [ 1 l] and Slama et al. [ 121 on Microalbutest in that the Micro-Bumintest is a reasonably safe screening method for microalbuminuria which would detect nearly all patients who need further investigation and who would escape detection using Albustix. The new tablet test has the advantage over the previously described Microalbutest in that a standardized color pattern chart is provided which makes the standardization of results from different laboratories possible and does not rely on freshly prepared tablet standards for color comparison. Although the reading of this test appeared straightforward when performed by laboratory workers who have undergone initial familiarization runs and passed the color vision test, its performance in a diabetic clinic or physician’s office by untrained personnel is currently being investigated by us. This study shows that the Micro-Bumintest with the new color pattern chart is convenient, rapid, acceptably precise and reproducible and will be useful as an initial screening tool for microalbuminuria.
Acknowledgements The authors wish to thank Anthea Kennelly and Margaret Tkachuk for their technical assistance, and Mr. L. Crowley and Dr. J.M. Botero of Ames Laboratory for their cooperation and advice.
References Mogensen, C. E., Chachati, A., Christensen, C. K. et al. (1986) Microalbuminuria: an early marker of renal involvement in diabetes. Uremia Investigation 9, 85-95. Mogensen, C. E. and Christensen C. K. (1984) Predicting diabetic nephropathy in insulin-dependent patients. New Engl. J. Med. 311, 89-93. Viberti, G. C., Hill, R. D., Jarrett, R. J., Argyropoulos, A., Mahmud, U. and Keen, H. (1982) Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet i, 1430-1432. Parving, H. H., Oxenboll, B., Svendsen, P., Christensen, J. S. and Anderson, A. R. (1982) Early detection of
142
5
6 7
8
patients at risk of developing diabetic nephropathy. A longitudinal study of urinary albumin excretion. Acta Endocrinol 100, 550-555. Viberti, G. C., Bilous, R. W., Mackintosh, D., Bending, J. J. and Keen, H. (1983) Long-term correction of hyperglycaemia and progression of renal failure in insulindependent diabetes. Br. Med. J. 286, 598-602. Mogensen, C. E. (1988) Management of diabetic renal involvement and disease. Lancet i, 867-869. Brodows, R. G., Nichols, D., Shaker, G. and Kubasik, N. P. (1986) Evaluation of a new radioimmunoassay for urinary albumin. Diabetes Care 9, 189-193. Mohamed, A., Wilkin, T., Leatherdale, B. and Davies, R. (1984) A microenzyme-linked immunosorbent assay for
urinary albumin, and its comparison with radioimmunoassay. J. Immunol. Methods 74, 17-22. 9 Mogensen, C. E. (1984) Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabets. New Engl. J. Med. 310, 356-360. 10 Feldt-Rasmussen, B. and Mathiesen, E. R. (1984) Variability of urinary albumin excretion in incipient diabetic nephropathy. Diabet. Nephr. 4, 101-103. 11 Schmitz, A. (1986) Microalbutest: a new screening method for detecting microalbuminuria in diabetes mellitus. Uremia Investigation 9, 79-84. 12 Slama, G., Boillot, J., Desplanque, N. and Letanoux, M. (1985) Bedside estimation of microalbuminuria. Lancet i, 1338-1339.
137-142
137
Elsevier DIABET 00390
Evaluation of Micro-Bumintest reagent tablets for screening of microalbuminuria Joseph Tai and Wah Jun Tze Departments
of Pathology and Pediatrics, University of Britih
Columbia, Vancouver, BC. Canada
(Received 28 June 1989) (Revision received 21 November 1989) (Accepted 28 December 1989)
Summary The presence of significant microalbuminuria is an important predictor of early diabetic nephropathy. Currently methods available to detect microalbuminuria are not suitable for routine screening. This study assessed the performance of Micro-Bumintest reagent tablets (Ames Laboratory) as a visual screening test for qualitative measurement of urinary albumin. 150 urine specimens from type 1 diabetic patients with a disease duration of < 1-19 years were assayed over 15 runs using quantitative albumin (radioimmunoassay) and total protein assays. In parallel, 600 determinants were made with the tablets. Each sample was assessed by two readers using a color chart with patterns illustrating typical negative and positive color reactions with a grading from 1 to 7. Urinary protein was determined with an Ames Clinitek 10 urine chemistry analyzer and the Multistix 10 SG reagent strips. The results revealed that as the albumin concentration increased, the percentage of specimens detected visually as presumptive positive (grading = 3) reactions and positive reactions (grading > 3) increased. A positive Micro-Bumintest reaction was obtained with an albumin concentration of > 40 pg/ml. Presumptive positive reactions occur more than 50% of the time at protein concentrations > 14 mg/dl. The tablet test is more sensitive than the Clinitek lO/Multistix 10 SG strip system. These data show that the Micro-Bumintest reagent tablets are a sensitive and convenient screening test for detection of microalbuminuria. Key words: Microalbuminuria;
Protein;
Children
Introduction It has been shown that a urine albumin concentration between 15 and 200 pg/ml, termed Address for correspondence: Dr. W.J. Tze, Dept. of Paediatrics, University of British Columbia, 4480 Oak Street, Room lA46, Vancouver, B.C., Canada V6H 3V4. 0168-8227/90/$03.50
0 1990 Elsevier Science Publishers
microalbuminuria, is an early sign of presymptomatic diabetic nephropathy [ 1,2]. Two studies showed that about 80% of adult insulindependent diabetic patients with 24-h urinary albumin excretion rates over 50 mg/24 h will develop clinical macroproteinuria and some degree of renal failure over the next 6-24 years and only 4% with albumin excretion rates below
B.V. (Biomedical
Division)
138
50 mg/24 h develop macroproteinuria [ 3,4]. In patients with overt diabetic nephropathy, improved metabolic control had no effect on diabetic nephropathy [ 51. However, recent studies suggest that early aggressive therapy of the diabetes and any associated raised blood pressure will arrest the progression or even reverse the incipient diabetic nephropathy [ 61. Therefore, the detection of microalbuminuria is important in the clinicalmanagement ofdiabetes mellitus. Methods for quantitative measurement of urinary albumin are available, but they are all rather expensive or require further experimentation in individual laboratories and results are not obtained immediately [7,8]. The Albustix test can only detect albumin concentrations above approximately 150-200 pg/ml and is not sensitive enough for the detection of microalbuminuria. A quick and sensitive screening method would therefore be helpful. The purpose of the study was to test the performance of the Ames Micro-Bumintest Tablet relative to radioimmunoassay and Multistix 10 SG test strips for detecting microalbuminuria. Materials and methods 150 urine specimens from IDDM patients with a disease duration of < 1-19 years were tested over 15 runs, using quantitative albumin (double antibody radioimmunoassay kit KHAD, Diagnostic Products Corp., Los Angeles, CA) and total protein (Bio-Rad Protein Assay Kit II, Mississauga, Ontario) assays, Ames Multistix 10 SG urinalysis reagent strips with a Clinitek Instrument and two lots of Micro-Bumintest reagent tablets (Ames Division, Miles Laboratories Ltd., Rexdale, Ontario). Specimen collection and preparation
24-h urine samples were collected without addition of preservative and the specimens were stored frozen at - 20°C until use. Specimens were removed from the freezer and stored overnight in a refrigerator to thaw. Three ml of each sample was centrifuged at 1500 x g for 30 min to
remove any precipitation. The supernatant was decanted into fresh tubes, mixed by inversion, and used for the Micro-Bumintest and the comparative assays. The urine specimens were tested in duplicates for albumin by radioimmunoassay and for total protein according to the manufacturer’s instructions. A standard curve was prepared each day using human serum albumin (CAP, UPRM, 2.0 g/l) diluted in 0.9% saline. To confirm assay performance of the albumin radioimmunoassay and total protein assay, control charts were established before clinical testing by testing in duplicate control samples over 20 individual runs. The coefficient of variation of radioimmunoassay for 20 runs was 4.4 7.. The mean and established control limits were established and were adhered to during the study. Micro-Bumintest
Two Micro-Bumintest readers assessed each specimen run and both readers passed a short test for defective red-green color vision before the beginning of the tablet evaluation. In addition, each reader completed a 5-run familiarization study using Ames supplied urine controls before testing patient specimens. The testings were performed under laboratory lighting and each specimen was tested with two lots of Micro-Bumintest tablets for a total of four results. For testing, a tablet was placed on a clean paper towel. Using a disposable dropper, about 30 ~1 (1 drop) of urine was added to the centre of the tablet which was allowed to be absorbed. With a clean dropper, 1 drop of distilled water was added on the tablet. A second drop was added after the water had been absorbed. After the absorbance of the second drop the readers visually compared the reacted tablet color to the three reference color patterns (levels 2, 4 and 6) and recorded the number (l-7) that most closely matched the color intensity. A negative reaction with Micro-Bumintest reagent tablets is one in which there is no defined bluish-green spot left on the tablet. A positive reaction is defined as any trace of a bluish-green spot or ring that is seen on the tablet surface after the water has been
139
absorbed. Micro-Bumintest results reported as 1 or 2 represent a negative reaction, a result reported as 3 shows a presumptive positive reaction, and results reported as 4 to 7 are positive reactions with increasing color intensity. The urine specimens were also tested with the Ames Multistix 10 SG reagent strip to measure the presence or absence of analytes and/or metabolites. Also, a specific gravity measurement with a clinical refractometer was obtained.
Results 150 urine specimens were tested resulting in 600 Micro-Bumintest determinations. The albumin and total protein assay mean results are compared to Micro-Bumintest visual results in Tables 1 and 2, respectively. Micro-Bumintest results are compared to Multistix 10 SG reagent strip results in Table 3. Table 1 illustrates the sensitivity of MicroBumintest for albumin as determined relative to the results of the albumin radioimmunoassay. As the albumin concentration increased, the percentage of specimens detected visually as presumptive positive reactions and positive reactions increased. A consistent positive Micro-Bumintest reaction was obtained with an albumin concentration of greater than 40 pg/ml, with observer concordance of 95 %. Table 2 shows the sensitiviTABLE
1
Comparison of urinary albumin concentration by radioimmunoassay and Micro-Bumintest frequency distribution Albumin @g/ml)
Micro-Bumintest <3
0 - 20 20.1- 40 40.1- 60 60.1- 80 80.1-100 > 100
158 (50.0) 10 (9.3) 0 (0) 0 (0) 0 (0) 0 (0)
a Percent is shown in brackets.
frequency” 3
144 (45.6) 71 (65.7) 6 (10.7) 1 (3.6) 0 (0) 0 (0)
>3 14 (4.4) 27 (25) 50 (89.3) 27 (96.4) 16 (100) 76 (100)
TABLE
2
Comparison of urinary total protein Micro-Bumintest frequency distribution Protein (mg/dl)
o- 1.0 l.l- 3.0 3.1- 4.0 4.1- 5.0 5.1- 6.0 61.- 7.0 7.1- 8.0 8.1- 9.0 9.1-10.0 10.1-11.0 11.1-12.0 12.1-13.0 13.1-14.0 14.1-15.0 15.1-16.0 16.1-17.0 17.1-18.0 >18
concentration
Micro-Bumintest
frequency”
<3
3
12 (100) 19 (67.9) 46 (82.1) 26 (46.4) 30 (48.4) 5 (25.0) 10 (26.3) 10 (20.8) 2 (7.1) 2 (4.5) 2 (10.0) 0 0 0 0 0 0 0
(0) (0) (0) (0) (0) (0) (0)
0 (0)
and
23
0 (0)
6 (21.4)
3 (10.7)
10 (17.9) 19 (33.9) 31 (50.0) 11 (55.0) 22 (57.9) 27 (56.3) 17 (60.7) 23 (52.3) 5 (25.0) 14 (38.9) 10 (50.0) 3 (18.8) 2 (25.0) 0 (0)
0 (0) 11 (19.7) 1 (1.6) 4 (20.0) 6 (15.8) 11 (22.9) 9 (32.2) 19 (43.2) 13 (65.0) 22 (61.1) 10 (50.0) 13 (81.2) 6 (75.0) 12 (100)
4 (100) 0 (0)
0 (0) 92 (100)
a Percent is shown in brackets.
ty of Micro-Bumintest for total protein in urine relative to the comparative total protein assay. Micro-Bumintest visual presumptive positive reactions occur more than 50% of the time in the protein range of 6-13 mg/dl and positive reactions occur more than 50% of the time at protein concentrations 2 14 mg/dl. Table 3 shows the findings of Multistix teststrips and Micro-Bumintest tablets classified by the albumin concentration as determined by radioimmunoassay. The urine samples are classified into four groups according to albumin concentrations (Group 1: O-20 pg/ml, n = 79; Group 2: 20.1-40 pg/ml, n = 27 ; Group 3 : 40.1-100 pg/ml, n = 25; Group 4: > 100 pg/ml, n = 19). As the urinary albumin concentration increased the percentage of Micro-Bumintest results giving presumptive positive or positive reactions also increased. Therefore for all urine samples with albumin concentration of > 40.1 pg/ml, 96.0% (169/ 176)
140 TABLE 3 Comparison
of Micro-Bumintest Number
Albumin cont.
tablet, Multistix test strip readings and urinary albumin by radioimmunoassay Mean”
Range
S.G.”
Glucose”
PH”
Micro-Bumintest <3/nb
O-20
79
20.1-40
27
40.1-100
25
> 100
19
5.92 4.55 32.15 5.35 61.26 14.16 383.51 244.65
a Mean f SD. b Number of observations
O.l18.9 20.339.65 42.1591.50 lOO.i999.0
+ & f f
TABLE 4 Comparison of Clinitek 10 protein results with albumin concentration by radioimmunoassay Clintek reading”
@g/ml) Neg.
Trace
0 - 20 20.1- 40
67 (84.8) 12 (44.4)
40.1-100 > 100
11 (44.0) 0 (0)
+ + f i
6.63 1.12 6.48 0.89 6.24 0.71 6.08 0.56
+ k f k
689.24 420.67 522.20 460.84 526.92 420.77 305.56 424.92
f k f f
> 3/n
158/316 141316 144/3 16 lo/108 27,‘108 711108 o/100 93/100 7/100 O/76 76176 O/76
per total number of observations.
of the Micro-Bumintest readings were > 3 (positive). With urine albumin concentration of > 100 pg/ml, 100% gave readings of > 3 (positive). Table 3 also shows that the specific gravity, pH and ketone content of urine samples did not influence the outcome of the Micro-Bumintest results. The urine glucose content of Group 1 samples was significantly higher than that of Group 4 samples (P = 0.0019) but not different from the rest. Table 4 shows that when the results of protein determination by Multistix strips were correlated with albumin concentration as determined by radioimmunoassay the Multistix did not detect microalbumin in 1 l/25 cases or 44% of the Group
Albumin
1.028 0.007 1.027 0.007 1.022 0.008 1.021 0.009
3/n
100
2300
8 (10.1) 4 (5.1) 11 (40.7) 3 (11.1)
0 (0) 0 (0)
0 (0) 1 (3.7)
14 (56.0) 0 (0) 4 (21.0) 4 (21.0)
0 (0) 9 (47.4)
0 (0) 2 (10.5)
a Percent is shown in brackets.
30
3 urine samples with 40.1-100 pg/ml range.
albumin
in
the
Discussion Microalbuminuria, a marker for predicting overt clinical nephropathy is of clinical importance in and insulin-dependent both maturity-onset diabetes mellitus [l-4,9]. Unfortunately, lack of a simple procedure to detect albumin in urine at concentrations not detectable by conventional dipstick methods has limited this assessment to a few research laboratories. The aim of the present study was to test the performance of Micro-Bumintest. In this study Micro-Bumintest detected all samples with albumin concentration > 40 pg/ml as positive with 96% sensitivity (n = 44). The remaining samples were detected as presumptive positive. Observer concordance (n = 2) for a positive test with Micro-Bumintest is 95 %. It is more sensitive than the dipstick method which has a sensitivity of 75% (33/44 determinations). In the 20.1-40 pg/ml range the test is less certain, with 65.7 and 25% of the results giving presumptive positive and positive readings, respectively. At the O-20 pg/ml level, 45.6% of urine samples gave reactions at presumptive positive level and 4.4% false-positive reactions.
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The rather high incidence of presumptive positive results could be due to the color characteristics of the tables at low urinary protein concentrations. This is compounded by the fact that the color pattern for level 3 is absent from the color pattern chart. The Micro-Bumintest reaction detects urinary albumin at concentrations 2 40 pg/ml and urinary total protein concentrations of 2 14 mg/dl. The metabolic condition of the diabetic patient did not affect the outcome of the Micro-Bumintest results. However, how the sample is prepared is important, since frozen-thawed samples which were not centrifuged or filtered with filter paper to remove precipitates consistently gave false positive readings, despite low albumin and protein concentrations as determined by the comparative assays. Despite the minor problems, MicroBumintest could be used to identify individuals with microalbuminuria and clinical albuminuria, but further quantitative assessment would be necessary in positive cases to allow close monitoring of these patients. Also the fact that day-to-day variation in urinary albumin excretion is around 40-50x would suggest that patients with urine samples giving presumptive positive tablet reactions should be investigated within reasonably short intervals of 3-6 months [lo]. If a second reading is positive then a quantitative measurement should be made by radioimmunoassay or other quantitative procedure. A group of 15 diabetic patients with urinary albumin concentrations of > 20.1 pg/ml was followed for a year. Urinary albumin concentration was measured on 21 separate occasions and Micro-Bumintest assessments also performed. The urine concentration fluctuated in these patients but remained above 20.1 pug/ml in all cases, with 12 samples in the 20.1-40 pg/ml range and 18 samples >40.1 pg/ml. One sample in the tirst group gave a negative reading and 6 and 5 samples gave presumptive positive and positive readings, respectively. One sample in the second group gave a presumptive positive reading and the remainder gave positive readings. This follow-up observation confirmed that Micro-Bumintest as-
sessment is quite reproducible. The present investigation agrees with the earlier studies of Schmitz [ 1 l] and Slama et al. [ 121 on Microalbutest in that the Micro-Bumintest is a reasonably safe screening method for microalbuminuria which would detect nearly all patients who need further investigation and who would escape detection using Albustix. The new tablet test has the advantage over the previously described Microalbutest in that a standardized color pattern chart is provided which makes the standardization of results from different laboratories possible and does not rely on freshly prepared tablet standards for color comparison. Although the reading of this test appeared straightforward when performed by laboratory workers who have undergone initial familiarization runs and passed the color vision test, its performance in a diabetic clinic or physician’s office by untrained personnel is currently being investigated by us. This study shows that the Micro-Bumintest with the new color pattern chart is convenient, rapid, acceptably precise and reproducible and will be useful as an initial screening tool for microalbuminuria.
Acknowledgements The authors wish to thank Anthea Kennelly and Margaret Tkachuk for their technical assistance, and Mr. L. Crowley and Dr. J.M. Botero of Ames Laboratory for their cooperation and advice.
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