Setting reference intervals, urine dipstick confirmation, and background stain on Western blotting

Aug. 1, 2002
Edited by Daniel M. Baer, M.D.

Setting reference intervals

Does every laboratory need to set its own reference values, or can it use values from the literature? Do you know any article about making reference values for blood gas analyses? How many patients need to be included in a study of reference values? 

A: The concept of reference intervals is evolving. Although traditionally laboratories have based reference intervals on values obtained from healthy individuals, for an increasing number of tests (such as cholesterol, glucose, and PSA), reference limits are based on risk of disease as determined from outcome studies. At present, however, most reference limits are still defined in the traditional fashion.

The most widely accepted document of reference values is that published by the NCCLS (Wayne, PA).1 To establish reference limits with reasonable accuracy, it is necessary to use a sample size of at least 120 individuals. If it is necessary to partition the reference interval (for example, for children and adults, or for males and females), it is necessary to use 120 individuals for each group. In the United States, regulatory agencies do not require laboratories to establish their own reference intervals, but to validate the values that they use (these can be derived from publications, the manufacturer, or other laboratories in the area). The NCCLS document suggests the use of 20 individuals to validate reference limits; if no more than two are outside the reference interval, then the proposed limits are valid for the laboratory. We have found that in our laboratory, which serves a somewhat unique population, that we can validate most reference limits, but we need to establish our own in about 10 percent to 20 percent of cases.

With respect to blood gases, there have been relatively few studies cited over the last 30 years in Index Medicus covering reference values in populations other than neonates. One recent study by Crapo, et al. compared values at sea level and at an altitude of 1,400 m, using a total sample of 339 adults.2 They found that partial pressure of oxygen declines gradually with increasing age. 

Another study by Dong, et al. of arterialized capillary blood gases evaluated 712 normal individuals between the ages of 1 month and 24 years.3 They found that partial pressures of oxygen and carbon dioxide were lower in infants and young children, reaching adult levels by 7 to 12 years of age.

D. Robert Dufour, M.D.
Chief of Pathology
Veterans Affairs Medical Center
Washington, DC


1. Sasse, E (ed.). How to define and determine reference intervals in the clinical laboratory: approved guideline, 2nd Edition. Wayne, PA: NCCLS, 2000.

2. Crapo RO, Jensen RL, Hegewald M, Tashkin DP. Arterial blood gas reference values for sea level and an altitude of 1,400 meters. Am J Respir Crit Care Med 1999;160:1525-31.

3. Dong SH, Liu HM, Song GW, Rong ZP, Wu YP. Arterialized capillary blood gases and acid-base studies in normal individuals from 29 days to 24 years of age. Am J Dis Child 1985;139:1019-22.

Urine dipstick confirmation

Q: Our laboratory performs urinalyses on the Clinitek Atlas. All urines with a positive glucose require confirmation by Clinitest. According to the section supervisor, Clinitest confirmation is a CAP requirement. Our protocol states that due to differences in sensitivity between the chemstrip and Clinitest, the latter is required. The Atlas CliniTek booklet indicates that the analyzers chemstrips are sensitive to 100 mg/dL glucose, while the Clinitest is sensitive to 250 mg/dL glucose thus it is possible to have a positive glucose with a negative Clinitest. Does CAP truly require this redundant testing, and what would be the clinical significance if any?

We also still perform confirmatory tests on the urine dipstick positive reactions for protein (sulfosalicylic acid), and bilirubin (Ictotest). Is this the standard operating procedure in most labs, or has the quality of test strips advanced enough that confirmation is not needed?

We also have questioned orders for acetone qualitative (serum or urine). Is the standard method for testing Acetest tablets or urine dipstick? (N-Multistix are the only urine dipsticks we have in stock).

A: Modern urinalysis reagent strips are generally sufficiently reliable and semi-quantitative so that chemical confirmation is not necessary. Possible exceptions are tests for protein and
bilirubin. The College of American Pathologists has no requirements for confirmatory testing of urine reagent strips, either manual or automated. 

Confirmatory tests may be defined as a test used to confirm the accuracy or correctness of a procedure. According to NCCLS, Confirmatory chemical urinalysis tests detect the same substance with the same or greater sensitivity and/or specificity, or they use a different reaction or methodology to detect that substance.1 

The Clinitest Tablet Test (Bayer, Tarrytown, NY) is based on a different principle than the reagent strip tests for glucose. The tablets sensitivity is less than the reagent strip tests. The reagent strips will react with 100 mg/dL glucose, while the Clinitest tablet test is sensitive to 250 mg/dL glucose, or nonglucose-reducing substances. The reagent strip tests for glucose are all specific for glucose, as they are based on the use of the enzyme glucose oxidase. Although Clinitest can be used as a semi-quantitative test for glucose for up to 5,000 mg/dL (5 g/dL or 5 g percent) when the two-drop method is employed, it is not specific for glucose. It is a copper reduction test and measures any of the reducing sugars, which may be found in urine. It is for this reason that the Clinitest Tablet Test is used to test the urine of pediatric patients for nonglucose reducing sugars such as galactose. 

To indicate the extent of glucosuria as an indirect indication of the blood glucose level, it may be desirable to further semi-quantitate urine specimens that show values greater than 2,000
mg/dL (Multistix/Bayer) or greater than 1,000 mg/dL (Chemstrip/Roche, Indianapolis, IN). In this case, either the two-drop Clinitest method, or Chemstrip uG or uGK (Roche) might be used. Both products will semi-quantitate glucose values to 5 g/dL (5,000 mg/dL). The presence of ketones with glucose further implies inadequate diabetic control.

For confirmatory testing of other reagent strip constituents, commonly used tests include the sulfosalicylic acid test (SSA) for protein, and the Ictotest Tablet Test (Bayer) for bilirubin. 

In the case of the SSA test for protein, confirmatory testing continues to be performed by many laboratories due to the clinical importance of proteinuria. 

In addition, it must be remembered that the reagent strips are most sensitive to the presence of albumin, while SSA and other protein precipitation tests react with any protein that might be present in the urine, such as the light chain immuno-globulins seen in multiple myeloma. An alternative to SSA protein confirmation might be to use a quantitative protein chemistry method. This should be performed when confirmatory testing is specifically requested by the clinician, or when the color of the urine specimen obscures proper reading of the color reaction on the reagent pad. In addition, when large quantities of protein are seen on the reagent strip, the technologist should search for the presence of casts, fat, and oval fat bodies (renal tubular fat) in the urine sediment.

In the case of bilirubin, the Ictotest Tablet Test (Bayer) is significantly more sensitive than reagent strip tests. Therefore, when the presence of bilirubin is suspected, but negative or trace by reagent strip, the Ictotest Tablet test may be of use. Since the reagent strip colors are difficult to interpret and subject to masked or false positive reactions, it may be desirable to confirm all positive reactions with another method, such as the Ictotest Tablet Test.

Regarding testing for blood, plasma or serum ketones, reagent strips and the Acetest Tablet Test (Bayer) have been used. However, the reagent strips correlate only moderately well with quantitative acetoacetic in plasma and poorly with total blood ketones.2 The Acetest Tablet Test is preferred when testing blood, plasma, or serum for ketones. Manufacturers directions differ for each specimen type, and should be followed.

Karen M. Ringsrud MT(ASCP)
Assistant Professor
Department of Laboratory Medicine
and Pathology
University of Minnesota Medical School


1. NCCLS, Urinalysis and Collection, Transportation, and Preservation of Urine Specimens. Approved Guideline GP16-A, Wayne, PA, National Committee for Clinical Laboratory Standards, 1995, p.7.

2. Fuller CE, Threatte GA, Henry JB. Basic Examination of Urine. In Henry JB (ed): Clinical Diagnosis and Management by Laboratory Methods, 20th Ed, Philadelphia, WB Saunders Co. 2001, p. 380.

Background staining on Western blotting

Q: On several occasions, we found a strong background staining (after incubation with alkaline
phosphatase-labeled anti-human IgG and substrate) of Western blot strips that can hinder the interpretation of the test for HIV antibodies. Recently, we found that serum from a patient produced this background stain for only a half of the Western blot strip with B. burgdorferi antigens. The phenomenon was found repeatedly with the same serum and the bands appear as white bands on the dark background (like a negative image of the usual bands). I do not remember the cause of this phenomenon and how it can be avoided.

A: In the usual procedure for Western blotting, one can stain the sheet or strip with various protein binding agents. The blocking agents are usually are detergents and nonspecific protein or sera. If a specific antibody probe is to be applied, the matrix should first be incubated with a blocking agent to saturate all remaining protein binding sites, which will prevent the non-specific binding of the protein/antibody probe. Caution must be exercised in the selection of the protein to be used for the blocking. Powdered dried milk (bovine) has been widely used; however, because a significant number of patients have anti-bovine protein antibodies in their sera, a heavy background staining can occur. The solution to this dilemma is to replace the inexpensive milk proteins with commercial human fraction V (serum albumin).1

The background staining problem may be addressed by the above techniques. However, I can not fully explain the negative image of the usual bands from a patient tested for antibodies to the B. burgdorferi antigens.

Robert M. Nakamura, M.D.
Chairman Emeritus and Senior Consultant
Department of Pathology
Scripps Clinic
La Jolla, CA 


1. Craig WY, Poulin SE, Collins MF, Ledue TB, Ritchie RF. Background staining in immunoblotting assays: Reduction of signal caused by cross-reactivity with blocking agents. J of Immunol. Methods.158:67-76,1993.

Daniel M. Baer is professor emeritus of laboratory medicine at Oregon Health and Science University in Portland, OR, and a member of MLOs editorial advisory board.

© 2002 Nelson Publishing, Inc. All rights reserved.