3.4: Red Cells or Hemoglobin in urine

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RELATED READING: Chapter 26 SEE URINALYSIS INFOBASE.

Red cells are not found in the urine of healthy individuals. Individuals with some forms of glomerular damage allow red cells to pass through the glomerular barrier. The dipstick is a rapid method of determining the presence of red cells in urine. If positive, microscopic evaluation of the urine sediment is usually performed.

Principle of the Method

The dipstick uses the peroxidase-like activity of hemoglobin (in the red cells or free) to convert a benzidine dye into a chromogen. The amount of color change in the dye is proportional to the amount of red cells present on the dipstick pad.

The reactions are as follows:

Intact red blood cells are lysed on the pad to release hemoglobin.

\[\text{Hemoglobin} + \text{complexed peroxide} \rightarrow \text{free}\; H_{2} O_{2}\]

\[H_{2} O_{2} + \text{benzidine dye} \rightarrow H_{2} O + \text{oxidized chromogen (color change)}\]

The dye reaction requires time to become visible. Each red cell on the pad gives a visible reaction. If only a few cells are present, the reaction gives a speckled appearance on the pad. There is also a maximum color change that can be obtained with the strip reagent. This is commonly set at 0.0150.062 mg/dl of hemoglobin, equivalent to 5-20 intact red blood cells per high powered field.

Reagents

All the reagents for the reaction are embedded in the pad of the dipstick. As with all reagents stored in a dried form, their stability is affected by moisture. Therefore the reagents must be protected from moisture. In addition, careful attention must be given to the manufacturer’s shelf life, which should appear on the label of every dipstick container.

Specimen

Freshly voided urine is the preferred specimen. The first urine specimen of the day is considered the most desirable, because it is the most concentrated. Urine specimens are acceptable up to four hours after voiding. Refrigerated specimens are acceptable up to 24 hours after excretion.

Procedure

Collect the urine in an appropriate specimen container. After verifying that the strips are working (see quality control) quickly dip the strip in the urine, removing excess liquid by moving the edge of the strip against the rim of the container as you remove the strip from the container. After this initial pass to remove excess liquid, remove any remaining liquid by touching the entire edge of the strip to a gauze pad or a paper towel. There should be no visible liquid on the strip except for that on the pad.

Results

Start timing the reaction as soon as the strip is placed in the urine. After 60 seconds read the strip visually. Match the observed color of the pad with the chart color on the bottle of strips or with a color chart, if that is available. Record your observation. If a Clinitek or other instrument is available, place the strip in the device as soon as you dip the strip and record the printed result. If reading visually, continue reading the results after 60 seconds, 120 seconds, and 300 seconds.

Calculations

No calculations are necessary for the visual readings. If instrument readings are made, the calibrated instrument calculates the hemoglobin concentration. If an instrument is used, correlate the visual reading with that of the instrument.

Quality Control

When visual readings are taken, be certain that the reader is not color blind. Before testing a test sample, take two strips and test a positive and a negative control sample. These results should be within accepted values. Ideally, a positive and negative control should be tested along with each batch of patient specimens tested.

Expected values

Urine from healthy individuals should give negative results. Urine from known patients with kidney disease may give positive results depending upon the extent of glomerular damage present.

STUDENT REPORT

Solution	Visual Color Score
Time	30 sec.	60 sec.	300 sec.
QC negative
QC positive
1 g/L
500 mg/L
250 mg/L
125 mg/L
62 mg/L
31 mg/L

Test solution 1
Test solution 2
Test solution 3

Discussion Questions

What is the range of linearity of the visual method?
What is the range of linearity of the instrument?
Does the test result change with time?
If the result does change with time, Why?
Does the range of linearity include all healthy and disease conditions?