Urine occult blood test is an essential item in urine testing. Commonly used testing methods include urine analyzer occult blood reaction and urine microscopic examination of red blood cells (microscopic examination of RBC).
Because microscopy has important clinical value in distinguishing glomerular and non-glomerular hematuria, some scholars have proposed that microscopy is the "gold standard" for urine red blood cell testing and cannot be replaced by a
urine analyzer. However, in recent years, with the rapid popularization of urine analyzers, measurement parameters have developed in a direction that is fast, simple, and accurate, providing better diagnostic basis for clinical use; microscopic examination is time-consuming and laborious, and many people are unwilling to do urine sediment examine. Therefore, in some hospitals, urine microscopy has a tendency to be gradually replaced by urine analyzers. To this end, YSENMED tested random urine samples from 400 outpatients using two methods: occult blood reaction of urine analyzer and red blood cell examination under urine microscope, and compared the degree of agreement between the two methods.
1. Principle
The urine analyzer uses the dry chemical test paper method, which detects certain chemical substances in urine based on the color change of each module on the multi-link test strip after the chemical reaction with the urine components, such as the direct detection of red blood cells and white blood cells. Cytoplasmic contents indirectly infer the presence or absence of cells. The principle of occult blood reaction in urine analyzer is: the peroxidase-like activity of heme in hemoglobin (Hb) can decompose peroxide to release new ecology [O], which oxidizes the substrate o-toluidine into o-toluidine. The color of benzidine changes from yellow → grass green → dark blue. Urine microscopy uses the magnification of the microscope to directly and intuitively present cells and other tangible components under the microscope, which is a true reflection of the substrate.
2. Method
(1) Urine analyzer detects urinary occult blood. Dip the urine test strip into 10ml of mixed fresh urine for 1 second, take it out, put it on the instrument for measurement, and automatically print the results.
(2) Microscopy: Take 10ml of urine in a test tube, centrifuge at 1500r/min for 5min, discard the supernatant, and leave 0.2ml of sediment. After mixing thoroughly, take out about 20 μl on a glass slide, add a cover slip (2.0cm × 2.0cm), and then use weaker light to observe the whole picture with a low-magnification lens to avoid missing small but meaningful objects, and then Use a high-power microscope to identify cells. Results are reported as: х/high power field (HP).
Since the principles of the two inspection methods are different and the factors that affect them are also different, there are certain differences in the results of the two inspection methods.
1. False positive. That is, the occult blood reaction of the urine analyzer is positive and the microscopic examination is negative. The common reasons are:
(1) Hemoglobin (Hb) The occult blood reaction of the urine analyzer can have a positive reaction with both intact RBCs and Hb released by dissolution of RBCs, while a microscope can only detect undissolved RBCs in urine. The Hb content in the urine of healthy people is very small and is qualitatively negative. During disease, there are two sources of Hb in urine: First, intravascular hemolysis occurs, plasma Hb exceeds the binding capacity of haptoglobin, and free Hb is excreted in the urine. The other is bleeding from the urinary tract (especially the upper urinary tract), where RBC destroys the escape of Hb when it is hypertonic or/and hypotonic. At this time, the occult blood reaction of the urine analyzer is positive, but no RBCs or only RBC fragments are seen in the urine sediment microscopy.
(2) Myoglobin (Mb) molecules also contain heme groups and have peroxidase activity. Mb mainly exists in cardiac muscle and skeletal muscle tissue. The content in normal human urine is very small and cannot be detected. When severe damage occurs to myocardium or skeletal muscle, plasma Mb increases and is excreted by the kidneys, causing an increase in urine Mb content. Therefore, the occult blood reaction is positive, and microscopic examination of RBC is negative.
(3) The urine of some patients contains heat-labile enzymes, which can also cause the color of the reagent block to change, resulting in a positive occult blood reaction and a negative microscopic examination.
(4) Certain oxidative contaminants, such as hypochlorite, can cause false positive occult blood reactions.
(5) Bacteriuria: Urine is a good culture medium for bacteria. During bacteriuria, some bacteria will produce oxidative substances, causing the color of the reagent block to change, resulting in a positive occult blood reaction and negative RBC microscopy. In addition, the invasion of microbial peroxidase can also cause false positives.
(6) Long-term storage and high-temperature experiments have proven that if the specimen is not fresh, long-term storage or high-temperature storage, the positive rate of occult blood reaction will relatively increase, while RBC microscopy will decrease accordingly, and the test results are likely to be false positive. Therefore, it is recommended that urine specimens should not be stored for more than 4 hours.
(7) Expired use or contamination of urine test strips as well as improper storage and incorrect operation methods can cause false positive test results.
(8) The sensitivity of the urine analyzer is too high (++ appears at 0.2 mg/dl), and the "grade difference" range is too large (+++ is found at 0.8-50 mg/dl). It is often encountered that the instrument test is ++, but the mirror test is ++. The red blood cell test was negative, and no clinical cause of hemoglobinuria could be found.
2. False negative. That is, the microscopic examination is positive and the occult blood reaction of the urine analyzer is negative. The common reasons are:
(1) Influence of food or drugs: When urine becomes alkaline due to diet or drugs, RBCs dissolve and rupture, forming brown particles. The occult blood reaction is positive, but the microscopic examination is negative.
(2) When VitC exists in large amounts in urine, it can compete for oxygen produced by the reaction, causing false negative reactions.
(3) High specific gravity and high protein urine samples reduce the sensitivity of the reagent block occult blood reaction, causing false negatives in the occult blood reaction.
(4) The increase in mucus content in the urine causes the RBCs to be wrapped and the reagent cannot come into contact with Hb, causing the occult blood reaction to appear falsely negative.
3. The impact of centrifugation on the results.
If the centrifugation speed is too fast, the formed components may be destroyed; but if the speed is too slow, when the RBC per milliliter of urine is below 5,000, RBC may not be found in the sediment, and latent glomerulonephritis may be missed. , so when there is doubt about the test results, the occult blood reaction can be used to confirm.
The urine analyzer method for detecting red blood cells in urine is extremely sensitive and may sometimes be different from the results of microscopy. Factors affecting the measurement results must be fully considered and a comprehensive analysis must be conducted in conjunction with clinical practice. In principle, positive results of occult blood reaction in urine analyzers should be determined by microscopy. Legal re-examination is an important link and key to ensuring the quality of urine testing. If the negative result determined by the urine analyzer method is inconsistent with the microscopic examination, it should also be reexamined to ensure the accuracy and reliability of the measurement results. So far, the test results of no instrument can completely replace the microscope. Urine sediment microscopy is still an indispensable examination method in urinalysis due to its unique clinical value.
