Today, a patient came to the outpatient department of our hospital to see a doctor. He complained of constipation recently. After using Kaiseru in the morning, his stool first turned hard black and then soft yellow. At the end, there was blood in the stool, so he came to see a doctor. After the outpatient doctor received the patient, he prescribed a stool regular occult blood test for testing. After receiving the specimen, we selected a proper amount of specimen and placed it in a Hitachi cup filled with normal saline, inserted the stool occult blood test strip, and waited for the reaction result (colloidal gold method was used to measure stool occult blood). The specimens were then diluted with saline and smeared for microscopic observation.
　　
1 Observation under the mirror
　　
Under the microscope, we saw a large number of irregular round substances with burrs. Since we are not sure what it is, please check the identification of teachers in the medical group. Some teachers think it is shrunken red blood cells. I immediately searched the Internet for relevant pictures, and it was. Under the microscope, the volume of red blood cells is larger, and the cell membrane is serrated (or burr-like), which is smaller than the average red blood cell, and there are more in the field of view. This is the first time I have seen such characteristic red blood cells, so I am very impressed, as shown in the following figure:
　　
The picture was not typical enough to express what I saw. I searched a very close picture on the internet to make everyone feel more impressed (if there is infringement, please contact delete, thank you) as shown in the figure:
　　
(The cell membrane in the figure shrinks, becomes smaller in volume and irregular in shape, which is called crumpled red blood cells)
　　
2 Furine occult blood (FOB) test strip observation
　　
After the microscope observation, almost 4 minutes passed. We went to look at the condition of the stool occult blood test strip, but miraculously found that it was a bar (if positive, it was two bars), which was very different from what we saw under the microscope. The same is also inconsistent with the blood in the stool seen by the naked eye. In this case, we went to study the reagent instructions. The notes in the instructions said: "When tarry stool or visible blood occurs, the hemoglobin concentration exceeds the detection range of 4000ug/mL, which may lead to HOOK effect. At this time, the specimen should be diluted 50-100 before testing." According to the instructions, we diluted the specimen 50 times and 100 times and retested it. Sure enough, both results showed occult blood positive. The following figure:
　　
3 Confirmatory test
　　
In order to verify the false negative result of fecal occult blood in this case, we tested the original specimen (diluted fecal fluid) with urine dry chemical test strip and found occult blood, pH 7.5. Because the detection of urine occult blood is based on the principle of hemoglobin contact activity method, through the role of hemoglobin peroxidase catalytic decomposition of peroxide, so that o-linked toluene ammonia oxidation color. This result shows that it is the methodology of fecal occult blood test paper that leads to false negative results.
　　
4 Case analysis
　　
Detection principle of colloidal gold method: the use of double antibody sandwich method to detect human Hb in feces. During the test, due to the siphon effect of the material, the specimen moves forward slowly. When a sufficient amount of Hb exists, it combines with colloidal gold-labeled anti-Hb monoclonal antibody to form a "gold-labeled anti-Hb monoclonal antibody-Hb" complex, continues to crawl to the detection area (T) and then interacts with the solid anti-Hb monoclonal antibody to form a "gold-labeled anti-Hb monoclonal antibody complex-Hb monoclonal antibody" double-Hb monoclonal antibody sandwich deposit, that is, a red band (detection line-T line) appears, thus indicating that the Hb content in the specimen exceeds the threshold. If the specimen does not contain Hb or the Hb content is lower than the detection threshold, the colloidal gold-labeled anti-Hb monoclonal antibody will cross the detection area (T) during chromatography and directly combine with the solid phase anti-mouse IgG in the control area (C) to form a red band (I .e. control line-C line).
　　
The HOOK effect is the hook effect: it refers to the phenomenon of false negative due to the inappropriate ratio of antigen to antibody, in which excess antibody is called the pre-band effect; Excessive antigen is called the post-band effect. And this case is the back band phenomenon in the HOOK effect.
　　
The phenomenon of abnormal red blood cells observed under the microscope is affected by physiological saline. The PH of normal saline is 7, which is relatively high. The osmotic pressure of sodium chloride solution on red blood cells is the cause of red blood cell shrinkage deformation.
　　
5 Summary and inspiration
　　
This case reminds us that when the naked eye sees, the microscope observation is inconsistent with the test paper, we should find the reason and find the solution, instead of being so-so, we should report what we see, which is easy to cause clinical doctors to misdiagnose and even medical disputes. The instructions of the kit are a good solution. We usually have to read the instructions of the reagent frequently, which explains all the situations we can encounter, which can save a lot of time and avoid detours.
　　
The methods of HOOK effect in immunological detection are: time-resolved immunofluorescence analysis, rate scattering immunoturbidimetry, ELISA (one-step method, two-step method), immune permeation chromatography, immune colloidal gold method, etc. The items that are prone to HOOK effect in clinical testing are: alpha-fetoprotein (AFP), hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), immunoglobulin (Ig), carbohydrate antigen 125(CA125), human chorionic gonadotropin (HCG), fecial occult blood (FOB), etc. How to deal with the HOOK effect? The most direct is to retest after dilution. Of course, we should also pay more attention to the patient's clinical symptoms and diagnosis, combined with the patient's other laboratory test results, do not let go of any abnormal measurement values, and use other methods to detect.