The hemoglobin–haptoglobin complex test (HHKT) is a diagnostic method used in gastroenterology to detect intestinal (within organ) bleeding. The basic principle of this immunological diagnostic technique for detecting intestinal bleeding is based on the biochemical properties of hemoglobin or haptoglobin (protein present in blood plasma). Because of this, increased hemoglobin–haptoglobin complexes can be detected in the intestinal lumen in cases of intestinal bleeding. Detection of these complexes is then performed using a stool sample. The hemoglobin-haptoglobin complex test is of important significance in the detection of diseases associated with bleeding in the intestinal tract. Based on this, for example, hemorrhoids, autoimmune diseases affecting the intestine, or various tumors (intestinal polyps; colon cancer) can be detected with the test method.
The procedure
An important factor for the success in the early detection of, for example, tumors in the intestinal area represents the low required amount of blood that can be detected in the stool with the help of this immunological examination. The hemoglobin-haptoglobin complex test and the hemoglobin test are performed by ELISA. ELISA stands for Enzyme Linked Immunosorbent Assay and refers to an immunological detection method in which the specific binding reaction between antibodies and antigens is used to visualize viruses, proteins or hormones. ELISA uses the property of the specific antibodies used that they bind to the substance (antigen) to be detected. Depending on the ELISA test, it is possible to label antibody or antigen beforehand with an enzyme (potentially active protein structure). The reaction accelerated by the enzyme serves as evidence for the presence of the antigen. The substrate (starting material) is converted by the respective enzyme, so that subsequently the reaction product is usually detected by a color change or by the presence of fluorescence. The strength of the color change is directly dependent on the amount of antigen in the wells and thus simultaneously serves the quantitative assessment (assessment of the mass or concentration present) of the test sample. The hemoglobin-haptoglobin complex is permanently formed intravascularly (within blood vessels) by the leakage of hemoglobin from aged erythrocytes (red blood cells). In this process, the hemoglobin binds to the β-subunit of α-haptoglobin (special structure of haptoglobin). This complex is rapidly eliminated from the blood by the reticulohistiocyte system. The reticulohistiocytic system includes all phagocytic cells (scavenger cells), which are also part of the cellular immune response. Due to rapid elimination (removal), the half-life (time at which the amount is halved) of this complex is approximately ten to 30 minutes. In comparison, the half-life of free haptoglobin is about five hours. In healthy individuals, the hemoglobin-haptoglobin level is less than two micrograms per milliliter of blood. If this value is significantly elevated, there is a 95% probability of intestinal bleeding. Transport/storage: transport within 24 h, intermediate storage in refrigerator (4 – 8 °C) up to 1 day possible.If special collection systems are used, the material is stable for 5 days after sample collection at room temperature.
Indications (areas of application)
- Hemorrhoids – depending on the stage of hemorrhoids, it is a clinically relevant and possibly leading to symptoms vascular cushion.
- Crohn’s disease – chronic inflammatory bowel disease.
- Ulcerative colitis – unlike Crohn’s disease, ulcerative colitis can be identified as a continuous spread of inflammatory infiltrates across the digestive tract.
- Tumors – the hemoglobin-haptoglobin complex test serves here as a specific and sensitive method for diagnosing colorectal adenomas and carcinomas. Here, the hemoglobin-haptoglobin complex test detects increased adenomas and is therefore often combined with the immunological fecal hemoglobin test, which is more sensitive and specific for colon carcinomas.
Advantages of the hemoglobin-haptoglobin complex test.
- Unlike other detection methods, the hemoglobin-haptoglobin complex test is less sensitive to vitamin C and meat, so there can be no falsification of test results if previously consumed.
- Due to the high sensitivity (percentage of diseased patients in whom the disease is detected by the use of the test, i.e., a positive test result occurs) and specificity (probability that actually healthy people who do not have the disease in question are also detected as healthy in the test) of these immunological methods, the method represents the means of choice for a detection of blood in the stool, as it is both easy to perform and can provide clinically meaningful results.
- Furthermore, this method has the advantage that the patient does not have to follow a special diet. This advantage arises from the fact that immunological methods work exclusively on the principle of binding highly specific antibodies.
- Immunological testing methods based on the specific detection of hemoglobin in stool have a significant increase in sensitivity and specificity compared to other immunological testing methods. In various clinical studies, this immunological test has been shown to detect approximately 95% of colon carcinomas (colorectal cancers) and 70% of colon polyps (tissue protrusions into the lumen of the colon; some types of colon polyps are precancerous lesions for colon carcinoma). A disadvantage is bacterial degradation of hemoglobin in the intestine, which can result in false-negative values in older (> 24 hr) stool samples.
Disadvantages of the hemoglobin-haptoglobin complex test.
- When evaluating the immunological method, it should be noted that bacterial degradation of hemoglobin occurs in the intestine. Because of this, there is a possibility of false negative values in older (more than 24 hours old) stool samples.
Interpretation
A positive test result requires endoscopic examination of the entire colon (colonoscopy). Colonoscopic workup should be performed within 31 days according to a European quality guideline. Evaluations by Kaiser Permanente’s Research Institute of patients with a positive test result showed that the risk of colon cancer being detected during colonoscopy increased by 3% with each month. However, a significantly increased tumor rate (compared with patients who had a colonoscopy appointment in the first month) was seen only after a 10-month delay in colonoscopy.