Frequently Asked Questions - HemDirect Test

  • No difficult manipulation of sample material
    The sample is transferred into the extraction buffer in the collection tube and extracted/dissolved by shaking. After that three drops of the liquid are added directly to the test.
  • Rapid generation of the test result
    Results should be read within a ten minute period after addition of the sample.
  • High sensitivity
    The lower detection limit is at least 40 ng/ml hemoglobin. This corresponds to human blood that has been diluted to 10-6 - 10–7 (average hemoglobin content of blood: 120-180 mg/ml).
  • High specificity for human hemoglobin
    Due to the specificity of the antibodies, positive test results allow the direct conclusion that the sample most probably contains blood of human origin. A cross reactivity is observed for blood of primates and ferret blood.
  • The test contains two monoclonal anti-hemoglobin antibodies as active compounds. One of the antibodies has been immobilized as line in the test result region (T) of the membrane. The other antibody is gold-labeled. It is placed downstream of the membrane on a glass fibre pad and becomes mobilized by the addition of the liquid. If hemoglobin is present in the sample it will bind to the gold-labeled antibody forming a complex. Through the capillary effect of the membrane, the liquid moves across the membrane towards the test result region. Here the immobilized antibody that recognizes another epitope of hemoglobin will bind the hemoglobin complexed with the gold labelled antibody (formation of a sandwich complex). This can be seen by the appearance of the red test result line.
    The control line is composed of a different antibody pair (rabbit antibodies/goat anti-rabbit antibodies) one of which is immobilized at the control region. The other one is gold-labelled and mobile. As these antibodies interact directly with each other, the control line is formed independent of the presence of hemoglobin. The appearance of the control line serves as an internal control for the correct performance of the test.

    A high dose hook effect is observed, if too much free hemoglobin that is not bound to the gold-labeled antibody reaches the test result region. In this case the antibody immobilized at the test result region becomes saturated with free hemoglobin. This prevents the binding of the hemoglobin complexed with the gold-labeled antibody, thus interfering with the formation of the test result line. The test result appears negative in spite of the presence of hemoglobin in the sample.
    The high dose hook effect can be avoided using the color of the sample as a guide. The visual detectable color caused by hemoglobin vanishes between a 10-3 and 10-4 dilution. At this concentration range there is no danger of a high dose hook effect. In contrast samples that are clearly colored due to hemoglobin are likely to cause false negative results because of the high dose hook effect. Good results are obtained when the extract has a "straw" color.
    If you are concerned that a negative result is from High Dose Hook Effect, then a simple remedy is to dilute the extract and re-run the sample.

    Blood samples stored at room temperature for 33 years have given positive results.

    Please find more data in the validation study of the Seratec HemDirect by Dale L. Laux, Forensic Scientist at the Ohio BCI and Amanda Misencik:
    Validation Study of the Seratec HemDirect Hemoglobin Assay for the Forensic Identification of Human Blood.

    With strong samples, good results are obtained after 10 minutes extracting. For very weak samples, we recommend to extract overnight in smaller volumes of buffer.

    The buffer and the tests can be stored at room temperature or in the fridge, between 2 and 30°C.

    The SERATEC HemDirect immunoassays enables the user to identify traces of human blood in forensic sample material. In a specific antigen/antibody reaction, the presence of human hemoglobin can be determined within minutes. The test result is interpreted visually by the appearance of a red test result line in hemoglobin positive samples. The assay is rapid and easy to perform and requires no difficult manipulation of the sample material. If necessary, it can be performed directly at the crime scene.