Coomb test can be direct & indirect
(A) Direct coomb test: This is done to detect any coated RBCs with antibodies (autoimmune haemolytic anaemia) in vivo (inside the body). Patient coomb positive suspected RBCs washed 3 times - add 3-4 drops of blood in a tube and then add Normal saline (fill the tube) and centrifuge at 3000 r.p.m for 3 minutes. Repeat this 3 times and then decant any remaining saline by putting the tube upside down on a tissue. Add AHG reagent or coomb reagent, centrifuge for a minute and see any agglutination for positive coomb test.

(B) Indirect Coomb test (Indirect antihuman globulin test - IAT): This test is done to detect any IgG antibodies in-vitro (outside the body). For example it is used for cross match, detect any antibodies in pregnant lady as prenatal care to know if the mother have any antibodies that can cross the placenta and attack baby RBCs. Thus it detects antibodies in the serum that are unbound to RBCs (apposite Direct coomb test which detects antibodies that are bound to RBCs). In cross matching, Pateint serum in Which presence of IgG antibodies is suspected incubated with Donors RBCs washed 3 times with Saline or LIS (Low Ionized Saline). If specific antibodies need to be detected then Ready made RBCs with known Antigen used, so pateint serum with known RBCs incubated. If RBcs washed with Saline the incubation period then is 1 hour which can be reduced to 20-30 minutes by washing with LIS. After incubation, Centrifuge and see any agglutination.
Note: Indirect Coomb test can also be used for incompatible ABO grouping which are IgM antibosies.

Hope this helps

Forgot to add that Incubation temperature is 37 degree

after washing, you will take one drop of RBCs, and add the AHG reagent, prefreably 2 drops.
then you will centrifuge and read for agglutination, if it is negative then add check cells, by now you should obtain positive test, otherwize your test is invalid and should be repeated.
This is for the Direct cooms test, in which you testing whether the patient RBCs are sensitized in vivo or not, in other words, does the RBCs of the patient have antibodies attached to it in vivo.

While for the indirect coombs, you are testing whether the patient have antibodies in the serum or not, her you will use reagent RBCs from the lab, and add patient serum to it. if the test is also negative then you have to add check cells the test should turn to positive, if not then repeat the test from the start point.
Thank you very much

Thank you very much for the worthy information
Please can you explain in more detail the washing process. I mean what kind of tissue I use and how many times I add saline.

Red blood cells only concerning the type of tissue is blood, but RBCs what we are concerned with.

Thanks for this information

واو كتابه مفصله جدا اخوي medical lab specialist ويعطيك الف عافيه باين عليك متعملق في بنك الدم

سوال على الطاير : في اي الحالات (يجب) عمل كروس ماتش في ال room temperature ولمادا !!؟؟*

Hope this helps

شكرا لكم

على فكرة ال X-match في ال room temperature يطلق كان يطلق عليه عندنا في امريكا ب incomplete crossmatch وهو يعمل كما قال اخي medical lab specialist
شكرا لكم جميعا

مشكور اخي medical lab والدكتور اشرف على ردكم الجميل لكن سوالي لكما ليش احنا في الاصل بنعمل في ال Cross match at room temperature ؟؟وشي الي يهمنا في ال IgM مادام نحن بنقل الدم في الجسم في 37 ؟؟؟ بنعمل الاختبار في 37 وخلاص , هل يوجد حالات معينه او امراض معينه يتم نقل الدم الى الجسم بصوره بارده مرضى القلب مثلا كما سمعت ؟؟؟؟!!

تحياتي :sm171:

اخي حسين اذا انت كنت عامل خطا في تحديد فصيلة المريض يمكن كشفها في ال incomplete crossmatch ببساطة
شكرااااااااااااااا لك اخي حسين

Thanks brother Hussen99 & your question is fantastic. The answer is as follows:

As I said previously that this test is done for ABO incompatibility & for cold agglutinin antibodies which are IgM type. You thought it otherway round, imagine what is easier to you as laboratory technologist to perform a test at room temperature (natural temperature at room) or at 37 which requires water bath (special instrument), offcourse i would say room temperature but that doesnt mean that ABO incompatibility studies can not performed at 37, in fact it can be. Going back to the structure of IgM (remember Immunology lecture) - IgM has 5 Ig sites with 10 antigen binding sites (see attached picture) which means it doesnt require any coomb reagent to help visulaisation of agglutination. In fact if you would suspect IgG antibodies then you need coomb reagent help with 37 degree incubation (Indirect coombs test). ABO incompatibility studies can be performed in 37 as i said, however as far as gold antibodies are concerned you can not detect them at 37. This is because they react best at temperature between 28-31 degree which is quite similar to room temperature and if the test is performed at 37 you will miss to identify them. Inside the body cold antibodies are not reactive, however the need of detection of these antibodies is very important as they can give false ABO grouping & X-match incompatibility. For example, imagine you do a slide ABO grouping which is usaualy done at room temperature. Patients who have these cold antibodies will give agglutination with all anti-sera giving false AB Rh(D) positive blood group. Hope this helps

مشكور اخي medical lab كل الشكر على هدا المجهود والتفسير العلمي الممتاز ويعطيك الف عافيه ورحم الله والديك
واتعبانك اخي (( ولكن)) عندي نقطعه بسيطه ضائعه في الموضوع وهي اني مررت على بعض بنوك الدم لا تعمل اختبار( ICT) الي هو الكروس ماتش والسكرينغ في الروم تيمبرتشر ابدا فقط ب *37 , (( الا)) في حالات (تشخيصيه معينه) يهمنا فيها IGM بصوره (رئيسيه) اثناء نقل الدم وكما دكرت في ردي السابق (بعض)(( مرضى القلب)) اثناء حاجتم الى دم يتم نقل الدم اليهم بصوره(( بارده )) لا اعلم الى اي حد !!!!!!والله اعلم

ومشكور مره اخرى اخي وانتضرني عندي عدة اسئله في بنك الدم

تحياتي:sm171: