Photography
Today I bring to you Direct Antiglobulin Testing (DAT). This is a pretty simple immunohematology test performed in certain circumstances. Some Blood Banks still include them with all crossmatches, but that's really an outdated practice. So, players in this test are of course the red blood cell (RBC) and IgG Antibodies. The DAT detects RBC's that are bound with IgG Antibodies. One drop of the patients RBC's are placed into a glass test tube. They are then washed 3 to 4 times with normal saline. (Yes, quite literally washed, okay more like rinsed since there's no soap involved) One drop of Anti-human globulin is added. The tube is then centrifuged for 15 seconds and then "read". (Using a mirror we re-suspend the cell button and visually observe the presence or absence of agglutination [clumping]). If agglutination is observed the test is positive. If negative a drop of Coombs Check cells are added. The tube is centrifuged again for 15 sec. and then read again. Agglutination must be observed at this point or the test is invalid. So, say I've just gotten a positive DAT, what does that mean? Well, here's the thing... A DAT is a nonspecific test. It can mean alot of things. Other tests and some vital patient information must be taken into account. If this patient is a newborn, the most likely cause is that some of the mothers cells have gotten into the babies blood stream and the babies immune system is attacking moms cells. If this is an adult with a negative antibody screen, its most likely drug induced. For our purposes in Blood Bank, we run DAT's when a Transfusion Reaction is reported and when we have positive Antibody Screens (not with every one) A positive DAT along with a positive ASN and several (or all) positive panel cells usually indicates a Warm Auto-Antibody or a Cold Auto-Antibody. Warm Autos are far more common. So, what does a warm auto mean. It means the patients red cells have been sensitised to their own immune system and this reaction is taking place at 37 degrees C (body temp). Warm autos are generally seen in Hemolytic Anemia. So as far a Blood Bank is concerned what does a positive DAT mean for us? (Not, give it to Sal to work up) It means there is other testing we cant' perform based on the nature of our testing practices and there is alot of testing we will have to perform in order to provide crossmatch compatible blood. (Actually it does mean give it to Sal to work up.) Essentially we have to revert back to another style of testing all together. Most labs now use gel testing in blood bank. There are a couple automated platforms now that use MTS gel technology. Gel is in layman's terms, a multiphase microwell system that streamlines antiglobulin testing. (As in, does everything we used to do in tube, faster and with less specimen.) Washing is not necessary in gel and results are consistent, reproducible and now automated. With a positive DAT, since there is no washing with gel, all testing done in gel will be positive because the patients cells are already sensitized to themselves. (Washing the cells cleans away the access antibody so only IgG bound to the patients cells are tested.) We use LISS technique when the DAT is positive) LISS itself stands for Low Ionic Strength Solution. There is an incubation step and lots of washing involved. This allows us to work around the problem. Patients with Warm autos give us headaches in the Blood Bank, they aren't necessarily difficult, the work ups are tedious and take extra time. We, are of course, are always on the clock so this always becomes a problem with unstable or critical admissions. Okay, I'm getting far too in depth for most of you! Anyone still awake?
So this whole thing is supposed to be about pictures right, right. This is all really vague unless you've had some immunology classes, so I've made some visual aids.
Red Blood Cell
IgG Antibody
Here is the RBC with some IgG bound to the surface (note safety pin as bond) and some free floating IgG from the plasma.
Here is the RBC after the washing step, the free floating IgG has been removed and the bound IgG is all that remains
Today I bring to you Direct Antiglobulin Testing (DAT). This is a pretty simple immunohematology test performed in certain circumstances. Some Blood Banks still include them with all crossmatches, but that's really an outdated practice. So, players in this test are of course the red blood cell (RBC) and IgG Antibodies. The DAT detects RBC's that are bound with IgG Antibodies. One drop of the patients RBC's are placed into a glass test tube. They are then washed 3 to 4 times with normal saline. (Yes, quite literally washed, okay more like rinsed since there's no soap involved) One drop of Anti-human globulin is added. The tube is then centrifuged for 15 seconds and then "read". (Using a mirror we re-suspend the cell button and visually observe the presence or absence of agglutination [clumping]). If agglutination is observed the test is positive. If negative a drop of Coombs Check cells are added. The tube is centrifuged again for 15 sec. and then read again. Agglutination must be observed at this point or the test is invalid. So, say I've just gotten a positive DAT, what does that mean? Well, here's the thing... A DAT is a nonspecific test. It can mean alot of things. Other tests and some vital patient information must be taken into account. If this patient is a newborn, the most likely cause is that some of the mothers cells have gotten into the babies blood stream and the babies immune system is attacking moms cells. If this is an adult with a negative antibody screen, its most likely drug induced. For our purposes in Blood Bank, we run DAT's when a Transfusion Reaction is reported and when we have positive Antibody Screens (not with every one) A positive DAT along with a positive ASN and several (or all) positive panel cells usually indicates a Warm Auto-Antibody or a Cold Auto-Antibody. Warm Autos are far more common. So, what does a warm auto mean. It means the patients red cells have been sensitised to their own immune system and this reaction is taking place at 37 degrees C (body temp). Warm autos are generally seen in Hemolytic Anemia. So as far a Blood Bank is concerned what does a positive DAT mean for us? (Not, give it to Sal to work up) It means there is other testing we cant' perform based on the nature of our testing practices and there is alot of testing we will have to perform in order to provide crossmatch compatible blood. (Actually it does mean give it to Sal to work up.) Essentially we have to revert back to another style of testing all together. Most labs now use gel testing in blood bank. There are a couple automated platforms now that use MTS gel technology. Gel is in layman's terms, a multiphase microwell system that streamlines antiglobulin testing. (As in, does everything we used to do in tube, faster and with less specimen.) Washing is not necessary in gel and results are consistent, reproducible and now automated. With a positive DAT, since there is no washing with gel, all testing done in gel will be positive because the patients cells are already sensitized to themselves. (Washing the cells cleans away the access antibody so only IgG bound to the patients cells are tested.) We use LISS technique when the DAT is positive) LISS itself stands for Low Ionic Strength Solution. There is an incubation step and lots of washing involved. This allows us to work around the problem. Patients with Warm autos give us headaches in the Blood Bank, they aren't necessarily difficult, the work ups are tedious and take extra time. We, are of course, are always on the clock so this always becomes a problem with unstable or critical admissions. Okay, I'm getting far too in depth for most of you! Anyone still awake?
So this whole thing is supposed to be about pictures right, right. This is all really vague unless you've had some immunology classes, so I've made some visual aids.
Antihuman Globulin has now been added. In this instance the Antihuman globulin is not binding to the IgG bound to the RBC, so the DAT is negative.
Antihuman Globulin has been added and it has bound to the IgG bound to the RBC, so this DAT is positive.
I hope the visual aids have made the DAT slightly clearer. (Yea, clear as mud) I want to dedicate this post to National Laboratory Professionals Week, which is also this week. I also want to hail all my colleagues both past and present. We very rarely get appreciated for all the work we do. I don't have the statistics in front of me but its something like lab work makes up 70% of a patients chart. Over 70 billion lab tests are run each year. The lab plays a very crucial role in the diagnosis and care of patients, but no doctor will ever admit that, in this day in age, without a lab he/she wouldn't be able to adequately care for said patients. Alright stepping off the soap box and putting my white coat back on... Happy Lab Week and Happy Crafting!
-Q
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