Where do ABO Isoagglutinin Antibodies Originate?

 Where do human ABO isoagglutinins originate?

Where do they come from? Why are they here?

We innately know as part of our Blood Bank training that ABO isoagglutinins exist and they play a part in how we perform a patient's blood type. O patients will have Anti-A and Anti-B against the A and B antigens in their plasma. A patients will have Anti-B, B patients will have Anti-A, and AB patients will have no isoagglutinins. How did these antibodies get there? If their immune system has never seen incompatible blood types, how does the immune system know to make these antibodies?

As we understand from the adaptive immune system, antibodies are created through prior antigen sensitization. ABO isoagglutinins, on the other hand, are consistently dubbed "naturally occurring". Karl Landsteiner was of the opinion that these antibodies were naturally and spontaneously produced based on red cell antigen makeup.

Any isoagglutinins in a newborn's plasma is that of the mother. This is typically why neonates are given O cells, to prevent any type mismatches with the mothers plasma still within the neonate. As time goes on the neonate will begin to form its own immune system and the mothers antibodies will disappear in favor of the neonate's. Months into life we may start to see the formation of their own isoagglutinins. But why? Did the mother's immune system provide instructions on how to create isoagglutinins based on a certain blood type result?

It is actually not cemented in science or completely agreed to as to where ABO isoagglutinin antibodies come from. One of the most commonly held thoughts is that the antibodies DO arise as a result of antigenic stimulation, just not from blood, but from the environment.

Environmental antigens such as from food, dirt, and especially gut bacteria may contain cross-reactive antigens that cause ABO isoagglutinins to form. A popular study showed that chickens kept in a sterile environment would produce anti-B when presented with bacteria known to produce high-levels of B or B-like antigen. They were able to get the chickens to produce anti-A, but the strength of the antibody never amounted to anything more than weak. This kept researchers on their toes about whether there was more out there to learn.

It wasn't until the discovery of B lymphocyte subsets that more information and research began to focus on the ABO isoagglutinin origins. It is now known that there are different subsets of B cells. B1 cells and B2 cells offer different functions. B1 cells function as an arm of innate immunity and do NOT require antigenic stimulation to be produced. B2 cells are the more well known "adaptive" immunity cells that arise as a result of antigenic stimulation and provide lasting immunological memory.

It is believed there is a link between ABO isoagglutinins and B1 cells and that the ABO linked B1 cells are created as a result of genetic information being passed down through generations.

It is now thought that there are two phases to ABO isoagglutinin creation. When a baby is born they will soon create the B1 cells, capable of creating Anti-A and/or Anti-B isoagglutinins, usually always IgM.  It has been shown that newborns do make B1 cells almost exclusively that early on in age. As they age, and their adaptive immune system begins to flourish, B2 cells will begin to be made, which are the typical Memory B cells we are used to that are created in response to an antigenic stimulation. This is where the environmental factors, especially gut bacteria which have A and B like antigens can stimulate the production of additional anti-A or anti-B antibodies. 

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Daratumumab + Isoagglutinin Titers

Daratumumab

Daratumumab (Darzalex, Anti-CD38) is a known troublemaker for Blood Bankers everywhere. It typically causes panagglutination in antibody screens and panels but thankfully does not interfere with Blood Typing (I'm looking at you Anti-CD47/Magrolimab). Daratumumab typically mimics the appearance of a high-titer low-avidity (HTLA) antibody and as a result does not always disappear when moving to less sensitive methods, such as a "non-additive" or "non-enhanced" antibody screen/panel. 

DTT (Dithiothreitol) Testing

The most common method of removing Daratumumab interference is to use DTT (Dithiothreitol). DTT denatures CD38 by disrupting integral disulfide bonds within the CD38 glycoprotein ensuring that Daratumumab (Aka. Anti-CD38) cannot attach to your testing cells (screen and panel) and cause interference. 

ABO Titers

As mentioned before...this does not interfere with ABO/Rh blood typing. But that doesn't mean Dara is completely off the hook just yet. Blood Typing is done at immediate spin with no extra help but cells and antisera. But what if an Anti-A1 or Anti-B titer needs to be performed? Many titer procedures follow the same course whether for an alloantibody or an isoagglutinin. As such, a titer would be prepared using non-enhanced saline only medium. It would then be washed x4, have anti-IgG added, and read as you would anything else. This presents a problem though...if the patient is on Daratumumab, it would be attaching to the A1 reagent red cell you are using for your titer. Upon adding anti-IgG it would bind the Daratumumab bound to the CD38 on the cell and you will visualize the positive reaction. How do you know if it's the Daratumumab reacting or the Anti-A1 reacting? Without modifying your titer procedures, this would present the possibility of over-reporting titer results if you aren't careful. Daratumumab can potentially titer out to the thousands. 

In this specific circumstance, a DTT treated titer would likely be warranted. A study showed the difference between Anti-A1 titers in a patient actively on Daratumumab. The untreated titer went out to >2048 at AHG with the study patient. The DTT treated titer showed a more modest titer of 32 at AHG phase.

Daratumumab makes us have to look at a lot of our procedures differently. 

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Should Cord Blood Results Be Used As An Official Type on File with Blood Bank?

Umbilical Cord Blood Testing

Umbilical Cord Blood testing is a commonly performed procedure immediately after giving birth. It is indicated for mothers who are RhD negative as well as mothers who type as O. Some physicians may request Cord Blood testing on mother's of other types but it is not common unless Fetal/Maternal type mismatch related hemolysis is suspected (ex. Baby is A, Mom is B)

In some Blood Banks/Transfusion Centers, Umbilical Cord Blood is a "for diagnostic purposes only" sample type. Considering this, the Blood Type that is traditionally done as part of a Cord Blood Workup is not treated as an official Blood Type on file for the patient. Per AABB Standard 5.14.1 two blood types on file are required to receive Type Specific and Electronically Crossmatched PRBCs. As such, even if a blood type was performed on the Cord Blood, two separate heelsticks would need to be performed as part of the pretransfusion process to meet this standard. 

I can't find any indication from AABB that Cord Blood is unacceptable for use as an official type. It's likely up to the individual medical director to make the call. I've heard of institutions even performing Type and Screens from the Cord Blood. Why so against using Cord Blood for pretransfusion needs?

Outside of the US according to the National Pathology Accreditation Advisory Council for Australia in their Requirements for Transfusion / Laboratory Practice they outright say "Cord blood must not be used for pretransfusion testing." in C8.3(i)

In the United States, Christiana Care out of Wilmington, DE DOES allow for cord blood specimens to be used per according to their specimen acceptability.

"For neonates, a properly labeled EDTA cord blood or pediatric venous specimen is required"

Cord Blood Contamination during Collection

I think the answer generally lies within the higher possibility of Cord Blood contamination with maternal Red Blood Cells. During the collection of Umbilical Cord blood immediately after birth, it is possible that maternal blood surrounding the cord/placenta may inadvertently and unknowingly drip into the sample tube. Enough contamination could affect the outcome of the Rh interpretation or the entire Blood Type determination. There is also possibility that during birth a retroplacental hemorrhage occurs, which may allow maternal blood to enter the space of the umbilical cord circulation. These sources of potential area are nearly completely removed simply by performing a heelstick. 

I have seen this situation arise previously. The Blood Bank received a Cord Blood and typed O pos (D4 typing: 2+ positive). The baby was subsequently transferred to the NICU where it is commonplace to order "For Possible Transfusion" Type and Screen's on the patients being admitted. A heelstick was performed to collect the Type and Screen and was sent to Blood Bank. The heelstick specimen was typing as a perfect O negative. The Du (weak D) testing was also negative indicating a true O neg. A second heelstick was performed, and revealed O negative once again. What happened? Mom was O positive. It is extremely likely that the collection was contaminated somehow by maternal red blood cells. 

This is certainly not a common occurrence however. This study shows near perfect matches between Umbilical Cord Blood and heelstick specimens. The only divergence was between a few DAT results. The Blood Types matched 100% between samples. I think though that this simply shows that both sample types are valid for use, it doesn't rule out the chance for user error within the system, such as a nurse haphazardly drawing the Cord Blood. 

For those that do accept Cord Blood as official it seems to actually have its benefits. There are studies that have shown if the laboratory can use Cord Blood for initial admission testing, rather than using heelsticks, the clinical outcome of the neonate seems improved. This is especially apparent for low and very low birth weight neonates. It saves from having to take what little blood might be in circulation in these neonates. It can prevent the need for excessive vasopressors and even delay or remove the need for PRBC transfusion.   

How does your Transfusion service handle this?

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Anti-G and Pregnant Women

"I just learned about Anti-H in Bombay phenotype patients, now you're telling me there's an anti-G??" 

What is Anti-G?

Anti-G is an antibody deriving relevance in pregnant or TTC (trying to conceive) prenatal women. On a normal everyday antibody screen and panel, Anti-G generally looks identical to that of a patient presenting with a combined anti-D and anti-C, in that anti-G will cause agglutination with anti-D AND anti-C positive cells. For males showing reactivity with D and C antigens, after all else has been ruled out, they will likely been resulting in their Blood Bank file as Anti-D and Anti-C. The investigation ends here. 

Whether it is Anti-D and Anti-C or Anti-G does not matter. The patient will receive D and C negative blood, and will thus likely have a compatible AHG crossmatch. Remember, Anti-G agglutinates with D and/or C positive blood. As long as the blood is D and C negative, they will be receiving the proper antigen negative blood regardless. 

In females of childbearing age, the story is a bit different. We really want to know what is going down serologically. For a multitude of reasons. Anti-D and Anti-C can cause severe HDFN, whereas anti-G, although capable of HDFN, tends to be milder in presentation. Additionally, differentiating between Anti-D/Anti-C and Anti-G is extremely important in terms of Anti-D prophylaxis. 

There are many possibilities that can take place in terms of what is really reacting when we see what looks like anti-D and anti-C reactivity on a screen/panel. 

The patient could have (1) Anti-D, Anti-C, and Anti-G,  (2) Anti-D and Anti-G,  (3) Anti-C and Anti-G, (4) Anti-D and Anti-C, or simply (5) Anti-G. In any situation where the patient DOES NOT have an actual anti-D and is Rh negative, the patient should still follow guidelines for receiving Rh immunoglobulin at the proper intervals. Thus a patient with Anti-C and G or just anti-G is STILL A CANDIDATE for RhIG in a effort to prevent additional anti-D alloimmunization. A mother with a confirmed anti-D with or without an additional anti-G would thus not be a candidate for RhIG, allowing for the detection of any un-needed injections. 

 As with many transfusion centers, maternal antibodies are usually serially titered at certain intervals throughout the pregnancy as well. If a REAL anti-D or a REAL anti-C is present, it is likely that the pathologist or patient's clinicians would want to monitor the titer of these antibodies for HDFN and fetal viability purposes. 

Unfortunately, most hospital blood banks do not have the capability of differentiating between AntiD/C and an Anti-G in house.  The only potential clue would be if the patient had a true Anti-D and Anti-C, it's possible that a Blood Bank Technologist could potentially notice a difference in reaction strength between the two antibodies. Perhaps the anti-D is reacting at 4+ on the panel but the anti-C is only reacting at 2+. If an anti-G, it's likely that the D+ and C+ cells would react in a similar manner. This isn't concrete though, as we know. 

Send Out / Immunohematology Reference Lab testing is the most common way of differentiating between these antibodies. There usually isn't any specialized testing involved...it's usually a combination of adsorptions (DOUBLE adsorptions)/elutions. It's just a very time consuming process that most hospital Blood Banks do not have the budgeted FTEs for. 

Check out these links for more info on lab testing that differentiates between G and D/C

AJTS

Blood Bank Guy

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Can I Request Unvaccinated Blood For Transfusion?

 For myself or my loved one in need? 

No. No you cannot. Are you going to request blood from the Flu unvaccinated? HPV? Hepatitis? TDap? No, just the COVID vaccine? 

COVID Vaccine and Blood

There is no way for a transfusion center Blood Bank to know the vaccination status of the donors behind the blood products on the shelf. When we receive the blood products in house it is understood, per contract, and up-to-date facility standards that the blood is tested for Trypanosoma cruzi, Hepatitis B, Hepatitis C, HIV, Human T-Lymphotropic virus (HTLV), Treponema pallidum (aka Syphilis), Zika virus, West Nile Virus, and Babesia. Individual blood centers may or may not test for the last three listed depending on geographical location or standard operating procedure. It is also possible that the blood center tests for CMV (Cytolomegalovirus). The Blood Bank does not receive a donor history form or any information about the donor whatsoever from the donation center. Additionally, it is extremely unlikely the Red Cross, or any other donation center would give up this information, as it is irrelevant and a violation of the donors medical privacy. 

The Red Cross, for example, does not routinely test blood products for COVID, nor does it routinely test plasma for antibodies to COVID, likely as a result of increased vaccination rates, widespread COVID infection rates, and decreased need for convalescent plasma. 

The only real way to assuredly get "COVID unvaccinated" blood products would be to set up a directed donation process in which the recipient chooses a person to donate blood for them if they were to require blood products. Directed Donation is very uncommon and usually requires a consult with a Transfusion Medicine physician prior to entertaining the process. There are many reasons why Directed Donation is not recommended (possibly another post about this). Choosing blood products from the random donor pool on a Blood Bank's shelf is generally regarding as safer than choosing Directed Donation. A Pathologist would likely, in kind terms, try to almost... talk people out of Directed Donation. Several times a year we have patient's family members looking to do a Directed Donation. After consulting with a Blood Bank physician, it almost NEVER happens. 

Also, if you're receiving Red Blood Cells, there's no vaccine in this product anyway. Any antibody or other protein that you're afraid of would be in the plasma, the liquid portion of blood. 

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