The Super Coombs

 

When the regular Coombs doesn't cut it, reach for the Super Coombs!

What is a Super Coombs?

It's a silly term (that may sometimes be clinically used) for the testing performed for "DAT negative autoimmune hemolytic anemia" investigation. A physician may request this workup when traditional methods of performing the Direct Coombs (or Direct Antiglobulin Test) turn up negative, but the patient is still exhibiting signs of Hemolytic Anemia and cannot be explained by other reasons. Typically these patients may have lower hemoglobin and hematocrit and see inappropriate responses to transfusion, have an extremely low or undetectable Haptoglobin, elevated LDH and bilirubin, etc. 

As we may know, negative doesn't always mean "Definitely not there". Negative simply means below the detectable limits of the testing system. In many cases, negative does mean "not there" and we can otherwise trust the testing system, but sometimes analytes exist in such small quantities that normal testing systems may not exhibit positive reactivity. This is true for patients with "DAT negative autoimmune hemolytic anemia". As such, a negative routinely performed DAT does not technically rule out AIHA.

But What Entails A Super Coombs? You Still Didn't Say.

 A Super Coombs, Enhanced DAT, or DAT Negative AIHA workup is actually a battery of tests performed on a patient sample to detect Red Blood Cell bound antibodies or complement (IgG/C3) on the cell surface that may be missed by traditional DAT testing. More often than not, this testing is performed at an Immunohematology Reference lab and is NOT performed at routine hospital Blood Bank laboratory. Physicians interested in this testing should expect a several day turn-around-time due to the need to ship out the patient's specimens to an outside lab. 

There are no rigid guidelines on how to perform, or WHAT to perform for a DAT negative AIHA workup, but several tests do exist to try and coax out some reactivity. Indeed, many scientists believe the number one reason for DAT negative AIHA is simply RBC-bound IgG antibody being below the threshold of detectability in a normal Some tests look for weak / low affinity reactivities, others look for possible non IgG causes such as IgM or IgA bound antibodies.

Polybrene 

The Direct Polybrene Test is a rapid method, but its efficacy hinges on the technician's expertise. Polybrene, scientifically known as hexadimethrine bromide, causes a general aggregation of healthy RBCs, facilitating the cross-linking of antibody molecules. It's a fascinating phenomenon: after introducing sodium citrate solution, general aggregates disperse, but any antibody-driven agglutination remains, undeterred by the neutralization of the Polybrene effect.

Should antibodies remain undetected post-dispersion, the test can segue into an antiglobulin procedure, specifically targeting anti-IgG. Prior research has illuminated the Direct Polybrene Test's prowess: its sensitivity is nearly on par with flow cytometry and equals the ELAT in detecting minuscule concentrations of RBC-associated IgG. The test has also proven invaluable for samples exhibiting DAT-negative AIHA.

Let's delve into Polybrene's mechanics. By counterbalancing the negative charge gracing the RBC surface, Polybrene optimizes antibody adherence. This action is instrumental when striving to pinpoint antibodies that exist in trace amounts or are traditionally elusive.

Blood bank specialists might appreciate this efficient manual Polybrene procedure for discerning RBC antibodies, tailored for routine laboratory apparatus:

• Combine RBCs with test sera in a low-ionic medium, keeping them at room temperature for approximately one minute.
  
  
• Introduce Polybrene, an integral quaternary ammonium polymer, prompting RBCs to aggregate nonspecifically.
  
  
• Following centrifugation, decant the cell-free supernatant.
  
  
• Neutralize the Polybrene-induced effect on the cells using a sodium citrate-glucose solution, and subsequently, assess the hemagglutination outcomes both macroscopically and under a microscope.

Remarkably, this entire protocol can be executed in a mere three minutes. Within the Rh system, its sensitivity surpasses the antiglobulin reaction by significant folds. Most antigenic systems, save for Kell, exhibit this heightened sensitivity. Nonetheless, Kell system sensitivity can be augmented significantly via a supplemental antiglobulin reaction (on the sensitized polybrene processed cells), provided the utilized antiglobulin reagent is devoid of anti-C4 and anti-C3 activities. For those aiming to boost the test's sensitivity to cold-reactive antibodies, a brief pre-treatment chill (about 30 seconds) before introducing the citrate-glucose solution is recommended.

Summary -- Polybrene induces aggregation of RBCs. Aggregation is dispersed by sodium citrate. However, if RBC bound antibody is present, RBCs will NOT disperse, thus the test would then be positive for RBC bound antibody below the detectable limit of routine testing.

4°C LISS Wash

Chill out!

The 4°C LISS wash is a specialized technique developed to heighten the sensitivity of the DAT, especially when the standard DAT might not pick up on lower levels of bound antibodies or complement proteins.

Washing RBCs with a cold LISS can slow down the dissociation of some antibodies from the RBCs, which could otherwise be missed at room temperature.

So, Why is This Relevant to DAT-negative AIHA?


In some AIHA cases, the antibodies bound to RBCs might be in very low quantities or might dissociate easily from the RBC surface. The standard DAT could miss these antibodies, resulting in a false-negative result. By using the 4°C LISS wash, we can amplify the chances of detecting these otherwise elusive antibodies, ensuring that cases of AIHA don't go undetected.

Micro-Column Agglutination 

Micro-Column agglutination, commonly referred to as "gel testing," is a technique in immunohematology that harnesses microtubes filled with a specialized gel (usually dextran-acrylamide) to detect interactions between red blood cells (RBCs) and antibodies. The gel acts as a physical barrier, sorting RBCs based on whether they've agglutinated (clumped together) due to the presence of bound antibodies.

How Does It Differ from Traditional Methods?

Traditional testing often involves looking for visible clumping of RBCs in liquid. In contrast, column agglutination uses the gel's properties to enhance the visibility and clarity of agglutination. If RBCs are bound together by antibodies, they'll form a line or clump within the gel, while unbound RBCs will travel to the bottom of the column during centrifugation. This gives a clear visual indicator of the presence of antibodies.

Column Agglutination's Role in DAT-negative AIHA:

AIHA (Autoimmune Hemolytic Anemia) is characterized by the body's immune system mistakenly targeting its RBCs. The Direct Antiglobulin Test (DAT) is a primary tool to diagnose this by detecting antibodies bound to RBCs. However, sometimes AIHA can be elusive, presenting as DAT-negative.

In such cases, the heightened sensitivity of column agglutination becomes invaluable. Here's why:

• Enhanced Detection: The gel matrix can trap even weakly agglutinated RBCs, making it more likely to spot cases where antibody binding is minimal.

• Clear Results: By physically separating clumped and non-clumped cells, the gel method provides a stark visual distinction, reducing interpretative ambiguities.

• Reduced False Positives: Gel testing minimizes the effects of rouleaux (a phenomenon where RBCs stack like coins, sometimes mimicking genuine agglutination).

In Essence:

Column agglutination provides a refined lens for spotting antibody-RBC interactions, especially when traditional methods like the DAT fall short. For cases of suspected AIHA where the DAT is negative, this method acts as a powerful secondary tool, ensuring that even subtle cases don't escape notice.

Flow Cytometry 

How Does Flow Cytometry Work in AIHA Context?

1. Labeling Cells: In the context of AIHA, RBCs are often labeled with fluorescently-tagged antibodies that specifically bind to human antibodies or complement proteins on the RBC surface.

2. Analysis: Once labeled, the RBCs are passed through the flow cytometer. When these fluorescently-tagged RBCs pass through the laser beam, they emit light, which is detected and analyzed.

3. Data Interpretation: The resulting data is then displayed in histograms or scatter plots, showing the presence or absence (and often the amount) of antibodies or complement on the surface of RBCs.

Why Use Flow Cytometry for DAT-negative AIHA?

The Direct Antiglobulin Test (DAT) is a cornerstone in diagnosing AIHA, as it detects antibodies or complement proteins bound to RBCs. However, not all AIHA cases show a positive DAT, even when the condition is present.

Flow cytometry comes into play in such challenging cases for several reasons:

• Increased Sensitivity: Flow cytometry can detect even small amounts of antibodies or complement on RBCs, making it particularly valuable for cases with low-level binding that might be missed by traditional methods.

• Quantitative Analysis: Besides just identifying the presence of antibodies, flow cytometry can give a quantitative assessment, indicating the density of antibody binding on RBCs.

• Comprehensive Profiling: With the capability to use multiple fluorescent tags, flow cytometry can help discern the specific types of antibodies or complement proteins present, offering a more detailed profile of the immune response.

In a study, Flow Cytometry detected more AIHA cases than the Gel Card Test, reaffirming its sensitivity. Several reasons, including the low level of sensitizing red cell autoantibody or the involvement of low-affinity antibodies, can result in false negatives with traditional testing methods.

Anti-IgA and Anti-IgM

Routine DAT testing involves the use of Anti-IgG. However, some cases of AIHA may involve Anti-IgA or Anti-IgM, thus appearing negative through the use of Anti-IgG DAT testing. 

BioRad Gel Testing card with IgA and IgM included!

IgA:

• IgA-mediated AIHA is rare compared to its IgG counterpart.
• In these instances, IgA autoantibodies target antigens on the red blood cell surface. The subsequent immune response leads to red cell destruction.

IgM: 

• IgM is a large pentameric molecule. When IgM autoantibodies bind to red blood cells, they can sometimes trigger the classical complement pathway leading to hemolysis.
• At colder temperatures, IgM can be particularly effective at binding to red cells, leading to cold agglutinin disease (CAD), a subset of AIHA. In these cases, the hemolysis is often exacerbated by cold exposure.
• Again, standard DAT may not always detect IgM, and specialized reagents would be needed.

Have you performed a DAT negative AIHA workup? Let us know!