The Super Coombs

 

When the Regular Coombs Doesn't Cut It: The “Super Coombs”

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

Okay, not exactly.

There isn't actually one universally standardized laboratory test called the Super Coombs.

The term is sometimes used informally for an enhanced direct antiglobulin test (DAT) or DAT-negative autoimmune hemolytic anemia workup performed when a patient has convincing evidence of immune-mediated hemolysis despite a negative routine DAT.

And yes:

DAT-negative autoimmune hemolytic anemia is real.

A negative DAT means that the testing method did not detect enough immunoglobulin or complement on the patient's red blood cells to produce a positive reaction.

It does not necessarily mean that absolutely nothing is bound to those cells.

What Is the Direct Antiglobulin Test Looking For?

The direct antiglobulin test determines whether a patient's red blood cells are coated in vivo with immunoglobulin and/or complement.

Depending on the laboratory's method, routine testing may begin with a polyspecific antiglobulin reagent capable of detecting IgG and complement such as C3d, or it may use separate monospecific reagents.

So the routine DAT isn't simply an “anti-IgG test.”

A positive DAT can occur in autoimmune hemolytic anemia, hemolytic transfusion reactions, hemolytic disease of the fetus and newborn, drug-related immune hemolysis, and several other settings. And importantly, a positive DAT by itself does not prove that immune hemolysis is occurring. The result has to be interpreted alongside the patient's clinical and laboratory evidence of hemolysis.

Typical laboratory evidence supporting hemolysis can include:

• falling hemoglobin

• increased reticulocytes

• increased indirect bilirubin

• increased LDH

• decreased or undetectable haptoglobin

• spherocytes on the peripheral smear in warm AIHA

• hemoglobinuria in some forms of intravascular hemolysis

So what happens when all of that points toward AIHA...

...and the DAT is negative?

DAT-Negative Autoimmune Hemolytic Anemia

A small minority of patients with otherwise convincing autoimmune hemolytic anemia have a negative DAT by routine methods.

Several mechanisms can explain this.

1. Too Little IgG Is Bound to the RBC

The most common explanation is simply quantity.

The patient's RBCs may be coated with IgG, but the number of IgG molecules per cell is below the sensitivity threshold of the routine DAT.

That does not necessarily mean there is too little antibody to produce biologically important red-cell destruction.

It just means there is too little for that particular assay to detect reliably.

More sensitive methods can sometimes demonstrate RBC-bound IgG in these patients.

2. The IgG Has Low Affinity

Another possibility is a low-affinity IgG autoantibody.

The antibody may bind the RBC well enough in the patient's circulation to contribute to hemolysis but dissociate from the red-cell membrane during laboratory processing.

Remember what happens during a traditional tube DAT:

The patient's RBCs are washed repeatedly before antiglobulin reagent is added.

That washing is necessary to remove unbound immunoglobulin from the plasma.

But if the antibody has unusually low affinity for its target, some of the antibody you are trying to detect may wash right off the RBC.

By the time AHG is added:

Congratulations. You washed away your positive DAT.

Cold washing and low-ionic-strength washing techniques can help preserve some low-affinity antibody binding and reveal sensitization that is missed by conventional processing.

3. The Autoantibody Isn't IgG

IgG is the classic immunoglobulin associated with warm AIHA.

But it's not the only possibility.

Rare patients may have RBC-bound IgA, and unusual cases involving RBC-bound IgM have also been described.

If the routine method does not contain the appropriate reagent to recognize the immunoglobulin coating the cells, the DAT can remain negative.

Current reference-laboratory DAT-negative AIHA evaluations may therefore include testing specifically capable of detecting IgA in addition to conventional IgG and C3 testing. Versiti's current “Super Coombs” evaluation specifically includes enhanced DAT testing with IgA detection.

So What Exactly Is a “Super Coombs”?

There is no universal menu.

Different immunohematology reference laboratories may use different combinations of methods according to their validated procedures and the results obtained during the investigation.

For example, Versiti's current DAT-Negative Hemolytic Anemia Evaluation, also listed under the names Super Coombs and Micro Coombs, can include:

• ABO/Rh typing

• conventional DAT testing for IgG and C3

• enhanced DAT testing

• IgA detection

• routine and enhanced elution

• routine and enhanced antibody-detection testing

• additional studies selected by the reference laboratory when indicated.

The American Red Cross Immunohematology Reference Laboratories likewise offer investigation specifically for DAT-negative autoimmune hemolytic anemia.

So “Super Coombs” is better understood as:

“The normal DAT didn't answer the question, so we're going looking with more sensitive and specialized tools.”

Cold Wash / Low-Ionic-Strength DAT

Low-affinity antibodies present a particular technical problem because they can dissociate from the RBC while the specimen is being washed.

One approach is to wash the patient's RBCs under cold conditions and/or using a low-ionic-strength solution (LISS) before performing the DAT.

The goal isn't to “amplify” the antibody.

It's to reduce dissociation of weakly bound antibody during specimen processing, so more antibody remains attached to the RBC when the antiglobulin reagent is finally added.

Cold washing and LISS-based approaches have been used specifically to investigate suspected low-affinity IgG in DAT-negative AIHA.

That makes this test useful when the biology is essentially:

Antibody is present on the RBC in the patient → routine washing knocks it off → routine DAT becomes negative.

Change the washing conditions, and the antibody may become detectable.

Column Agglutination

Column-agglutination methods—usually called gel testing in everyday Blood Bank language—can also provide greater sensitivity than some conventional tube techniques.

In column testing, sensitized or agglutinated RBCs become trapped within or near the top of the gel or microcolumn after centrifugation, while unsensitized cells migrate toward the bottom.

Because different DAT platforms have different sensitivities, a sample that is negative using one methodology may occasionally become reactive using another.

But gel isn't automatically a magical “DAT-negative AIHA detector.”

A patient with sufficiently low-density IgG, a low-affinity antibody, or an immunoglobulin class not detected by the reagent can still test negative.

That's why reference-laboratory investigations generally combine methods rather than simply repeating the DAT in gel and calling it a day.

Flow Cytometry

If you're trying to find a very small amount of antibody on a red cell, why rely entirely on visible agglutination?

You can label it fluorescently.

Flow cytometry can detect very small quantities of RBC-bound immunoglobulin by incubating the patient's red cells with fluorescently labeled anti-human immunoglobulin reagents and measuring fluorescence on individual cells.

This can provide substantially greater analytical sensitivity for low levels of RBC-bound IgG than conventional DAT methods.

Flow cytometry also allows the amount of bound immunoglobulin to be assessed semi-quantitatively rather than simply calling the reaction positive or negative.

Studies comparing methods have demonstrated that flow cytometry can identify RBC-bound IgG in samples that are negative by routine DAT methods.

The tradeoff?

Most routine hospital Blood Banks don't have a validated red-cell flow-cytometry assay sitting around waiting for a DAT-negative AIHA workup.

That's reference-laboratory territory.

What About Polybrene?

Polybrene, or hexadimethrine bromide, has also historically been used in specialized investigation of DAT-negative immune hemolysis.

Polybrene is a positively charged polymer that reduces the normal negative surface charge surrounding RBCs and promotes close interaction between red cells.

A specialized direct Polybrene test can provide greater sensitivity to small amounts of RBC-bound antibody in some circumstances and has been used in investigations of DAT-negative AIHA. Published diagnostic approaches have included Polybrene among several techniques for detecting sensitization missed by routine DAT testing.

But I wouldn't present Polybrene as the standard modern “Super Coombs.”

It's better thought of as one specialized tool that may be used by laboratories with a validated method.

Anti-IgA and Anti-IgM Testing

This part is easy to overlook.

Suppose the patient's RBCs are coated predominantly with IgA.

If your routine antiglobulin system is designed to detect IgG and complement but doesn't detect IgA adequately, the result may be negative.

Using monospecific anti-IgA can uncover these rare cases.

The same general concept applies to unusual IgM-mediated disease that isn't being captured by the routine system.

Modern diagnostic studies of DAT-negative AIHA have demonstrated several distinct categories, including:

• low levels of RBC-bound IgG

• low-affinity IgG

• IgA-associated disease

• IgM-associated disease

although the first two account for the majority of cases.

Enhanced Elution

Another tool is elution.

Instead of trying only to demonstrate antibody attached to the RBC by agglutination, an eluate attempts to remove immunoglobulin from the patient's red cells so that the recovered antibody can be investigated separately.

Reference-laboratory DAT-negative hemolytic anemia evaluations may include both routine and enhanced elution techniques, particularly when very small amounts of RBC-bound antibody are suspected.

Again, that's why “Super Coombs” is really a workup rather than a single test.

You might need several different ways of asking the same basic question:

Is there evidence that this patient's RBCs are being immunologically sensitized even though my routine DAT can't demonstrate it?

A Negative Super Coombs Doesn't Completely Rule It Out Either

Here's another important limitation.

Even specialized testing isn't perfect.

Versiti specifically cautions that a negative enhanced evaluation does not completely exclude immune-mediated hemolytic anemia, and a positive enhanced result by itself is also not sufficient to diagnose AIHA.

The diagnosis still depends on the entire picture.

Does the patient actually have hemolysis?

Have other causes been excluded?

Does the serologic investigation make physiologic sense?

Does the clinical course fit?

DAT-negative AIHA should not become a label applied to every unexplained anemia simply because somebody ordered a more sensitive Coombs test.

Don't Forget the Other Causes of Hemolysis

Before settling on DAT-negative AIHA, clinicians have to consider other explanations for hemolytic anemia.

Depending on the presentation, the differential can include:

• delayed hemolytic transfusion reactions

• drug-induced immune hemolysis

• microangiopathic hemolytic anemia

• mechanical hemolysis

• hereditary membrane disorders

• RBC enzyme deficiencies

• paroxysmal nocturnal hemoglobinuria

• infections

• other acquired and congenital causes of hemolysis

That's one reason DAT-negative AIHA can be such a challenging diagnosis.

The laboratory isn't merely trying to prove that the DAT was “wrong.”

It's trying to determine whether immune red-cell destruction is actually the best explanation for the patient's hemolysis.

The Takeaway

The Direct Antiglobulin Test is extremely useful.

It is not infinitely sensitive.

A patient can have autoimmune hemolytic anemia despite a negative routine DAT because:

There may be too little IgG on the RBC.

The IgG may have low enough affinity that it dissociates during washing.

The coating antibody may be IgA or, less commonly, IgM rather than conventional IgG.

Enhanced techniques—including altered wash conditions, more sensitive antiglobulin methods, specialized immunoglobulin reagents, enhanced elution, Polybrene-based testing, and flow cytometry—can sometimes reveal what the routine DAT missed.

So when the patient is clearly hemolyzing and the DAT comes back negative:

Don't immediately conclude that the immune system is off the hook.

Sometimes you just need a bigger Coombs?