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Tech Tip: ”HTLA” antibodies revisited

A Blog from Eric Ching:

In the fall of 2017, I blogged on titration under today’s heading of Tech Tip. In it, I talked about the use of titration to demonstrate the titration characteristic of the so-called High Titre, Low Avidity (HTLA) antibodies and the use of Ch(a-) and Rg(a-) plasma to identify anti-Chido and anti-Rodgers antibody by plasma inhibition and titration. These antibodies have also been referred to as serologically difficult antibodies (SDA).

 The “low avidity” is a misnomer as the weak reactions typical to this group of antibodies is due to the paucity of the antigen sites rather than the decreased affinity of the defining antibodies. In addition, some of these antibodies could be at low titre when first presented.

Let’s review the HTLA titration characteristic:

Titre                      1    2    4    8   16   32    64    128   256    512
Alloantibody        4    4    3    2     1     0      0       0      0         0
HTLA                     1    1    1   1      1     1      1       0      0         0

Again, the reason for this phenomenon is that HLTA antibodies are directed against antigens of low density; there is still antibody excess when diluted many folds to saturate all antigen sites. As a result, there are only a few of the IgG’s Fc to be agglutinated by anti-IgG in the indirect antiglobulin test to give equal weak reactions for four or more tubes.

In this blog, I am going to expound on this subject:

Historically, HTLA antibodies have been described as nebulous, grubby, fragile, hard to reproduce, troublesome and nuisance interfering antibodies. Although the great majority these antibodies are clinically insignificant, their presence negatively impacts the timely delivery and safety of blood transfusion. In one report, the incidence of significant alloantibody in the presence of HTLA antibody is 25%! (Moulds MK. AJMT 47:10, 789-795)

Since most of the antigens are of high frequencies, inconclusive panel results may lead to suspicion of borderline warm auto or alloantibody combinations; their interference may also mask clinically significant alloantibody(ies). In urgent request of transfusion, they may create anxiety to both technical and medical staff.

In prenatal testing, we should pay attention to a false high titre of anti-D if the prenatal sample also contains an HTLA antibody, for example:

                        Titre       1    2    4    8    16   32    64    128   256    512
 R2R2 Bg-    Anti-D      3    2    1    1      0     0     0        0       0        0
R2R2 Bg+    Anti-D      3    2    1    1     1    1    1        0      0        0     
Please take note that some clinically significant alloantibodies may exhibit the HTLA titration characteristicAnti-Yta, anti-Ytb, anti-Lub, anti-Cra, anti-Jsb, anti-Vel, anti-Jra, anti-Lan and anti-Cob.

The “black list” of HTLA
The blood group systems of Kna, McCa, Sla and Yka in Knops, Chido/Rodgers and JMH, Gya and Hy in Dombrock

Antigens Bga, Bgb, Bgc; Csa ? Era

HTLA characteristics
Except for one case of anti-Hy and a few cases of anti-Bg’s causing hemolytic transfusion reactions, the great majority of HTLA antibodies have not been reported to cause hemolytic transfusion reactions and HDFN.

Antigens are of high frequencies (>90%) except the Bg’s and Sla amongst Blacks (60%).

Antibodies are non-complement binding IgG. (The IgM anti-Sid is not mentioned here as it is rarely detected in tubeless methodologies.) Most anti-JMH are IgG4 which do not cause hemolysis.

Anti-Cha and -Rga are inhibited by pooled plasma and are adsorbed by C4 coated red cells (Transfusion22:243,1982), I refer the latter as sample remains undiluted. The definitive proof of anti-Chor Rgmust be governed by the fact that antibody activity failed to be neutralized by known Ch(a-) or Rg(a-) plasma.

Ficin and papain inhibit reactivities of anti-Cha,-Rga and JMH; these enzymes also weaken Knops antibodies while anti-Gya, anti-Hy and anti-Csa are not affected.

Cord red cells have weaker expressions of most HTLA defined antigens except JMH and Csa.

The commercially available soluble complement receptor 1 (CR1) can be used to neutralize the Knops antibodies such as anti-Kna, -McCa, -Sla, -Yka, etc.

Use of 0.2M DTT and 6% AET will weaken or abolish their reactivity except Cha, Rga and Csa but be aware the fact that these reducing agents also destroy Kell and weaken Lutheran blood group antigens.

Bga, Bgb and Bgc are stripped by incubating red cells with 20% CDP at room temperature for 30 minutes. Bg antibodies can be adsorbed by lyophilized human platelet concentrate HPCTM.

I came across a chart stating that anti-Era, an alloantibody to a high frequency antigen, may exhibit HTLA titration characteristic but I couldn’t find any reference. Please let me know if you have a reference for me. BTW, Rosebush antigens are destroyed by EDTA Glycine Acid  other than the Kell system antigens.
Comments are encouraged! 


So helpful and straightforward. Thanks so much
5/13/2023 8:54:57 PM

rachel kudrow
thank you for sharing this informative article
8/31/2020 2:34:48 AM

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