Diagnosis of AHA Requires Laboratory Testing – What Are Its Key Specifics?

Introduction

Acquired hemophilia A is caused by spontaneously developed specific antibodies against the coagulation factor VIII (FVIII) in individuals without a congenital FVIII defect. Clinically, it manifests mostly (in up to 87% of patients) through dramatic, even life-threatening bleeding. It is detected in 1-2 persons per 1 million inhabitants annually, but the mortality rate reaches up to 22%. It occurs significantly more often in older age (in the population aged 65-89 years, up to 4 times more frequently, i.e., up to 8 patients per 1 million inhabitants annually), with other risk factors including autoimmune diseases, malignancy, recent pregnancy, and even reactions to medications. Correct diagnosis requires sufficient information and experience.

Laboratory and Screening

In the context of laboratory diagnostics, a prolonged activated partial thromboplastin time (aPTT) is typically indicated as a marker. In this context, Dr. Hrachovinová clarified that “a prolonged aPTT in patients who have congenital defects is normal, but in people who have never had prolonged aPTT and have not bled, it is a different situation – there it is necessary to differentially diagnose why the prolongation occurred. It can be disseminated intravascular coagulation, the presence of lupus anticoagulant, the presence of DOACs (direct inhibitor of coagulation factor II), heparin, or acquired hemophilia A.”

She further emphasized that it is important to always perform a complete laboratory screening, i.e., measure coagulation time PT/INR, thrombin time (TT), fibrinogen level, D-dimers, and possibly anti-Xa. This allows the exclusion of disseminated intravascular coagulation, heparin contamination, or the effects of anticoagulants whose use was not known in the patient. After excluding these, suspicions of lupus anticoagulant or AHA remain. In this context, the speaker added an observation that, according to her own patient group, 80% of individuals with AHA have significantly elevated D-dimer levels despite it being a bleeding disorder.

Examination Procedure

In cases of suspected acquired FVIII inhibitor, the following steps are applied:

1. aPTT – mix of patient and normal plasma before and after incubation at 37°C
2. exclusion of lupus anticoagulant (LA)
3. determination of FVIII activity (and possibly other coagulation factors)
4. determination of FVIII inhibitor titer

Mixing Tests Based on aPTT

The mixing test is performed by mixing the patient's plasma and control (normal) plasma in different ratios (1:4; 1:1; 4:1) and measuring aPTT for all these mixes before incubation at 37°C and after 2 hours of incubation. At a 1:4 ratio (i.e., the smallest proportion of patient plasma), the aPTT value after 2 hours is significantly prolonged in AHA. The curve has a distinctly different shape than in the presence of lupus anticoagulant or a coagulation factor deficiency.

Determination of FVIII Level

FVIII activity depends on the type of antibody and usually does not correspond to the bleeding risk. The coagulation test for FVIII is influenced by the aPTT reagent used – it is necessary to use reagents insensitive to LA (overlap of direct and indirect FVIII inhibitors). The author of the announcement considered the chromogenic determination of FVIII optimal, noting that it is a time-consuming and economically demanding method – here, at least an initial correlation of chromogenic and one-phase FVIII values is recommended to definitively determine that a deficit is present.

Determination of Inhibitor Presence

The classic Bethesda method is used to determine the FVIII inhibitor titer, defined as 1 Bethesda unit per milliliter of plasma (BU/ml), which is the antibody concentration that causes 50% residual FVIII activity in a defined incubation mixture (after 2 hours of incubation at 37°C).

The source of FVIII is normal plasma, where FVIII must be present at least 80%. The mixture of normal and patient plasma (1:1) is incubated at 37°C, and after 2 hours, the FVIII value is measured. The inhibitor neutralizing 50% activity from the reaction mixture corresponds to 1 BU. The inhibitor calculation is performed as follows:

1. Measure the residual FVIII value of the control (e.g., 40%).
2. Measure the residual FVIII value of the patient (e.g., 24%).
3. From both residual values, obtain the so-called corrected FVIII level (in this case, 24/40 × 100 = 60%).
4. Subsequently, the corrected FVIII level is read from the graph.

However, in acquired hemophilia A there is a complication because the inhibiting antibodies in this disease have complex kinetics (second order), so the Bethesda method cannot be used identically as in hemophiliacs with congenital FVIII deficiency, whose antibodies have first-order kinetics. Thus, when calculating the inhibitor value in AHA, the basic principle is that the corrected FVIII activity should be around 50%.

There can also be the following unusual situations when determining the FVIII inhibitor titer:

• Higher residual FVIII value in AHA patients already under treatment – the patient's FVIII “interferes” with the FVIII supplied in the laboratory, so it is necessary to degrade the patient's FVIII by incubation at 56°C before measuring the FVIII inhibitor level.
• Bleeding in patients taking direct oral anticoagulants (DOACs) or warfarin – in the case of warfarin, the aPTT-to-INR ratio can help, in the case of DOACs, plasma adjustment is necessary.
• Patient is taking emicizumab.
• Patient has a combination of lupus anticoagulant and FVIII inhibitor in AHA.

Summary and Conclusion

In conclusion, Dr. Hrachovinová summarized that AHA is a very serious disease and every significant unexpected prolongation of aPTT must be analyzed. She reassured that the method of mixing tests based on aPTT is available in all coagulation laboratories. Determination of the FVIII inhibitor requires experience. Correct and fast diagnosis of AHA then forms the basis for timely and effective treatment.

Eva Srbová
editorial staff proLékaře.cz

Source: Hrachovinová I. Laboratory diagnostics of AHA and its specifics. XXVIII. Czech-Slovak Conference on Thrombosis and Hemostasis and XX. Czech-Slovak Conference on Laboratory Hematology, Hradec Králové, September 14, 2022.