What causes mixed-field agglutination encountered in ABO (Anti-B and Anti-A) forward grouping?

Causes of Mixed-Field Agglutination in ABO Forward Grouping

Cold reactive autoagglutinins are the most common cause of mixed-field agglutination encountered in ABO forward grouping. This pattern can be misleading and must be carefully distinguished from other causes to ensure accurate blood typing.

Primary Causes of Mixed-Field Agglutination

Cold Reactive Autoagglutinins

• Cold reactive autoagglutinins cause red cells to spontaneously agglutinate at lower temperatures, creating a mixed-field pattern when testing with anti-A and anti-B reagents 1
• These antibodies react with red cell antigens at room temperature or below, causing some cells to clump while others remain free in suspension
• This creates the characteristic mixed-field appearance in the reaction tube or column agglutination test

Other Important Causes

1. A3 Subgroup

   • While A3 subgroup has traditionally been associated with mixed-field agglutination, research shows this is not always the case
   • Mixed-field agglutination observed in column agglutination testing (CAT) may be incorrectly attributed to A3 subgroup 1
   • PCR-sequence-based typing has shown that many cases presumed to be A3 are actually other ABO variants

2. Chimerism

   • Dispermic chimerism (fusion of two different zygotes) can present with mixed-field agglutination during routine ABO typing 2, 3
   • These individuals have two distinct cell populations with different ABO types
   • Can be confirmed through genetic testing showing multiple alleles at various loci

3. Hematological Disorders

   • Patients with acute myeloid leukemia and other malignant hemopathies may show mixed-field agglutination 4
   • This occurs due to decreased activity of A or B glycosyltransferases in affected red cell populations
   • The enzyme activity is reduced in cell subpopulations that have lost A or B antigens

4. Polyagglutination

   • Bacterial infections or certain hematological diseases can expose normally hidden red cell antigens (cryptoantigens) 5
   • Most normal sera contain natural antibodies to these antigens, causing widespread agglutination
   • This can create apparent discrepancies in ABO typing and mixed-field patterns

Diagnostic Approach

When mixed-field agglutination is observed:

1. Repeat testing using both column agglutination and tube methods with different antibody clones
2. Perform testing at different temperatures to identify cold-reactive antibodies
3. Consider molecular testing (PCR-SBT of ABO gene) if subgroup is suspected
4. Evaluate patient for underlying conditions:
   • Recent infections
   • Hematological disorders
   • History of transplantation
   • Twin pregnancy or chimeric possibility

Clinical Significance

• Mixed-field agglutination can lead to ABO typing discrepancies and potential transfusion complications if misinterpreted
• Proper identification of the underlying cause is essential for accurate blood typing
• In cases where cold autoagglutinins are suspected, warming the sample to 37°C before testing may help resolve the issue

Mixed-field agglutination is not typically caused by abnormal concentrations of serum proteins, Para-Bombay phenotype, or decreasing Rh type. The pattern requires thorough investigation to determine its true etiology, with cold-reactive autoagglutinins being the most common culprit in routine blood banking practice.