In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labelled probe

An in situ hybridization assay using a digoxigenin-labelled probe was developed to detect B19 DNA in bone marrow erythroid elements of immunodeficient patients with hypoplastic anaemia. A 700 bp Bam HI-Hin dIII fragment of B19 DNA was used to construct the probe by incorporating deoxyuridine triphosphate labelled with digoxigenin. The in situ hybridized B19 DNA probe was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. Dark blue coloured inclusions at the enzyme site were detected in the nuclei of B19 infected erythroid cells at different stages of cell differentiation. Six out of the nine patients studied showed a positive reaction by in situ hybridization assay. The assay we developed proved highly specific and sensitive and it appears to be a suitable diagnostic test for investigating the possible role of B19 infection as a cause of haematopoietic disorders in immunocompromised hosts.