SHORT REPORT Detection of microsatellite markers in leukaemia using DNA from archival bone marrow smears T HOMAS PABST, 1 J U ¨ RG S CHWALLER , 1 A NDREAS T OBLER 1,3 AND MARTIN F. F EY 1,2 1 Laboratory for Clinical and Experimental Research, 2 Institute of Medical Oncology, and 3 Central Haematology Laboratory, University of Berne, Switzerland Received 2 April 1996; accepted for publication 12 July 1996 Summary. We describe a simple PCR method to detect highly polymorphic repetitive DNA markers (microsatellites) in leukaemia using DNA from fresh and archival bone marrow smears. Neither the period of storage nor staining of the slides affected microsatellite PCR. Comparison of slide DNA with constitutive DNA from buccal epithelium revealed loss of heterozygosity (LOH) with at least one marker in four of 11 leukaemias. Marrow smears offer an attractive archival source of DNA for the detection of microsatellite markers. Applications include detection of LOH or tracing the origin of cell populations, e.g. in bone marrow transplant recipients. Keywords: microsatellites, leukaemia, slides, clonality, PCR. Microsatellites composed of di-, tri- or tetranucleotide repeats are highly polymorphic DNA markers dispersed in the human genome (Weber & May, 1989). They are used in genetic linkage studies and gene mapping, or as markers to discover loss of heterozygosity (LOH) in cancer and genetic instability in replication-error positive (RER ) tumours (Mao et al, 1994; Wooster et al, 1994). Microsatellite allelotyping of tumours by PCR has greatly facilitated the identification of novel tumour suppressor genes by mapping specific genomic regions (Takeuchi et al, 1995; Devilder et al, 1995). Such analyses are performed mostly with high molecular weight DNA from fresh malignant cells. Degraded DNA from archival material such as formalin-fixed and paraffin- embedded tissue or air-dried bone marrow smears might represent another useful substrate (Fey et al, 1987; Gru ¨ newald et al, 1991). Gene amplification by the poly- merase chain reaction (PCR) has greatly facilitated the analysis of degraded DNA (Impraim et al, 1987). We now describe a simple method of microsatellite detection using DNA from archival bone marrow slides stored for up to 16 years. MATERIALS AND METHODS Samples. 100 smears of 50 leukaemia cases (two per case) comprising 25 acute myeloid leukaemias (AML), 20 acute lymphoid leukaemias (ALL), three chronic lymphoid leukae- mias (CLL) and two chronic myeloid leukaemias (CML) in chronic phase were studied. From each case, one air-dried unfixed stained (May-Gru ¨ nwald-Giemsa) and one unstained marrow smear were used. The 50 leukaemia cases are divided in two groups: 50 slides from 25 cases had been stored in paper envelopes at room temperature for 8–16 years (group I; mean 11 . 1 years); 50 slides from fresh marrow specimens of 25 leukaemias had been stored between 1 and 5 months (group II; mean 82 . 6 d). In 11 of the 50 leukaemia cases, DNA from archival (five cases) or fresh smears (six cases), from corresponding fresh leukaemic blasts isolated by Ficoll-Hypaque density gradient centrifugation, and from buccal epithelium (as a source of constitutional DNA) were available for comparative analysis. DNA extraction. Smears were scraped off the glass slides in a laminar air flow hood with a sterile scalpel blade and the resulting powdered material transferred to Eppendorf tubes. DNA was extracted in phenol–chloroform as described (Fey et al, 1987). Microsatellite amplification. (CA) n microsatellite loci from five different chromosomes that are tightly linked to chromosomal breakpoints frequently involved in leukaemia were selected (Table I) (Gyapay et al, 1994). PCR was performed in a 30 l volume containing 100ng of DNA template, 50 mmol KCl, 10 mmol Tris-HCl (pH 8 . 3), 1 . 5 mmol MgCl 2 ,0 . 27 M of each primer, 100 M of each nucleotide, and 0 . 8 U of Taq DNA Polymerase (Boehringer, Mannheim, Germany). Samples were processed through 30–35 cycles at British Journal of Haematology , 1996, 95, 135–137 135 1996 Blackwell Science Ltd Correspondence: Dr M. F. Fey, Institute of Medical Oncology, University of Berne/Inselspital, CH-3010 Berne, Switzerland.