The effect of freezing, washing and high-speed centrifugation on the outcome of BD ProbeTec Chlamydia analysis on urine samples
Abstract number: 1134_03_277
Lisby G., Westh H.
When urine samples for Chlamydiatrachomatis (CT) are not analysed immediately, it has been standard procedure to freeze the samples at 20°C until analysis. On known CT-positive and CT-negative patient samples, we valuated the impact of freezing, washing or not washing the unfrozen samples as well as the impact of centrifugating frozen samples at 10.000 xg prior to analysis.
All urine samples were analysed (no freezing) with the BD ProbeTec system. The remaining sample was frozen at 20 C for inclusion in this study. We carried out three arms of investigation: Arm 1: Frozen positive samples and frozen negative samples were thawed and reanalysed after washing. Arm 2:Refrigerated positive and negative samples were analysed without washing. Arm 3: frozen positive and frozen negative samples were thawed and analysed after centrifugation at 10.000 xg and washing. We tested the effect of centrifugation at 10.000 xg to determine whether 10.000 xg is more efficient than the traditional 3,000 xg to sediment the CT organisms. Due to cellular lysis caused by the freeze/thaw cyclus, the intracellular CT will be found in the extracellular phase, and might not sediment completely at 3,000 xg.
In arm 1, 181 positive samples were frozen, thawed and reanalysed after washing. 162 (89.5%) remained positive, 13 (7.2%) became negative and six (3.3%) showed inhibition. Of the 182 negative samples, 166 (91.2%) produced a negative result, while 15 (8.2%) showed inhibition and one (0.6%) was borderline positive. In arm 2, of the 90 positive samples analysed without a washing step, 85 (94.5%) remained positive and five (5.5%) became negative. In 91 negative samples, 70 (76.9%) produced a negative result, while 21 (23.1%) showed inhibition. In arm 3, data are pending.
Compared to standard testing of urine samples by the BD ProbeTec system, (unfrozen, washed/centrifugated (3,000 xg) samples), freezing at 20C followed by washing caused loss of signal in 7.2% of positive samples as well as inhibition in 3.3% of positive samples and 8.2% of negative samples. Also compared to standard testing, omitting the washing step caused 5.5% of the known positive samples to become negative and of the known negative samples, 23.1% showed inhibition.
1. Freezing samples reduces sensitivity and causes low-grade inhibition. 2. Omitting the washing step in unfrozen samples causes extensive inhibition.
|Session name:||XXIst ISTH Congress|
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