Poster Only Presentations

Stability of thiopurine S-methyltransferase activity in whole blood.

Rachel Marrington and Craig Webster.

Department of Clinical Biochemistry and Immunology, Birmingham Heart of England NHS Foundation Trust

The determination of the activity of thiopurine S-methyltransferase (TPMT) is important in identifying those patients at risk of developing severe side effects if treated with drugs such as azothioprine; 6-mercaptopurine and 6-thioguanine.  TPMT activity should be measured prior to commencing these drugs, and a deficiency of the TPMT enzyme could result in the patient becoming seriously ill.  Methods for TPMT phenotyping have been in place for a number of years.  The stability of the enzyme has always been an issue and it has been shown that storage at room temperature significantly reduces the activity even after a week.  Current recommendations are storage refrigerated but stability of only up to 8 days has been reported.   A phenotyping method described by Ford et al (Ann Clin biochem 2006; 43:354€“360), uses whole blood lysates which are prepared by freezing whole blood at €“80 °C.

We have studied the stability of TPMT under these conditions (both in bulk and in aliquots of the required volume), comparing results to samples refrigerated at 4 °C for a period of a month.  A single donation of EDTA whole blood was mixed and stored as aliquots of 800 µL at 4€“8 °C and €“ 80 °C.  Aliquots of 200 µL were also stored at €“80 °C.  On the day of analysis the 800 µL aliquots were mixed and three aliquots of 200 µL were taken from each 600 µL stock sample.  The average of triplicate measurements for each storage condition for each time point (0, 7, 14, 21 and 28 days) showed thiopurine S-methyltransferase activity to be stable for four weeks at €“80 °C, with no statistical difference between the blood being stored frozen in bulk or in aliquots of 200 µL.  However, a decrease in activity was observed after four weeks in the sample stored refrigerated.

Evaluation of an Enzyme-Linked Immunosorbent Assay for Laboratory Testing of Erythropoietin Levels.

Robert Payne

Erythropoietin (EPO) is the major in vivo regulator of red cell production (erythropoiesis), and is produced primarily in the kidney in response to tissue hypoxia via a complex homeostatic feedback mechanism. Raised serum EPO concentrations can be seen in most types of anaemia and polycythaemia and in the presence of certain tumours. Low EPO concentrations can be seen in anaemia of renal insufficiency and anaemia of chronic disorders. The use of recombinant human erythropoietin in therapy of anaemia associated with end stage renal failure is a now well established route of treatment, as it can elevate haemoglobin levels without the need for transfusion. With the development of increasingly standardized and accurate techniques for the determination of EPO concentration in serum, the use of EPO as tool in diagnosis of disorders such as polycythaemia is becoming more popular. Recent years have seen the development of enzyme-linked immunosorbent assays (ELISAs) for EPO. These rely on the use of one or more monoclonal anti-EPO antibodies. ELISAs are highly sensitive and are less time consuming than many other EPO test methods available. This study is concerned with the possibilities for the development of an in-house method for measuring erythropoietin levels in serum at Birmingham Heartlands Hospital NHS Trust Haematology Laboratory. Currently, requests for erythropoietin testing are sent away to Leeds General Infirmary. Consequently, EPO results can take some time to be returned, and this may affect laboratory users desire to use erythropoietin levels in diagnosis. This study concerns initial steps that are taken in development of a new technique by evaluation of an EPO ELISA developed by IBL Hamburg, assayed on a new Triturus® analyzer. Serum samples were obtained from UK NEQAS for haematinics with mean results determined from other participant laboratories around the country. Although the assay yielded promising results following the same pattern as the NEQAS values, the results were inaccurate. This was deemed to be due to grossly elevated optical density values.  After review, it has been decided that washing and/or incubation steps will need adjusting to bring the O.D. values down to normal. Shortening of the incubation period will prevent over-incubation of the samples. This will involve the processing of small runs, to allow constant adjustment of the methodology to bring results into line. The study is continuing. It is considered by the laboratory that an in-house method of erythropoietin testing could greatly enhance the service offered by the laboratory to users and open up useful channels in patient diagnosis (e.g. polycythaemia) and in the monitoring of patient response to treatment with rHuEPO.

Development and evaluation of a new diagnostic assay to detect resistance to the new HIV antiviral class of integrase inhibitors.

PM Cook and E Smit.

Antiviral Resistance Testing Service, Health Protection Agency,
West Midlands Public Health Laboratory.

Clinical trials for Elvitegravir(GS9137) and Raltegravir(MK-158) have shown inhibition of HIV-1 integration and a significant drop in the viral load (VL) of patients with multi-drug resistant HIV-1. Raltegravir had been approved by the FDA for use in these patients. Resistance to integrase inhibitors has been described and an assay to detect those mutations has been developed.

88 specimens were tested. The VL of the specimens selected ranged from 1081 to >1,000000 copies/ml. The overall PCR success rate was 70/88 (80%). The mean VL of the samples amplified was 110305 copies/ml. The lowest viral dilution that amplified was 600 copies/ml using the NIBSC 2nd International Standard 97/650

Samples from a variety of representative subtypes of HIV-1 group M were tested. 23/29(79%) subtype B and 21/27(77%) subtype C samples were amplified, as were 2/3(66%)subtype D, 50%(1/2) subtype H and  80% CRF01-AE and CRF02_AG subtypes. And all A, F,G, J ,K, CRF06_cpx and CRF011_cpx samples. 90% of the amplified samples were successfully sequenced.

Sequences were examined for resistance mutations. Primary resistance wasn€™t found, so the patients€™ HIV would be susceptible to either integrase inhibitor. However, secondary resistance mutations were discovered, as natural variants in these sequences, showing the importance of baseline resistance testing.