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Technical Briefs |
Department of Clinical Biochemistry, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom
aauthor for correspondence: fax 44-117-928-3107, e-mail ann.bowron{at}ubht.swest.nhs.uk
Homocystinuria is an autosomal recessive disorder usually caused by deficiency of cystathionine ß-synthase, leading to grossly increased plasma and urine concentrations of homocysteine. There is considerable evidence that early detection and treatment can prevent the clinical consequences of the enzyme deficiency (1)(2); therefore, screening for the disorder has been advocated (1). Many cases of homocystinuria have secondary hypermethioninemia. Neonatal screening for homocystinuria by measurement of increased methionine concentrations in dried blood spots (DBS) has been performed in some centers but has poor sensitivity. Approximately 20% of cases are missed, partly because of low methionine concentrations in breast milk and some infant formulas (3). The measurement of homocysteine in DBS has not been used as a screen for homocystinuria, partly because of uncertainty about the suitability of DBS samples. Homocysteine concentrations are unstable in whole blood stored at room temperature and increase by 
1.0 µmol/L per hour (4). This is attributable to in vitro erythrocyte transmethylation reactions that lead to continuous production and release of homocysteine (5). It is not known whether homocysteine is released from erythrocytes in blood that has been spotted on filter paper and dried, either during the spotting and drying process or during storage at room temperature. A recent report assessed stability in screening cards that were stored at 4 °C, which does not reflect routine practice (6). Another investigated the stability of DBS samples, using whole blood to which large concentrations of aqueous homocysteine calibrator had been added, thus potentially masking any increase in homocysteine attributable to release from erythrocytes (7). The lack of information on the stability of homocysteine in DBS as applied to neonatal screening prompted the following investigation.
Blood samples were collected from adult inpatients into tubes containing potassium EDTA. Samples were mixed gently, and blood was spotted on filter paper cards (standard neonatal screening cards) and dried in air. The cards were stored at room temperature until analysis. Immediately after the blood spots were prepared, the remaining samples were centrifuged, and the plasma was removed and stored at –20 °C until analysis. Ethical approval was obtained from the UK South West Local Research Ethics Committee.
We measured total homocysteine in DBS by HPLC, using a modification of a method for plasma total homocysteine (8). We punched 6-mm DBS into flat-bottomed tubes, added 80 µL of 2.16 µmol/L cysteamine (internal standard) and 15 µL of 100 mL/L tributylphosphine in dimethylformamide to the tubes, and incubated the tubes at 4 °C for 30 min. We used 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfuric acid to derivatize the thiols. Separation was performed by reversed-phase HPLC with fluorometric detection (Shimadzu RF-10AXL). The method was standardized by use of 15 µL of an aqueous homocysteine calibrator (20 µmol/L) in place of DBS. The DBS method was linear up to 140 µmol/L, and the lower limit of detection was 4 µmol/L. The within-batch imprecision (CV) of the assay was 5%, and the between-batch CV was 8% (11.9 µmol/L homocysteine). Plasma homocysteine was analyzed by HPLC (8). The assay was linear up to 200 µmol/L with a within-batch CV of 1% and between-batch CV of 4% (12.2 µmol/L homocysteine). Plasma total homocysteine values were compared with DBS results from the same samples.
Total homocysteine in DBS from 29 patients was analyzed when the filter paper was dry (1 h), at 4 h, and at 24 h. Longer-term stability was then assessed by repeating the experiment on samples from another group of patients (n = 27), measuring total homocysteine in DBS samples at 1 h, then after 1, 2, 3, 7, 14, and 28 days. Homocysteine concentrations in stored DBS samples were compared with 1 h samples by use of the Student paired t-test. The homocysteine concentrations in plasma and DBS were compared by linear regression.
There was no difference between DBS homocysteine concentration in samples analyzed at 1 h [mean (SD), 8.9 (3.2) µmol/L] and those stored for 4 h [8.5 (3.1) µmol/L; P = 0.64] or 24 h [8.3 (3.7) µmol/L; P = 0.51]. In the long-term stability study, there was no significant change in homocysteine concentrations from baseline [14.5 (4.7) µmol/L] for samples stored for 24 h [14.0 (4.6) µmol/L; P = 0.17]. There was a small consistent decrease over the next 2 days [day 2, 13.7 (4.6) µmol/L (P <0.05); day 3, 13.4 (4.5) µmol/L (P <0.05)], with a mean total decrease of 7%. This small consistent decrease in homocysteine concentration continued at a slower rate over the remainder of the 28-day period: day 7, 14.3 (4.9) µmol/L (P = 0.62); day 14, 13.8 (4.6) µmol/L (P = 0.001); day 28, 13.2 (4.7) µmol/L (P = 0.001). The total decrease in homocysteine over 28 days was 1.3 µmol/L, or 9%. The change in homocysteine was not related to the baseline concentration.
DBS homocysteine concentrations at 1 h correlated very well with plasma values (r2 = 0.88); the homocysteine concentrations measured in DBS were 60% of concentrations in the corresponding plasma samples (Fig. 1
).
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This investigation into homocysteine stability used a method standardized with 15 µL of aqueous calibrator, based on an estimation of the volume of sample in the DBS. If this method was to be used for quantification of DBS homocysteine, more accurate standardization would be required, such as the use of a calibration curve constructed with use of blood samples to which homocysteine had been added (9)(10). DBS homocysteine showed a very good relationship with the plasma values over a wide range of concentrations, suggesting that this is an acceptable sample to use to estimate plasma homocysteine. Lower homocysteine concentrations in DBS compared with plasma samples are attributable to the dilution caused by hemolysis of whole blood during the drying process. Erythrocytes contain very low concentrations of homocysteine, 10% of plasma values (11). The DBS homocysteine assay showed considerably poorer precision than the analysis of plasma by the same method, but the CV of the DBS assay compared well with other DBS homocysteine assays (5)(6). Increased variability may be attributable to the process of blood-spot preparation, the small sample volume, elution of homocysteine from the blood spot, and the greater sample dilution. The assay may not be able to quantify DBS homocysteine in all individuals because the lower limit of detection is above the concentrations found in some healthy neonates (7)(12).
The results of this study show that homocysteine is stable in DBS at room temperature for 24 h, followed by a small but consistent decrease by day 3 and a more modest reduction after 28 days of storage. This is acceptable for the purposes of a screening program in which the samples are sent by mail to the laboratory. A possible explanation for the stability is that during the process of drying the blood on the filter paper, the enzymes in the sample become denatured as a result of dehydration. The continuous enzymatic production of homocysteine that occurs in vitro may therefore be inhibited in dried whole blood. The slight decrease in homocysteine concentration between days 1 and 28 may be attributable to bacterial breakdown of amino acids in the sample.
Further investigation into the analysis of homocysteine in the diagnosis of neonatal homocystinuria is required; for example, the concentration of homocysteine in blood of neonates with CBS deficiency needs to be determined followed by establishment of appropriate diagnostic cutoff values. However, for neonatal screening of homocystinuria by measurement of blood spot homocysteine to be considered, it is important to establish the suitability of these samples for homocysteine analysis. We have described a method that is robust, reproducible, and suitable for the detection of increased homocysteine concentrations and have demonstrated that homocysteine is sufficiently stable in DBS to meet the requirements of a screening program.
References
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