Post-mortem stability and redistribution of carbohydrate-deficient transferrin (CDT)

Forensic Science International 174 (2008) 161–165 www.elsevier.com/locate/forsciint

Post-mortem stability and redistribution of carbohydrate-deficient transferrin (CDT) Juha Rainio a, Giorgia De Paoli b, Henrik Druid c, Riitta Kauppila c, Fabio De Giorgio a, Federica Bortolotti b, Franco Tagliaro b,* b

a Institute of Legal Medicine, Catholic University of the Sacred Heart School of Medicine, Rome, Italy Department of Medicine and Public Health, Unit of Forensic Medicine, University of Verona, Policlinico G.B. Rossi, Piazzale L.A. Scuro, 1, 37134 Verona, Italy c Department of Forensic Medicine, Karolinska Institutet, Stockholm, Sweden

Received 13 November 2006; received in revised form 22 March 2007; accepted 25 March 2007 Available online 1 May 2007

Abstract Post-mortem diagnosis of chronic alcohol abuse is a challenge for forensic experts due to the lack of pathognomonic morphological findings and often also inadequate background information. Objective methods demonstrating chronic excessive alcohol consumption would therefore be a useful tool for forensic pathologists. In clinical practice, several markers of chronic alcohol abuse have recently been introduced, among which carbohydrate-deficient transferrin (CDT) is the most accepted, but the use of these markers in autopsy has not yet been established. We examined post-mortem stability and possible post-mortem redistribution of CDT and compared two analytical methods, capillary zone electrophoresis and high-performance liquid chromatography. According to our results, CDT remains stable for an appreciable time after death. The results further indicate that CDT is not subject to major post-mortem redistribution. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Carbohydrate-deficient transferrin; Chronic alcohol abuse; Autopsy; Post-mortem diagnosis; Capillary zone electrophoresis; High-performance liquid chromatography

1. Introduction Chronic alcohol abuse is a major issue in legal medicine, involving investigation of both living and dead individuals. In a significant number of forensic autopsies, death is directly or indirectly related to excessive alcohol consumption. In addition to cause-of-death determination, alcohol is an important background factor in, for example, homicides, suicides, traffic accidents and other misfortunes. The post-mortem diagnosis of chronic alcohol abuse remains a challenge for forensic experts due to the lack of pathognomonic morphological findings and often inadequate background information. It has been estimated that half of all alcoholics die with a negative blood alcohol concentration [1], and alcohol-related deaths tend to be under-reported in official

* Corresponding author. Tel.: +39 045 8074618; fax: +39 045 505259. E-mail address: [email protected] (F. Tagliaro). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.03.020

mortality statistics [2]. For these reasons, objective methods for identifying chronic alcohol abuse would be useful for forensic pathologists. In clinical practice, several new markers of chronic alcohol abuse have been introduced with some efforts to apply them to autopsy material, including fatty acid ethyl esters [3,4], ethyl glucuronide [5–9], phosphatidylethanol [10] and carbohydratedeficient transferrin (CDT). Of these, CDT is the most frequently used and the most extensively studied. It also covers longer time periods than most other markers. Serum transferrin (Tf) is an iron-transporting glycoprotein synthesized mainly by hepatocytes that occurs in at least seven isoforms: hexa-, penta-, tetra-, tri-, di-, mono- and asialotransferrin. In healthy subjects, the tetrasialo isoform accounts for about 80% of all circulating transferrin. Chronic ethanol intake results in decreased hepatic glycosylation of transferrin and, consequently, in increased amounts of less glycosylated transferrin isoforms (mainly asialo- and disialotransferrin), referred to as CDT. According to the literature, cut-off values

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used for analyses with capillary electrophoresis of samples of living individuals have ranged between 1.8 and 4.5% [11–13]. The most frequently used methods for CDT analysis include immunometric assays, capillary zone electrophoresis and highperformance liquid chromatography. Thus far, relatively little has been published concerning CDT determination in post-mortem samples. Sadler et al. [1] studied 32 alcoholics and an equal number of age- and sexmatched controls, comparing the diagnostic value of serum CDT, g-glutamyltransferase, alcohol liver disease and external examination findings as markers of heavy continuous drinking. They concluded that CDT is a useful post-mortem marker of chronic alcohol abuse with the specificity of 25% and sensitivity of 91% in RIA. Malcolm et al. [14] published a study on determination of CDT levels in serum samples collected from 25 autopsies, finding no evidence of degradation of the CDT level in the period in which the samples were taken (2–36 h). Simonnet et al. [15] investigated the effects of haemolysis on CDT levels in samples taken from living persons, as well as the influence of blood collection site, blood storage and body decomposition in samples from 10 autopsies, and concluded that haemolysis or repeated freezing and thawing may decrease CDT concentration. Osuna et al. [16] and Berkowicz et al. [17,18] reported determination of CDT level in vitreous humour of 66 and 28 cadavers, respectively, and concluded that CDT analysis is a promising method for postmortem diagnosis of chronic alcohol abuse. According to these articles, specificity of vitreous humour CDT analysis ranged between 71 and 92% and sensitivity between 28 and 95% [16– 18]. In this study, we examined post-mortem stability and possible post-mortem redistribution of CDT. In addition, we compared two analytical methods, capillary zone electrophoresis and high-performance liquid chromatography. The study was a joint research project between the Institute of Legal Medicine at the Catholic University in Rome, the Unit of Forensic Medicine at the University of Verona, Italy, and the Department of Forensic Medicine at the Karolinska Institutet, in Stockholm, Sweden. 2. Materials and methods Blood samples were obtained from 70 corpses at the Department of Forensic Medicine of the Karolinska Institutet. Four blood samples from each case were collected as follows: a femoral blood sample (sample P) was taken transcutaneously with a single-use syringe and needle as soon as possible after the arrival of the body to the mortuary. At autopsy, performed from 1 to 6 days later, blood samples from the left (sample FBL) and right (sample FBR) femoral veins and the inferior vena cava (sample VC) were collected in separate tubes by cutting the vessels. The total number of samples thus was 280. Each blood sample was centrifuged and the serum was stored in Eppendorf tubes without preservative and kept at 20 8C until analysis, which was carried out at the Unit of Forensic Medicine, University of Verona, approximately 2 months later. All serum samples were analysed, evaluating the amount of disialotransferrin with capillary zone electrophoresis (CZE). Moreover, 76 samples, comprising samples from 19 individuals, were analysed in parallel with high-performance liquid chromatography (HPLC). Multicapillary CZE was performed on a commercially available CE system for routine serum protein analysis (CapillarysTM, Sebia, Evry, France), featuring

eight uncoated fused silica capillaries operated in parallel (effective length 17.5 cm  i.d. 25 mm; bubble cell with a diameter of 100 mm at the detection window) and UV detection at a wavelength of 200 nm. All reagents were provided in a commercial kit (CapillarysTM CDT assay), which included buffer solution (pH 8.8), washing solution, sample diluent and plastic consumables. Data analysis was performed with a proprietary software (CapillarysTM, Sebia). HPLC analysis was performed using a gradient HPLC analyser with a UV– vis detector (Shimadzu Europe, Duisburg, Germany) set at a wavelength of 460 nm. The separation procedure is described elsewhere [13]. The analytical anion exchange column (length 65 mm  i.d. 4.6 mm), the pre-column and the mobile phase were provided in a commercial kit, ClinRep1 (CDT in Serum), from Recipe (Munich, Germany). Sample preparation required iron saturation (with ferric solution) and lipoprotein precipitation with CaCl2. CDT isoform quantification was based on calculation of the ratio of the disialo-Tf peak area to the sum of the peak areas for all isotransferrins from asialo-Tf to pentasialo-Tf (CDT index). Information about time of death, time of arrival at the morgue, and time to autopsy was retrieved. Based on this information, the period between death and collection of sample P and the period between collection of samples P and FBL (or FBR) were calculated. General data, such as cause of death, age and sex, were also recorded. Paired Student’s t-test was used to evaluate possible differences between CDT concentrations in the first and second samples. Analysis of variance (ANOVA) was used to compare the analytical results in samples from different sampling sites. Regression analysis was applied to assess the correlation between the concentrations obtained with the two analytical methods. Calculations were performed with Microsoft Excel.

3. Results Of the 280 serum samples analysed with CZE and the 76 samples analysed with HPLC, an interpretable result was obtained for 115 (41%) and 41 (54%), respectively. The largest amount of non-interpretable (NI) results, i.e. results in which peaks of CDT isoforms could not be identified and, consequently, the amount of disialo-CDT quantified, was obtained in analysis of samples from the vena cava (34% of all NIs). In HPLC analysis, the result was NI in 24 samples (32%) and no result was yielded by 11 samples (14%) because the sample had already been exhausted in previous analyses. In total, at least one interpretable result was achieved in 57 of the 70 autopsies studied (81%). Table 1 presents changes in CDT values in femoral blood samples during cold storage of the body at the morgue using the CZE (Table 1a) and HLPC (Table 1b) methods. The CDT concentration in the first sample (P) was compared with the concentrations in the autopsy samples (FBL or FBR) in 29 cases. The difference in CDT values obtained with the two methods between the P and FBL (or FBR) samples ranged from +0.9 to 0.8 and +0.4 to 0.6%, respectively. The standard deviation for CZE and HPLC results was +0.4 and +0.3 and the median 0.1 and 0.2, respectively. The total time between the two samples varied between 24.8 and 145.3 h (median 73 h). The difference was evaluated using the Student’s t-test for paired data, and no significant difference was found. p-value <0.05 was considered significant. ‘‘Warm time’’, i.e. estimated time during which the corpses had been in room temperature conditions before transportation to the mortuary, ranged from 1.7 to 38.0 h (median 7.3 h), whereas total post-mortem time before the

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Table 1 Changes in disialotransferrin values detected by CZE (a) and by HPLC (b) between femoral blood samples (unless otherwise indicated) collected before autopsy (1st sample) and during autopsy (2nd sample) Case no. (a) CZE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Cause of death Warm 1st sample 2nd sample Difference between Time between death Time between 1st (%) a 1st and 2nd samples and 1st sample (h) and 2nd samples (h) timeb (h) (%) a method 0.4 0.4 0.8 0.2 0.2 2.8 1.3 0.5 1.6 0.5 1.5 0.9 2.2 0.7 1.9 1.1 0.7 0.9 3.8 0.5 1.5 6.4 3.4 0.8 0.7

0.3 0.1 1.1 0.2 c 0.1 2.8 1.2 0.6 c 1.2 1.0 1.3 1.1 2.5 1.6 c 1.3 c 1.6 0.5 c 1.1 3.0 0.3 1.0 d 6.0 4.0 1.1 0.3

S.D. Median (b) HPLC method 1 0.5 2 1.4 3 6.0 4 1.1 5 0.8 6 2.4 7 3.6 8 1.0 9 0.9 10 2.4 11 5.5 S.D. Median a b c d

0.7 1.4 5.8 0.5 0.6 1.8 4.0 0.9 0.7 2.4 5.0

0.1 0.3 +0.3 0 0.1 0 0.1 +0.1 0.4 +0.5 0.2 +0.2 +0.3 +0.9 0.6 +0.5 0.2 +0.2 0.8 0.2 0.5 0.4 +0.6 +0.3 0.4

7.5 18.5 17.8 11.2 4.8 15.1 15.2 57.8 50.2 16.2 14.5 24.5 7.8 27.4 7.2 4.5 16.3 15.6 12.5 36.5 42.0 16.8 10.7 17.6 13.1

+0.4 0.1

15.9

+0.2 0 0.2 0.6 0.2 0.6 +0.4 0.1 0.2 0 0.5

7.5 10.1 66.1 17.8 4.8 18.6 20.1 14.5 24.5 7.8 16.8

+0.3 0.2

16.8

Age (y) Sex

64.6 89.3 64.4 50.6 88.8 48.4 44.8 99.5 73.7 72.2 123.8 92.7 98.3 50.2 145.3 138.3 72.7 24.8 139.7 49.6 50.5 73.0 66.8 27.3 25.8

7.3 17.3 16.4 13.9 4.0 30.5 13.4 38.0 10.0 3.4 3.2 23.1 5.2 14.3 14.8 1.7 3.2 4.1 11.5 1.8 6.2 7.1 10.5 5.1 6.0

Acute myocardial infarct Acute myocardial infarct Hypothermia Gunshot wound to head Hanging Acute myocardial infarct Urosepsis Hypothermia Cerebral haemorrhage Drowning Amphetamine intoxication Vertebral fractures Drug poisoning Drug poisoning Acute myocardial infarct Carbon monoxide poisoning Hanging Hanging Coronary sclerosis Cervical fracture Hanging Drowning Acute pancreatitis Drug poisoning Coronary sclerosis

62 74 78 73 59 63 84 81 79 83 59 76 39 57 63 98 65 75 57 85 31 69 56 85 42

F M M M M M M F M M M F M F M F M M M M M M M M M

64.6 73.8 26.9 64.4 88.8 75.2 92.7 123.8 92.7 98.3 73.0

7.3 5.3 28.0 16.4 4.0 9.5 18.7 3.2 11.2 5.2 7.1

Acute myocardial infarct Acute myocardial infarct Ventricular ulcer Hypothermia Hanging Drowning Subdural bleeding Amphetamine poisoning Vertebral fractures Drug poisoning Drowning

83 50 66 78 59 69 56 59 76 39 69

M M F M M M M M F M M

Proportion of disialotransferin of total transferrin. Estimated post-mortem time in warm conditions before transport to mortuary. Side opposite to 1st sample. From the inferior vena cava.

1st sample ranged from 4.5 to 66.1 h (median 15.9 h). These data as well as cause of death, age and sex of all cases are provided in Table 1. Table 2 displays CDT values of samples taken at autopsy from the left and right femoral veins and the inferior vena cava. ANOVA revealed no significant differences in results between samples from the three sampling sites, and variation in results within each sample group was similar. Comparison of CDT values analysed with CZE and HPLC showed a significant correlation (R2: 0.9128) (see Fig. 1).

4. Discussion Sensitivity and specificity of CDT for the diagnosis of excessive alcohol consumption have been demonstrated in numerous clinical studies. Previous publications on CDT analysis in post-mortem samples have yielded similar findings. The specificity and sensitivity in post-mortem serum samples have been estimated to vary from 9 to 63 and 88 to 100%, respectively [1,14], and the corresponding parameters in samples of vitreous humour from 71 to 92 and 28 to 95% [16–18].

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Table 2 Disialotransferrin values detected by CZE (a) and by HPLC (b) in post-mortem serum samples collected from various veins at autopsy Case no.

Left femoral vein (%)

Right femoral vein (%)

Inferior vena cava (%)

Total post-mortem intervala (days)

(a) CZE method 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1.1 0.1 2.4 1.2 4.5 1.2 1.3 1.1 2.5 2.0 1.6 1.1 3.0 0.3 6.0 4.0 0.3

0.9 0.1 NI NI 4.7 1.7 1.6 1.2 1.8 1.4 1.1 0.8 3.0 0.3 5.6 3.8 0.2

0.9 NI 2.1 1.2 4.5 NI 1.6 1.1 NI NI NI 1.7 NI NI 4.7 NI NI

3.4 3.9 3.9 2.5 4.7 4.8 5.8 4.9 4.4 4.4 6.0 1.7 6.4 3.6 3.7 3.2 1.6

(b) HPLC method 1 2 3 4 5 6 7 8 9 10

0.7 1.4 5.8 0.5 1.8 4.0 0.9 0.7 2.4 5.0

0.7 1.0 5.5 0.9 2.3 4.5 NI 0.7 2.2 5.0

1.0 1.6 6.0 0.6 2.4 NI 0.5 IA NI 4.2

3.0 3.5 3.8 3.4 3.9 4.7 5.8 4.9 4.4 3.7

NI, non-interpretable; IA, insufficient amount. a Combined time from death to arrival at morgue and from arrival to autopsy.

However, CDT analysis in these studies has been performed with immunoassay methods, which can be regarded as less reliable than HPLC or CZE methods [13]. The scarce selectivity of immunoassay methods could explain the wide range in reported diagnostic sensitivity and specificity.

Fig. 1. Correlation between disialotransferrin results obtained with the CZE method and the HPLC method.

We investigated the viability of performing CDT analyses on post-mortem samples by instrumental separative techniques, namely HPLC and CZE, and obtained interpretable results for 81% of cases. In remaining cases results were non-interpretable most likely due to haemolysis. Since haemolysis is a major problem in post-mortem blood analysis, other types of specimens, suitable for CDT analysis but less susceptible to post-mortem degradation, need to be found. This is probably not very simple and requires systematic research. As the scope of our study was not to analyse specificity and sensitivity of CDT in post-mortem samples but to examine post-mortem stability and potential post-mortem redistribution of CDT, we did not compare CDT concentrations with background information or with autopsy findings related to alcohol use. The material consisted of consecutive cases referred to the Department of Forensic Medicine, Stockholm, and the proportion of alcoholics in these cases is generally about 50%. As stated by Simonnet et al. [15], routine use of CDT in medico-legal casework requires an exhaustive study on the suitability of the samples collected in forensic autopsies and on the stability of the marker in poor storage conditions. Results of Sadler et al. [1] and Simonnet et al. [15] suggest that the variations in CDT concentrations were within the range

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of analytical variability, thus excluding interference of haemolysis or collection site on CDT concentration. Our findings show that CDT concentrations remained fairly unchanged after several days of cold storage of the body, implying that post-mortem time does not influence analytical results to any significant degree. Previous publications have demonstrated that CDT stays stable in post-mortem serum samples up to 96 h [1,14]. The maximum post-mortem interval in our study ranged between 39.2 and 152.4 h, being on average 95 h at the moment of autopsy. Moreover, comparison of CDT values in samples from three different sites suggests that CDT is not subject to substantial post-mortem redistribution. The parallel analysis of 30 samples with CZE and HPLC revealed a highly significant correlation, as displayed in Fig. 1. The inter-method variation was randomly distributed, and hence, no systematic difference was seen. Our results indicate that CDT stays stable for an appreciable time after death. Further, CDT does not appear to be subject to major post-mortem redistribution. In conclusion, additional studies on use of CDT in autopsy diagnostics with CZE and HPLC are justified because the methods are robust and the results apparently are not influenced by post-mortem changes.

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