Background Drug-induced agranulocytosis, a severe side effect marked by a deficit or absolute lack of granulocytic white blood cells, is a rare side-effect of the anti-inflammatory drug sulphasalazine. Mutations in the human neutrophil elastase gene ( ELA2 ), causing increased intracellular concentration of this serine protease, inhibits neutrophil differentiation in severe congenital neutropenia (SCN). Since the clinical symptoms of agranulocytosis and SCN are similar, we hypothesized that it may origin from a common genetic variation in  ELA2  or that sulphasalazine may affect human neutrophil elastase activity and protein expression.
Background
Drug-induced agranulocytosis, a severe side effect marked by a deficit or absolute lack of granulocytic white blood cells, is a rare side-effect of the anti-inflammatory drug sulphasalazine. Mutations in the human neutrophil elastase gene ( ELA2 ), causing increased intracellular concentration of this serine protease, inhibits neutrophil differentiation in severe congenital neutropenia (SCN). Since the clinical symptoms of agranulocytosis and SCN are similar, we hypothesized that it may origin from a common genetic variation in  ELA2  or that sulphasalazine may affect human neutrophil elastase activity and protein expression.ELA2ELA2Methods We screened for genetic differences in  ELA2  in DNA from 36 patients who had suffered from sulphasalazine-induced agranulocytosis, and compared them with 72 patients treated with sulphasalazine without blood reactions. We also performed in vitro studies of the blood cell lines HL60 and U937 after sulphasalazine exposure with respect to cell survival index, neutrophil elastase protein expression and activity.
Methods
We screened for genetic differences in  ELA2  in DNA from 36 patients who had suffered from sulphasalazine-induced agranulocytosis, and compared them with 72 patients treated with sulphasalazine without blood reactions. We also performed in vitro studies of the blood cell lines HL60 and U937 after sulphasalazine exposure with respect to cell survival index, neutrophil elastase protein expression and activity.ELA2Results None of the mutations in  ELA2 , which previously have been reported to be associated with SCN, was found in this material. Protein expression of human neutrophil elastase in lymphoma U937 cells was not affected by treatment with concentrations equivalent to therapeutic doses. Cell survival of lymphoma U937 and promyelocytic leukemia HL-60 cells was not affected in this concentration range, but exhibited a decreased proliferative capacity with higher sulphasalazine concentrations. Interestingly the promyelocytic cells were more sensitive to sulphasalazine than the lymphoma cell line.
Results
None of the mutations in  ELA2 , which previously have been reported to be associated with SCN, was found in this material. Protein expression of human neutrophil elastase in lymphoma U937 cells was not affected by treatment with concentrations equivalent to therapeutic doses. Cell survival of lymphoma U937 and promyelocytic leukemia HL-60 cells was not affected in this concentration range, but exhibited a decreased proliferative capacity with higher sulphasalazine concentrations. Interestingly the promyelocytic cells were more sensitive to sulphasalazine than the lymphoma cell line.ELA2Conclusion Neutrophil elastase expression and  ELA2  mutations do, however, not seem to be involved in the etilogy of sulphasalazine-induced agranulocytosis. Why sulphasalazine is more toxic to promyelocytes than to lymphocytes remains to be explained.
Conclusion
Neutrophil elastase expression and  ELA2  mutations do, however, not seem to be involved in the etilogy of sulphasalazine-induced agranulocytosis. Why sulphasalazine is more toxic to promyelocytes than to lymphocytes remains to be explained.ELA2

Background Sulphasalazine (SA) has anti-inflammatory, immunosuppressive and antibiotic actions, and is a component in the therapy of Crohn's disease, ulcerative colitis and rheumatoid arthritis. Bacterial enzymes in the colon split sulphasalazine into sulphapyridine and 5-aminosalicylic acid before it is absorbed. Sulphapyridine acts as a sulphonamide antibiotic, whereas 5-aminosalicylic acid is believed to be the anti-inflammatory metabolite. Common side/toxic effects are vomiting, skin rash and headache. The incidence of the hematological adverse effects associated with sulphasalazine is generally low, but the reactions can be severe and sometimes fatal. The risk of sulphasalazine-induced agranulocytosis, i.e. profoundly depressed circulating neutrophils is highest within the first three months of sulphasalazine-treatment, with a fatality rate of 6.5 % [ 1 ]. Clinical symptoms of agranulocytosis include fever, malaise and susceptibility to infections. Patients with arthritic disorders have a greater risk of developing sulphasalazine-induced agranulocytosis than patients with inflammatory bowel diseases. Severe congenital neutropenia (SCN) and cyclic neutropenia (CN) occur both as inherited and as sporadic diseases. SCN has a constant low neutrophil number if left untreated, whereas CN manifests with cyclic oscillations of neutrophil number with a 21-day cycle. Recently, diverse heterozygous mutations in  ELA2 , encoding human neutrophil elastase, have been identified in a majority of the cases with CN and two-thirds of the cases with SCN [ 2 ]. In this study, we hypothesized that sulphasalazine-induced agranulocytosis, with clinical symptoms similar to congenital neutropenia, may arise from genetic variation in the human neutrophil elastase gene. We genotyped 108 sulphasalazine-treated patients for  ELA2 , one third which of had experienced sulphasalazine-induced agranulocytosis. We, furthermore, tested for cytotoxic doses of sulphasalazine, and studied protein expression of human neutrophil elastase in sulphasalazine-treated blood cell lines.
Background
Sulphasalazine (SA) has anti-inflammatory, immunosuppressive and antibiotic actions, and is a component in the therapy of Crohn's disease, ulcerative colitis and rheumatoid arthritis. Bacterial enzymes in the colon split sulphasalazine into sulphapyridine and 5-aminosalicylic acid before it is absorbed. Sulphapyridine acts as a sulphonamide antibiotic, whereas 5-aminosalicylic acid is believed to be the anti-inflammatory metabolite. Common side/toxic effects are vomiting, skin rash and headache. The incidence of the hematological adverse effects associated with sulphasalazine is generally low, but the reactions can be severe and sometimes fatal. The risk of sulphasalazine-induced agranulocytosis, i.e. profoundly depressed circulating neutrophils is highest within the first three months of sulphasalazine-treatment, with a fatality rate of 6.5 % [ 1 ]. Clinical symptoms of agranulocytosis include fever, malaise and susceptibility to infections. Patients with arthritic disorders have a greater risk of developing sulphasalazine-induced agranulocytosis than patients with inflammatory bowel diseases.Severe congenital neutropenia (SCN) and cyclic neutropenia (CN) occur both as inherited and as sporadic diseases. SCN has a constant low neutrophil number if left untreated, whereas CN manifests with cyclic oscillations of neutrophil number with a 21-day cycle. Recently, diverse heterozygous mutations in  ELA2 , encoding human neutrophil elastase, have been identified in a majority of the cases with CN and two-thirds of the cases with SCN [ 2 ].ELA2In this study, we hypothesized that sulphasalazine-induced agranulocytosis, with clinical symptoms similar to congenital neutropenia, may arise from genetic variation in the human neutrophil elastase gene. We genotyped 108 sulphasalazine-treated patients for  ELA2 , one third which of had experienced sulphasalazine-induced agranulocytosis. We, furthermore, tested for cytotoxic doses of sulphasalazine, and studied protein expression of human neutrophil elastase in sulphasalazine-treated blood cell lines.ELA2Methods Subjects Patients were treated with sulphasalazine (Salazopyrin, Pharmacia, Sweden) for inflammatory joint diseases and inflammatory bowel disease. The cases with sulphasalazine-induced agranulocytosis were originally collected through the Swedish Medical Products Agency's register of adverse side effects [ 3 ]. The control group had been treated with sulphasalazine without adverse effects for at least 3 months. From the original patient material consisting of 39 cases and 75 controls, DNA was available for 36 cases and 72 controls. The patient journals were studied for information concerning neutrophil differentiation in bone marrow aspirates. The study was approved by the Ethics Committee of the Medical Faculty at Uppsala University, registration number 95–200. Mutation analysis Genomic DNA was extracted from whole blood using standard techniques. Fragments covering exons 2–5 of  ELA2  were amplified by PCR using primer pairs listed in Table  1 . The selection of exons 2–5 and some of the flanking intron sequences was based on previously reported mutations in cases with SCN and CN [ 2 , 4 ], as outlined in Figure  1 . Products for exon 2–5 were amplified with 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), activated by 15 min at 95°C followed by 4 cycles 94°C 30 sec, 65°C 30 sec, 72°C 1.5 min and 35 cycles of 94°C 30 sec, 67°C 30 sec, 72°C 1.5 min with a final extension of 10 min at 72°C. The exception was amplification of exon 5, where a 2°C lower annealing temperature was used. All primers contained a consensus M13 sequence to enable sequencing with the same primer, included in BigDye Primer sequencing kit from Applied Biosystems, Stockholm, Sweden. Applied Biosystems 310 analyzer and Sequence Analysis software was used for all sequencing. Thus, the 36 cases and 72 controls were analyzed for genetic mutations in  ELA2 . Table 1 Primer sequences for PCR amplification of  ELA2  exon 2–5  ELA2  target sequence Primers Exon 2 F 5'-tgtaaaacgacggccagtgggaggggacaggctccttgg-3' Exon 2 R 5'-caggaaacagctatgaccaccgggacgcggggtccgagc-3' Exon 3 F 5'-tgtaaaacgacggccagtcaggcccgtcgccggatggg-3' Exon 3 R 5'-caggaaacagctatgacctccgtcgcagcctccaccct-3' Exon 4 F 5'-tgtaaaacgacggccagtgtgacgcgctgacgatctgt-3' Exon 4 R 5'-caggaaacagctatgaccgcagtaccgggctgggagcg-3' Exon 5 F 5'-tgtaaaacgacggccagtcagtccagcttccccacctt-3', Exon 5 R 5'-caggaaacagctatgaccgacctactgaccattttcaac-3' PCR primers sequences for  ELA2  exon 2–5, for following sequencing reactions with BigDye primer, Applied Biosystems. Figure 1 Outline of reported mutations in ELA2 exon-sequences in patients with severe congenital neutropenia and cyclic neutropenia  Outline of mutations previously reported [2, 4] in  ELA2  exons 1–5. The SNP S173 [6] is indicated as an extended arrow and represents base number 4890 in accession number Y00477 and is a base C→A substitution. Cell culture The lymphoma cell line U937 and the promyelocytic cell line HL-60 (American tissue culture collection) were cultured in Dulbeccos modified Eagles Medium, DMEM (Sigma) supplemented with 10 % fetal bovine serum (SVA, Uppsala, Sweden), L-glutamine and penicillin-streptomycin (Sigma). Western blot Equal numbers (8 × 10 6 ) of U937 cells were grown in 75 cm 2  dishes in complete medium containing 0, 125 and 250 μM sulphasalazine for 24 h. For protein isolation, cells were washed in PBS and lysed in buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 8.0 and protease inhibitor cocktail (Sigma) and were kept on ice for 30 min. Lysates were centrifuged for 10 min at 10 000 × g, and protein concentration was determined using BioRad protein assay. Criterion precast gels (BioRad, Sweden) were used to perform SDS-page with 20 μg protein loaded per well. After gel transfer to a nitrocellulose membrane, the membranes were blocked over night in 5 % dry milk in TBS-Tween. Primary antibody against human neutrophil elastase (Calbiochem, Sweden) was diluted 1:1000 in 5 % dry milk in TBS-T. After 2 h incubation, and four sets of washing, a secondary antibody was added (1:5000) and blots were developed using ECL (ECL Western blotting system, Amersham, Sweden). Western blot analysis of human neutrophil elastase expression was performed twice. Elastase activity assay Cells (HL-60 and U937) treated with 0, 125, 250 and 500 μM of for 24 h were lysed with 100 μl of buffer containing 100 mM Tris-HCl pH 7.4, 1 mM MgCl 2 , 0.1 % Triton X-100. After homogenization, 300 μl of 1.4 M NaCl in 0.1 % Triton X-100 was added and samples were centrifuged at 15 000 × g, for 15 min at 4°C. The supernatants were transferred to new tubes and assayed for elastase activity using Suc-Ala-Ala-Ala-pNA (Sigma) as a substrate. For each assay we took 25 μl sample, mixed with 100 μl buffer containing 100 mM Tris-HCl pH 8.5, 1 M NaCl, 500 mM MgCl 2  and 0.1 % Triton X-100. To this, 50 μl of substrate was added, to a final concentration 1 μM. After 30 min of incubation in room temperature, absorbance was read at 405 nm and the concentration was calculated from a standard curve of elastase (Sigma). Cell survival index For the cell viability assay, we used a fluorometric microculture cytotoxicity assay (FMCA) previously described by Larsson et al [ 5 ]. Briefly, 20 000 cells/well were plated in 96-well plates (NUNC, DK) in complete medium with addition of increased concentrations of sulphasalazine (0, 125, 250, 500, 750 and 1000 μM) and incubated for 72 h in a humidified atmosphere used in regular cell culturing. All samples were plated in triplicates and three wells with cell culture medium served as blanks. As controls we had cells without additions and cells only with solvent, in this case 0.5 M NaOH, with equal molarities as in the wells with the highest sulphasalazine-concentration. At the end of the 72 h incubation period, plates were centrifuged (200 × g, 5 min) and medium was aspirated in a microtitre plate washer, washed with PBS and 100 μl of 10 μg/ml of fluorescein diacetate (Sigma, Sweden), was added. This dye exclusively binds intact cell membranes of viable cells. After 1 h incubation at 37°C, the fluorescence was read in the Fluoroscan 2 (Labsystems OY, Finland) at 480 nm excitation and 530 nm as emission. The results are presented as survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100. Thus, a low numerical value indicates high sensitivity to the cytotoxic effect of sulphasalazine. Effective concentration is defined as the concentration when 50 % of the cells are viable (EC 50 ). Statistics Two-tailed Student's t-test was used to compare subject characteristics and results from cell culture between cases and controls. Frequencies of subject characteristics male versus females was tested with Chi 2 -test with one degree of freedom, using Minitab 14. A  p -value less than 0.05 was denoted with (*),  p  < 0.01 with (**) and was considered as statistically significant.
Methods
Subjects Patients were treated with sulphasalazine (Salazopyrin, Pharmacia, Sweden) for inflammatory joint diseases and inflammatory bowel disease. The cases with sulphasalazine-induced agranulocytosis were originally collected through the Swedish Medical Products Agency's register of adverse side effects [ 3 ]. The control group had been treated with sulphasalazine without adverse effects for at least 3 months. From the original patient material consisting of 39 cases and 75 controls, DNA was available for 36 cases and 72 controls. The patient journals were studied for information concerning neutrophil differentiation in bone marrow aspirates. The study was approved by the Ethics Committee of the Medical Faculty at Uppsala University, registration number 95–200.
Subjects
Patients were treated with sulphasalazine (Salazopyrin, Pharmacia, Sweden) for inflammatory joint diseases and inflammatory bowel disease. The cases with sulphasalazine-induced agranulocytosis were originally collected through the Swedish Medical Products Agency's register of adverse side effects [ 3 ]. The control group had been treated with sulphasalazine without adverse effects for at least 3 months. From the original patient material consisting of 39 cases and 75 controls, DNA was available for 36 cases and 72 controls. The patient journals were studied for information concerning neutrophil differentiation in bone marrow aspirates. The study was approved by the Ethics Committee of the Medical Faculty at Uppsala University, registration number 95–200.Mutation analysis Genomic DNA was extracted from whole blood using standard techniques. Fragments covering exons 2–5 of  ELA2  were amplified by PCR using primer pairs listed in Table  1 . The selection of exons 2–5 and some of the flanking intron sequences was based on previously reported mutations in cases with SCN and CN [ 2 , 4 ], as outlined in Figure  1 . Products for exon 2–5 were amplified with 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), activated by 15 min at 95°C followed by 4 cycles 94°C 30 sec, 65°C 30 sec, 72°C 1.5 min and 35 cycles of 94°C 30 sec, 67°C 30 sec, 72°C 1.5 min with a final extension of 10 min at 72°C. The exception was amplification of exon 5, where a 2°C lower annealing temperature was used. All primers contained a consensus M13 sequence to enable sequencing with the same primer, included in BigDye Primer sequencing kit from Applied Biosystems, Stockholm, Sweden. Applied Biosystems 310 analyzer and Sequence Analysis software was used for all sequencing. Thus, the 36 cases and 72 controls were analyzed for genetic mutations in  ELA2 . Table 1 Primer sequences for PCR amplification of  ELA2  exon 2–5  ELA2  target sequence Primers Exon 2 F 5'-tgtaaaacgacggccagtgggaggggacaggctccttgg-3' Exon 2 R 5'-caggaaacagctatgaccaccgggacgcggggtccgagc-3' Exon 3 F 5'-tgtaaaacgacggccagtcaggcccgtcgccggatggg-3' Exon 3 R 5'-caggaaacagctatgacctccgtcgcagcctccaccct-3' Exon 4 F 5'-tgtaaaacgacggccagtgtgacgcgctgacgatctgt-3' Exon 4 R 5'-caggaaacagctatgaccgcagtaccgggctgggagcg-3' Exon 5 F 5'-tgtaaaacgacggccagtcagtccagcttccccacctt-3', Exon 5 R 5'-caggaaacagctatgaccgacctactgaccattttcaac-3' PCR primers sequences for  ELA2  exon 2–5, for following sequencing reactions with BigDye primer, Applied Biosystems. Figure 1 Outline of reported mutations in ELA2 exon-sequences in patients with severe congenital neutropenia and cyclic neutropenia  Outline of mutations previously reported [2, 4] in  ELA2  exons 1–5. The SNP S173 [6] is indicated as an extended arrow and represents base number 4890 in accession number Y00477 and is a base C→A substitution.
Mutation analysis
Genomic DNA was extracted from whole blood using standard techniques. Fragments covering exons 2–5 of  ELA2  were amplified by PCR using primer pairs listed in Table  1 . The selection of exons 2–5 and some of the flanking intron sequences was based on previously reported mutations in cases with SCN and CN [ 2 , 4 ], as outlined in Figure  1 . Products for exon 2–5 were amplified with 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), activated by 15 min at 95°C followed by 4 cycles 94°C 30 sec, 65°C 30 sec, 72°C 1.5 min and 35 cycles of 94°C 30 sec, 67°C 30 sec, 72°C 1.5 min with a final extension of 10 min at 72°C. The exception was amplification of exon 5, where a 2°C lower annealing temperature was used. All primers contained a consensus M13 sequence to enable sequencing with the same primer, included in BigDye Primer sequencing kit from Applied Biosystems, Stockholm, Sweden. Applied Biosystems 310 analyzer and Sequence Analysis software was used for all sequencing. Thus, the 36 cases and 72 controls were analyzed for genetic mutations in  ELA2 .ELA2ELA2Primer sequences for PCR amplification of  ELA2  exon 2–5ELA2ELA2Exon 2 FExon 2 RExon 3 FExon 3 RExon 4 FExon 4 RExon 5 FExon 5 RPCR primers sequences for  ELA2  exon 2–5, for following sequencing reactions with BigDye primer, Applied Biosystems.ELA2Cell culture The lymphoma cell line U937 and the promyelocytic cell line HL-60 (American tissue culture collection) were cultured in Dulbeccos modified Eagles Medium, DMEM (Sigma) supplemented with 10 % fetal bovine serum (SVA, Uppsala, Sweden), L-glutamine and penicillin-streptomycin (Sigma).
Cell culture
The lymphoma cell line U937 and the promyelocytic cell line HL-60 (American tissue culture collection) were cultured in Dulbeccos modified Eagles Medium, DMEM (Sigma) supplemented with 10 % fetal bovine serum (SVA, Uppsala, Sweden), L-glutamine and penicillin-streptomycin (Sigma).Western blot Equal numbers (8 × 10 6 ) of U937 cells were grown in 75 cm 2  dishes in complete medium containing 0, 125 and 250 μM sulphasalazine for 24 h. For protein isolation, cells were washed in PBS and lysed in buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 8.0 and protease inhibitor cocktail (Sigma) and were kept on ice for 30 min. Lysates were centrifuged for 10 min at 10 000 × g, and protein concentration was determined using BioRad protein assay. Criterion precast gels (BioRad, Sweden) were used to perform SDS-page with 20 μg protein loaded per well. After gel transfer to a nitrocellulose membrane, the membranes were blocked over night in 5 % dry milk in TBS-Tween. Primary antibody against human neutrophil elastase (Calbiochem, Sweden) was diluted 1:1000 in 5 % dry milk in TBS-T. After 2 h incubation, and four sets of washing, a secondary antibody was added (1:5000) and blots were developed using ECL (ECL Western blotting system, Amersham, Sweden). Western blot analysis of human neutrophil elastase expression was performed twice.
Western blot
Equal numbers (8 × 10 6 ) of U937 cells were grown in 75 cm 2  dishes in complete medium containing 0, 125 and 250 μM sulphasalazine for 24 h. For protein isolation, cells were washed in PBS and lysed in buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 8.0 and protease inhibitor cocktail (Sigma) and were kept on ice for 30 min. Lysates were centrifuged for 10 min at 10 000 × g, and protein concentration was determined using BioRad protein assay. Criterion precast gels (BioRad, Sweden) were used to perform SDS-page with 20 μg protein loaded per well. After gel transfer to a nitrocellulose membrane, the membranes were blocked over night in 5 % dry milk in TBS-Tween. Primary antibody against human neutrophil elastase (Calbiochem, Sweden) was diluted 1:1000 in 5 % dry milk in TBS-T. After 2 h incubation, and four sets of washing, a secondary antibody was added (1:5000) and blots were developed using ECL (ECL Western blotting system, Amersham, Sweden). Western blot analysis of human neutrophil elastase expression was performed twice.Elastase activity assay Cells (HL-60 and U937) treated with 0, 125, 250 and 500 μM of for 24 h were lysed with 100 μl of buffer containing 100 mM Tris-HCl pH 7.4, 1 mM MgCl 2 , 0.1 % Triton X-100. After homogenization, 300 μl of 1.4 M NaCl in 0.1 % Triton X-100 was added and samples were centrifuged at 15 000 × g, for 15 min at 4°C. The supernatants were transferred to new tubes and assayed for elastase activity using Suc-Ala-Ala-Ala-pNA (Sigma) as a substrate. For each assay we took 25 μl sample, mixed with 100 μl buffer containing 100 mM Tris-HCl pH 8.5, 1 M NaCl, 500 mM MgCl 2  and 0.1 % Triton X-100. To this, 50 μl of substrate was added, to a final concentration 1 μM. After 30 min of incubation in room temperature, absorbance was read at 405 nm and the concentration was calculated from a standard curve of elastase (Sigma).
Elastase activity assay
Cells (HL-60 and U937) treated with 0, 125, 250 and 500 μM of for 24 h were lysed with 100 μl of buffer containing 100 mM Tris-HCl pH 7.4, 1 mM MgCl 2 , 0.1 % Triton X-100. After homogenization, 300 μl of 1.4 M NaCl in 0.1 % Triton X-100 was added and samples were centrifuged at 15 000 × g, for 15 min at 4°C. The supernatants were transferred to new tubes and assayed for elastase activity using Suc-Ala-Ala-Ala-pNA (Sigma) as a substrate. For each assay we took 25 μl sample, mixed with 100 μl buffer containing 100 mM Tris-HCl pH 8.5, 1 M NaCl, 500 mM MgCl 2  and 0.1 % Triton X-100. To this, 50 μl of substrate was added, to a final concentration 1 μM. After 30 min of incubation in room temperature, absorbance was read at 405 nm and the concentration was calculated from a standard curve of elastase (Sigma).Cell survival index For the cell viability assay, we used a fluorometric microculture cytotoxicity assay (FMCA) previously described by Larsson et al [ 5 ]. Briefly, 20 000 cells/well were plated in 96-well plates (NUNC, DK) in complete medium with addition of increased concentrations of sulphasalazine (0, 125, 250, 500, 750 and 1000 μM) and incubated for 72 h in a humidified atmosphere used in regular cell culturing. All samples were plated in triplicates and three wells with cell culture medium served as blanks. As controls we had cells without additions and cells only with solvent, in this case 0.5 M NaOH, with equal molarities as in the wells with the highest sulphasalazine-concentration. At the end of the 72 h incubation period, plates were centrifuged (200 × g, 5 min) and medium was aspirated in a microtitre plate washer, washed with PBS and 100 μl of 10 μg/ml of fluorescein diacetate (Sigma, Sweden), was added. This dye exclusively binds intact cell membranes of viable cells. After 1 h incubation at 37°C, the fluorescence was read in the Fluoroscan 2 (Labsystems OY, Finland) at 480 nm excitation and 530 nm as emission. The results are presented as survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100. Thus, a low numerical value indicates high sensitivity to the cytotoxic effect of sulphasalazine. Effective concentration is defined as the concentration when 50 % of the cells are viable (EC 50 ).
Cell survival index
For the cell viability assay, we used a fluorometric microculture cytotoxicity assay (FMCA) previously described by Larsson et al [ 5 ]. Briefly, 20 000 cells/well were plated in 96-well plates (NUNC, DK) in complete medium with addition of increased concentrations of sulphasalazine (0, 125, 250, 500, 750 and 1000 μM) and incubated for 72 h in a humidified atmosphere used in regular cell culturing. All samples were plated in triplicates and three wells with cell culture medium served as blanks. As controls we had cells without additions and cells only with solvent, in this case 0.5 M NaOH, with equal molarities as in the wells with the highest sulphasalazine-concentration. At the end of the 72 h incubation period, plates were centrifuged (200 × g, 5 min) and medium was aspirated in a microtitre plate washer, washed with PBS and 100 μl of 10 μg/ml of fluorescein diacetate (Sigma, Sweden), was added. This dye exclusively binds intact cell membranes of viable cells. After 1 h incubation at 37°C, the fluorescence was read in the Fluoroscan 2 (Labsystems OY, Finland) at 480 nm excitation and 530 nm as emission. The results are presented as survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100. Thus, a low numerical value indicates high sensitivity to the cytotoxic effect of sulphasalazine. Effective concentration is defined as the concentration when 50 % of the cells are viable (EC 50 ).Statistics Two-tailed Student's t-test was used to compare subject characteristics and results from cell culture between cases and controls. Frequencies of subject characteristics male versus females was tested with Chi 2 -test with one degree of freedom, using Minitab 14. A  p -value less than 0.05 was denoted with (*),  p  < 0.01 with (**) and was considered as statistically significant.
Statistics
Two-tailed Student's t-test was used to compare subject characteristics and results from cell culture between cases and controls. Frequencies of subject characteristics male versus females was tested with Chi 2 -test with one degree of freedom, using Minitab 14. A  p -value less than 0.05 was denoted with (*),  p  < 0.01 with (**) and was considered as statistically significant.ppResults Subjects The characteristics of the subjects are presented in Table  2 . The agranulocytosis cases were significantly older than the control patients ( p  = 0.023). The white blood cell count (WBC) before sulphasalazine-treatment did not differ between cases and controls, nor did the dose of sulphasalazine. Bone marrow aspirates had been taken from 10 patients (cases). In all samples, the myelopoesis was seriously reduced and a maturation arrest at the promyelocyte-myelocyte stage of neutrophilic differentiation was seen. Table 2 Characteristics of subjects  Cases (n = 36) Controls (n = 72) p -value Age range (median) 11–77 (55) 13–90 (47) 0.023 WBC before  a 9.3 ± 4.7 8.5 ± 2.5 0.26 Dose of sulphasalazine (gram/day) 2.2 ± 0.6 2.0 ± 0.4 0.13 Male : Female 17 : 19 33 : 39 0.891 a  WBC data only available for 28 cases and 67 controls. Cases are defined as the patients treated with sulphasalazine, who were diagnosed with agranulocytosis and controls were defined as patients treated with sulphasalazine without hematological side effects within the first three months of sulphasalazine treatment. The statistics of differences between cases and controls in age, white blood cell count (WBC) before sulphasalazine treatment and dose of sulphasalazine was calculated with 2-tailed Student's t-test. The frequency of males versus females was calculated with Chi 2  test, one degree of freedom. Significance level was set at 0.05. Mutation analysis None of the previously reported mutations in  ELA2  was found in this material, although we found a silent single nucleotide polymorphism, called S173 [ 6 ] that corresponds to a C4890A substitution in Genbank accession number Y00477 (marked with extended arrow in Figure  1 ). The incidence of the S173 polymorphism did not differ between controls and cases, 0.31 for both, and S173 has previously been detected in healthy subjects [ 6 ]. No correlation between the S173 polymorphism and white blood count before sulphasalazine-treatment was found (Table  3 ). Table 3 White blood count (WBC), before sulphasalazine treatment, in subjects with or without the S173 polymorphism  WBC Subjects with S173 (n = 34) 8.74 ± 2.51 Subjects without S173 (n = 74) 8.66 ± 3.51 White blood count (WBC) was estimated by the local physicians before starting with the sulphasalazine treatment. The average WBC in subjects with or without S173 is presented as the mean value ± SD. It was no statistical difference between the groups. Elastase expression, elastase activity and cell survival after sulphasalazine exposure to HL-60 and U937 By western blot analysis, we analyzed neutrophil elastase protein expression in U937 cells. No difference in human neutrophil elastase expression was detected after treatment with 125 and 250 μM sulphasalazine (Figure  2 ), compared to controls. The elastase activity in HL-60 and U937 cells was not affected by increasing sulphasalazine concentration, ranging from 0 to 500 μM, and expressed as elastase activity/μg protein (data not shown). For the cell survival index, each FMCA experiment was performed three times separately with similar results (inter-assay variation less than 10 %). Concentrations below 250 μM sulphasalazine did not affect the survival index of U937 and HL-60 cells (Figure  3A,3B ), but at 500 μM of sulphasalazine, the survival index of HL-60 cells decreased to a third (Figure  3A ). The U937 was only marginally affected at 500 μM sulphasalazine-concentration, but cellular survival decreased with approximately 40 % at 750 μM of sulphasalazine (Figure  3B ). The effective concentration (EC 50 ) of sulphasalazine was approximately 370 μM for HL-60 cells and 820 μM for U937 cells. Figure 2 Western blot analysis of human neutrophil elastase (hNE) expression after sulphasalazine exposure  U937 cells were incubated for 24 h with 0, 125 and 250 μM sulphasalazine, followed by cell lysis and protein isolation. 20 μg of protein was applied in each lane, transferred to nitrocellulose membrane and incubated with human neutrophil elastase antibody. Figure 3 Survival index of HL-60 and U937 cells, incubated with increasing concentrations of sulphasalazine  Survival index of HL-60 (A) and U937 cells (B) treated with increasing concentrations of sulphasalazine (0–1000 μM) for 72 h and measured with FMCA. Survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100.
Results
Subjects The characteristics of the subjects are presented in Table  2 . The agranulocytosis cases were significantly older than the control patients ( p  = 0.023). The white blood cell count (WBC) before sulphasalazine-treatment did not differ between cases and controls, nor did the dose of sulphasalazine. Bone marrow aspirates had been taken from 10 patients (cases). In all samples, the myelopoesis was seriously reduced and a maturation arrest at the promyelocyte-myelocyte stage of neutrophilic differentiation was seen. Table 2 Characteristics of subjects  Cases (n = 36) Controls (n = 72) p -value Age range (median) 11–77 (55) 13–90 (47) 0.023 WBC before  a 9.3 ± 4.7 8.5 ± 2.5 0.26 Dose of sulphasalazine (gram/day) 2.2 ± 0.6 2.0 ± 0.4 0.13 Male : Female 17 : 19 33 : 39 0.891 a  WBC data only available for 28 cases and 67 controls. Cases are defined as the patients treated with sulphasalazine, who were diagnosed with agranulocytosis and controls were defined as patients treated with sulphasalazine without hematological side effects within the first three months of sulphasalazine treatment. The statistics of differences between cases and controls in age, white blood cell count (WBC) before sulphasalazine treatment and dose of sulphasalazine was calculated with 2-tailed Student's t-test. The frequency of males versus females was calculated with Chi 2  test, one degree of freedom. Significance level was set at 0.05.
Subjects
The characteristics of the subjects are presented in Table  2 . The agranulocytosis cases were significantly older than the control patients ( p  = 0.023). The white blood cell count (WBC) before sulphasalazine-treatment did not differ between cases and controls, nor did the dose of sulphasalazine. Bone marrow aspirates had been taken from 10 patients (cases). In all samples, the myelopoesis was seriously reduced and a maturation arrest at the promyelocyte-myelocyte stage of neutrophilic differentiation was seen.pCharacteristics of subjectspAge range (median)WBC before  aDose of sulphasalazine (gram/day)Male : Femalea  WBC data only available for 28 cases and 67 controls.Cases are defined as the patients treated with sulphasalazine, who were diagnosed with agranulocytosis and controls were defined as patients treated with sulphasalazine without hematological side effects within the first three months of sulphasalazine treatment. The statistics of differences between cases and controls in age, white blood cell count (WBC) before sulphasalazine treatment and dose of sulphasalazine was calculated with 2-tailed Student's t-test. The frequency of males versus females was calculated with Chi 2  test, one degree of freedom. Significance level was set at 0.05.Mutation analysis None of the previously reported mutations in  ELA2  was found in this material, although we found a silent single nucleotide polymorphism, called S173 [ 6 ] that corresponds to a C4890A substitution in Genbank accession number Y00477 (marked with extended arrow in Figure  1 ). The incidence of the S173 polymorphism did not differ between controls and cases, 0.31 for both, and S173 has previously been detected in healthy subjects [ 6 ]. No correlation between the S173 polymorphism and white blood count before sulphasalazine-treatment was found (Table  3 ). Table 3 White blood count (WBC), before sulphasalazine treatment, in subjects with or without the S173 polymorphism  WBC Subjects with S173 (n = 34) 8.74 ± 2.51 Subjects without S173 (n = 74) 8.66 ± 3.51 White blood count (WBC) was estimated by the local physicians before starting with the sulphasalazine treatment. The average WBC in subjects with or without S173 is presented as the mean value ± SD. It was no statistical difference between the groups.
Mutation analysis
None of the previously reported mutations in  ELA2  was found in this material, although we found a silent single nucleotide polymorphism, called S173 [ 6 ] that corresponds to a C4890A substitution in Genbank accession number Y00477 (marked with extended arrow in Figure  1 ). The incidence of the S173 polymorphism did not differ between controls and cases, 0.31 for both, and S173 has previously been detected in healthy subjects [ 6 ]. No correlation between the S173 polymorphism and white blood count before sulphasalazine-treatment was found (Table  3 ).ELA2White blood count (WBC), before sulphasalazine treatment, in subjects with or without the S173 polymorphismWBCWhite blood count (WBC) was estimated by the local physicians before starting with the sulphasalazine treatment. The average WBC in subjects with or without S173 is presented as the mean value ± SD. It was no statistical difference between the groups.Elastase expression, elastase activity and cell survival after sulphasalazine exposure to HL-60 and U937 By western blot analysis, we analyzed neutrophil elastase protein expression in U937 cells. No difference in human neutrophil elastase expression was detected after treatment with 125 and 250 μM sulphasalazine (Figure  2 ), compared to controls. The elastase activity in HL-60 and U937 cells was not affected by increasing sulphasalazine concentration, ranging from 0 to 500 μM, and expressed as elastase activity/μg protein (data not shown). For the cell survival index, each FMCA experiment was performed three times separately with similar results (inter-assay variation less than 10 %). Concentrations below 250 μM sulphasalazine did not affect the survival index of U937 and HL-60 cells (Figure  3A,3B ), but at 500 μM of sulphasalazine, the survival index of HL-60 cells decreased to a third (Figure  3A ). The U937 was only marginally affected at 500 μM sulphasalazine-concentration, but cellular survival decreased with approximately 40 % at 750 μM of sulphasalazine (Figure  3B ). The effective concentration (EC 50 ) of sulphasalazine was approximately 370 μM for HL-60 cells and 820 μM for U937 cells. Figure 2 Western blot analysis of human neutrophil elastase (hNE) expression after sulphasalazine exposure  U937 cells were incubated for 24 h with 0, 125 and 250 μM sulphasalazine, followed by cell lysis and protein isolation. 20 μg of protein was applied in each lane, transferred to nitrocellulose membrane and incubated with human neutrophil elastase antibody. Figure 3 Survival index of HL-60 and U937 cells, incubated with increasing concentrations of sulphasalazine  Survival index of HL-60 (A) and U937 cells (B) treated with increasing concentrations of sulphasalazine (0–1000 μM) for 72 h and measured with FMCA. Survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100.
Elastase expression, elastase activity and cell survival after sulphasalazine exposure to HL-60 and U937
By western blot analysis, we analyzed neutrophil elastase protein expression in U937 cells. No difference in human neutrophil elastase expression was detected after treatment with 125 and 250 μM sulphasalazine (Figure  2 ), compared to controls. The elastase activity in HL-60 and U937 cells was not affected by increasing sulphasalazine concentration, ranging from 0 to 500 μM, and expressed as elastase activity/μg protein (data not shown). For the cell survival index, each FMCA experiment was performed three times separately with similar results (inter-assay variation less than 10 %). Concentrations below 250 μM sulphasalazine did not affect the survival index of U937 and HL-60 cells (Figure  3A,3B ), but at 500 μM of sulphasalazine, the survival index of HL-60 cells decreased to a third (Figure  3A ). The U937 was only marginally affected at 500 μM sulphasalazine-concentration, but cellular survival decreased with approximately 40 % at 750 μM of sulphasalazine (Figure  3B ). The effective concentration (EC 50 ) of sulphasalazine was approximately 370 μM for HL-60 cells and 820 μM for U937 cells.Discussion Idiosyncratic drug-induced agranulocytosis can be due to several different mechanisms of action, including immunological, toxic and genetic [ 7 , 8 ]. Toxic drug-induced neutropenia is often dose-dependent, whereas immunological and genetic causes are less related to dose. In our study, bone marrow aspirates from patients with sulphasalazine-induced agranulocytosis revealed maturation arrest of neutrophils at the promyelocyte-myelocyte stage. These findings resemble promyelocytic maturation arrest seen in severe congenital neutopenia (SCN) and cyclic neutropenia (CN) [ 9 ]. In the majority of cases with SCN and CN, germline mutations in the human neutrophil elastase gene ( ELA2 ) are implicated as the primary abnormality [ 2 , 4 ]. The focus of this study is therefore on the human neutrophil elastase gene as a possible cause of sulphasalazine-induced agranulocytosis. We found a coding synonymous polymorphism in  ELA2 , which, however, was equally represented among cases and controls. Heterozygous mutations in  ELA2  act in a dominant manner, interfering with sub-cellular trafficking of neutrophil elastase, and leading to an accumulation of neutrophil elastase in the cytosol [ 10 ]. For normal neutrophil cell maturation, the proliferative action of the granulocyte colony stimulating factor (G-CSF) is necessary [ 11 ]. When G-CSF is exposed to active elastase enzyme in vitro, G-CSF is rapidly cleaved and rendered inactive [ 11 ]. In theory, SCN and CN are caused by an accumulation of neutrophil elastase, leading to an inactivation of G-CSF and a negative feedback on granulopoiesis, which causes neutropenia. Other proteins, connected to expression and transportation of human neutrophil elastase, have also been linked to SCN disease. In canine cyclic hematopoieses, lack of the intracellular transport protein AP3β causes accumulation of canine neutrophil elastase in the cytosolic compartments [ 12 ], and mutations in  ELA2  may disrupt the AP3β-recognition site [ 13 ]. Furthermore, mutations in the proto-oncogene  GFI1 , a transcriptional repressor of  ELA2 , causes over-expression of neutrophil elastase in mice, thus, making them neutropenic [ 14 ]. During maintenance therapy with sulphasalazine, trough serum sulpha concentration is on average approximately 100 μM at the Department of Clinical chemistry and pharmacology, Uppsala University hospital. To avoid toxic effects, trough serum concentration of sulpha should stay below 600 μM [ 15 ]. Our in vitro data suggest a decreased cell survival of sulphasalazine at concentrations around 500 μM. Interestingly promyelocytic leukemia HL-60 cells were more sensitive to sulphasalazine than lymphoma U937 cells, with EC 50  values of 370 μM and 820 μM, respectively. Human neutrophil elastase expression in lymphoma U937 cells did not differ after sulphasalazine at 125 and 250 μM, indicating that human neutrophil elastase production is not affected by sulphasalazine at subtoxic levels.
Discussion
Idiosyncratic drug-induced agranulocytosis can be due to several different mechanisms of action, including immunological, toxic and genetic [ 7 , 8 ]. Toxic drug-induced neutropenia is often dose-dependent, whereas immunological and genetic causes are less related to dose. In our study, bone marrow aspirates from patients with sulphasalazine-induced agranulocytosis revealed maturation arrest of neutrophils at the promyelocyte-myelocyte stage. These findings resemble promyelocytic maturation arrest seen in severe congenital neutopenia (SCN) and cyclic neutropenia (CN) [ 9 ]. In the majority of cases with SCN and CN, germline mutations in the human neutrophil elastase gene ( ELA2 ) are implicated as the primary abnormality [ 2 , 4 ]. The focus of this study is therefore on the human neutrophil elastase gene as a possible cause of sulphasalazine-induced agranulocytosis. We found a coding synonymous polymorphism in  ELA2 , which, however, was equally represented among cases and controls.ELA2ELA2Heterozygous mutations in  ELA2  act in a dominant manner, interfering with sub-cellular trafficking of neutrophil elastase, and leading to an accumulation of neutrophil elastase in the cytosol [ 10 ]. For normal neutrophil cell maturation, the proliferative action of the granulocyte colony stimulating factor (G-CSF) is necessary [ 11 ]. When G-CSF is exposed to active elastase enzyme in vitro, G-CSF is rapidly cleaved and rendered inactive [ 11 ]. In theory, SCN and CN are caused by an accumulation of neutrophil elastase, leading to an inactivation of G-CSF and a negative feedback on granulopoiesis, which causes neutropenia. Other proteins, connected to expression and transportation of human neutrophil elastase, have also been linked to SCN disease. In canine cyclic hematopoieses, lack of the intracellular transport protein AP3β causes accumulation of canine neutrophil elastase in the cytosolic compartments [ 12 ], and mutations in  ELA2  may disrupt the AP3β-recognition site [ 13 ]. Furthermore, mutations in the proto-oncogene  GFI1 , a transcriptional repressor of  ELA2 , causes over-expression of neutrophil elastase in mice, thus, making them neutropenic [ 14 ].ELA2ELA2GFI1ELA2During maintenance therapy with sulphasalazine, trough serum sulpha concentration is on average approximately 100 μM at the Department of Clinical chemistry and pharmacology, Uppsala University hospital. To avoid toxic effects, trough serum concentration of sulpha should stay below 600 μM [ 15 ]. Our in vitro data suggest a decreased cell survival of sulphasalazine at concentrations around 500 μM. Interestingly promyelocytic leukemia HL-60 cells were more sensitive to sulphasalazine than lymphoma U937 cells, with EC 50  values of 370 μM and 820 μM, respectively. Human neutrophil elastase expression in lymphoma U937 cells did not differ after sulphasalazine at 125 and 250 μM, indicating that human neutrophil elastase production is not affected by sulphasalazine at subtoxic levels.Conclusions In conclusion, neutrophil elastase does not appear to be involved in the etiology of sulphasalazine-induced agranulocytosis. No causative  ELA2  mutations were found, and therapeutic concentrations of sulphasalazine did not increase the expression of human neutrophil elastase. High concentrations of sulphasalazine were toxic to white blood cells in vitro; however, there is no evidence that this toxicity is mediated through human neutrophil elastase. Promyelocytic cells were more sensitive to sulphasalazine than lymphoma cells, and the reason for this difference may also explain sulphasalazine-induced agranulocytosis.
Conclusions
In conclusion, neutrophil elastase does not appear to be involved in the etiology of sulphasalazine-induced agranulocytosis. No causative  ELA2  mutations were found, and therapeutic concentrations of sulphasalazine did not increase the expression of human neutrophil elastase. High concentrations of sulphasalazine were toxic to white blood cells in vitro; however, there is no evidence that this toxicity is mediated through human neutrophil elastase. Promyelocytic cells were more sensitive to sulphasalazine than lymphoma cells, and the reason for this difference may also explain sulphasalazine-induced agranulocytosis.ELA2Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions AJ carried out the molecular genetic studies, participated in the sequence alignment, drafted the manuscript and carried out the in vitro experiments. MW participated in the design of the study and performed the statistical analysis. HM conceived the study, and AJ, MW and HM participated in its design and coordination. All authors read and approved the final manuscript.
Authors' contributions
AJ carried out the molecular genetic studies, participated in the sequence alignment, drafted the manuscript and carried out the in vitro experiments. MW participated in the design of the study and performed the statistical analysis. HM conceived the study, and AJ, MW and HM participated in its design and coordination. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The United Kingdom National Service Framework for coronary heart disease (CHD) suggested that patients with CHD are likely to benefit from cardiological supervision. We set out to assess the effect of access to cardiologists on survival among AMI patients admitted in two UK hospitals.
Background
The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The United Kingdom National Service Framework for coronary heart disease (CHD) suggested that patients with CHD are likely to benefit from cardiological supervision. We set out to assess the effect of access to cardiologists on survival among AMI patients admitted in two UK hospitals.Methods The study was conducted in a university hospital and a district general hospital in England. Information was obtained on age, sex, ethnicity, Carstairs socioeconomic deprivation category derived from postcode of residence, comorbidity, distance from hospital and medication from all patients admitted with acute myocardial infarction in two acute trusts between July 1999 and June 2000. Record linkage to subsequent Hospital Episode Statistics and Registrar General's death records provided follow up information on procedures and mortality up to eighteen months after admission. Cox proportional hazard models were used to investigate the main hypothesis controlling for confounding. The main outcome measure was 18-month survival after myocardial infarction.
Methods
The study was conducted in a university hospital and a district general hospital in England. Information was obtained on age, sex, ethnicity, Carstairs socioeconomic deprivation category derived from postcode of residence, comorbidity, distance from hospital and medication from all patients admitted with acute myocardial infarction in two acute trusts between July 1999 and June 2000. Record linkage to subsequent Hospital Episode Statistics and Registrar General's death records provided follow up information on procedures and mortality up to eighteen months after admission. Cox proportional hazard models were used to investigate the main hypothesis controlling for confounding. The main outcome measure was 18-month survival after myocardial infarction.Results Access to a cardiologist was univariately associated with improved survival (hazard ratio 0.16, 95% CI 0.10 to 0.25). This effect remained after controlling for the effect of patient characteristics (hazard ratio 0.22, 95% CI 0.14 to 0.25). The effect disappeared after controlling for access to effective medication (hazard ratio 0.70, 95% CI 0.33 to 1.46).
Results
Access to a cardiologist was univariately associated with improved survival (hazard ratio 0.16, 95% CI 0.10 to 0.25). This effect remained after controlling for the effect of patient characteristics (hazard ratio 0.22, 95% CI 0.14 to 0.25). The effect disappeared after controlling for access to effective medication (hazard ratio 0.70, 95% CI 0.33 to 1.46).Conclusions Access to a cardiologist is associated with better survival compared to no access to a cardiologist among a cohort of patients already admitted with AMI. This effect is mainly due to the more frequent use of effective medicines by the group referred to cardiologists. Hospitals may improve survival by improving access to effective medicines and by coordinating care between cardiologists and general physicians.
Conclusions
Access to a cardiologist is associated with better survival compared to no access to a cardiologist among a cohort of patients already admitted with AMI. This effect is mainly due to the more frequent use of effective medicines by the group referred to cardiologists. Hospitals may improve survival by improving access to effective medicines and by coordinating care between cardiologists and general physicians.

Background The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The effect of the speciality of the attending physician on mortality in AMI has been studied mainly in the United States (US) with conflicting results arising from the studies [ 3 - 6 ]. The explanations considered for the difference in mortality observed in some of the studies include patient's condition (case mix and comorbidity)[ 3 ], volume of workload[ 5 , 7 ] and treatment given[ 3 ]. Previous studies show that knowledge and use of effective medicines is better among cardiologists compared with general physicians in the US [ 8 - 10 ]. Co-ordination of care between cardiologists and non-cardiologists also improves survival of patients seen by non-cardiologists [ 11 ]. The National Service Framework (NSF) for coronary heart disease (CHD) published in March 2000, suggested that patients with CHD are likely to benefit from cardiological supervision. To provide this level of care, all acute hospitals will eventually need a minimum of two cardiologists [ 1 ]. A recent survey by the Royal College of Physicians found an average of 1.7 whole time equivalent (WTE) cardiologists in the 211 hospitals receiving patients with AMI in the UK [ 2 ]. Eight hospitals did not have a cardiologist. There is a need to establish whether the involvement of a cardiologist in the management of AMI patients affects the quality and outcome of care, and if so to identify ways to improve the outcome of care for patients unable to gain access to a cardiologist. This study aims to assess the effect of access to cardiologists on survival among AMI patients accounting for access to effective investigation, medication, procedures and the underlying condition of the patient at presentation.
Background
The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The effect of the speciality of the attending physician on mortality in AMI has been studied mainly in the United States (US) with conflicting results arising from the studies [ 3 - 6 ]. The explanations considered for the difference in mortality observed in some of the studies include patient's condition (case mix and comorbidity)[ 3 ], volume of workload[ 5 , 7 ] and treatment given[ 3 ]. Previous studies show that knowledge and use of effective medicines is better among cardiologists compared with general physicians in the US [ 8 - 10 ]. Co-ordination of care between cardiologists and non-cardiologists also improves survival of patients seen by non-cardiologists [ 11 ].The National Service Framework (NSF) for coronary heart disease (CHD) published in March 2000, suggested that patients with CHD are likely to benefit from cardiological supervision. To provide this level of care, all acute hospitals will eventually need a minimum of two cardiologists [ 1 ]. A recent survey by the Royal College of Physicians found an average of 1.7 whole time equivalent (WTE) cardiologists in the 211 hospitals receiving patients with AMI in the UK [ 2 ]. Eight hospitals did not have a cardiologist.There is a need to establish whether the involvement of a cardiologist in the management of AMI patients affects the quality and outcome of care, and if so to identify ways to improve the outcome of care for patients unable to gain access to a cardiologist. This study aims to assess the effect of access to cardiologists on survival among AMI patients accounting for access to effective investigation, medication, procedures and the underlying condition of the patient at presentation.Methods A retrospective cohort design was used. Study population and inclusion criteria All patients admitted to two hospitals in Eastern England between 1 st  July 1999 and 31 st  June 2000 with a discharge diagnosis of AMI were included in the analysis. A diagnosis of AMI was based on evidence of raised cardiac enzymes and/or other indicators of myocardial necrosis and on a physician's judgement of ECG changes indicative of AMI. Data collection and analysis Hospital Episode Statistics were used to extract records of all patients admitted with the ICD10 diagnostic codes for Acute Myocardial Infarction (I21, I22, I23) in any diagnostic field. The records were restructured to produce one record per patient using the new NHS number. For records with missing NHS number, a unique identifier was created using date of birth, sex and post code. The unique identifier was also used as a check for the records with NHS numbers. Records of all Finished Consultant Episodes with OPCS 4 codes of either Coronary Artery Bypass Grafting (K40–46) or Percutaneous Transluminal Coronary Angiography (K49–50) were extracted. The process was repeated for angiography (K63 – 65). Record linkage with subsequent Hospital Episode Statistics and Registrar General's records provided follow up information on procedures and death up to eighteen months after the index AMI. All cases were reviewed by examining the case notes. Detailed information on variables summarised below was obtained. List of variables extracted from patients' case notes • Hospital where patient was first admitted • Whether the patient was seen by a cardiologist or not, determined by evidence of a cardiology review in a patients case note • The average distance from residence to hospital first seen was determined by mapping the distance in miles using the RAC™ website distance calculator • A history of comorbidity – defined as the presence of diabetes, metastatic malignancy, cerebrovascular accident (CVA), asthma, chronic obstructive airway disease, renal impairment, endocrine disorder, chronic infection, dementia • Physical impairment with poor mobility • Procedures – exercise testing, angiography, CABG, PTCA • Appropriate medication – β blockers, aspirin, ACE inhibitors, thrombolysis (table  2 ) Table 1 Patient characteristics, and whether seen by cardiologist or by general physician Patient characteristic Physician Cardiologist (N = 275) Non Cardiologist (N = 201) Mean age (SD) 64.4 (SD 12.8) 74.8 (SD 11.4) Sex n (%) Male 275 (71.4) 201 (52.8) History of:   Congestive heart failure % 11.3 29.6   Angina % 94.9 86.7   Hypertension % 41.8 46.6   Diabetes mellitus % 12.1 17.1   Impaired mobility % 7.8 17.7   Smoking % 36.0 49.6 Impaired left ventricular function % 24.6 25.0 Table 2 Odds ratios for drug and procedure use after AMI by physician (cardiologist relative to non-cardiologist) Intervention Number (Percent) Odds ratio (95% CI) Cardiologist (275) Non Cardiologist (201) Thrombolytic therapy* 140 (51%) 66 (33%) 2.08 (1.35 – 3.23) β blocker on discharge* 206 (75%) 90 (45%) 3.70 (2.38 – 5.56) Aspirin* 264 (96%) 160 (80%) 6.67 (3.23 – 14.29) ACE inhibitors 124 (45%) 80 (40%) 1.27 (0.01 – 1.92) Exercise testing 192 (70%) 46 (23%) 7.70 (4.76 – 12.50) Coronary angiography 151 (55%) 12 (6%) 4.76 (3.22 – 7.14) Coronary angioplasty 74 (27%) 30 (15%) 1.92 (1.28 – 2.94) Coronary artery bypass grafting 66 (24%) 30 (15%) 1.75 (1.15 – 2.70) * See text for eligibility criteria • A history of  Smoking  Previous MI  Hypertension • Impaired left ventricular function based on echocardiography result or angiography • Severity of AMI using the number of vessels affected (vessels were considered affected if a lesion of 50% or more was noted in the angiography report) • Demographic factors: age, sex, ethnicity, socioeconomic status and Carstairs socio-economic deprivation category derived from postcode of residence Baseline patient characteristics were tabulated. Categorical variables were tested for statistical significance using the χ 2  test. Continuous variables were tested using the Student t test comparing patients seen by cardiologists and those seen by other physicians. The relative odds of drug and procedure use by speciality of physician were calculated. Specific variables included the use of thrombolytic therapy, β blockers, aspirin, ACE inhibitors among clinically appropriate groups, and the use of exercise testing, coronary angiography, angioplasty and coronary artery bypass grafting (CABG). Cox proportional hazards models were used to investigate the effect on survival of seeing a cardiologist. Multivariate models were fitted to control for the effect of patient and hospital characteristics. Controlled factors included: age, sex, comorbidity, hospital, distance from patients residence to hospital and Carstairs socio-economic deprivation category in the first instance. The effect of angiography, revascularisation and the use of effective medicines (aspirin and/or β blockers and/or thrombolysis – eligibility criteria listed below) were subsequently introduced into the model to investigate whether these affected outcome. The factors were included in the model either because they are known to confound the association between type of physician and survival or are known to be associated with survival and were not comparable between the two groups. Eligibility criteria for appropriate use of effective medicines [ 13 ]. Thrombolytic therapy • No warfarin therapy on admission • ST-segment elevation on initial ECG • Less than 12 hours since onset of chest pain • Systolic BP less than 180 mmHg and diastolic BP less than 110 mmHg at presentation • No severe CVA, gastrointestinal disease or chronic liver disease Aspirin • No haemorrhagic complication • No severe CVA, gastrointestinal disease or chronic liver disease or renal failure • No warfarin therapy on admission β blockers • No chronic lung disease • No cardiogenic shock, hypotension, complete heart block or decompensated heart failure The characteristics of the two hospitals are summarised figure  1 . The results from the two hospitals were combined in order to make our findings more generalisable. The general characteristics did not show important differences in the organisation of care between the hospitals. It is unlikely there are important unmeasurable factors associated with a particular hospital that might cause confounding, but hospital of care is still included as a variable in the multivariate analysis to control for centre effect. Patients from the two hospitals undergo procedures in the same specialist cardiothoracic hospital. Figure 1 Characteristics of study hospitals. The main outcome was eighteen-month survival defined as time between date of AMI and death. Statistical analysis was carried out using Stata (Version 6.0). In general for Cox regression analysis, the number of events i.e. deaths (and similarly the number of non-events) per variable modelled should be at least 10 [ 12 ]. Ethical approval was obtained from the two local research ethics committees of the hospitals involved in the study. Permission from the director of research and Caldicott guardians of data in each hospital was also obtained.
Methods
A retrospective cohort design was used.Study population and inclusion criteria All patients admitted to two hospitals in Eastern England between 1 st  July 1999 and 31 st  June 2000 with a discharge diagnosis of AMI were included in the analysis. A diagnosis of AMI was based on evidence of raised cardiac enzymes and/or other indicators of myocardial necrosis and on a physician's judgement of ECG changes indicative of AMI.
Study population and inclusion criteria
All patients admitted to two hospitals in Eastern England between 1 st  July 1999 and 31 st  June 2000 with a discharge diagnosis of AMI were included in the analysis. A diagnosis of AMI was based on evidence of raised cardiac enzymes and/or other indicators of myocardial necrosis and on a physician's judgement of ECG changes indicative of AMI.Data collection and analysis Hospital Episode Statistics were used to extract records of all patients admitted with the ICD10 diagnostic codes for Acute Myocardial Infarction (I21, I22, I23) in any diagnostic field. The records were restructured to produce one record per patient using the new NHS number. For records with missing NHS number, a unique identifier was created using date of birth, sex and post code. The unique identifier was also used as a check for the records with NHS numbers. Records of all Finished Consultant Episodes with OPCS 4 codes of either Coronary Artery Bypass Grafting (K40–46) or Percutaneous Transluminal Coronary Angiography (K49–50) were extracted. The process was repeated for angiography (K63 – 65). Record linkage with subsequent Hospital Episode Statistics and Registrar General's records provided follow up information on procedures and death up to eighteen months after the index AMI. All cases were reviewed by examining the case notes. Detailed information on variables summarised below was obtained. List of variables extracted from patients' case notes • Hospital where patient was first admitted • Whether the patient was seen by a cardiologist or not, determined by evidence of a cardiology review in a patients case note • The average distance from residence to hospital first seen was determined by mapping the distance in miles using the RAC™ website distance calculator • A history of comorbidity – defined as the presence of diabetes, metastatic malignancy, cerebrovascular accident (CVA), asthma, chronic obstructive airway disease, renal impairment, endocrine disorder, chronic infection, dementia • Physical impairment with poor mobility • Procedures – exercise testing, angiography, CABG, PTCA • Appropriate medication – β blockers, aspirin, ACE inhibitors, thrombolysis (table  2 ) Table 1 Patient characteristics, and whether seen by cardiologist or by general physician Patient characteristic Physician Cardiologist (N = 275) Non Cardiologist (N = 201) Mean age (SD) 64.4 (SD 12.8) 74.8 (SD 11.4) Sex n (%) Male 275 (71.4) 201 (52.8) History of:   Congestive heart failure % 11.3 29.6   Angina % 94.9 86.7   Hypertension % 41.8 46.6   Diabetes mellitus % 12.1 17.1   Impaired mobility % 7.8 17.7   Smoking % 36.0 49.6 Impaired left ventricular function % 24.6 25.0 Table 2 Odds ratios for drug and procedure use after AMI by physician (cardiologist relative to non-cardiologist) Intervention Number (Percent) Odds ratio (95% CI) Cardiologist (275) Non Cardiologist (201) Thrombolytic therapy* 140 (51%) 66 (33%) 2.08 (1.35 – 3.23) β blocker on discharge* 206 (75%) 90 (45%) 3.70 (2.38 – 5.56) Aspirin* 264 (96%) 160 (80%) 6.67 (3.23 – 14.29) ACE inhibitors 124 (45%) 80 (40%) 1.27 (0.01 – 1.92) Exercise testing 192 (70%) 46 (23%) 7.70 (4.76 – 12.50) Coronary angiography 151 (55%) 12 (6%) 4.76 (3.22 – 7.14) Coronary angioplasty 74 (27%) 30 (15%) 1.92 (1.28 – 2.94) Coronary artery bypass grafting 66 (24%) 30 (15%) 1.75 (1.15 – 2.70) * See text for eligibility criteria • A history of  Smoking  Previous MI  Hypertension • Impaired left ventricular function based on echocardiography result or angiography • Severity of AMI using the number of vessels affected (vessels were considered affected if a lesion of 50% or more was noted in the angiography report) • Demographic factors: age, sex, ethnicity, socioeconomic status and Carstairs socio-economic deprivation category derived from postcode of residence Baseline patient characteristics were tabulated. Categorical variables were tested for statistical significance using the χ 2  test. Continuous variables were tested using the Student t test comparing patients seen by cardiologists and those seen by other physicians. The relative odds of drug and procedure use by speciality of physician were calculated. Specific variables included the use of thrombolytic therapy, β blockers, aspirin, ACE inhibitors among clinically appropriate groups, and the use of exercise testing, coronary angiography, angioplasty and coronary artery bypass grafting (CABG). Cox proportional hazards models were used to investigate the effect on survival of seeing a cardiologist. Multivariate models were fitted to control for the effect of patient and hospital characteristics. Controlled factors included: age, sex, comorbidity, hospital, distance from patients residence to hospital and Carstairs socio-economic deprivation category in the first instance. The effect of angiography, revascularisation and the use of effective medicines (aspirin and/or β blockers and/or thrombolysis – eligibility criteria listed below) were subsequently introduced into the model to investigate whether these affected outcome. The factors were included in the model either because they are known to confound the association between type of physician and survival or are known to be associated with survival and were not comparable between the two groups. Eligibility criteria for appropriate use of effective medicines [ 13 ]. Thrombolytic therapy • No warfarin therapy on admission • ST-segment elevation on initial ECG • Less than 12 hours since onset of chest pain • Systolic BP less than 180 mmHg and diastolic BP less than 110 mmHg at presentation • No severe CVA, gastrointestinal disease or chronic liver disease Aspirin • No haemorrhagic complication • No severe CVA, gastrointestinal disease or chronic liver disease or renal failure • No warfarin therapy on admission β blockers • No chronic lung disease • No cardiogenic shock, hypotension, complete heart block or decompensated heart failure The characteristics of the two hospitals are summarised figure  1 . The results from the two hospitals were combined in order to make our findings more generalisable. The general characteristics did not show important differences in the organisation of care between the hospitals. It is unlikely there are important unmeasurable factors associated with a particular hospital that might cause confounding, but hospital of care is still included as a variable in the multivariate analysis to control for centre effect. Patients from the two hospitals undergo procedures in the same specialist cardiothoracic hospital. Figure 1 Characteristics of study hospitals. The main outcome was eighteen-month survival defined as time between date of AMI and death. Statistical analysis was carried out using Stata (Version 6.0). In general for Cox regression analysis, the number of events i.e. deaths (and similarly the number of non-events) per variable modelled should be at least 10 [ 12 ]. Ethical approval was obtained from the two local research ethics committees of the hospitals involved in the study. Permission from the director of research and Caldicott guardians of data in each hospital was also obtained.
Data collection and analysis
Hospital Episode Statistics were used to extract records of all patients admitted with the ICD10 diagnostic codes for Acute Myocardial Infarction (I21, I22, I23) in any diagnostic field. The records were restructured to produce one record per patient using the new NHS number. For records with missing NHS number, a unique identifier was created using date of birth, sex and post code. The unique identifier was also used as a check for the records with NHS numbers. Records of all Finished Consultant Episodes with OPCS 4 codes of either Coronary Artery Bypass Grafting (K40–46) or Percutaneous Transluminal Coronary Angiography (K49–50) were extracted. The process was repeated for angiography (K63 – 65). Record linkage with subsequent Hospital Episode Statistics and Registrar General's records provided follow up information on procedures and death up to eighteen months after the index AMI.All cases were reviewed by examining the case notes. Detailed information on variables summarised below was obtained.List of variables extracted from patients' case notes• Hospital where patient was first admitted• Whether the patient was seen by a cardiologist or not, determined by evidence of a cardiology review in a patients case note• The average distance from residence to hospital first seen was determined by mapping the distance in miles using the RAC™ website distance calculator• A history of comorbidity – defined as the presence of diabetes, metastatic malignancy, cerebrovascular accident (CVA), asthma, chronic obstructive airway disease, renal impairment, endocrine disorder, chronic infection, dementia• Physical impairment with poor mobility• Procedures – exercise testing, angiography, CABG, PTCA• Appropriate medication – β blockers, aspirin, ACE inhibitors, thrombolysis (table  2 )Patient characteristics, and whether seen by cardiologist or by general physicianCongestive heart failure %Angina %Hypertension %Diabetes mellitus %Impaired mobility %Smoking %Odds ratios for drug and procedure use after AMI by physician (cardiologist relative to non-cardiologist)* See text for eligibility criteria• A history ofSmokingPrevious MIHypertension• Impaired left ventricular function based on echocardiography result or angiography• Severity of AMI using the number of vessels affected (vessels were considered affected if a lesion of 50% or more was noted in the angiography report)• Demographic factors: age, sex, ethnicity, socioeconomic status and Carstairs socio-economic deprivation category derived from postcode of residenceBaseline patient characteristics were tabulated. Categorical variables were tested for statistical significance using the χ 2  test. Continuous variables were tested using the Student t test comparing patients seen by cardiologists and those seen by other physicians.The relative odds of drug and procedure use by speciality of physician were calculated. Specific variables included the use of thrombolytic therapy, β blockers, aspirin, ACE inhibitors among clinically appropriate groups, and the use of exercise testing, coronary angiography, angioplasty and coronary artery bypass grafting (CABG).Cox proportional hazards models were used to investigate the effect on survival of seeing a cardiologist. Multivariate models were fitted to control for the effect of patient and hospital characteristics. Controlled factors included: age, sex, comorbidity, hospital, distance from patients residence to hospital and Carstairs socio-economic deprivation category in the first instance. The effect of angiography, revascularisation and the use of effective medicines (aspirin and/or β blockers and/or thrombolysis – eligibility criteria listed below) were subsequently introduced into the model to investigate whether these affected outcome. The factors were included in the model either because they are known to confound the association between type of physician and survival or are known to be associated with survival and were not comparable between the two groups.Eligibility criteria for appropriate use of effective medicines [ 13 ].Thrombolytic therapy• No warfarin therapy on admission• ST-segment elevation on initial ECG• Less than 12 hours since onset of chest pain• Systolic BP less than 180 mmHg and diastolic BP less than 110 mmHg at presentation• No severe CVA, gastrointestinal disease or chronic liver diseaseAspirin• No haemorrhagic complication• No severe CVA, gastrointestinal disease or chronic liver disease or renal failure• No warfarin therapy on admissionβ blockers• No chronic lung disease• No cardiogenic shock, hypotension, complete heart block or decompensated heart failureThe characteristics of the two hospitals are summarised figure  1 . The results from the two hospitals were combined in order to make our findings more generalisable. The general characteristics did not show important differences in the organisation of care between the hospitals. It is unlikely there are important unmeasurable factors associated with a particular hospital that might cause confounding, but hospital of care is still included as a variable in the multivariate analysis to control for centre effect. Patients from the two hospitals undergo procedures in the same specialist cardiothoracic hospital.The main outcome was eighteen-month survival defined as time between date of AMI and death.Statistical analysis was carried out using Stata (Version 6.0).In general for Cox regression analysis, the number of events i.e. deaths (and similarly the number of non-events) per variable modelled should be at least 10 [ 12 ].Ethical approval was obtained from the two local research ethics committees of the hospitals involved in the study. Permission from the director of research and Caldicott guardians of data in each hospital was also obtained.Results Medical records were obtained for 94% of eligible patients (476 of 506 patients). Four patients did not have enzyme changes of myocardial infarction or were clear cases of miscoding. The mean age of the cohort was 69.3 years (SD 13.2) and 34.5% were female. The patients with missing records did not differ from those with records in terms of age or sex using information from routine data (mean age was 70.4 years with SD 11.1; 36.2% were female). There were no significant differences in the proportion of data missing between the two hospitals including those for eligibility to effective medications and co morbidities. General characteristics Patients in our sample seen by a cardiologist were younger, with a higher proportion of males to females. They were more likely to have a history of CHD prior to myocardial infarction, but less likely to have a history of congestive heart failure or hypertension (Table  1 ). Patients with impaired mobility or who smoked were less likely to be seen by a cardiologist. The difference in the proportion of patients with echocardiographic evidence of impaired left ventricular function complicating their myocardial infarction by physician seen was not significant. Drug and procedure use The likelihood of receiving effective medication (thrombolytic therapy, β blockers, aspirin) was higher among patients seen by a cardiologist compared with those not seen by a cardiologist. Furthermore, patients seen by a cardiologist were more likely to have undergone exercise testing, angiography and revascularisation procedures (Table  2 ). The use of ACE inhibitors was not more frequent among cardiologists. Survival Univariate analysis indicated that patients seen by a cardiologist had better survival (hazard ratio 0.16, 95% CI 0.10 to 0.25). Effect of seeing a cardiologist after controlling for patient characteristics The survival of patients seen by a cardiologist was still better than that for those not seen by a cardiologist (hazard ratio 0.22, 95% CI 0.14 to 0.25) after controlling for the confounding effect of pre-existing comorbidity, age, sex, hospital first seen, deprivation and distance (Table  3 ). The effect of seeing a cardiologist remained after excluding patients that died within a week of MI. The hazard ratio was 0.21 (95% CI 0.12 – 0.37). Table 3 Cox regression analysis for eighteen month survival comparing cardiologists and non-cardiologist physicians and controlling for potential confounders. Variable Mortality (%) Adjusted hazard ratio (CI) * Adjusted hazard ratio (CI) # Seen by a Cardiologist Yes 30 (11%) 0.22 (0.14 – 0.38) 0.70 (0.33 – 1.46) No 88 (44%) Comorbidity Yes 15 (25%) 1.11 (0.62 – 1.99) 0.98 (0.49 – 1.98) No 107 (26%) Hospital A 58 (24%) 1.01 (0.67 – 1.51) 1.08 (0.71 – 1.73) B 65 (28%) Age (years) 30 – 40 1 (8%) 1.05 (1.03 – 1.08) 1.03 (0.99 – 1.06) 41 – 60 8 (7%) 61 – 70 18 (17%) 71 – 99 96 (40%) Sex Males 64 (21%) 0.91 (0.62 – 1.35) 0.83 (0.44 – 1.55) Females 58 (34%) Distance 1.0 (0.97 – 1.04) 0.96 (0.88 – 1.03) Angiography Yes 12 (7%) - 0.87 (0.24 – 3.17) No 112 (36%) Revascularisation Yes 14 (13%) - 0.25 (0.04 – 1.52) No 110 (30%) Effective medicines γ Yes 48 (14%) - 0.15 (0.07 – 0.29) No 76 (56%) * Adjusted for patient characteristics, hospital # Adjusted for patient characteristics, hospital and the use of effective medicines and procedures  γ  Access to aspirin, and/or β blockers and/ or thrombolysis if appropriate Effect of seeing a cardiologist after controlling for patient characteristics and use of effective intervention The effect of seeing a cardiologist on survival became non-significant once access to angiography, revascularisation procedures and effective medicines (aspirin and/or β blockers and/or thrombolysis) had been adjusted for. The hazard ratio was 0.70 (95% CI 0.33 – 1.46). The fully adjusted model indicated that the most important factor affecting survival was access to effective medication. (Table  3 ).
Results
Medical records were obtained for 94% of eligible patients (476 of 506 patients). Four patients did not have enzyme changes of myocardial infarction or were clear cases of miscoding. The mean age of the cohort was 69.3 years (SD 13.2) and 34.5% were female. The patients with missing records did not differ from those with records in terms of age or sex using information from routine data (mean age was 70.4 years with SD 11.1; 36.2% were female). There were no significant differences in the proportion of data missing between the two hospitals including those for eligibility to effective medications and co morbidities.General characteristics Patients in our sample seen by a cardiologist were younger, with a higher proportion of males to females. They were more likely to have a history of CHD prior to myocardial infarction, but less likely to have a history of congestive heart failure or hypertension (Table  1 ). Patients with impaired mobility or who smoked were less likely to be seen by a cardiologist. The difference in the proportion of patients with echocardiographic evidence of impaired left ventricular function complicating their myocardial infarction by physician seen was not significant.
General characteristics
Patients in our sample seen by a cardiologist were younger, with a higher proportion of males to females. They were more likely to have a history of CHD prior to myocardial infarction, but less likely to have a history of congestive heart failure or hypertension (Table  1 ). Patients with impaired mobility or who smoked were less likely to be seen by a cardiologist. The difference in the proportion of patients with echocardiographic evidence of impaired left ventricular function complicating their myocardial infarction by physician seen was not significant.Drug and procedure use The likelihood of receiving effective medication (thrombolytic therapy, β blockers, aspirin) was higher among patients seen by a cardiologist compared with those not seen by a cardiologist. Furthermore, patients seen by a cardiologist were more likely to have undergone exercise testing, angiography and revascularisation procedures (Table  2 ). The use of ACE inhibitors was not more frequent among cardiologists.
Drug and procedure use
The likelihood of receiving effective medication (thrombolytic therapy, β blockers, aspirin) was higher among patients seen by a cardiologist compared with those not seen by a cardiologist. Furthermore, patients seen by a cardiologist were more likely to have undergone exercise testing, angiography and revascularisation procedures (Table  2 ). The use of ACE inhibitors was not more frequent among cardiologists.Survival Univariate analysis indicated that patients seen by a cardiologist had better survival (hazard ratio 0.16, 95% CI 0.10 to 0.25).
Survival
Univariate analysis indicated that patients seen by a cardiologist had better survival (hazard ratio 0.16, 95% CI 0.10 to 0.25).Effect of seeing a cardiologist after controlling for patient characteristics The survival of patients seen by a cardiologist was still better than that for those not seen by a cardiologist (hazard ratio 0.22, 95% CI 0.14 to 0.25) after controlling for the confounding effect of pre-existing comorbidity, age, sex, hospital first seen, deprivation and distance (Table  3 ). The effect of seeing a cardiologist remained after excluding patients that died within a week of MI. The hazard ratio was 0.21 (95% CI 0.12 – 0.37). Table 3 Cox regression analysis for eighteen month survival comparing cardiologists and non-cardiologist physicians and controlling for potential confounders. Variable Mortality (%) Adjusted hazard ratio (CI) * Adjusted hazard ratio (CI) # Seen by a Cardiologist Yes 30 (11%) 0.22 (0.14 – 0.38) 0.70 (0.33 – 1.46) No 88 (44%) Comorbidity Yes 15 (25%) 1.11 (0.62 – 1.99) 0.98 (0.49 – 1.98) No 107 (26%) Hospital A 58 (24%) 1.01 (0.67 – 1.51) 1.08 (0.71 – 1.73) B 65 (28%) Age (years) 30 – 40 1 (8%) 1.05 (1.03 – 1.08) 1.03 (0.99 – 1.06) 41 – 60 8 (7%) 61 – 70 18 (17%) 71 – 99 96 (40%) Sex Males 64 (21%) 0.91 (0.62 – 1.35) 0.83 (0.44 – 1.55) Females 58 (34%) Distance 1.0 (0.97 – 1.04) 0.96 (0.88 – 1.03) Angiography Yes 12 (7%) - 0.87 (0.24 – 3.17) No 112 (36%) Revascularisation Yes 14 (13%) - 0.25 (0.04 – 1.52) No 110 (30%) Effective medicines γ Yes 48 (14%) - 0.15 (0.07 – 0.29) No 76 (56%) * Adjusted for patient characteristics, hospital # Adjusted for patient characteristics, hospital and the use of effective medicines and procedures  γ  Access to aspirin, and/or β blockers and/ or thrombolysis if appropriate
Effect of seeing a cardiologist after controlling for patient characteristics
The survival of patients seen by a cardiologist was still better than that for those not seen by a cardiologist (hazard ratio 0.22, 95% CI 0.14 to 0.25) after controlling for the confounding effect of pre-existing comorbidity, age, sex, hospital first seen, deprivation and distance (Table  3 ). The effect of seeing a cardiologist remained after excluding patients that died within a week of MI. The hazard ratio was 0.21 (95% CI 0.12 – 0.37).Cox regression analysis for eighteen month survival comparing cardiologists and non-cardiologist physicians and controlling for potential confounders.* Adjusted for patient characteristics, hospital # Adjusted for patient characteristics, hospital and the use of effective medicines and procedures  γ  Access to aspirin, and/or β blockers and/ or thrombolysis if appropriate* Adjusted for patient characteristics, hospital # Adjusted for patient characteristics, hospital and the use of effective medicines and proceduresAccess to aspirin, and/or β blockers and/ or thrombolysis if appropriateEffect of seeing a cardiologist after controlling for patient characteristics and use of effective intervention The effect of seeing a cardiologist on survival became non-significant once access to angiography, revascularisation procedures and effective medicines (aspirin and/or β blockers and/or thrombolysis) had been adjusted for. The hazard ratio was 0.70 (95% CI 0.33 – 1.46). The fully adjusted model indicated that the most important factor affecting survival was access to effective medication. (Table  3 ).
Effect of seeing a cardiologist after controlling for patient characteristics and use of effective intervention
The effect of seeing a cardiologist on survival became non-significant once access to angiography, revascularisation procedures and effective medicines (aspirin and/or β blockers and/or thrombolysis) had been adjusted for. The hazard ratio was 0.70 (95% CI 0.33 – 1.46). The fully adjusted model indicated that the most important factor affecting survival was access to effective medication. (Table  3 ).Discussion This study improves our understanding of the care of AMI by cardiologists and general physicians in UK hospitals. Access to a cardiologist was univariately associated with better survival. This effect remained after controlling for the effect of patient characteristics, including the presence of comorbidity, but disappeared when the confounding effect of access to effective medicines was controlled for. As noted in the analysis, patients seen by a cardiologist were more likely to have been prescribed these medicines and to have had exercise testing, angiography and revascularisation. This implies that the survival advantage associated with being seen by a cardiologist is due to the more frequent use of effective medicines and is similar to findings by Chen J et al in the United States [ 3 ]. Previous studies have shown a high level of miscoding in routine data for AMI patients (RM Norris cited in [ 2 ]). The use of a case note review improved the findings of this study by assisting in ascertaining the diagnosis of myocardial infarction and ensuring the accuracy and completeness of the data used in the analysis. The possibility remains that some cases not seen through the cardiology department and not recorded in the patient administration system were missed. This is likely to be a small number and unlikely to invalidate the findings. Identifying all patients with infarction is a major shortcoming of many studies that estimate mortality among patients seen by cardiologists [ 2 ]. This study includes all patients irrespective of where they were managed in the hospital; however patients dying from myocardial infarction before arriving at the hospital were not included. All cases diagnosed by the managing clinician as AMI with ECG changes and found to have enzyme changes indicating myocardial necrosis were included in the analysis after the case notes were reviewed. This may have involved some misclassification due to misdiagnosis, but this is likely to occur at random, affecting only a small number of cases, and can only underestimate the effect of each factor. Limitations of this study include potential for bias in the allocation of patients to treatments and physicians (ie cardiologist/non cardiologist) that were not measured. Another possible limitation is bias arising from medical case notes which were unobtainable. Records from routine data were used to examine whether those with missing records differed in any systematic way from those with available records; it was found that they did not differ in age or sex. The study did not use any criteria for judging clinical appropriateness of the procedures used. Incomplete recording of information in the case notes hampered the determination of appropriateness for effective medication. However there was no difference in the proportion of missing data between the two hospitals. Since doctors may selectively be referring younger patients with lower comorbidity to a cardiologist, we controlled for age and comorbidity in the analysis. However, residual confounding from age and comorbidity could still account for some of the observed difference in survival. All patients admitted to the Coronary Care Unit (CCU) in one of the hospitals are seen by a cardiologist and have a higher chance of accessing thrombolysis, angiography and revascularisation. A cardiologist also sees the majority of patients admitted to the CCU in the second hospital. The protocol for the management of AMI in both hospitals stipulates a cardiology review of all AMI patients. Access to a cardiologist may be a proxy measure of access to effective treatment and it may not be the trigger for effective treatment. These limitations mean that the findings should be interpreted with a degree of caution. In addition our inability to use formal appropriateness criteria limits the interpretation of findings. Despite these limitations the results indicate that, in the short term, acute trusts can improve survival of patients by increasing the use of effective medicines among general physicians.
Discussion
This study improves our understanding of the care of AMI by cardiologists and general physicians in UK hospitals. Access to a cardiologist was univariately associated with better survival. This effect remained after controlling for the effect of patient characteristics, including the presence of comorbidity, but disappeared when the confounding effect of access to effective medicines was controlled for. As noted in the analysis, patients seen by a cardiologist were more likely to have been prescribed these medicines and to have had exercise testing, angiography and revascularisation. This implies that the survival advantage associated with being seen by a cardiologist is due to the more frequent use of effective medicines and is similar to findings by Chen J et al in the United States [ 3 ].Previous studies have shown a high level of miscoding in routine data for AMI patients (RM Norris cited in [ 2 ]). The use of a case note review improved the findings of this study by assisting in ascertaining the diagnosis of myocardial infarction and ensuring the accuracy and completeness of the data used in the analysis. The possibility remains that some cases not seen through the cardiology department and not recorded in the patient administration system were missed. This is likely to be a small number and unlikely to invalidate the findings. Identifying all patients with infarction is a major shortcoming of many studies that estimate mortality among patients seen by cardiologists [ 2 ]. This study includes all patients irrespective of where they were managed in the hospital; however patients dying from myocardial infarction before arriving at the hospital were not included.All cases diagnosed by the managing clinician as AMI with ECG changes and found to have enzyme changes indicating myocardial necrosis were included in the analysis after the case notes were reviewed. This may have involved some misclassification due to misdiagnosis, but this is likely to occur at random, affecting only a small number of cases, and can only underestimate the effect of each factor.Limitations of this study include potential for bias in the allocation of patients to treatments and physicians (ie cardiologist/non cardiologist) that were not measured. Another possible limitation is bias arising from medical case notes which were unobtainable. Records from routine data were used to examine whether those with missing records differed in any systematic way from those with available records; it was found that they did not differ in age or sex. The study did not use any criteria for judging clinical appropriateness of the procedures used. Incomplete recording of information in the case notes hampered the determination of appropriateness for effective medication. However there was no difference in the proportion of missing data between the two hospitals.Since doctors may selectively be referring younger patients with lower comorbidity to a cardiologist, we controlled for age and comorbidity in the analysis. However, residual confounding from age and comorbidity could still account for some of the observed difference in survival.All patients admitted to the Coronary Care Unit (CCU) in one of the hospitals are seen by a cardiologist and have a higher chance of accessing thrombolysis, angiography and revascularisation. A cardiologist also sees the majority of patients admitted to the CCU in the second hospital. The protocol for the management of AMI in both hospitals stipulates a cardiology review of all AMI patients. Access to a cardiologist may be a proxy measure of access to effective treatment and it may not be the trigger for effective treatment. These limitations mean that the findings should be interpreted with a degree of caution. In addition our inability to use formal appropriateness criteria limits the interpretation of findings.Despite these limitations the results indicate that, in the short term, acute trusts can improve survival of patients by increasing the use of effective medicines among general physicians.Conclusions We observed better survival among patients seen by a cardiologist compared with patients with no access to a cardiologist, among a cohort of patients already admitted with AMI. This effect was entirely explained by the more frequent use of effective medicines by cardiologists in the multivariate analysis. About eight hospitals in the UK Royal College of Physicians survey have no cardiologist and another 30% have a single cardiologist [ 2 ]. It will take time to provide the minimum of two cardiologists per hospital as recommended in the CHD NSF. There are several reasons why a hospital may benefit from appointing a cardiologist, ranging from the treatment of a specific subgroup of patients that will benefit from revascularisation to prompt management of angina patients via rapid access chest clinics. However, in the short term hospitals can improve the survival of patients admitted with AMI by improving access to effective medicines. Coordination of care between cardiologists and general physicians and targeted interventions using feedback from audit, research and peer education are likely to lead to more frequent use of effective cardiovascular medicines by general physicians.
Conclusions
We observed better survival among patients seen by a cardiologist compared with patients with no access to a cardiologist, among a cohort of patients already admitted with AMI. This effect was entirely explained by the more frequent use of effective medicines by cardiologists in the multivariate analysis. About eight hospitals in the UK Royal College of Physicians survey have no cardiologist and another 30% have a single cardiologist [ 2 ]. It will take time to provide the minimum of two cardiologists per hospital as recommended in the CHD NSF. There are several reasons why a hospital may benefit from appointing a cardiologist, ranging from the treatment of a specific subgroup of patients that will benefit from revascularisation to prompt management of angina patients via rapid access chest clinics. However, in the short term hospitals can improve the survival of patients admitted with AMI by improving access to effective medicines. Coordination of care between cardiologists and general physicians and targeted interventions using feedback from audit, research and peer education are likely to lead to more frequent use of effective cardiovascular medicines by general physicians.Competing interest None declared
Competing interest
None declaredAuthors' contributions All authors contributed in writing the paper, in addition, IA collected the data and conducted the analysis, DK conceived the idea, BA provided advice on statistical analysis, JP provided input in the conduct of the study, PW contributed in refining the research question and discussing the findings of paper.
Authors' contributions
All authors contributed in writing the paper, in addition, IA collected the data and conducted the analysis, DK conceived the idea, BA provided advice on statistical analysis, JP provided input in the conduct of the study, PW contributed in refining the research question and discussing the findings of paper.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: