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Methylenetetrahydrofolate reductase C677T and A1298C mutations: are they responsible for decreased in utero fetal viability?
44TH ANNUAL CSCC-CAMB-AACC CONFERENCE
product. Furthermore, polarographic studies have identified that the isolated red Jaffe´ product is similar to the conventional product but no longer contains a nitro anion species. Studies in the presence of either excess base or excess creatinine result in the formation of orange 2:1 product formation with additional nitro anion formation. Orange products consist of two creatinine molecules, each reacted at a meta position of the alkaline picrate or two hydroxy groups or a mixture of one hydroxy group and one creatinine group attached to the meta positions of the aromatic ring. In all cases, the corresponding nitro anion formation may be monitored by observing a characteristic decrease in the nitro group reduction waves combined with an increase in the nitro anion reduction wave.
4 ETHNIC DIFFERENCES IN ALLELE FREQUENCIES AT POLYMORPHIC SITES OF THE PARAOXONASE GENES, PON1 AND PON2 Chan, P.C., Wong, B.Y.L., and Cole, D.E.C. Department of Pathobiology and Laboratory Medicine, University of Toronto and The Toronto General Hospital Genetic Repository, Toronto, Ontario, Canada M5G 2C4 Polymorphisms of the PON1 (L55M & R192Q) and PON2 (A148G & S311C) genes have been shown to be associated with coronary heart disease, plasma lipoprotein levels, and diabetes. Objective: The current study was designed to determine if allele frequencies for these genetic variations differed significantly among healthy, adult individuals self-reporting different ethnic origins. Methods: Samples from 45 Afro-Americans (B), 50 Caucasian Canadians (C) and 46 Asian Canadians (A) were included in the study. Genotyping was performed using a novel allele-specific PCR protocol developed in our laboratory. Results: MM
ML
LL
QQ
QR
RR
(B) (C) (A)
2 4 0
16 23 2
27 23 44
5 20 3
23 26 27
17 4 16
PON2
AA
AG
GG
SS
SC
CC
(B) (C) (A)
28 31 29
13 14 15
4 5 2
28 31 29
14 14 15
3 5 2
PON1
Analysis: Genotypes in all 3 ethnic groups were in Hardy-Weinberg equilibrium. Frequencies for PON1L55M in Asians and R192Q in Caucasians were different from their respective groups (Fisher, p ⬍ 0.0001), with L55and Q192 being more common in Caucasians. No difference in PON2 allelic frequencies exists among the 3 populations. Conclusion: Ethnicity is a more important potential confounder in studies of the PON1 polymorphism and its relationship to disease than in studies with PON2. 226
5 COST OF EMERGENCY DEPARTMENT CARDIAC MARKER TESTING AT A UNIVERSITY TEACHING HOSPITAL Collier, C.P.,1 Raymond, M.J.,1 Dagnone, L.E.,2 Departments of Pathology1 and Emergency Medicine2, Queen’s University and Kingston General Hospital, 76 Stuart Street, Kingston, Ontario, Canada K7L 2V7 Objective: To compare the actual cost of laboratory testing for cardiac markers in the emergency department to the cost of following a specific decision algorithm. Design: A retrospective analysis of laboratory data was compared to Medical Records data for a 9 month period (98.09 –99.05) in order to estimate 12 month utilization rates and costs. For this collection period, the recommended investigation for acute myocardial infarction (AMI) was serial sampling for myoglobin (MYO) and creatine kinase (CK) (at least 2 hours apart) with either a CKMB or troponin I (cTnI) for confirmation. Results: Annual testing for the Emergency Department (ED) cardiac marker testing included 7,436 CKs, 3013 MYOs and 3489 CKMB/cTnIs for a total cost of $39,471. Based on a recent utilization study at our institution, MYO testing is no longer available. Our new cardiac marker testing recommendations for the ED includes: CK for ECG positive AMI patients; 2 serial CKs, preferably 3– 4 hours apart (minimum 2 hours), with cTnI (or CKMB) confirmatory testing for patients with non-diagnostic ECGs or for ruling out AMI. The cost of this algorithm based on our current volumes would be $56,804 for the ED. Conclusions: By using serial CK testing instead of MYO testing, approximately $13,800 can be saved annually. If this savings is applied to enhanced cTnI testing based on our new algorithm, the overall increase in the cost of cardiac marker testing in the ED will be only $20,000. 6 METHYLENETETRAHYDROFOLATE REDUCTASE C677T AND A1298C MUTATIONS: ARE THEY RESPONSIBLE FOR DECREASED IN UTERO FETAL VIABILITY? Donnelly, J.G.1,2 and Isotalo, P.A.1 Dept. of Pathology and Laboratory Medicine1, University of Ottawa, 451 Smyth Rd, Ottawa, Canada K1H 8M5, Div. of Biochemistry2, Ottawa Hospital, Civic Campus, 1053 Carling Ave, Ottawa, Canada K1Y 4E9 Objectives: Methylenetetrahydrofolate reductase (MTHFR) mutations, through their defects in homocysteine metabolism, have been implicated as risks for neural tube defects. To investigate the relationship between MTHFR mutations and fetal viability, we examined the prevalence of MTHFR C677T and A1298C mutations in fetal, neonatal and adult groups to determine if certain MTHFR genotype combinations were under-represented in any group. Methods: Samples of fetal tissue (n ⫽ 161), neonatal cord blood (n ⫽ 119) and adult whole blood (n ⫽ 129) were assessed for MTHFR C677T and A1298C mutations by CLINICAL BIOCHEMISTRY, VOLUME 33, APRIL 2000
POSTER ABSTRACTS
polymerase chain reaction and restriction fragment length polymorphism analysis. The Fisher’s exact test was used to compare observed MTHFR genotype frequencies. Statistical significance was a p ⬍ 0.05. Results: Cis and trans mutations were identified in all groups. Combined 677CT/1298CC and 677TT/1298CC genotypes, that correspond to triple and quadruple mutations, respectively, were not observed in either the neonatal (p ⫽ 0.0402) or adult (p ⫽ 0.0353) groups, while they were identified in low frequency in the fetal group. In addition, the combined 677TT/1298AC genotype occurred in 10.9% of adults, 1.7% of neonates (p ⫽ 0.0035) and 1.2% of fetuses (p ⫽ 0.0004). Conclusions: Decreased in utero viability is suggested by the absence of 677CT/1298CC and 677TT/1298CC genotypes in both the adult and neonatal groups, while being identified in the fetal group. The difference in 677TT/ 1298ACgenotype representation between groups also suggests that MTHFR mutations may carry a selective disadvantage in genetically predisposed fetuses. The recognition that combined MTHFR genotypes may influence pregnancy outcomes further demonstrates the clinical significance of inherited homocysteine metabolic defects. 7 COMPARISON OF THE PROCEDURES FOR MEASURING HBA1C Fu, L., Lafferty, J., Vacovsky, M., and Luxton, G. Hamilton Regional Laboratory Medicine Program, Dept. of Path. and Mol. Med., McMaster Univ., Hamilton, Ontario, Canada L8L 2X2 Objective: Lack of standardization and multiple methodologies for HbA1c have resulted in variable reference ranges, target values and reporting protocols making interpretation difficult. This study compared HbA1c results from three commonly used automated chromatographic systems. Methods: The three analyzers used in this study were Chiron Glycomat 765, Biorad Variant II and Tosoh A1c 2.2 Plus chromatographic systems. The analyzers were set up and the analyses were done according to each manufacturer’s directions. Quality control (QC) material for Chiron and Biorad analyzers was Biorad Lyphocheck Diabetes QC, and for the Tosoh analyzer was provided by its own manufacturer. 20 patient samples were analyzed by all three systems. 25 additional patient samples were analyzed on both the Biorad and Glycomat systems. Specimens with urea concentration up to 24.2mmol/L and bilirubin up to 346 mol/L were analyzed on both the Biorad and Tosoh systems. Results: For low level QC, the %CVs of Glycomat, Biorad and Tosoh were 5.6, 2.3 and 2.4, respectively. For high level QC, %CVs were 3.0, 1.8 and 1.2, respectively. Comparing the observed values of patient samples, the correlation coefficients of Biorad vs Glycomat, Tosoh vs Glycomat and Tosoh vs Biorad were 0.972, 0.986 and 0.998, respectively. There was no statistically significant difference between the Biorad and Glycomat, or Tosoh and Glycomat. Also there was no significant difference of the CLINICAL BIOCHEMISTRY, VOLUME 33, APRIL 2000
measurements between Biorad and Tosoh due to carbamylation and hyperbilirubinemia. Conclusion: All three analytical methods performed well with the QC materials, and the HbA1c values of the patient samples as measured by the three systems are highly correlated. 8 EVALUATION OF THE PERFORMANCE OF BIORAD VARIANT II FOR HBA1C, HBF AND HBA2 Fu, L., Lafferty, J., Vacovsky, M., and Luxton, G. Hamilton Regional Laboratory Medicine Program, Dept. of Path. and Mol. Med., McMaster Univ., Hamilton, Ontario, Canada L8L 2X2 Objective: To evaluate the performance of the Biorad Variant II HbA1c program and the -thalassemia short program. Precision, linearity and interference in the presence of abnormal hemoglobin (Hb) variants were studied. Method: HbA1c program and -thalassemia short program are HPLC cation exchange chromatography systems. They were set up and operated on Biorad Variant II analyzer according to the manufacturer’s directions. Precision was measured within run, between runs and between days. Interference of HbF, HbS and HbC on HbA1c was checked by mixing up to 50% cord blood or HbSC disease blood with low and high level HbA1c samples. The accuracy was also compared with in-house methods (Glycomat, HPLC and FAD). Results: %CVs of HbA1c, HbF and HbA2 measurement: HbA1c HbF HbA2
Within run
Day to day
0.5–1.5%, n ⫽ 5 ⫻ 12 0.7–39.7%*, n ⫽ 12 ⫻ 5 0.9–7.0%, n ⫽ 12 ⫻ 5
1.3–5.8%, n ⫽ 20 ⫻ 12 0.9–51.2%*, n ⫽ 13 ⫻ 4 3.7–10.9%, n ⫽ 13 ⫻ 4
*CVs higher than 10% were due to very low HbF level.
There was no statistical difference between runs and different methods. The recovery of HbA1c was 98%. There was no interference on HbA1c with mixed cord blood as high as 50%, also no interference at decision levels with mixed HbSC. Both common and rare Hb variants were detected by the system. Conclusion: The HbA1c program has good precision, accuracy and minimal interference from other Hb fractions. The imprecision and inaccuracy of HbF and HbA2 measurements using -thalassemia short program is also acceptable for diagnostic decision making. The system can detect all common and a large number of rare Hb variants. 9 EVALUATION OF A URINARY ASSAY FOR NUCLEAR MATRIX PROTEIN (NMP22). A MARKER FOR TRANSITIONAL CELL CARCINOMA OF THE BLADDER Gornall, D.A., and Toguri, A. Departments of Laboratory and Urology, The Scarborough Hospital—General Division, 3050 Lawrence Avenue East, Scarborough, Ontario, Canada M1P 2V5 The Nuclear Matrix Protein (NMP22) has a high sensitivity for the detection of transitional cell carcinoma (TCC) 227
product. Furthermore, polarographic studies have identified that the isolated red Jaffe´ product is similar to the conventional product but no longer contains a nitro anion species. Studies in the presence of either excess base or excess creatinine result in the formation of orange 2:1 product formation with additional nitro anion formation. Orange products consist of two creatinine molecules, each reacted at a meta position of the alkaline picrate or two hydroxy groups or a mixture of one hydroxy group and one creatinine group attached to the meta positions of the aromatic ring. In all cases, the corresponding nitro anion formation may be monitored by observing a characteristic decrease in the nitro group reduction waves combined with an increase in the nitro anion reduction wave.
4 ETHNIC DIFFERENCES IN ALLELE FREQUENCIES AT POLYMORPHIC SITES OF THE PARAOXONASE GENES, PON1 AND PON2 Chan, P.C., Wong, B.Y.L., and Cole, D.E.C. Department of Pathobiology and Laboratory Medicine, University of Toronto and The Toronto General Hospital Genetic Repository, Toronto, Ontario, Canada M5G 2C4 Polymorphisms of the PON1 (L55M & R192Q) and PON2 (A148G & S311C) genes have been shown to be associated with coronary heart disease, plasma lipoprotein levels, and diabetes. Objective: The current study was designed to determine if allele frequencies for these genetic variations differed significantly among healthy, adult individuals self-reporting different ethnic origins. Methods: Samples from 45 Afro-Americans (B), 50 Caucasian Canadians (C) and 46 Asian Canadians (A) were included in the study. Genotyping was performed using a novel allele-specific PCR protocol developed in our laboratory. Results: MM
ML
LL
QR
RR
(B) (C) (A)
2 4 0
16 23 2
27 23 44
5 20 3
23 26 27
17 4 16
PON2
AA
AG
GG
SS
SC
CC
(B) (C) (A)
28 31 29
13 14 15
4 5 2
28 31 29
14 14 15
3 5 2
PON1
Analysis: Genotypes in all 3 ethnic groups were in Hardy-Weinberg equilibrium. Frequencies for PON1L55M in Asians and R192Q in Caucasians were different from their respective groups (Fisher, p ⬍ 0.0001), with L55and Q192 being more common in Caucasians. No difference in PON2 allelic frequencies exists among the 3 populations. Conclusion: Ethnicity is a more important potential confounder in studies of the PON1 polymorphism and its relationship to disease than in studies with PON2. 226
5 COST OF EMERGENCY DEPARTMENT CARDIAC MARKER TESTING AT A UNIVERSITY TEACHING HOSPITAL Collier, C.P.,1 Raymond, M.J.,1 Dagnone, L.E.,2 Departments of Pathology1 and Emergency Medicine2, Queen’s University and Kingston General Hospital, 76 Stuart Street, Kingston, Ontario, Canada K7L 2V7 Objective: To compare the actual cost of laboratory testing for cardiac markers in the emergency department to the cost of following a specific decision algorithm. Design: A retrospective analysis of laboratory data was compared to Medical Records data for a 9 month period (98.09 –99.05) in order to estimate 12 month utilization rates and costs. For this collection period, the recommended investigation for acute myocardial infarction (AMI) was serial sampling for myoglobin (MYO) and creatine kinase (CK) (at least 2 hours apart) with either a CKMB or troponin I (cTnI) for confirmation. Results: Annual testing for the Emergency Department (ED) cardiac marker testing included 7,436 CKs, 3013 MYOs and 3489 CKMB/cTnIs for a total cost of $39,471. Based on a recent utilization study at our institution, MYO testing is no longer available. Our new cardiac marker testing recommendations for the ED includes: CK for ECG positive AMI patients; 2 serial CKs, preferably 3– 4 hours apart (minimum 2 hours), with cTnI (or CKMB) confirmatory testing for patients with non-diagnostic ECGs or for ruling out AMI. The cost of this algorithm based on our current volumes would be $56,804 for the ED. Conclusions: By using serial CK testing instead of MYO testing, approximately $13,800 can be saved annually. If this savings is applied to enhanced cTnI testing based on our new algorithm, the overall increase in the cost of cardiac marker testing in the ED will be only $20,000. 6 METHYLENETETRAHYDROFOLATE REDUCTASE C677T AND A1298C MUTATIONS: ARE THEY RESPONSIBLE FOR DECREASED IN UTERO FETAL VIABILITY? Donnelly, J.G.1,2 and Isotalo, P.A.1 Dept. of Pathology and Laboratory Medicine1, University of Ottawa, 451 Smyth Rd, Ottawa, Canada K1H 8M5, Div. of Biochemistry2, Ottawa Hospital, Civic Campus, 1053 Carling Ave, Ottawa, Canada K1Y 4E9 Objectives: Methylenetetrahydrofolate reductase (MTHFR) mutations, through their defects in homocysteine metabolism, have been implicated as risks for neural tube defects. To investigate the relationship between MTHFR mutations and fetal viability, we examined the prevalence of MTHFR C677T and A1298C mutations in fetal, neonatal and adult groups to determine if certain MTHFR genotype combinations were under-represented in any group. Methods: Samples of fetal tissue (n ⫽ 161), neonatal cord blood (n ⫽ 119) and adult whole blood (n ⫽ 129) were assessed for MTHFR C677T and A1298C mutations by CLINICAL BIOCHEMISTRY, VOLUME 33, APRIL 2000
POSTER ABSTRACTS
polymerase chain reaction and restriction fragment length polymorphism analysis. The Fisher’s exact test was used to compare observed MTHFR genotype frequencies. Statistical significance was a p ⬍ 0.05. Results: Cis and trans mutations were identified in all groups. Combined 677CT/1298CC and 677TT/1298CC genotypes, that correspond to triple and quadruple mutations, respectively, were not observed in either the neonatal (p ⫽ 0.0402) or adult (p ⫽ 0.0353) groups, while they were identified in low frequency in the fetal group. In addition, the combined 677TT/1298AC genotype occurred in 10.9% of adults, 1.7% of neonates (p ⫽ 0.0035) and 1.2% of fetuses (p ⫽ 0.0004). Conclusions: Decreased in utero viability is suggested by the absence of 677CT/1298CC and 677TT/1298CC genotypes in both the adult and neonatal groups, while being identified in the fetal group. The difference in 677TT/ 1298ACgenotype representation between groups also suggests that MTHFR mutations may carry a selective disadvantage in genetically predisposed fetuses. The recognition that combined MTHFR genotypes may influence pregnancy outcomes further demonstrates the clinical significance of inherited homocysteine metabolic defects. 7 COMPARISON OF THE PROCEDURES FOR MEASURING HBA1C Fu, L., Lafferty, J., Vacovsky, M., and Luxton, G. Hamilton Regional Laboratory Medicine Program, Dept. of Path. and Mol. Med., McMaster Univ., Hamilton, Ontario, Canada L8L 2X2 Objective: Lack of standardization and multiple methodologies for HbA1c have resulted in variable reference ranges, target values and reporting protocols making interpretation difficult. This study compared HbA1c results from three commonly used automated chromatographic systems. Methods: The three analyzers used in this study were Chiron Glycomat 765, Biorad Variant II and Tosoh A1c 2.2 Plus chromatographic systems. The analyzers were set up and the analyses were done according to each manufacturer’s directions. Quality control (QC) material for Chiron and Biorad analyzers was Biorad Lyphocheck Diabetes QC, and for the Tosoh analyzer was provided by its own manufacturer. 20 patient samples were analyzed by all three systems. 25 additional patient samples were analyzed on both the Biorad and Glycomat systems. Specimens with urea concentration up to 24.2mmol/L and bilirubin up to 346 mol/L were analyzed on both the Biorad and Tosoh systems. Results: For low level QC, the %CVs of Glycomat, Biorad and Tosoh were 5.6, 2.3 and 2.4, respectively. For high level QC, %CVs were 3.0, 1.8 and 1.2, respectively. Comparing the observed values of patient samples, the correlation coefficients of Biorad vs Glycomat, Tosoh vs Glycomat and Tosoh vs Biorad were 0.972, 0.986 and 0.998, respectively. There was no statistically significant difference between the Biorad and Glycomat, or Tosoh and Glycomat. Also there was no significant difference of the CLINICAL BIOCHEMISTRY, VOLUME 33, APRIL 2000
measurements between Biorad and Tosoh due to carbamylation and hyperbilirubinemia. Conclusion: All three analytical methods performed well with the QC materials, and the HbA1c values of the patient samples as measured by the three systems are highly correlated. 8 EVALUATION OF THE PERFORMANCE OF BIORAD VARIANT II FOR HBA1C, HBF AND HBA2 Fu, L., Lafferty, J., Vacovsky, M., and Luxton, G. Hamilton Regional Laboratory Medicine Program, Dept. of Path. and Mol. Med., McMaster Univ., Hamilton, Ontario, Canada L8L 2X2 Objective: To evaluate the performance of the Biorad Variant II HbA1c program and the -thalassemia short program. Precision, linearity and interference in the presence of abnormal hemoglobin (Hb) variants were studied. Method: HbA1c program and -thalassemia short program are HPLC cation exchange chromatography systems. They were set up and operated on Biorad Variant II analyzer according to the manufacturer’s directions. Precision was measured within run, between runs and between days. Interference of HbF, HbS and HbC on HbA1c was checked by mixing up to 50% cord blood or HbSC disease blood with low and high level HbA1c samples. The accuracy was also compared with in-house methods (Glycomat, HPLC and FAD). Results: %CVs of HbA1c, HbF and HbA2 measurement: HbA1c HbF HbA2
Within run
Day to day
0.5–1.5%, n ⫽ 5 ⫻ 12 0.7–39.7%*, n ⫽ 12 ⫻ 5 0.9–7.0%, n ⫽ 12 ⫻ 5
1.3–5.8%, n ⫽ 20 ⫻ 12 0.9–51.2%*, n ⫽ 13 ⫻ 4 3.7–10.9%, n ⫽ 13 ⫻ 4
*CVs higher than 10% were due to very low HbF level.
There was no statistical difference between runs and different methods. The recovery of HbA1c was 98%. There was no interference on HbA1c with mixed cord blood as high as 50%, also no interference at decision levels with mixed HbSC. Both common and rare Hb variants were detected by the system. Conclusion: The HbA1c program has good precision, accuracy and minimal interference from other Hb fractions. The imprecision and inaccuracy of HbF and HbA2 measurements using -thalassemia short program is also acceptable for diagnostic decision making. The system can detect all common and a large number of rare Hb variants. 9 EVALUATION OF A URINARY ASSAY FOR NUCLEAR MATRIX PROTEIN (NMP22). A MARKER FOR TRANSITIONAL CELL CARCINOMA OF THE BLADDER Gornall, D.A., and Toguri, A. Departments of Laboratory and Urology, The Scarborough Hospital—General Division, 3050 Lawrence Avenue East, Scarborough, Ontario, Canada M1P 2V5 The Nuclear Matrix Protein (NMP22) has a high sensitivity for the detection of transitional cell carcinoma (TCC) 227