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11. Ishihara N, Matsuo H, Murakoshi H, Fernandez JBL, Samoto T, Maruo T. Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation. Am J Obstet Gynecol 2002;186:158 – 66. 12. Redman CWG, Sacks GP, Sargent IL. Preeclampsia, an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180:499 – 506. 13. Smarason AK, Sargent IL, Starkey PM, Redman CWG. The effect of placental syncytiotrophoblast microvillous membranes from normal and pre-eclamptic women on the growth of endothelial cells in vitro. Br J Obstet Gynaecol 1993;100:943–9. 14. Knight M, Redman CWG, Linton EA, Sargent IL. Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies. Br J Obstet Gynaecol 1998;105:632– 40. 15. Gupta AK, Rusterholz C, Huppertz B, Malek A, Schneider H, Holzgreve W, et al. A comparative study of the effect of three different syncytiotrophoblast micro-particles preparations on endothelial cells. Placenta 2004; in press. 16. Lo YMD, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999;45:184 – 8. 17. Zhong XY, Laivuori H, Livingston JC, Ylikorkala O, Sibai BM, Holzgreve W, et al. Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol 2001;184:414 –9. 18. Shibasaki T, Odagiri E, Shizume K, Ling N. Corticotropin-releasing factor-like activity in human placental extracts. J Clin Endocrinol Metab 1982;55: 384 – 6. 19. Ng EK, Leung TN, Tsui NB, Lau TK, Panesar NS, Chiu RW, et al. The concentration of circulating corticotropin-releasing hormone mRNA in maternal plasma is increased in preeclampsia. Clin Chem 2003;49:727–31. 20. Li Y, Zhong XY, Kang A, Troeger C, Holzgreve W, Hahn S. Inability to detect cell free fetal DNA in the urine of normal pregnant women nor in those affected by preeclampsia associated HELLP syndrome. J Soc Gynecol Investig 2003;10:503– 8. 21. Huppertz B, Kingdom J, Caniggia I, Desoye G, Black S, Korr H, et al. Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation. Placenta 2003;24:181–90. Previously published online at DOI: 10.1373/clinchem.2004.040196
High Ischemia-Modified Albumin Concentration Reflects Oxidative Stress But Not Myocardial Involvement in Systemic Sclerosis, Didier Borderie,1†* Yannick Allanore,2† Christophe Meune,3 Jean Y. Devaux,3 Ohvanesse G. Ekindjian,1 and Andre´ Kahan2 (Departments of 1 Biochemistry A, 2 Rheumatology A, and 3 Nuclear Medicine, Paris V University, Assistance Publique-Hoˆpitaux de Paris, Cochin Hospital, Paris, France; † these authors contributed equally to this work; * address correspondence to this author at: Service de Biochimie A, 27 rue du faubourg Saint-Jacques, 75014 Paris, France; fax 33-1-5841-1585, e-mail
[email protected]) Systemic sclerosis (SSc) is a connective tissue disease characterized by widespread vascular lesions and fibrosis of the skin and internal organs. In SSc, vasospasm causes frequent episodes of reperfusion injury and free-radicalmediated endothelial disruption. Primary myocardial involvement is far more common than initially suspected on clinical grounds (1–5 ) and affects survival rates because it is associated with a poor prognosis (6, 7 ). Myocardial fibrosis is thought to occur secondarily to repeated focal ischemia in the coronary microcirculation as a result of abnormal vasoreactivity, with or without associated structural vascular disease (4, 5 ). The early and accurate
identification of cardiac involvement is therefore of paramount clinical importance. The concentration of ischemia-modified albumin (IMA), as measured by the albumin cobalt binding test (Ischemia Technologies, Inc.), is a new marker to rule out transient myocardial ischemia (8, 9 ). This test measures the binding of exogenous cobalt to the NH2 terminus of human albumin. In the presence of myocardial ischemia, structural changes occur in the NH2 terminus of albumin, rapidly reducing its capacity to bind transition metal ions after an ischemic event (10 ). We assessed the accuracy of the albumin cobalt binding test for detecting ischemia in SSc patients and investigated the roles of myocardial ischemia and peripheral oxidative stress in this condition. We also considered carbonyl residues and advanced oxidation protein products (AOPP) as factors indicative of protein oxidation. We included consecutive patients hospitalized for systematic follow-up who fulfilled the American Rheumatism Association preliminary criteria for SSc. The exclusion criteria were pregnancy; symptoms of heart failure, including class III or IV dyspnea (New York Heart Association); venous distension and recent major lower limb edema; pulmonary arterial hypertension (systolic arterial pressure ⬎40 mmHg and/or mean artery pressure ⬎25 mmHg, determined by echocardiography); severe pulmonary involvement (forced vital capacity or carbon monoxide diffusing capacity ⬍50% of the predicted normal value); renal involvement (creatinine concentration ⬎ 106 mol/L); or severe disease complications such as cancer or gangrene. At the time of the study, none of the patients was taking medication for cardiac or vascular disease. If previously treated with vasodilators, patients were asked to stop taking these drugs 3 days before admission. This interruption period corresponds to five times the half-life of calcium channel blockers and angiotensin-converting enzyme. All patients gave informed consent for all procedures, and the study was approved by the local ethics committee (Paris, Cochin). We assessed the following in all patients: blood cell count, Westergren erythrocyte sedimentation rate, serum creatinine concentration, and anti-centromere and antitopoisomerase I antibody concentrations. The concentration of high-sensitivity C-reactive protein was measured by immunoturbidimetry on a Roche modular PP instrument using the CRP latex Tina-quant® assay (Roche Diagnostics). Pulmonary involvement was assessed by computed tomography scan, forced vital capacity, and the ratio of carbon monoxide diffusion capacity to hemoglobin concentration. Pulmonary arterial systolic pressure was determined by Doppler echocardiography at rest. The thickness of the skin was quantified on a scale of 0 –3, by use of the modified Rodman skin scoring technique, for each of 17 body surface areas (11, 12 ). All patients underwent thallium-201 myocardial singlephoton-emission computerized tomography at rest, using a gamma camera (Starport 400AT; General Electric) interfaced with an ADAC computer (DPS 3300). Myocardial perfusion was assessed semiquantitatively by two expe-
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rienced practitioners, using a blind protocol and a 17segment model as follows: score 4 for normal, 3 for mild reduction (not definitely abnormal), 2 for moderate reduction (definitely abnormal), 1 for severe reduction, and 0 for absence of uptake (13 ). We determined both a “global perfusion score” (sum of the mean perfusion scores for each of the 17 segments) and the “number of perfusion defects” (with a perfusion defect defined as a score ⱕ2). The controls were healthy laboratory staff who agreed to provide blood. Blood samples (10 mL) were collected in tubes without anticoagulant. The samples were centrifuged at 3000g for 10 min within 1 h of collection, and the resulting sera were stored at ⫺80 °C until use. The interval between sample collection and analysis was ⬍10 weeks. Cardiac troponin I (cTnI) was measured by an immunochemiluminescent assay on an ACS180 analyzer (Bayer Diagnostics). In accordance with the manufacturer’s data, the lower limit detection of this assay was 0.03 g/L; the total imprecision (CV) was 10% and 20%, respectively, at 0.4 and 0.1 g/L. Serum IMA was measured on a Roche Modular PP instrument. Because no cutoff value was validated for this analyzer, 85 kilounits/L, the upper limit of the range of IMA concentrations for a reference population (95th percentile of a population of 283 apparently healthy individuals), was used as a cutoff point for ischemia, in accordance with data reported by the manufacturer and determined with the Roche Modular P analyzer. The inter- and intraassay CVs were ⬍3.7% and 4.3%, respectively, at IMA concentrations of 106 and 60 kilounits/L (n ⫽ 20). Protein carbonyl groups were determined by ELISA (14 ), and the AOPP concentrations were determined as described by Witko-Sarsat et al. (15 ). The lowest concentrations determined in our laboratory with CVs of 10% and 20% were, respectively, 0.20 and 0.12 mol/g for protein carbonyl groups and 25 and 17 mol/L for AOPP. Concentrations of IMA and markers of oxidative stress are expressed as medians with ranges. Data were analyzed by the Mann–Whitney test for group comparisons and the Spearman rank correlation test for assessment of the relationship between quantitative variables. P values ⬍0.05 were considered significant. We investigated 32 consecutive SSc patients [mean (SD) age, 54.1 (11.6) years], including 26 women. The clinical and laboratory data for these patients are presented in Table 1. None of the following variables was correlated with IMA values: age, pulmonary fibrosis, carbon monoxide diffusion, autoantibody status, high-sensitivity Creactive protein, and erythrocyte sedimentation rate. In SSc patients, the median global myocardial perfusion score was 37 (range, 8 – 47; scores ⬍41 were considered abnormal), and the median of number of perfusion defects was 11 (range, 5–17). All patients had a cTnI concentration ⬍0.4 g/L (10% CV); two patients had a cTnI value ⬎0.1 g/L (20% CV). The median (range) IMA concentration was 87 kilounits/L (55–115 kilounits/L; Fig. 1). Nineteen patients (59%) had IMA concentrations ⱖ85 kilounits/L, exceeding the 95th percentile for a population of 283 apparently healthy individuals in ac-
Table 1. Clinical and biological characteristics of patients with SSc. Characteristics
Mean (SD) disease duration 关range兴, years Cutaneous form, limited/diffuse Mean (SD) skin score (Rodman’s score) Raynaud syndrome, n (%) Lung fibrosis on computed tomography scan, n (%) Forced vital capacity ⬍75% of predicted value, n (%) Ratio of carbon monoxide diffusion capacity to hemoglobin ⬍80% of predicted value, n (%) Pulmonary hypertension (systolic pulmonary arterial pressure ⬎40 mmHg), n (%) Positive for anti-topoisomerase I antibodies, n (%) Positive for anti-centromere antibodies, n (%) Mean (SD) serum creatinine, mol/L Mean (SD) erythrocyte sedimentation rate, mm/h Mean (SD) C-reactive protein, mg/L Mean (SD) cTnI, g/L Mean (SD) IMA, kilounits/L Mean (SD) serum albumin, g/L Patients receiving low-dose prednisone (ongoing treatment), n (mean mg/day) Patients receiving angiotensin-converting enzyme inhibitor, n (%) Patients receiving D-penicillamine, n (%) Patients receiving omeprazole, n (%)
SSc patients (n ⴝ 32)
6.5 (4.8) 关1–20兴 15/17 12 (7.3) 32 (100) 16 (50) 8 (25) 23 (72) 8 (25) 15 (47) 4 (12) 78.8 (13.1) 20.2 (6) 8.7 (6.6) 0.07 (0.06) 89 (13) 39.2 (4.3) 9 (7.4) 9 (21) 8 (19) 32 (100)
cordance with the manufacturer’s data determined with the Roche Modular P analyzer. The IMA concentration was not correlated with the global myocardial perfusion score (r ⫽ 0.13; P ⫽ 0.48) or the number of perfusion defects (r ⫽ 0.23; P ⫽ 0.2). SSc patients diagnosed less than 5 years previously had higher median IMA concentrations [93 (74 –115) kilounits/L] than did patients with longer disease durations [83 (55–106) kilounits/L; P ⬍0.05; Fig. 1]. IMA concentrations were inversely correlated with disease duration (r ⫽ ⫺0.48; P ⬍0.01) and positively correlated with skin score (r ⫽ 0.54; P ⫽ 0.002). Concentrations of serum markers of oxidative stress were significantly higher in SSc patients than in controls: carbonyl residues, 0.82 (0.37–1.09) mol/g vs 0.34 (0.26 – 0.64) mol/g (P ⬍0.001); AOPP, 95.1 (36.6 –280) mol/L vs 78.2 (43.2–129) mol/L (P ⬍0.05). The IMA concentration was correlated with carbonyl residue concentration (r ⫽ 0.59; P ⫽ 0.002) but not with AOPP concentrations. However, neither carbonyl residues nor AOPP were correlated with disease duration. SSc patients had high IMA concentrations, but the IMA concentration was not correlated with global myocardial perfusion score or the number of perfusion defects, despite functional impairment of the coronary microvasculature (16 ). The IMA concentration was associated with
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involve hypoxia, acidosis, or free radical damage, most of which occur within minutes. IMA seems to have a short half-life, returning to baseline values in 6 –12 h, as shown recently in patients with stable angina pectoris after transient ischemia induced during elective percutaneous transluminal angioplasty (9 ). However, it is unclear whether the changes in the N-terminal region of albumin are reversible or lead to preferential degradation by proteolytic systems, as reported for other oxidized proteins (22 ). Nevertheless, the high values obtained in the albumin cobalt binding test for SSc patients reflect a succession of ischemic-reperfusion episodes, given the short half-life of IMA. References Fig. 1. Comparison of IMA values (kilounits/L) as a function of SSc disease duration (n ⫽ 32): recent (⬍5 years) or longer duration (⬎5 years). Horizontal bars indicate median values. The dashed line indicates the 95th percentile for a population of 283 apparently healthy individuals.
disease duration and skin score in SSc patients, reflecting the strong dependence of activity on the intensity of free radical reactions in the first few years of the disease, especially in patients with diffuse forms, who have high skin scores early in their disease (17, 18 ). Free radicals generated by reperfusion injury and the inflammatory process may be of major importance in SSc patients (19 ). This study confirms that SSc is associated with excessive protein oxidative stress, as reflected by the high concentrations of carbonyl groups and AOPP. These results suggest that protein oxidation may occur early in the pathogenesis of SSc and may indicate underlying subclinical disease (oxidative stress or vascular dysfunction). IMA has been studied primarily in selected populations thought to display myocardial involvement only in the absence of confounding clinical conditions. However, other organs seem to be responsible for the increase in IMA. Apple et al. (20 ) reported high concentrations of IMA 24 – 48 h after endurance exercise and suggested that this was attributable to delayed gastrointestinal or skeletal muscle ischemia. High IMA concentrations do not seem to depend purely on myocardial involvement. Thus, IMA may increase during ischemia-reperfusion, affecting any organ, and cannot be considered a specific cardiac marker in diseases associated with oxidative stress. The IMA concentration was closely related to the concentration of carbonyl groups but not AOPP concentrations. This result is not surprising because these markers do not provide the same information concerning the extent of oxidative damage to proteins (15 ), the half-lives of these damaged proteins, and/or their clearance rate. Albumin is the most abundant serum protein, with a mean concentration of 0.63 mmol/L, and is a powerful extracellular antioxidant. The biochemical mechanism modifying the N-terminal region of albumin during ischemia is unclear, but reperfusion after an ischemic event may damage serum albumin as much as, if not more than, ischemia itself (21 ). These modifications to albumin may
1. D’Angelo WA, Fries JF, Masi AT, Schulman LE. Pathologic observations in systemic sclerosis (scleroderma): a study of fifty-eight autopsy cases and fifty-eight matched controls. Am J Med 1969;46:428 – 40. 2. Bulkley BH, Ridolfi RL, Salyer WR, Hutchins GM. Myocardial lesions of progressive systemic sclerosis: a cause of cardiac dysfunction. Circulation 1976;53:483–90. 3. Kahan A, Devaux JY, Amor B, Menkes CJ, Weber S, Ntenberg A et al. Nifedipine and thallium-201 myocardial perfusion in progressive systemic sclerosis. N Engl J Med 1986;314:1397– 402. 4. Follansbee WP, Curtiss EI, Medsger TA Jr, Steen VD, Uretsky BF, Owens GR, et al. Physiologic abnormalities of cardiac function in progressive systemic sclerosis with diffuse scleroderma. N Engl J Med 1984;310:142– 8. 5. Candell-Riera J, Armadans-Gil L, Simeon CP, Castell-Conesa J, Fonollosa-Pla V, Garcia-del-Castillo H, et al. Comprehensive noninvasive assessment of cardiac involvement in limited systemic sclerosis. Arthritis Rheum 1996;39: 1138 – 45. 6. Medsger TA, Masi AT, Rodnan GP, Benedek TG, Robinson H. Survival with systemic sclerosis (scleroderma): a life-table analysis of clinical and demographic factors in 309 patients. Ann Intern Med 1971;75:370 – 6. 7. Steen VD, Follansbee WP, Conte CG, Medsger TA Jr. Thallium perfusion defects predict subsequent cardiac dysfunction in patients with systemic sclerosis. Arthritis Rheum 1996;39:677– 81. 8. Christenson RL, Duh, SH, Sanhai WR, Wu AH, Holtman V, Painter P, et al. Characteristics of an albumin cobalt binding test for assessment of acute coronary syndrome patients: a multicenter study. Clin Chem 2001;47:464 –70. 9. Bar-Or D, Lau E, Winkler JV. A novel assay for cobalt-albumin binding and its potential as marker for myocardial ischemia—a preliminary report. J Emerg Med 2000;19:311–5. 10. Bar-Or D, Winkler JV, Van Bengthuysen K, Harris L, Lau E, Hetzel FW. Reduced albumin-cobalt binding with transient myocardial ischemia after elective percutaneous transluminal coronary angioplasty: a preliminary comparison to creatine kinase-MB, myoglobin and troponin I. Am Heart J 2001;131:985–91. 11. Clements P, Lachenbruch P, Siebold J, White B, Weiner S, Martin R, et al. Inter and intra-observer variability of total skin thickness score (modified Rodman TSS) in systemic sclerosis. J Rheumatol 1995;22:1281–5. 12. Clements PJ, Hurwitz EL, Wong WK, Siebold JR, Mayes M, White B, et al. Skin thickness score as a predictor and correlate of outcome in systematic sclerosis. Arthritis Rheum 2000;43:2445–54. 13. Cerqueira MD, Weissman NJ, Dilsizian V, Jacobs AK, Kaul S, Laskey WK, et al. Standardized myocardial segmentation and nomenclature for tomographic imaging of the heart: a statement for healthcare professionals from the cardiac imaging committee of the Council on Clinical Cardiology of the American Heart Association. Circulation 2002;105:539 – 42. 14. Buss H, Chan TP, Sluis KB, Domigan NM, Winterbourn CC. Protein carbonyl measurement by a sensitive ELISA method. Free Radic Biol Med 1997;23: 361– 6. 15. Witko-Sarsat V, Friedlander M, Capelillere-Blandin C, Nguyen-Khoa T, Nguyen AT, Zingraff J, et al. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int 1996;49:1304 –13. 16. Kahan A, Nitenberg A, Foult JM, Amor B, Menkes CJ, Devaux JY, et al. Decreased coronary reserve in primary scleroderma myocardial disease. Arthritis Rheum 1985;28:637– 46. 17. Sambo P, Jannino L, Candela M, Salvi A, Donini M, Dusi S, et al. Monocytes of patients with systemic sclerosis (scleroderma) spontaneously release in vitro increased amounts of superoxide anion. J Invest Dermatol 1999;112: 78 – 84. 18. Cracowski JL, Marpeau C, Carpentier PH, Imbert B, Hunt M, StankeLabesque F, et al. Enhanced in vivo lipid peroxidation in scleroderma spectrum disorders. Arthritis Rheum 2001;44:1143– 8.
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19. Allanore Y, Borderie D, Lemarechal H, Ekindjian OG, Kahan A. Acute and sustained effects of dihydropyridine-type calcium channel antagonists on oxidative stress in systemic sclerosis. Am J Med 2004;116:595– 600. 20. Apple FS, Quist HE, Otto AP, Mathews WE, Murakami MM. Release characteristics of cardiac biomarkers and ischemia modified albumin as measured by the albumin cobalt binding test after a marathon race. Clin Chem 2002;48:1097–100. 21. Edwards SW, Hallett MB, Campbell AK. Oxygen-radical production during inflammation may be limited by oxygen concentration. Biochem J 1984;217: 851– 4. 22. Chan B, Dodsworth N, Woodrow J, Tucker A, Harris R. Site specific N-terminal autodegradation of human serum albumin. Eur J Biochem 1995;227:524 – 8. DOI: 10.1373/clinchem.2004.034371
Evaluation of Imprecision for Analysis of Short Tandem Repeats by Use of Mixed Blood Cells in Variable Concentrations, Sun-Young Kong,1,2 Chang-Seok Ki,1 HeeJin Kim,1 Ki-o Lee,1 Jae-chun Bae,1 Sun-Hee Kim,1 and JongWon Kim,1* (1 Department of Laboratory Medicine, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, Korea; 2Department of Diagnostic Laboratory, Center for Clinical Services, National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea; * address correspondence to this author at: Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwondong, Gangnam-gu, Seoul 135-710, Korea; fax 82-2-34102719, e-mail
[email protected]) Monitoring of chimerism after allogeneic stem cell transplantation is important for the early diagnosis of graft failure or disease relapse. Two main approaches used for monitoring chimerism are fluorescence in situ hybridization and PCR of short tandem repeats (STRs) expressing high degrees of polymorphism (1 ). Although both methods are useful, the STR assay has been increasingly used because fluorescence in situ hybridization can be used only in cases with specific genetic aberrations or a sexmismatched donor (2– 4 ). The STR assay, which produces quantitative results within 1 day, uses fluorescence-labeled primers and a capillary electrophoresis system (5, 6 ). However, the precision of the assay’s performance with respect to the chimeric stages of hematopoietic cells has not been fully investigated. We therefore aimed to evaluate the assay imprecision (CV) for seven STRs, D7S820, D8S1179, D16S539, D18S51, D21S11, TH01, and TPOX, to determine whether the precision changes according to the degree of chimerism. We used cell mixtures at various concentrations to simulate hematopoietic chimerism, and we also determined the detection limit. In an initial screening to find adequate samples for evaluation, we obtained peripheral blood specimens from 96 volunteer donors. Genomic DNA was isolated by use of Wizard Genomic DNA purification reagents (Promega) and was assayed for allele determination for seven different STRs. PCRs were set up in a final volume of 25 L containing 10⫻ buffer, 200 M deoxynucleotide triphosphates, 5 pmol of each primer labeled with a fluorescence
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dye, 1 U of Taq polymerase (Roche), and template DNA. PCR was carried out in a GeneAmp PCR System 9600 (Applied Biosystems), and PCR products were analyzed by capillary electrophoresis on an ABI 3100 (Applied Biosystems) with performance-optimized polymer 4 (POP-4), a 47-cm capillary, and GA buffer plus EDTA. We mixed 1 L of PCR product with 12 L of deionized formamide containing 0.3 L of GeneScan-500 ROX Size Standard (Applied Biosystems). Each sample was heated at 93 °C for 3 min to denature the DNA, chilled for 3 min at 4 °C, and then separated on ABI 3100. The sizes of the PCR products were determined by use of GeneScan software (Applied Biosystems). After we determined the alleles for the 96 donors based on PCR product size, we calculated the number of alleles, the heterozygosity, and useful statistical values for application to STR analysis, by use of the PowerStat program (Promega). On the basis of the allele data, we chose two unrelated individuals among volunteers who shared only one allele for each STR. We excluded individuals who had stutter bands because interpretation could be difficult when stutter bands were present. We drew whole blood from the selected volunteers and determined leukocyte counts on the XE-2100 automated hematology analyzer (Sysmex). To simulate mixed chimerism, we calculated the volumes required to achieve a constant 107 leukocytes with a targeted proportion of each sample. For example, when A and B had 5000 and 8000 leukocytes/L, respectively and we had planned to make 1:1 mixtures of A and B, we took 1000 L of the well-mixed whole blood from A and 625 L from B. The mixture of the two would then contain 5 ⫻ 106 leukocytes from A and 5 ⫻ 106 leukocytes from B. After mixtures had been prepared, DNA extraction was performed as described above. Samples targeting five different concentrations (1%, 5%, 50%, 95%, and 99% of one selected donor) were used to calculate the precision of each STR assay. Each sample was processed separately, and the measurement protocol consisted of two runs per day for 7 days. The results are presented as the ratio of the donor peaks area, which was calculated as follows: ratio ⫽ area of donor peaks/area of donor and recipient peaks (7 ). To determine the detection limit, we prepared 17 samples with concentrations ranging from 0% to 100% and assayed them twice. The detection limit was defined as the lowest dilution concentration at which the peak-area ratio of the minor cell population was ⬎0.01 in each of two estimations. The information for the observed alleles and statistical values for the seven STRs are represented in Table 1. The detection limits of the seven STR assays were between 0.5% and 5%, and the imprecision results are shown in Fig. 1. The imprecision ranged from 5.4% for TPOX to 12% for D7S820, on average, for all concentrations, and was inversely related to the proportion to the concentration of cell mixtures. This study shows that the precision differed among STR assays and, for each marker, was related to the concentration of cell mixtures simulating chimeric stages of hematopoietic cells. This was especially evident at lowrange concentrations ⬍5%, where the imprecision of the
