Cangel Pui-yee Chan

Development of a quantitative lateral-flow assay for rapid detection of fatty acid-binding protein.

Using human heart-type fatty acid-binding protein (H-FABP) as an early cardiac marker to confirm or exclude a diagnosis of acute myocardial infarction (AMI) soon after admission requires a rapid assay system. Due to the requirement of skillful technicians and complicated assay procedures, most immunochemical assays for H-FABP are of limited use for routine clinical practice. In the present study, we describe a rapid lateral-flow assay for detection of H-FABP. Fifty-one human samples were evaluated using a conventional ELISA and a newly developed lateral-flow assay. A good agreement between the two methods was found according to Bland and Altman plot. The correlation found was y=0.9685 x -0.6270 (r(2)=0.9585). The detector antibody labeled with colloidal gold was mixed with those without label to extend the linear range of the calibration curve up to 125 microg/l H-FABP with r(2)=0.9832. The detection limit of the assay was 2.8 microg/l. The test-strips can be stored either at 4 degrees C and room temperature for up to 1 year without significant loss of activity. Finally, a one-step FABP test so-called CardioDetect(R), which was derived from the serum lateral-flow assay has been designed for qualitative determination of H-FABP in whole blood samples. It requires no sample pretreatment and gives results within 15 min. Thirty-eight patients presenting with chest pain and suspected AMI were studied. Using an upper reference level of 7 microg/l, the specificity of the rapid test was 94%. Both sensitivity and negative predictive value (NPV) were 100%, implying that 100% of non-AMI patients could be excluded with no false-negative results. With this rapid and sensitive immunotest, H-FABP could soon be introduced into clinical practice.

A superior early myocardial infarction marker. Human heart-type fatty acid-binding protein.

Aufgrund der höheren kardialen Spezifität scheint humanes kardiales FABP (Fettsäurebindungsprotein) im Vergleich zu Myoglobin ein vielversprechender Laborparameter zur frühen Herzinfarktdiagnostik zu sein. FABP ist ein niedermolekulares, zytoplasmatisches Protein (15 kDa) welches bereits sehr früh nach Einsetzen einer myokardialen Ischämie ins periphere Blut freigesetzt wird und daher zum biochemischen Nachweis oder Ausschluss eines akuten Myokardinfarktes (AMI) geeignet erscheint. Bei n = 218 Patienten (Pat) mit akuten Thoraxschmerzen oder dem Verdacht auf einen AMI wurden in seriellen Messungen im peripheren Blut FABP und troponin I immunologisch sowie Kreatinkinase (CK) enzymatisch bestimmt. Bei n = 94 Patienten mit nachgewiesenem AMI erreichten die FABP-Konzentrationen innerhalb von drei Stunden nach Schmerzbeginn einen maximalen Level (577,6 ± 43,8 μg/L) und fielen innerhalb von 30 Stunden auf einen Normalwert ab. Die maximale FABP-Konzentration (peak) wurde im Mittel sieben bis neun Stunden früher erreicht, als bei der CK-oder Troponin-I-Bestimmung (2288 ± 131 U/L bzw. 357,1 ± 23,9 μg/L). Die CK-Werte normalisierten sich innerhalb von 50–70 Stunden während Troponin-I-Konzentrationen erst nach mehr als 70 Stunden die Nachweisgrenze unterschritten. Die Fläche unter der Receiver-Operating-Characteristics (ROC)-Kurve für FABP wurde zum Zeitpunkt der Aufnahme mit 0,871 und eine Stunde nach Aufnahme mit 0,995 errechnet, während die entsprechenden Flächen für CK 0,711 und 0,856 und für Troponin I 0,677 und 0,845 betrugen. Die diagnostische Trennschärfe zum Aufnahmezeitpunkt war für FABP am höchsten und wurde erst durch Troponin I acht Stunden nach Aufnahme (0,995) erreicht. Anhand der FABP-Messwerte konnte innerhalb einer Stunde nach Aufnahm e eine Sensitivität von 100% als auch ein negativer Vorhersagewert von 100% erreicht werden. Anhand von nur zwei Blutentnahmen zum Aufnahmezeitpunkt und nach Ablauf einer Stunde konnte mit der FABP-Bestimmung die Diagnose eines AMI sicher gestellt werden, umgekehrt konnte bei den übrigen Patienten mit 100% diagnostischer Sicherheit ein AMI ausgeschlossen werden, falsch negative Resultate wurden nicht gefunden. Die sogenannten späten Marker Troponin I und CK erbrachten eine vergleichbare diagnostische Sicherheit erst nach Ablauf von weiteren sieben Stunden. Somit scheint anhand der Bestimmung von FABP im Blut der frühzeitige Nachweis oder Ausschluss eines AMI möglich zu sein. Human heart-type fatty acid-binding protein (FABP) has a high potential as an early marker for myocardial infarction (MI) being more specific than myoglobin. FABP is a low molecular mass cytoplasmic protein (15 kDa) that is released early after the onset of ischemia and it may be useful for rapid confirmation or exclusion of acute myocardial infarction (AMI). Immunochemically assayed FABP, cardiac troponin I (cTnI) and enzymatically assayed creatine phosphokinase (CPK) were determined serially in plasma and serum samples from 218 patients presenting with chest pain and suspected MI. In the 94 patients with confirmed MI, FABP rose to a maximum level (577.6 ± 43.8 μg/L) 3 hours after the onset of symptoms and returned to normal within 30 hours. The FABP level peaked 7–9 hours earlier than CPK (2288 ± 131 U/L) and cTnI (357.1 ± 23.9 μg/L). CPK took 50–70 hours to return to normal level and cTnI returned to normal level over 70 hours. The areas under the receiver operating characteristic (ROC) curves for FABP were calculated as 0.871 at admission and 0.995 one hour after admission, whereas for CPK the areas were 0.711 and 0.856 and for cTnI the areas were 0.677 and 0.845, indicating that the FABP test gave a better diagnostic classification at the early stage being reached by cTnI (0.995) only 8 hours after admission. For FABP, both sensitivity and negative predictive value (NPV) increased quickly to 100% for samples monitored just one hour after admission. By using only two samples, one at admission and one 1 hour post admission, sequential FABP monitoring can reliably diagnose AMI patients 1 hour after admission and 100% of non-AMI patients can be excluded with no false negative results. The late markers cTnI and CPK have the similar diagnostic performance only 7 hours later. Thus measurement of FABP in plasma or serum allows the earliest immunochemical confirmation or exclusion of AMI.

Evidence-Based Point-of-Care Diagnostics: Current Status and Emerging Technologies

Point-of-care (POC) diagnostics brings tests nearer to the site of patient care. The turnaround time is short, and minimal manual interference enables quick clinical management decisions. Growth in POC diagnostics is being continuously fueled by the global burden of cardiovascular and infectious diseases. Early diagnosis and rapid initiation of treatment are crucial in the management of such patients. This review provides the rationale for the use of POC tests in acute coronary syndrome, heart failure, human immunodeficiency virus, and tuberculosis. We also consider emerging technologies that are based on advanced nanomaterials and microfluidics, improved assay sensitivity, miniaturization in device design, reduced costs, and high-throughput multiplex detection, all of which may shape the future development of POC diagnostics.

A superior early myocardial infarction marker

Aufgrund der höheren kardialen Spezifität scheint humanes kardiales FABP (Fettsäurebindungsprotein) im Vergleich zu Myoglobin ein vielversprechender Laborparameter zur frühen Herzinfarktdiagnostik zu sein. FABP ist ein niedermolekulares, zytoplasmatisches Protein (15 kDa) welches bereits sehr früh nach Einsetzen einer myokardialen Ischämie ins periphere Blut freigesetzt wird und daher zum biochemischen Nachweis oder Ausschluss eines akuten Myokardinfarktes (AMI) geeignet erscheint. Bei n = 218 Patienten (Pat) mit akuten Thoraxschmerzen oder dem Verdacht auf einen AMI wurden in seriellen Messungen im peripheren Blut FABP und troponin I immunologisch sowie Kreatinkinase (CK) enzymatisch bestimmt. Bei n = 94 Patienten mit nachgewiesenem AMI erreichten die FABP-Konzentrationen innerhalb von drei Stunden nach Schmerzbeginn einen maximalen Level (577,6 ± 43,8 μg/L) und fielen innerhalb von 30 Stunden auf einen Normalwert ab. Die maximale FABP-Konzentration (peak) wurde im Mittel sieben bis neun Stunden früher erreicht, als bei der CK-oder Troponin-I-Bestimmung (2288 ± 131 U/L bzw. 357,1 ± 23,9 μg/L). Die CK-Werte normalisierten sich innerhalb von 50–70 Stunden während Troponin-I-Konzentrationen erst nach mehr als 70 Stunden die Nachweisgrenze unterschritten. Die Fläche unter der Receiver-Operating-Characteristics (ROC)-Kurve für FABP wurde zum Zeitpunkt der Aufnahme mit 0,871 und eine Stunde nach Aufnahme mit 0,995 errechnet, während die entsprechenden Flächen für CK 0,711 und 0,856 und für Troponin I 0,677 und 0,845 betrugen. Die diagnostische Trennschärfe zum Aufnahmezeitpunkt war für FABP am höchsten und wurde erst durch Troponin I acht Stunden nach Aufnahme (0,995) erreicht. Anhand der FABP-Messwerte konnte innerhalb einer Stunde nach Aufnahm e eine Sensitivität von 100% als auch ein negativer Vorhersagewert von 100% erreicht werden. Anhand von nur zwei Blutentnahmen zum Aufnahmezeitpunkt und nach Ablauf einer Stunde konnte mit der FABP-Bestimmung die Diagnose eines AMI sicher gestellt werden, umgekehrt konnte bei den übrigen Patienten mit 100% diagnostischer Sicherheit ein AMI ausgeschlossen werden, falsch negative Resultate wurden nicht gefunden. Die sogenannten späten Marker Troponin I und CK erbrachten eine vergleichbare diagnostische Sicherheit erst nach Ablauf von weiteren sieben Stunden. Somit scheint anhand der Bestimmung von FABP im Blut der frühzeitige Nachweis oder Ausschluss eines AMI möglich zu sein. Human heart-type fatty acid-binding protein (FABP) has a high potential as an early marker for myocardial infarction (MI) being more specific than myoglobin. FABP is a low molecular mass cytoplasmic protein (15 kDa) that is released early after the onset of ischemia and it may be useful for rapid confirmation or exclusion of acute myocardial infarction (AMI). Immunochemically assayed FABP, cardiac troponin I (cTnI) and enzymatically assayed creatine phosphokinase (CPK) were determined serially in plasma and serum samples from 218 patients presenting with chest pain and suspected MI. In the 94 patients with confirmed MI, FABP rose to a maximum level (577.6 ± 43.8 μg/L) 3 hours after the onset of symptoms and returned to normal within 30 hours. The FABP level peaked 7–9 hours earlier than CPK (2288 ± 131 U/L) and cTnI (357.1 ± 23.9 μg/L). CPK took 50–70 hours to return to normal level and cTnI returned to normal level over 70 hours. The areas under the receiver operating characteristic (ROC) curves for FABP were calculated as 0.871 at admission and 0.995 one hour after admission, whereas for CPK the areas were 0.711 and 0.856 and for cTnI the areas were 0.677 and 0.845, indicating that the FABP test gave a better diagnostic classification at the early stage being reached by cTnI (0.995) only 8 hours after admission. For FABP, both sensitivity and negative predictive value (NPV) increased quickly to 100% for samples monitored just one hour after admission. By using only two samples, one at admission and one 1 hour post admission, sequential FABP monitoring can reliably diagnose AMI patients 1 hour after admission and 100% of non-AMI patients can be excluded with no false negative results. The late markers cTnI and CPK have the similar diagnostic performance only 7 hours later. Thus measurement of FABP in plasma or serum allows the earliest immunochemical confirmation or exclusion of AMI.

Human heart-type fatty acid-binding protein for on-site diagnosis of early acute myocardial infarction

Human heart-type fatty acid-binding protein (H-FABP) has a high potential as an early marker for acute myocardial infarction (AMI) being more sensitive than current routine cardiac markers. Seventy-four patients presenting to hospital with a median symptom onset of 2.2 h (IQR 1.5-2.9 h) were enrolled in this study and 54 (73%) had AMI. At presentation, H-FABP gave the highest sensitivity of 83.3% (95% CI: 70.7-92.1) and troponin I (cTnI) gave the highest specificity of 50.0% (95% CI: 27.2-72.8). This study demonstrated that H-FABP immunotest gave a better diagnostic classification at the early stage. Also, AMI was identified significantly earlier by H-FABP than cTnI (17 vs. 6 patients, p<0.05).

InfectCheck CRP barcode-style lateral flow assay for semi-quantitative detection of C-reactive protein in distinguishing between bacterial and viral infections

In the present study, we describe an InfectCheck barcode-style lateral flow assay for semi-quantitative detection of CRP in distinguishing between bacterial and viral infections. The severity of bacterial infection can be assessed by simply counting the number of red lines developed at the CRP test zone of the test device. If only one visible line appeared at the CRP test zone, it represents a low or mild inflammation with CRP levels <10 mg/L. Two and three visible lines mean moderate (>or=10-25 mg/L) and severe (>or=25-50 mg/L) inflammations respectively while four visible lines stand for a very severe inflammation (>or=50-100 mg/L). If the visible lines become faint and the intensity of the first line is weaker than that of the control line and may even disappear, this outcome corresponds to the stage of having super severe inflammation (>or=100 mg/L). A total of 500 patients admitted to hospital through the Accident and Emergency Unit at the Prince of Wales Hospital were examined. The InfectCheck CRP barcode-style rapid test gave a high sensitivity of 88.7% and a high negative predictive value of 93.8%. This result indicates that the rapid test is reliable to exclude non-infected patients. The calculated intra- and inter-assay coefficient of variation for the five CRP concentration ranges was both within 20.0%. It is a one-step whole blood rapid test without any sample pre-treatment and the result is available within 20 min. This user friendly diagnostic tool can allow self-testing by interested individuals without any expensive reading device.

Early diagnosis of acute myocardial infarction using immunosensors and immunotests

Abstract Various biochemical markers are available to detect cardiac tissue injury after an acute myocardial infarction (AMI) and to estimate the extent of this injury. One of the markers used to detect AMI early after onset of symptoms is heart-type fatty acid-binding protein (H-FABP). To make it clinically applicable it is important to develop rapid diagnostic sensors to assess its concentration. Due to the requirement of skilled technicians and lengthy assay procedures, most immunochemical assays for H-FABP are of limited use for routine clinical practice. A rapid and quantitative immunotest for the detection of H-FABP in serum samples has been successfully developed with a performance time of 10 min. Fifty-one serum samples from patients were evaluated using a conventional ELISA and the newly developed test. A good correlation was found with r 2 = 0.9585. The detection limit of the test was 2.8 µg/L. For the pooled serum sample with a lower H-FABP concentration (4 µg/L), the calculated intra-assay coefficient of variation (CV) was 10%, and the inter-assay CV was 15%; for the pooled serum sample with a high H-FABP concentration (103 µg/L), the intra-assay CV was 8% and the inter-assay CV was 10%. Furthermore, the immunotest can be stored either at 4°C and room temperature for up to one year without significant loss of activity. Finally, a one-step FABP test so-called CardioDetect® which was derived from the serum immunotest has been designed for qualitative determination of H-FABP in whole blood samples. It requires no sample pretreatment and gives result within 15 min. Thirty-nine patients presenting with chest pain and suspected AMI were studied. Using an upper reference level of 7 µg/L, the specificity of the rapid test was 94%. Both sensitivity and negative predictive value (NPV) were 100% implying that 100% of nonAMI patients can be excluded with no false negative results. With this rapid and sensitive immunotest, H-FABP will be soon introduced to clinical practice.

Matrix metalloproteinase 9 mRNA: An early prognostic marker for patients with acute stroke

OBJECTIVES To investigate matrix metalloproteinase 9 (MMP9) mRNA as a prognostic marker in stroke. DESIGN AND METHODS MMP9 mRNA concentrations in 126 stroke patients were analyzed using quantitative reverse transcription-polymerase chain reaction. RESULTS The normalized MMP9 mRNA concentration was almost 3 times higher in non-survival patients compared to survival patients (P=0.0002); and 1.9-fold higher in patients with post-stroke modified Rankin score (mRS) >2 than patients with mRS≤2 (P<0.05). CONCLUSIONS MMP9 mRNA was a predictor of poor outcome and mortality in stroke.

Comparison of miR-124-3p and miR-16 for early diagnosis of hemorrhagic and ischemic stroke

BACKGROUND The present study aimed to investigate and compare plasma concentrations of miR-124-3p and miR-16 as diagnostic markers in acute stroke. METHODS miR-124-3p and miR-16 concentrations in 93 stroke patients were analyzed using real-time polymerase chain reaction. The primary outcome was the differentiation of hemorrhagic and ischemic stroke. RESULTS Of 93 patients, 74 (79.6%) were diagnosed as ischemic stroke (IS) and 19 (20.4%) were diagnosed as hemorrhagic stroke (HS). Median plasma 124-3p concentrations taken within 24h of symptom onset were higher in HS patients than that in IS patients (3.8×10(5) vs 2.0×10(5) copies/ml plasma, p=0.0109), while median miR-16 concentration in IS patients were higher than that in HS patients (1.6×10(9) vs 1.3×10(9) copies/ml plasma, p=0.0399). The odds ratio (OR) for discriminating HS and IS with miR-124-3p and miR-16 was 5.37 and 9.75 respectively. CONCLUSION Both miR-124-3p and miR-16 are diagnostic markers to discriminate HS and IS.

Detection of serum neopterin for early assessment of dengue virus infection

Summary Objective Neopterin is generated and released in increased amounts by macrophages upon activation by interferon-γ during Th1-type immune response. The potential usefulness of neopterin in early prognostic information of dengue virus infection was investigated. Methods Neopterin concentrations were determined in serum samples from 110 dengue fever (DF) patients. The neopterin levels were compared with those in 50 measles and 40 influenza patients; 155 healthy blood donors served as controls. Results In acute sera of DF patients mean neopterin concentration was 48.2nmol/L, which was higher than that in patients with measles (mean: 36.3nmol/L) and influenza (18.8nmol/L) and in healthy controls (6.7nmol/L; P <0.001). In the patients with confirmed DF, an early neopterin elevation was detected already at the first day after the onset of symptoms and rose to a maximum level of 54.3nmol/L 4days after the onset. Higher increase of neopterin level in DF patients was associated with longer duration of fever and thus predicted the clinical course of the disease. Conclusions Neopterin concentrations were found significantly higher in DF patients compared with healthy controls and also with other viral infections (P <0.001) and may allow early assessment of the severity of DF.