nCD64 Expression Research Articles

CD64 Expression on Neutrophils (nCD64) as a Biomarker in Adult Patients With Sepsis: A Cross-Sectional Study.

Introduction Neutrophils that are at rest exhibit very low expression of CD64, the high-affinity immunoglobulin fragment crystallizable γ receptor I, which is also found in monocytes. Neutrophils that have been exposed to endotoxins or are infected express more CD64. Objectives The aim of this study was to assess the CD64 biomarker expression on neutrophils using the flow cytometry method and its comparison with total leukocyte count, C-reactive protein (CRP) level, and blood culture sensitivity test in the diagnosis of sepsis in adults. Materials and methods Using the quick Sequential Organ Failure Assessment (qSOFA) scoring criteria, 94 blood samples from individuals with clinical indications of sepsis were included in this investigation. Samples were collected in K2 EDTA (ethylenediaminetetraacetic acid) vacutainers and analyzed for neutrophil CD64 (nCD64) levels using BD FACSLyricTM flow cytometer (BD, Franklin Lakes, NJ) within 24 hours of collection. Total leukocyte count, CRP levels, and blood culture sensitivity tests were also assessed simultaneously. Results Majority of the patients, i.e., 29 (30.9%), belonged to the age group of 19 to 35 years, with a female preponderance. The mean total leukocyte count was 19.49 ± 8.12 x 103/µL, mean CRP level was 81.19 ± 56.33 mg/dL, and the mean nCD64 expression - median fluorescence intensity was 197.26 ± 79.56. A positive blood culture was found in 59 (62.8%) cases. Neutrophils showed bright expression of nCD64 in 39 (41.5%) patients, dim expression in 35 (37.2%) patients, and moderate expression in 20 (21.3%) patients in the present study. The correlation between nCD64 expression with CRP, total leukocyte count, and blood culture sensitivity test was statistically significant (p=0.001). nCD64 expression showed a sensitivity of 93.2% and specificity of 91.4% for diagnosing sepsis, with an area under the curve (AUC) of 0.973. This proves nCD64 to be a highly effective biomarker compared to conventional methods. Conclusion nCD64 has a higher sensitivity and specificity as compared to total leukocyte count, CRP levels, and blood culture sensitivity test in the diagnosis of sepsis. It is an effective and rapid test that can enhance sepsis diagnostic accuracy, when combined with other biomarkers. Multicentric research needs to be conducted to validate these findings.

Neutrophil CD64 – A Reliable Predictive Marker of Sepsis in Adult Intensive Care Unit

Abstract BACKGROUND: Sepsis poses a significant threat to adult patients admitted to intensive care units (ICUs), contributing to substantial morbidity and mortality. Neutrophil CD64 (nCD64) expression has been linked to inflammatory responses during infection or tissue injury, suggesting its potential as a predictive marker for sepsis. MATERIALS AND METHODS: We conducted a prospective analytical study over 2 years at a tertiary care hospital, enrolling 91 sepsis cases from the adult ICU. Alongside routine laboratory parameters, including complete blood count, prothrombin time, activated partial thromboplastin time, fibrinogen, C-reactive protein, and procalcitonin, the expression of nCD64 was analyzed using a Beckman Coulter Navios flow cytometer. Median fluorescence intensity (MFI) was calculated for these cases. Statistical analysis was performed using SPSS version 25.0 software, with a significance threshold of P < 0.05. RESULTS: MFI scores demonstrated a notable increase in diagnosed sepsis cases, as determined by clinical and biochemical parameters. Moreover, changes in MFI scores on Day 4 exhibited a correlation with other clinical and biochemical parameters, strengthening the association between nCD64 expression and sepsis severity. CONCLUSION: Our findings suggest that nCD64 serves as an independent prognostic factor in adult ICU sepsis patients. It offers a promising alternative to traditional sepsis markers for predicting patient outcomes, emphasizing its potential clinical utility in guiding therapeutic interventions and improving patient care in ICU settings.

Standardization of neutrophil CD64 and monocyte HLA-DR measurement and its application in immune monitoring in kidney transplantation.

Infections cause high mortality in kidney transplant recipients (KTRs). The expressions of neutrophil CD64 (nCD64) and monocyte HLA-DR (mHLA-DR) provide direct evidence of immune status and can be used to evaluate the severity of infection. However, the intensities of nCD64 and mHLA-DR detected by flow cytometry (FCM) are commonly measured by mean fluorescence intensities (MFIs), which are relative values, thus limiting their application. We aimed to standardize nCD64 and mHLA-DR expression using molecules of equivalent soluble fluorochrome (MESF) and to explore their role in immune monitoring for KTRs with infection. The study included 50 KTRs diagnosed with infection, 65 immunologically stable KTRs and 26 healthy controls. The blood samples were collected and measured simultaneously by four FCM protocols at different flow cytometers. The MFIs of nCD64 and mHLA-DR were converted into MESF by Phycoerythrin (PE) Fluorescence Quantitation Kit. The intraclass correlation coefficients (ICCs) and the Bland-Altman plots were used to evaluate the reliability between the four FCM protocols. MESFs of nCD64 and mHLA-DR, nCD64 index and sepsis index (SI) with the TBNK panel were used to evaluate the immune status. Comparisons among multiple groups were performed with ANOVA one-way analysis. Receiver operating characteristics (ROC) curve analysis was performed to diagnose infection or sepsis. Univariate and multivariate logistic analysis examined associations of the immune status with infection. MESFs of nCD64 and mHLA-DR measured by four protocols had excellent reliability (ICCs 0.993 and 0.957, respectively). The nCD64, CD64 index and SI in infection group were significantly higher than those of stable KTRs group. Patients with sepsis had lower mHLA-DR but higher SI than non-sepsis patients. ROC analysis indicated that nCD64 had the highest area under the curve (AUC) for infection, and that mHLA-DR had the highest AUC for sepsis. Logistic analysis indicated that nCD64 > 3089 and B cells counts were independent risk factors for infection. The standardization of nCD64 and mHLA-DR made it available for widespread application. MESFs of nCD64 and mHLA-DR had good diagnostic performance on infection and sepsis, respectively, which could be promising indicators for immune status of KTRs and contributed to individualized treatment.

Utility of neutrophil CD64 in distinguishing bacterial infection from inflammation in severe alcoholic hepatitis fulfilling SIRS criteria

To assess utility of neutrophilCD64 (nCD64) expression in differentiating bacterial infection from inflammation in patients with severe alcoholic hepatitis (SAH) fulfilling systemic inflammatory response syndrome criteria. Patients with SAH and infection (n = 58), SAH without infection (n = 70), and healthy controls (n = 20) were included. Neutrophil CD64 expression by flowcytometry, serum Procalcitonin (ELISA) and C-reactive protein (Nephelometry) and neutrophil–lymphocyte ratio (NLR) were studied. Percentage of neutrophils with CD64 expression (nCD64%) was significantly higher in patients with SAH and infection than in those without infection and controls [76.2% (56.9–86.5) vs. 16% (12.6–23.1) vs. 7.05% (1.4–9.5), p < 0.05], as was their mean fluorescence intensity [MFI; 1431 (229–1828) vs. 853 (20–968) vs. 99.5 (54.7–140.7), p < 0.05]. Using a cut-off of 27%, the sensitivity and specificity of nCD64% to diagnose bacterial infection was 94% and 81%, respectively, with area under curve (AUC) of 0.95. At a cut-off value of 0.261 ng/ml, the sensitivity and specificity of serum procalcitonin was 83% and 72%, respectively, with AUC of 0.86. Serum CRP, total leukocyte count, NLR had AUCs of 0.78, 0.63 and 0.64, respectively. Quantitative measurement of nCD64 can better distinguish systemic bacterial infection and inflammation in SAH as compared to traditional biomarkers.

Effect of neutrophil CD64 for diagnosing sepsis in emergency department.

The aim of this study is to investigate the diagnostic and prognostic value of neutrophil CD64 (nCD64) as a novel biomarker in sepsis patients. One hundred fifty-one adult patients diagnosed with sepsis and 20 age-matched healthy controls were enrolled in the study. Patients with sepsis were further subdivided into a sepsis group and a septic shock group. nCD64 expression, serum procalcitonin (PCT) level, C-reactive protein (CRP) level, and white blood cell (WBC) count were obtained for each patient, and Sequential Organ Failure Assessment (SOFA) scores were calculated. nCD64 expression was higher in the sepsis group with confirmed infection than in the control group. The receiver operating characteristic (ROC) curve of nCD64 was higher than those of SOFA score, PCT, CRP and WBC for diagnosing infection. The area under the curve (AUC) of nCD64 combined with SOFA score was the highest for all parameters. The AUC of nCD64 for predicting 28-day mortality in sepsis was significantly higher than those of PCT, CRP, and WBC, but slightly lower than that of SOFA score. The AUC of nCD64 or PCT combined with SOFA score was significantly higher than that of any single parameter for predicting 28-day mortality. nCD64 expression and SOFA score are valuable parameters for early diagnosis of infection and prognostic evaluation of sepsis patients.

Smartphone-imaged microfluidic biochip for measuring CD64 expression from whole blood.

Sepsis, a life-threatening syndrome that contributes to millions of deaths annually worldwide, represents a moral and economic burden to the healthcare system. Although no single, or even a combination of biomarkers has been validated for the diagnosis of sepsis, multiple studies have shown the high specificity of CD64 expression on neutrophils (nCD64) to sepsis. The analysis of elevated nCD64 in the first 2-6 hours after infection during the pro-inflammatory stage could significantly contribute to early sepsis diagnosis. Therefore, a rapid and automated device to periodically measure nCD64 expression at the point-of-care (POC) could lead to timely medical intervention and reduced mortality rates. Current accepted technologies for measuring nCD64 expression, such as flow cytometry, require manual sample preparation and long incubation times. For POC applications, however, the technology should be able to measure nCD64 expression with little to no sample preparation. In this paper, we demonstrate a smartphone-imaged microfluidic biochip for detecting nCD64 expression in under 50 min. In our assay, first unprocessed whole blood is injected into a capture chamber to immunologically capture nCD64 along a staggered array of pillars, which were previously functionalized with an antibody against CD64. Then, an image of the capture channel is taken using a smartphone-based microscope. This image is used to measure the cumulative fraction of captured cells (γ) as a function of length in the channel. During the image analysis, a statistical model is fitted to γ in order to extract the probability of capture of neutrophils per collision with a pillar (ε). The fitting shows a strong correlation with nCD64 expression measured using flow cytometry (R2 = 0.82). Finally, the applicability of the device to sepsis was demonstrated by analyzing nCD64 from 8 patients (37 blood samples analyzed) along the time they were admitted to the hospital. Results from this analysis, obtained using the smartphone-imaged microfluidic biochip were compared with flow cytometry. Again, a correlation coefficient R2 = 0.82 (slope = 0.99) was obtained demonstrating a good linear correlation between the two techniques. Deployment of this technology in ICU could significantly enhance patient care worldwide.

Significance of neutrophilic CD64 as an early marker for detection of neonatal sepsis and prediction of disease outcome

Background: Neonatal sepsis (NS) is a global health problem owing to its significant contribution to morbidity and mortality. We evaluated the significance of neutrophilic CD64 (nCD64) expression as an early marker for diagnosis of NS compared to CRP and assessed its relation to disease outcome.Methods: High sensitivity CRP (hs-CRP) and nCD64 were measured in 60 neonates with symptoms and signs of sepsis (40% were culture-proved) and 30 age- and sex-matched controls.Results: Baseline hs-CRP and nCD64% were significantly higher among septic neonates compared with the controls (p < 0.05), while, no significant difference was found between the two septic groups (p > 0.05). nCD64 cutoff value > 34.1% was able to discriminate septic neonates from controls with higher sensitivity and specificity than hs-CRP. The mortality rate was 21.7% among septic neonates. Baseline nCD64% was significantly higher among died patients compared with recovered neonates (p = 0.009) while no significant difference was found between baseline hs-CRP and disease outcome (p = 0.117).Conclusion: Flow cytometric assessment of nCD64 was able to discriminate neonates with sepsis from controls with higher accuracy than hs-CRP; however, the combination of both nCD64% and hs-CRP enhances the ability to diagnose NS. Quantitative measurement of nCD64 can predict disease outcome in NS.

Analysis of the Physiological Variation in Neutrophil CD64 Expression during the Early Neonatal Period.

Background Several biomarkers for the diagnosis of sepsis are elevated during the early neonatal period due to physiological variations. The aim of this study was to investigate the physiological variation in neutrophil CD64 (nCD64) expression during the early neonatal period and the change in nCD64 expression in neonates with noninfectious diseases. Methods Of 71 neonates enrolled in this prospective study, 5 and 51 were diagnosed as having bacteremia and noninfectious diseases, respectively. Fifteen healthy neonates were enrolled as normal controls. Peripheral white blood cell counts, serum C-reactive protein and procalcitonin levels, and nCD64 expression were examined at birth and on the first and fifth day of life in neonates with noninfectious diseases and healthy neonates. In neonates with bacteremia, these markers were measured at onset. Results nCD64 expression was significantly higher in neonates with bacteremia (median, 1,992) than in those with noninfectious diseases (1,823, p < 0.001) and healthy neonates (1,848, p = 0.002). Unlike other biomarkers, no differences in nCD64 expression were observed on the same days between neonates with noninfectious diseases and healthy neonates. Conclusion nCD64 expression may be a useful marker for the diagnosis of bacterial infection in the early neonatal period, because it does not show any physiological variations.

Neutrophil CD64 expression is not a useful biomarker for detecting serious bacterial infections in febrile children at the emergency department

Background CD64 is expressed on the surface membrane of neutrophils (nCD64) in the presence of bacterial infection. Although initial studies in intensive care settings have been promising, only two small, methodologically flawed studies have been performed in feverish children presenting to the emergency departement (ED), both of which were showing a moderate diagnostic value of nCD64 to detect a serious bacterial infection (SBI). This study aimed to determine the diagnostic value of nCD64 in children presenting with fever to the ED for detecting SBI. Methods In this prospective observational multi-centre study previously healthy children aged 1 month–16 years with fever, presenting to the ED of two hospitals in the Netherlands in 2011–2012 were included. Standardised information on clinical features were collected and nCD64 and CRP were measured routinely. Multivariable logistic regression was used to determine the discriminative ability to detect SBI (ROC-area) of nCD64 compared with CRP. Diagnostic performance measures including sensitivity, specificity and likelihood ratios were calculated. Results In 392 children (45%) with both CRP and nCD64 determined, 52 children (13%) had an SBI. The AUC of the ROC curve for CD64 was 0.62 (95% CI = 0.54–0.70) and 0.75 (95% CI = 0.67-0.83) for CRP. Neither duration of fever nor deviated vital signs influenced the diagnostic performance of nCD64. Conclusion NCD64 expression has poor discriminative value to detect children with an SBI in a general population of febrile children at the ED. It has no superior value compared to CRP in this setting, neither in total nor in sub-populations.

Neutrophil CD64 expression as a diagnostic marker for sepsis in adult patients: a meta-analysis.

IntroductionNeutrophil CD64 (nCD64) expression appears to be a promising marker of bacterial infections. The aim of this meta-analysis was to assess the accuracy of nCD64 expression for the diagnosis of sepsis in critically ill adult patients.MethodsWe systematically searched PubMed, Embase, ISI Web of Knowledge, and the Cochrane Library for literature published between database inception and 19 May 2014, as well as reference lists of identified primary studies. Studies were included if they included assessment of the accuracy of nCD64 expression for sepsis diagnosis in adult patients and provided sufficient information to construct a 2×2 contingency table.ResultsA total of 8 studies comprising 1986 patients fulfilled the inclusion criteria for the final analysis. The pooled sensitivity and specificity were 0.76 (95 % confidence interval [CI], 0.73–0.78) and 0.85 (95 % CI, 0.82–0.87), respectively. The positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio were 8.15 (95 % CI, 3.82–17.36), 0.16 (95 % CI, 0.09–0.30), and 60.41 (95 % CI, 15.87–229.90), respectively. The area under the summary receiver operating characteristic curve of nCD64 expression with Q* value were 0.95 (Q* =0.89).ConclusionsOn the basis of our meta-analysis, nCD64 expression is a helpful marker for early diagnosis of sepsis in critically ill patients. The results of the test should not be used alone to diagnose sepsis, but instead should be interpreted in combination with medical history, physical examination, and other test results.

Diagnostic values of CD64, C-reactive protein and procalcitonin in ventilator-associated pneumonia in adult trauma patients: a pilot study.

Ventilator-associated pneumonia (VAP) is one of the most common nosocomial infections; however, its diagnosis remains difficult to establish in the critical care setting. We investigated the potential role of neutrophil CD64 (nCD64) expression as an early marker for the diagnosis of VAP. Forty-nine consecutive patients with clinically suspected VAP were prospectively included in a single-center study. The levels of nCD64, C-reactive protein (CRP), and serum procalcitonin (PCT) were analyzed for diagnostic evaluation at the time of intubation (baseline), at day 0 (time of diagnosis), and at day 3. The receiver operating characteristic curves were analyzed to identify the ideal cutoff values. VAP was confirmed in 36 of 49 cases. In patients with and without VAP, the median levels (interquartile range, IQR) of nCD64 did not differ either at baseline [2.4 (IQR, 1.8-3.1) and 2.6 (IQR, 2.3-3.2), respectively; p=0.3] or at day 0 [2 (IQR, 2.5-3.0) and 2.6 (IQR, 2.4-2.9), respectively; p=0.8]. CRP showed the largest area under the curve (AUC) at day 3. The optimum cutoff value for CRP according to the maximum Youden index was 133 mg/dL. This cutoff value had 69% sensitivity and 76% specificity for predicting VAP; the AUC was 0.73 (95% CI, 0.59-0.85). The nCD64 and PCT values could not discriminate between the VAP and non-VAP groups either at day 0 or day 3. The results of this pilot study suggest that neutrophil CD64 measurement has a poor role in facilitating the diagnosis of VAP and thus may not be practically recommended to guide the administration of antibiotics when VAP is suspected.

Serial Determinations of Neutrophil CD64 Expression for the Diagnosis and Monitoring of Sepsis in Critically Ill Patients

Early identification of sepsis is important to be able to initiate timely therapy and optimize survival. Neutrophil CD64 (nCD64) expression has been proposed as a potential marker of sepsis. In this prospective observational study, adult patients admitted to our 34-bed medico-surgical department of intensive care over a 3.5-month period were included. Neutrophil CD64 expression was measured by flow cytometry at admission and daily until discharge or death. Blood C-reactive protein (CRP) level was measured routinely. Diagnosis of sepsis was recorded and appropriateness of empirical antibiotic treatment was established post hoc. Of the 548 patients included, 468 had flow cytometry measurements within 24 hours after admission, of whom 103 had sepsis. Septic patients had higher admission nCD64 expression than did nonseptic patients (P < .001). A cutoff admission nCD64 expression of 230 median fluorescence intensity (MFI) identified sepsis with a sensitivity of 89% (81%-94%) and specificity of 87% (83%-90%). When combining CRP and nCD64 expression, an abnormal result for both was associated with a 92% probability of sepsis, whereas sepsis was ruled out with a probability of 99% if both were normal. Septic patients receiving inappropriate empirical antibiotics had persistently elevated nCD64 expression, whereas expression decreased over time in patients receiving appropriate antibiotics. In nonseptic patients, an increase in nCD64 expression ≥40 MFI predicted intensive care unit (ICU)-acquired infection (n = 29) with a sensitivity of 88% and specificity of 65%. Measurement of nCD64 expression at ICU admission, especially when combined with CRP concentrations, is useful in diagnosing sepsis. Serial determinations of nCD64 could be used for monitoring purposes.

CD64-Neutrophil expression and stress metabolic patterns in early sepsis and severe traumatic brain injury in children

BackgroundCritical illness constitutes a serious derangement of metabolism. The aim of our study was to compare acute phase metabolic patterns in children with sepsis (S) or severe sepsis/septic shock (SS) to those with severe traumatic brain injury (TBI) and healthy controls (C) and to evaluate their relations to neutrophil, lymphocyte and monocyte expressions of CD64 and CD11b.MethodsSixty children were enrolled in the study. Forty-five children with systemic inflammatory response syndrome (SIRS) were classified into three groups: TBI (n = 15), S (n = 15), and SS (n = 15). C consisted of 15 non- SIRS patients undergoing screening tests for minor elective surgery. Blood samples were collected within 6 hours after admission for flow cytometry of neutrophil, lymphocyte and monocyte expression of CD64 and CD11b (n = 60). Procalcitonin (PCT), C-reactive protein (CRP), glucose, triglycerides (TG), total cholesterol (TC), high (HDL) or low-density-lipoproteins (LDL) were also determined in all groups, and repeated on day 2 and 3 in the 3 SIRS groups (n = 150).ResultsCRP, PCT and TG (p < 0.01) were significantly increased in S and SS compared to TBI and C; glucose did not differ among critically ill groups. Significantly lower were the levels of TC, LDL, and HDL in septic groups compared to C and to moderate changes in TBI (p < 0.0001) but only LDL differed between S and SS (p < 0.02). Among septic patients, PCT levels declined significantly (p < 0.02) with time, followed by parallel decrease of HDL (p < 0.03) and increase of TG (p < 0.02) in the SS group. Neutrophil CD64 (nCD64) expression was higher in patients with SS (81.2%) and S (78.8%) as compared to those with TBI (5.5%) or C (0.9%, p < 0.0001). nCD64 was positively related with CRP, PCT, glucose, and TG (p < 0.01) and negatively with TC, LDL, and HDL (p < 0.0001), but not with severity of illness, hematologic indices, length of stay or mechanical ventilation duration.ConclusionsIn sepsis, the early stress-metabolic pattern is characterized by a high (nCD64, glucose, TG) - low (TC, HDL, LDL) combination in contrast to the moderate pattern of TBI in which only glucose increases combined with a moderate cholesterol - lipoprotein decrease. These early metabolic patterns persist the first 3 days of acute illness and are associated with the acute phase CD64 expression on neutrophils.

Evaluation of CD64 Expression on Neutrophils as an Early Indicator of Neonatal Sepsis

Enhanced neutrophil CD64 (nCD64) expression is likely to be useful in diagnosis of neonatal sepsis. This study evaluated the diagnostic efficacy of nCD64 expression as an early indicator of neonatal sepsis. Sixty neonates (culture positive, 24; negative, 36) with suspected sepsis and 30 controls were studied prospectively. CD64 expression was evaluated flow cytometrically on neutrophils and monocytes. Mean and median nCD64 expression, mean and median monocyte CD64/nCD64 (M/N CD64) ratios were computed. Results were correlated with blood culture and other conventional indices of sepsis. The sick neonates had significantly higher mean and median nCD64 expression compared with controls. Monocyte CD64 values did not differ significantly among the groups. Both mean and median M/N CD64 ratios were significantly lower in the former group. Culture-positive neonates had significantly higher mean and median nCD64 values and significantly lower mean and median M/N CD64 ratios than clinically indistinguishable but culture-negative neonates. Both groups were significantly different with respect to these indices from normal controls. Median M/N CD64 ratio was the best discriminant by virtue of highest area under the receiver operator characteristic curve (0.903), with sensitivity and specificity of 91.7% and 88.9%, respectively. Conventional indices were inferior, both singly and in combination. Enhanced nCD64 reported as median M/N CD64 ratio is a highly sensitive marker of culture-positive neonatal sepsis. It additionally identifies a separate group among culture-negative sick neonates and may be useful to guide antibiotic administration especially in these neonates.

Evaluation of neutrophilic CD64, interleukin 10 and procalcitonin as diagnostic markers of early- and late-onset neonatal sepsis

The assay of infection markers can improve diagnostic sensitivity in neonatal sepsis. We determined the levels of neutrophilic CD64 (nCD64), procalcitonin (PCT) and interleukin 10 (IL-10) in infants with neonatal sepsis. Forty-nine newborn infants who met the criteria of sepsis were subjected to a routine sepsis evaluation as well as measurement of PCT and IL-10 levels and nCD64 expression. Of these 49 'infected' infants, 16 had a positive blood culture (culture-positive sepsis) and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (culture-negative sepsis). Another 49 healthy newborn infants were included as a control group. The sensitivity, specificity, positive predictive value and negative predictive value of PCT, IL-10 and nCD64 for the diagnosis of sepsis were determined. IL-10 had the highest sensitivity of 92% and specificity of 84% using a cut-off of > or =17.3 pg/ml. For PCT, the highest sensitivity of 65% and specificity of 60% were found at a cut-off value of > or =36.4 pg/ml. nCD64 had a maximal sensitivity of 92% and specificity of 71% at a cut-off value of 2.6%. Combinations of different markers may improve the sensitivity and specificity of biomarker tests. We found that the best combination was IL-10 and nCD64, which together provided sensitivity of 95% and specificity of 83%, and a negative predictive value of 86%.