Original Contribution

Value of ischemia-modified albumin in the diagnosis of pulmonary embolism

Suleyman Turedi MD^a,*, Abdulkadir Gunduz MD^a, Ahmet Mentese^b, Suleyman Caner Karahan MD^b, Sennur Ekici Yilmaz MD^a, Oguz Eroglu MD^a,

Irfan Nuhoglu MD^c, Ibrahim Turan^b, Murat Topbas MD^d

^aDepartment of Emergency Medicine, Faculty of Medicine, Karadeniz Technical University, Trabzon 61080, Turkey ^bDepartment of Biochemistry, Faculty of Medicine, Karadeniz Technical University, Trabzon 61080, Turkey ^cDepartment of Internal Medicine, Faculty of Medicine, Karadeniz Technical University, Trabzon 61080, Turkey ^dDepartment of Public Health, Faculty of Medicine, Karadeniz Technical University, Trabzon 61080, Turkey

Received 22 November 2006; revised 17 December 2006; accepted 18 December 2006

Abstract

Background: Pulmonary embolism (PE) is a common condition, but the diagnostic strategy for the evaluation of suspected PE is somewhat controversial. Despite the use of various biochemical markers (such as D-dimer and C-reactive protein) and various probability calculation algorithms based on clinical findings for that purpose, there is still a need for more specific and practical markers in PE diagnosis. The aim of this study was to investigate the diagnostic value of ischemia-modified albumin levels in the diagnosis of PE.

Methods: This case-control study was performed in the emergency department between March and September 2006. The serum IMA levels of a total of 60 individuals, consisting of 30 PE patients who had been definitively diagnosed via spiral computed tomographic angiography and 30 healthy volunteers, were examined.

Results: The measurement of IMA levels in patient plasma yielded mean values of 0.724 F 0.122 absorbance unit (ABSU) in the PE group and 0.360 F 0.090 ABSU in the control group. When plasma IMA levels in the PE group were compared with those in the control group, statistically significant increases in IMA were observed in the former (t = 13.19, df = 56, P b.0005). The value of 0.540 ABSU was calculated as the upper limit of reference interval. In the PE group, 97.7% (n = 29) had values exceeding 0.540 ABSU; none of the Control subjects had values exceeding this cutoff value.

Conclusions: In conclusion, our data suggest that IMA levels may be useful as a discriminative marker to exclude pulmonary embolism.

D 2007

* Corresponding author. Acil TVp AD, Fakultesi Hastanesi, Karadeniz Teknik U? niversitesi TVp, Trabzon 61080, Turkey. Tel.: +90 0462 377 5715;

fax: +90 0462 325 12 46.

E-mail address: [email protected] (S. Turedi).

Introduction

Pulmonary embolism (PE) is a common condition, the diagnosis of which in the emergency department (ED) remains problematic [1]. Objective testing for PE is crucial,

0735-6757/$ - see front matter D 2007 doi:10.1016/j.ajem.2006.12.013

because clinical assessment alone has been found to be incorrect in up to 50% of cases, and the mortality rate of untreated PE is high. Incorrect diagnosis of PE and unnecessary treatment with anticoagulant therapy is associ- ated with a risk of bleeding. Algorithms used to rule out or diagnose PE often involve ventilation-perfusion scans, spiral Computerized tomography , or pulmonary angi- ography, which are invasive, complex, and time consuming. New, simple tests are therefore warranted to rule out PE and to reduce the number of sophisticated Imaging techniques needed. Despite the use of various biochemical markers (such as D-dimer and C-reactive protein) and probability calculation algorithms based on clinical findings for that purpose, there is still a need for more specific and practical markers in PE diagnosis [2].

Laboratory analysis“>Studies based on the principle of changes in albumin cobalt binding capacity were carried out in the late 1990s by researchers such as Bar-Or et al [3] and Bhagavan et al [4] for the analysis of myocardial ischemia. This test is based on the determination of a reduction in cobalt binding in the human serum albumin N-terminal region during myocardial ischemia. In a 2001 study, Bar-Or et al [5] observed that the albumin binding capacity (IMA) concentration in the blood of patients developing temporary ischemia with percutane- ous transluminal coronary angioplasty began rising within a few minutes, and that when reperfusion was later estab- lished through angioplasty IMA Blood concentrations dropped to the level expected in individuals without ischemia in approximately 6 hours.

Our study was intended to establish the diagnostic value of IMA levels for the diagnosis of pulmonary thromboembolism.

Materials and methods

Study design

This case-control study was performed in the ED of Karadeniz Technical University Hospital, Turkey. The protocol for the study was approved by the hospital’s local ethics committee. The inclusion period lasted from March through September 2006.

Patients and methods

Thirty patients (mean age, 65 F 12 years) presenting to the ED and definitively diagnosed with PE via CT angiography were enrolled in the study. Exclusion criteria were as follows: (1) other ischemic diseases, such as acute

Table 1 Baseline characteristics in the PE and control groups
Characteristics	PE group (n = 30)	Control group (n =30)
Age, mean F SD	65 F 12	68 F 9
Women (%)	73.3	70.0

Age (median) (y) Sex	44-87 (68) 8 (27.7%) men
	22 (73.3%) women
Clinical presentation
Dyspnea	11
Chest pain	7
Dyspnea + chest pain	5
Dyspnea + hemoptysis	2
Syncope	5
Symptoms of deep vein thrombosis	5
Risk factors Surgery within 1 month	8
Immobilization	11
Cancer	2
Chronic obstructive pulmonary disease	1
Trauma	1
Previous deep vein thrombosis	1

coronary syndrome (ACS), acute myocardiac infarction, acute ischemic cerebrovascular disease, peripheral vein occlusion, or Mesenteric ischemia; (2) an abnormal serum Albumin level making the determination of IMA levels impossible (normal level, 3.5-5.5 mg/dL); (3) advanced liver, kidney, or heart insufficiency; (4) troponin T and electrocardiogram (ECG) variations evaluated from the point of view of ACS; (5) age younger than 18 years); (6) allergy to Contrast material; (7) venous thromboembolism or PE in the past 6 months; and (8) refusal to participate in the study. A control group of 30 age-matched, healthy volunteers (age, 68 F 9 years) served as a reference for Biochemical parameters. The exclusion criteria applied during the enrollment of the control group were the same as those in the patient group.

Table 2 Clinical characteristics of patients in the PE group (n = 30)

Laboratory analysis

Blood samples were taken from the brachial vein by using the venopuncture technique at time of presentation. Vacutainer tubes without anticoagulants were used to obtain serum. Serum specimens were obtained after 15 minutes of centrifugation at 3000 rpm. Specimens to be used for measuring IMA blood concentrations were pipetted into Eppendorf tubes and stored at -80 8C.

Reduced cobalt to IMA level was analyzed by using the rapid and colometric method developed by Bar-Or et al [3]. We placed 200 AL of patient serum into glass tubes and added 50 AL of 0.1% CoCl₂d 6H₂O (Sigma, St Louis, Mo). After gentle shaking, this was set aside for 10 minutes to ensure sufficient cobalt albumin binding. We added 50 AL of 1.5 mg/mL dithiotheitol (DTT) as a coloring agent. After

2 minutes, 1 mL of 0.9% NaCl was added to halt the binding between cobalt and albumin. A blind specimen was prepared for every specimen. At the DTT addition stage,

50 AL of distilled water was used instead of 50 AL of

1.5 mg/mL DTT to obtain a blind specimen without DTT.

Fig. 1 Serum absorbance values of PE patients and healthy individuals.

Specimen absorbancies were analyzed at 470 nm under spectrophotometry (Shimadzu UV160U). Color formation in specimens with DTT was compared with color formation in the blind tubes, and the results were expressed as absorbance units (ABSUs).

This colometric method of measurement scanning is based on the principle of quantitative scanning of free cobalts present after cobalt binding has taken place. This means that high absorbance levels as a result of increased amounts of free cobalt in the environment can be determined.

Statistical analysis

Statistical analysis was performed with SPSS for Windows, release 11.0. Results are expressed as mean F SD. Statistical analysis was performed by using Student t test and the Kolmogorov-Smirnov test. The upper limit of reference interval was determined by adding 2 SD to the mean absorbance value of control group. Statistical signif- icance was assumed at P b .05.

Results

Baseline characteristics

A total of 60 individuals were investigated, consisting of 30 PE patients meeting the study criteria and definitively diagnosed via computed spiral tomographic angiography and 30 healthy volunteers. Baseline demographic character- istics were similar between the 2 study groups (Table 1). The general clinical characteristics of the 30 patients in the PE group are shown in Table 2.

Laboratory findings

The 30 healthy (control) individuals and 30 PE patients were examined with regard to whether there was a difference in IMA levels between the 2 groups.

The measurement of IMA levels in patient plasma yielded mean values of 0.724 F 0.122 ABSU in the PE group and

0.360 F 0.090 ABSU in the control group. When plasma IMA levels in the PE group were compared with those in the

control group, statistically significant increases in IMA were observed in the former (t = 13.19, df = 56, P b .0005).

The upper limit of reference interval was determined by adding 2 SD to the mean absorbance value (mean, 0.360 ABSU) of control group. Accordingly, the value of 0.540 ABSU was calculated as the upper limit of reference interval. In the PE group, 97.7% (n = 29) had values that exceeded 0.540 ABSU; none of the control subjects had values that exceeded this cutoff value.

The serum absorbance values of PE patients and healthy individuals are shown in Fig. 1.

Discussion

Clinical symptoms and findings, and conventional chest x-ray are not sufficiently specific for the diagnosis of PE. Algorithms used to rule out or diagnose PE often involve lung ventilation-perfusion scans, spiral CT, or pulmonary angiography, which are invasive, complex, and time consuming. Ventilation perfusion lung scanning has been 1 of the most widely used imaging techniques when there is a clinical suspicion of PE. However, the result is frequently nondiagnostic, and additional testing is needed to confirm the diagnosis. In the past 10 years, spiral CT has become an alternative imaging technique. The sensitivity and specific- ity of spiral CT range from 53% to 100% and from 81% to 100%, respectively, when lung ventilation-perfusion scan or pulmonary arteriography are used as reference methods [6]. Despite pulmonary angiography being the gold standard in diagnosis and spiral CT, which is being increasingly used, appearing to be diagnostically reliable, these Radiologic tests have the disadvantages in that they are relatively expensive and complications can arise as a result of the application of contrast. The fact that PE is observed in only 30% of patients with suspected PE who are exposed to such invasive measures as angiography demonstrates the impor- tance of adjunctive diagnostic methods in place of these scanning tools [7].

Various Clinical approach models and biochemical markers are currently used to rule out suspected PE in patients without using these invasive and costly scanning tools. The most frequently used among these biochemical markers is D-dimer measurement. D-dimer is a product of the fibrinolysis process and coagulation activation, and an upper limit of reference interval value of more than 500 Ag/L has been determined to be relatively sensitive for acute venous thromboembolism (97%).

The negative predictive value of D-dimer levels for ruling out a diagnosis of thromboembolic disease has been reported as high [8]. However, D-dimer is a rather low specific marker (39%). The positive predictive value is very low as D-dimer levels can also be elevated in Venous thromboembolic disease and other diseases such as heart failure, surgery, infections, connective tissue disorders, and cancer [9,10]. For that reason, a high positive D-dimer level

is almost entirely impractical for positive diagnosis [11]. Therefore, there is a need for more appropriate markers for use in PE diagnosis.

During acute ischemic conditions, the metal binding capacity of albumin to transition metals such as copper, nickel, and cobalt is reduced, generating a metabolic variant of the protein, commonly known as ischemia-modified albumin. As with PE, another ischemic disease in which diagnosis or exclusion is difficult is acute coronary syndrome. Ischemia-modified albumin measurement has recently been proposed as a sensitive marker for the diagnosis of myocardial ischemia presenting with typical acute chest pain [12]. When IMA results were used in combination with ECG and serial troponins, physicians achieved 70% accuracy in ruling out cardiac ischemia, compared to only 50% diagnostic accuracy when using only ECG and troponin data to exclude ischemia [13]. Ischemia- modified albumin, a Food and Drug Administration- approved serum biomarker of cardiac ischemia, is a risk stratification tool in patients who are suspected to have ACS. A negative triple prediction test of IMA, ECG, and troponin, measured within 3 hours of chest pain, performed by Peacock et al [14], showed that IMA has good sensitivity and a high negative predictive value for ruling out ACS. Ischemia-modified albumin is a promising candidate for this role, but several unresolved issues must be addressed before it becomes a routine test, including the effect of variation in patient albumin concentrations, lactic acid interference, and specificity for cardiac, as opposed to skeletal, muscle ischemia [15].

Our study demonstrated a statistically significant increase in serum IMA levels in PE. Serum IMA levels were significantly high in 97.7% of PE patients, in which an IMA upper limit of reference interval value for the exclusion of PE of 0.540 ABSU was determined. The finding that only 1 PE patient had an IMA level lower than this value is striking. In addition, no healthy individuals had IMA levels greater than the upper limit of reference interval level. Despite the absence of data to compare with D-dimer, which rises in many situations and has a very low positive predictive value in the diagnosis of PE, it is significant that IMA is more specific for ischemic episodes.

In conclusion, our data suggest that IMA levels may be useful as a discriminative marker to exclude PE. Ischemia-

modified albumin results may reduce the need for diagnostic imaging. Larger studies involving a wider patient selection are needed to further clarify result.

References

1. Palla A, Giuntini C. Diagnosis of pulmonary embolism: have we reached our goal? Respiration 2004;71:22 - 3.
2. Steeghs N, Goekoop RJ, Niessen RWLM, et al. C-reactive protein and D-dimer with clinical probability score in the exclusion of pulmonary embolism. Br J Haematol 2005;130:614 - 9.
3. Bar-Or D, Lau E, Winkler JV. A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia- a preliminary report. J Emerg Med 2000;19:311 - 5.
4. Bhagavan NV, Lai EM, Rios PA, et al. Evaluation of Human serum albumin cobalt binding assay for the assessment of myocardial ischemia and myocardial infarction. Clin Chem 2003;49:581 - 5.
5. Bar-Or D, Winkler JV, Vanbenthuysen K, et al. Reduced albumin- cobalt binding with transient myocardial ischemia after elective percutaneous transluminal coronary angioplasty: a preliminary com- parison to creatine kinase-MB, myoglobin, and troponin I. Am Heart J 2001;141:985 - 91.
6. Friera-Reyes A, Caballero P, Ruiz-Gimenez N, et al. Usefulness of fast ELISA determination of D-dimer levels for diagnosing pulmonary embolism in an emergency room. Arch Bronconeumol 2005;41:499 - 504.
7. Perrier A. Noninvasive diagnosis of pulmonary embolism. Haemato- logica 1997;82:328 - 31.
8. Perrier A, Desmarais S, Miron MJ, et al. Non-invasive diagnosis of venous thromboembolism in outpatients. Lancet 1999;353:190 - 5.
9. Caine GJ, Lip GY. D-dimer assessment of patients with suspected pulmonary embolism. Arch Intern Med 2003;163:243.
10. Froehling DA, Elkin PL, Swensen SJ, et al. Sensitivity and specificity of the semiquantitative latex agglutination D-dimer assay for the diagnosis of acute pulmonary embolism as defined by computed tomographic angiography. Mayo Clin Proc 2004;79:164 - 8.
11. Bounameaux H, de Moeloose P, Perrier A, et al. Plasma measurement of D-dimer as diagnostic aid in suspected venous thromboembolism: an overview. Thromb Haemost 1994;71:1 - 6.
12. Lippi G, Montagnana M, Guidi GC. Albumin cobalt binding and ischemia modified albumin generation: an endogenous response to ischemia? Int J Cardiol 2006;108:410 - 1.
13. Morrow DA, deLemos JA, Sabatine MS, et al. The search for a biomarker of cardiac ischemia. Clin Chem 2003;49:537 - 9.
14. Peacock F, Morris DL, Anwaruddin S, et al. Meta-analysis of ischemia-modified albumin to rule out acute coronary syndromes in the emergency department. Am Heart J 2006;152:253 - 62.
15. Refaai MA, Wright RW, Parvin CA, et al. Ischemia-modified albumin increases after skeletal muscle ischemia during arthroscopic knee surgery. Clin Chim Acta 2006;366:264 - 8.