Lactate dehydrogenase isoenzyme 1: its usefulness as a screening test in diagnosis of myocardial infarction

Lactate dehydrogenase isoenzyme 1: its usefulness as a screening test in diagnosis of myocardial infarction

306 Lactate Dehydrogenase Isoenzyme 1 I t s Usefulness as a Screening Test in Diagnosis of Myocardial Infarction LILIAN M. EWEN Department of Clinica...

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306

Lactate Dehydrogenase Isoenzyme 1 I t s Usefulness as a Screening Test in Diagnosis of Myocardial Infarction LILIAN M. EWEN Department of Clinical Biochemistry, Royal Columbian Hospital, New Westminster, B.C. V3L 3W7

Immunological assay of LD-1 activity provides a quantitative measurement of the type of lactate dehydrogenase (LD, EC 1.1.1.27) activity characteristic of myocardial origin, Using this test. a laboratory diagnosis of myocardial infarction can be either ruled out or confirmed in approximately 75% of patients in whom this diagnosis is suspected, without electrophoretic separation of creatine kinase (CK, EC 2.7.3.2.) and LD isoenzymes. Normal total CK and LD activities cannot be used to rule out myocardial infarction since CK-MB and LD-1 may have increased although total activities remain within their reference ranges. LD-1 activity increases as quickly as CK-MB following the onset of pain in the majority of patients but it remains elevated longer giving a greater period of time during which the diagnosis of myocardial infarction can be confirmed.

~ecent work 11-5, has indicated that an imnluonochemical technique for separating lactate dehyR drogenase ILD, EC 1.1.1.271 isoenzyme 1 ILD-1 ~is valuable when applied to laboratory assessment of patients with a suspected diagnosis of myocardial infarction iM.I.L The original work with this method I1) indicated that LD-1 was 94c; sensitive and 96% specific, and that in approximately 25';} of patients the test was diagnostic one day earlier than the L D - 1 / L D - 2 ratio. Liu et al. 12) found the method to be simple, more precise, more accurate, more sensitive and no less specific then electrophoresis for detection of M.I. Bruns el al. 13~ found LD-1 to be diagnostically useful on the first day of hospitalization. These workers indicated that the ratio of LD-1/total LD had greater diagnostic significance than LD-1 alone, with a predictive value of 95';; for M.I. Fike {4J suggested that immunochemical determination of LD-1 offers a suitable alternative to isoenzyme separation by electrophoresis. P r a g a y et al. I5~ cautioned t h a t while immunochemical determination of LD-1 is a good c o m p l e m e n t a r y diagnostic assay for M.I. it cannot replace electrophoretic separation entirely. This paper describes our experience with this test over a two-year period during a prospective study undertaken to assess its potential usefulness, and following adoption of a protocol subsequently developed in 1981 for routine use of the test in a general laboratory setting. We confirm that activities of LD-1 in serum are increased as early as and remain elevated longer than the MB isoenzyme of creatine kinase ICK, EC 2.7.3.21 in patients with M.I., giving a wider window of time during which the enzyme is diagnostic. Used as a screening

Correspondence: Dr. Lilian M. Ewen, Department of Clinical Biochemistry, Royal Columbian Hospital, New Westminster, B.C. V3L 3W7. Revised manuscript received March 25, 1983; accepted for publication April 14, 1983.

test, LD-1 renders electrophoretic CK and LD isoenzyme separation unnecessary in approximately 75c/, of specimens, but the test cannot be used to replace electrophoretic separation entirely. The test is simple, rapid and highly sensitive but because erythrocytes contain a high concentration of this isoenzyme, it is not completely specific and our findings with respect to this are also described. Materials and methods Reagents available in kit fbrm fi'om Roche Di,agnostics, Vaudreuil, Que. J7V 6B3 ICat. #431561 were used to separate LD-1 fi'om other LD isoenzymes. This method employs an antibody to bind all fractions containing the M monomer. Subsequent centrifugation separates bound fi'actions leaving only LD-1 in the s u p e r n a t a n t . In our work, the activity of this isoenzyme was then measured by a pyruvate-to-lactate assay using reagents fl'om Smith Kline I n s t r u m e n t s , Inc., Sunnyvale, CA 94086 (Cat. #86235) and a centrifugal analyser IMultistat llI. I n s t r u m e n t a t i o n Laboratories, Inc.. Lexington, MA, 02173L Reagents from Smith Kline Instruments were also used to measure total CK activities ICat. #86232). CK-MB was measured quantitatively on a Dupont ACA analyser IDupont I n s t r u m e n t s , Wilmington, DE 19803 ~, and electrophoretic separations of CK and LD isoenzymes were performed using the system of Helena Laboratories Corporation, Beaumont, Texas 77704. Lyophilised quality control material was obtained fl'om Hyland Diagnostics IDivision of Travenol Laboratories, Inc.~, Deerfield, IL 60015 ILots #18047001A and 18057001A were usedl.

Experimental design The work was conducted in four phases. 1. EVALUATION O1" THE PRECISION OF THE

LD

AND

LD-1

METHOI)S AND DETERMINATION OF THE REFERENCE RANGES

For between-run precision h u m a n lyophylised quality control material with both normal and elevated activities, reconstituted daily, was used. Results are shown in Table 1. The reference ranges were determined p a r a m e t r i c a l l y (mean _+ 2 S.D.! using preoperative specimens from 74 patients admitted for minor surgery including reconstruction of knee ligaments, t h u m b tendon graft, septoplasty, rhinoplasty, minor plastic surgery, inguinal hernia repair, dilatation and curettage, and tubal ligation. Results are shown in Table 2. The reference range for CK, used routinely in this laboratory and previously established

LACTATE DEHYDROGENASE ISOENZYME 1 IN MYOCARDIAL INFRACTION TABI,I.: 1 LD and LD-1 A c t i v i t y , - Determination of Between-Run Precision Using Human Lyophilised Quality Control Serum LD Activity (U/LI

S.D C.V. q':) n

307

3. SPECIFI(TI'Y OF TIlE ME'I'H()I) IN A (;ENERAL HOSPITAl, SETTIN(',: LD-1

INCII)EN('E ()F NON-MYO('ARDIAI, ELFVATIONS OF

LD-1 Activity IU/L)

Normal

Elevated

Normal

Elevated

156 4.0 2.6

417 11.2 2.7

57 5,0 9.1

418 10.0 2.4

36

38

36

38

TABI.E 2 Reference Ranges ILD and LD-1 Activitiesr' LD-1 IU/LI

LD qU/L~

LD-1 - 1 0 0 LD

20 - 70

1()3 - 232

20 - 40

:'Based on 74 samples.

Total LD and LD-1 activity were m e a s u r e d on 360 s a m p l e s in which total LD was requested by the physician from 262 p a t i e n t s a d m i t t e d to this g e n e r a l hospital. 4. APPIA{'ATI()N()F LD-1 A('TIVITY MEAStrREMENT T() R()UTINE USE Following completion of the above studies a protocol was developed for routine application of LD-1 a c t i v i t y m e a s u r e m e n t to testing, p a t i e n t s a d m i t t e d with a suspected diagnosis of M.I. ITable 3). Results SENSITIVITY C)F LD-1 IN t'ARI)IA(' PATIENTS Results are s u m m a r i z e d in Table 4. l a! P r e l i m i n a r y

TABI,E :3

Protocol tbr Routine Application of LD-I Activity Measurement 1. Assay total CK, total LD and LD-1 on admission and following two mornings. 2. If LD-1 - (;5 U/L in 2 samples 24 hours apart, M.I. ruled out. 3. IfLD-1 • 50'~ total LD, M.I. probable. 4. IfLD-1 . 6 5 U / L b u t .... 50'i totalLD, s e p a r a t e C K a n d L D isoenzymes by electrophoresis. 5. Only total LD and LD-1 needed to follow patients with evidence of M.I.

p a r a m e t r i c a l l y Imean -+ 2 S.D.) was 5 - 1 1 0 U/L. For CK-MB by Dupont ACA, the reference range recommended by the m a n u f a c t u r e r was used 1 0 - 8 U / L or 0-1.8(/( when total CK > twice the upper limit of its reference range). Only CK-MM was seen after electrophoresis of normal serum, and CK-MB was considered to be of m y o c a r d i a l origin if p r e s e n t as > 5';; of the total activity. Reference values for LD isoenzymes, expressed as p e r c e n t a g e s of the total, d e t e r m i n e d by i n t e g r a tion of the a r e a under the peak, were as published for the H e l e n a system. Visual inspection of scan peak h e i g h t s was also used in i n t e r p r e t a t i o n .

2. ASSESSMENTOF THE SENSITIVITYI)l~'THE LD-1 METHOI)IN CARI)IAC PATIENTS This p a r t of the study was conducted in two segments: (a) p r e l i m i n a ~ , - - to give an indication of w h e t h e r the test could be useful 165 specimens in which CK and LD isoenzymes were requested were utilised) and ¢bl c o n f i r m a t o ~ - - in which 3 or more serial specimens on 57 p a t i e n t s a d m i t t e d with a provisional diagnosis of m y o c a r d i a l infarction were assayed. In both s e g m e n t s of this phase CK, LD, LD-1, CK-MB Iby Dupont ACA), and CK and LD isoenzymes s e p a r a t e d by electrophoresis were measured.

The activities of 27 of' the 65 specimens (42';;) fell w i t h i n the reference range tbr LD-1. One of these had a q u e s t i o n a b l e trace of CK-MB by electrophoresis but all other tests were within normal limits. F i f t y - e i g h t percent of p a t i e n t s 138 of 65) showed an increase in serum LD-1 activity. Of these 38, 15 showed an increased CK-MB level by electrophoresis, 18 showed increased CK-MB by the Dupont ACA, and 24 p a t i e n t s showed an increase in LD-1/LD-2 ratio by electrophoresis. In 4 specimens in which CK-MB isoenzyme was present by electrophoresis, no CK-MB activity was detected by the Dupont ACA. In 3 specimens showing increased activity by the Dupont ACA no CK-MB band was detected by electrophoresis. There were no specimens in which CK-MB a c t i v i t y was detected a n d / o r in which the LD-1/LD-2 ratio was increased on electrophoresis, t h a t did not show an elevated LD-1 activity. This p r e l i m i n a r y phase indicated t h a t m e a s u r e m e n t of LD-1 by the method under study was more sensitive t h a n CK or LD isoenzyme d e t e r m i n a t i o n s by electrophoresis, or CK-MB d e t e r m i n a t i o n by Dupont ACA. t b) C o n f i r m a t o o ,

Of 182 specimens assayed, 73 I40'/; ) were within the reference range tbr LD-1 and isoenzyme a s s a y s showed normal p a t t e r n s . Sixty percent 1109) of the 182 specimens a s s a y e d showed an increase in LD-1 activity. Of these 109, 64 showed CK-MB by electrophoresis and 50 had an increased CK-MB activity by the Dupont ACA method. An increase in the LD-1/LD-2 ratio by electrophoresis was found in 102 specimens. Again, as in the p r e l i m i n a r y phase, there were no specimens in which CK-MB activity was detected or in which the LD-1/LD2 ratio by electrophoresis was increased which did not show an increased LD-1 activity by the immunological method. This phase confirmed the a d e q u a t e s e n s i t i v i t y of the test; there were no false n e g a t i v e s when compared with other c u r r e n t l y used tests.

308

EWEN TABI,E 4

Assessment of the Sensitivity of LD-1 Method in Cardiac Patients Ia ~ Prelim inurv

LD-I Activity Normal LD-1 Activity Increased CK-MB lelectrophoresis~

CK-MB t Dupont~ LD-1/LD-2 increased lelectrophoresis

I b I C o n f i r m a t t ~ J 3,

All samples 27/65 1 4 2 c ¢ } 73/182140':~1 38/65 1 5 8 ' 4 ) 109/18216()'~} Samples with increased activity 15/38" 139':~ t 64/109 159"; I 18/38" 147';~ 50/109 I46':/~ 24/38 163~ 102/109194':~}

*In 4 specimens in which CK-MB isoenzyme was present by electrophoresis no activity was detected by Dupont ACA. In 3 specimens showing increased CK-MB by Dupont ACA no MB band was detected by electrophoresis,

SPECIFICITY (}F LD-1 IN A GENERAL H{)SPI'I'ALSE'I~I'IN(;

LD-1 activity was assayed in 360 specimens from 262 unselected patients in which total LD activity was requested by the physician. Of these 262 patients, 77 (29~i~ showed 1 or more samples with elevated LD-1 activity. Forty-eight of these 77 samples were fbund to be from patients in cardiac wards, with a known cardiac problem, or in whom a cardiac origin could not be ruled out. LD-1 activites in 13 159~) of the r e m a i n i n g patients were between 70 and 80 U/L, < 2 S D . above the upper limit of the reference range, leaving samples from 16 patients t6~/~ ~showing a definite increase in LD-1 activity and an increase in total LD activity and in whom myocardial origin could not be implicated. Reasons for hospitalization in these patients included bowel resection, surgery for carcinoma of the gallbladder, squamous cell carcinoma of the oesophagus, surgery following fractured hip, p u l m o n a r y thromboembolism, surgical repair of broken patella and caesarian section. The conclusion from this phase was, therefore, t h a t a definite increase in LD-1 activity which was unlikely to be of myocardial origin could occur 6~ of the time in a general hospital population in which total LD activity was requested by the physician. Discussion While preoperative specimens from patients admitted for minor surgery are not ideal for d e t e r m i n i n g reference ranges, the upper limit found in this laboratory {70 U/L) coincides very closely with t h a t obtained using n o n - p a r a m e t r i c statistics by Bruns et al. Ill 173 U / L I using the same method for assaying LD-1, a similar assay for activity m e a s u r e m e n t IBoehringerM a n n h e i m UV system LDH-P) and sera from normal adult volunteers. The literature on the subject of laboratory confirmation in diagnosis of M.I. suggests t h a t samples should be drawn 3 - 6 h, 1 2 - 2 4 h and 3 6 - 4 8 h after the onset of chest pain and assayed for CK and LD isoenzymes. The time sequence of increase in enzyme activities following infarction d e m a n d s t h a t at least two samples be drawn 24 h apart, assayed for CK and LD isoenzymes and shown to be normal before a laboratory diagnosis of M.I. can be ruled out. As a routine in this hospital it was convenient for samples to be drawn at

intervals as described in the protocol given above ITable 3k In our study LD-1 determined by the immunological method under investigation was found to be the most sensitive of the tests performed, confirming the work of others ~1, 2~. In this study no specimen having a value in the reference range for LD-1 showed a CK-MB band or an increase in LD-1/LD-2 ratio when electrophoresis was done; therefore, electrophoretic separation of samples provided no additional diagnostic information and was redundant. During routine use of this test in this laboratory, specimens with an LD-1 value less t h a n 65 U / L are not assayed for CK or LD isoenzymes by electrophoresis. This criterion eliminates a p p r o x i m a t e l y 40~/~ of samples on which electrophoretic separation m i g h t otherwise be performed. l n a d d i t i o n , because a ratio of LD-1/LD-2 . 1 isconsidered diagnostic of M.I., it is unnecessary to do isoenzyme assays on any sample in which LD-1 is g r e a t e r t h a n 50~ of the total LD activity, since this sample would of necessity show an LD-1 activity > LD-2. In the study where three specimens from each patient were assayed this a m o u n t e d to 35c~ of all specimens. Use of these two criteria therefore eliminated 75~ of the isoenzyme analyses which would otherwise have been performed by electrophoresis. An additional m e a n s to reduce electrophoretic separation by a further 1-2(~ can be t a k e n by e l i m i n a t i n g subsequent samples from patients in whom a laboratory diagnosis of M.I. is made; i.e., where a m a r k e d increase in CK-MB a n d / o r an LD-1 > LD-2 activity is found in an early sample. M e a s u r e m e n t of LD-1 activity can be used to follow the progress of the patient. Normal total CK and LD activities cannot be used to rule out M.l. CK-MB and LD-1 m a y have increased although total activities r e m a i n within their reference ranges. Also, if the sample is drawn late following the episode of pain, the CK which m a y have been elevated, m a y h a v e returned to normal. Thus by m e a s u r e m e n t of LD-1 activity and use of the above criteria, samples on which the electrophoretic separation of CK and LD isoenzymes is required as an aid in the laboratory diagnosis of M.|. can be reduced to a p p r o x i m a t e l y one-quarter of those drawn under the t h r e e - s a m p l e regime n o r m a l l y recommended. B r u n s et at. (31 have suggested t h a t the ratio of LD-1/LD has diagnostic significance. We confirm this

LACTATE DEHYDROGENASE ISOENZYME 1 IN MYOCARDIALINFARCTION but caution that it cannot be applied in all patients. Where the ratio is greater than 1, i.e., LD-1 is greater than 50~ of total activity, it is diagnostic, but in patients with M.I. and liver involvement le.g., shock} where LD-5 is increased, although LD-1 is increased {and may exceed LD-21, the ratio does not exceed 1. In such patients the ratio of LD-1/total LD cannot be applied and electrophoresis is necessary to demonstrate the increased LD-1/LD-2 ratio and increased LD-5. In some patients, increased LD-5 may be the first change seen, before either CK-MB or LD-1 is increased. LD-1 activity was tbund to increase as quickly as CK-MB following the onset of pain and it remains elevated longer, giving a greater period of time during which diagnosis of M.I. can be confirmed. However, it suffers from the same disadvantages as the electrophoretic separation of isoenzymes with respect to nonspecificity; e.g., activities of LD-1 are increased in myocarditis. And the possibilities of a red cell origin for the extra LD-1 activity must always be kept in mind. Assay of LD-1 activity takes approximately halt' the time required for the electrophoretic separation of the isoenzymes. Measurement of LD-1 activity can serve, therefore, as a useful screening test and is especially applicable for small hospitals where isoenzyme fl'actionations are not carried out. The assay of LD-1

309

activity on sequential samples will provide a definitive answer on a significant proportion of specimens received.

References 1. Usategui-Gomez, M., Wicks, R. W., and Warshaw, W. Immunochemical determination of the heart isoenzyme of lactate dehydrogenase ILDH~) in human serum. Clin. Chem. 25, 729-734 c19791. 2. Liu, F., Belding, R., Usategui-Gomez, M.. and Reynoso, G. Immunochemical determination of LDH- 1. A met. J. Cl in. Path. 75,701 707f1981L 3. Bruns, D. E., Emerson, J. D., lntemann, S., and Bertholf, R. Lactate dehydrogenase isoenzyme-l: Changes during the first day after acute myocardial infarction. Clin. Chem. 27, 1821-1823 11981L 4. Fike, S. S. Immunochemical and electrophoretic methods for determination of lactate dehydrogenase isoenzyme 1 compared. Clin. Chem. 26, 794-795 ~1981L 5. Pragay, D. A., Fan, P., Toppin, M., and Gotthelf, J. A word of caution regarding the use of the Isomune LD1 kit IRoche DiagnosticsL Clin. Chem. 27. 365-366 11981L 6. Bruns, D. E., Emerson. J. C., Bertholf, R. L., Hill, K. E., and Savory. J. Lactate dehydroganse isoenzyme 1 with the centrifugal analyser after immunochemical removal of other isoenzymes. Clin. Chem. 27,922-923 119811.