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The quality control of serum lactate dehydrogenase isoenzyme separations using commercial quality control sera
53
Clinica Chimica Acta, 51 (1974) 53-66 0 Elsevier Scientific Publishing Company,
Amsterdam
-- Printed
in The Netherlands
CCA 6213
THE QUALITY CONTROL OF SERUM LACTATE DEHYDROGENASE ISOENZYME SEPARATIONS USING COMMERCIAL QUALITY CONTROL SERA
DORIS MCKENZIE
and A.R. HENDERSON
Department of Clinical Biochemistry, London, Ontario N6G 2K3 (Canada) (Received
University Hospital, 339 Windermere Road,
July 2, 1973)
Summary
The lactate dehydrogenase (L-1actate:NAD oxidoreductase, EC 1.1. 1.27; LDH) isoenzyme content of 31 commercially available quality control sera was determined using thin layer agarose and fluorimetry. Only one of these sera was specifically intended for isoenzyme quality control purposes. The group of human sera, with no additives, proved to be the only one having an LDH isoenzyme distribution suitable for quality control use. Within-batch and between-batch precision has been determined over a 2-3 month period for LDH isoenzyme separations using several human quality control sera. The control of quality of this procedure has been shown to be feasible.
Introduction
At a recent Symposium on Clinical Enzymology [l] Rosalki commented on the diverse methodologies currently available for the assay of the lactate dehydrogenase (LDH) isoenzymes and advocated a standardised technique as a means of improving intra- and inter-laboratory control of this assay. It is remarkable that 10 years after Tonks’ paper [2] there are still procedures which are carried out in clinical laboratories with little or no regard to the control of quality. Typical examples of these procedures are the omission of quality control materials and methods from chemical estimations on cerebrospinal fluid and urine, and from all electrophoretic procedures. This aspect of LDH isoenzyme methodology receives no attention in monographs [3-S] on the subject and an article [7] in the latest volume of Standard Methods of Clinical Chemistry although laying great stress on methodology, contains no mention of the quality control of LDH isoenzyme assays. Clearly, methods of quality control should be applied, if possible, to LDH isoenzyme assays and we have therefore examined the LDH isoenzyme content
of most of the commercially available sera with this in mind. The sera had levels of total LDH activity in either the normal or above normal range. None of the samples were regarded, by their manufacturers, as being an LDH isoenzyme assay control serum, with one exception, but as these sera are generally available in most laboratories, we decided to examine them for LDH isoenzyme content. It would certainly be an added advantage for a commonly used serum to be also available for LDH isoenzyme control. An acceptable normal human LDH isoenzyme pattern displays, on densitometry, symmetrical peaks and good separation of each of the five bands. In our experience, and also of others [8], about 70% of aI1 LDH isoenzyme patterns have either a normal distribution, an isomorphic increase or an increased LDHi and LDH? . These patterns are basically similar, in that the majority of LDH activity resides in the fast moving isoenzymes, and it therefore seemed reasonable to select quality control sera with a similar isoenzyme pattern. We therefore examined samples of commercial quality control sera for LDH isoenzyme patterns with these properties. Our findings suggest that material is currently available for the introduction of a quality control programme for LDH isoenzyme assays. Materials and Methods
Quality control
sera
The source, supplier (if different) and the types and serial numbers of the sera used are as follows: 1. BDH Chemicals, Toronto, Ontario. Seronorm {Lot 113). 2. BMC (Boehringer Mannheim Corporation) Diagnostics & Biochemicals Ltd., St. Laurent, Quebec, Precinorm E (Lot No. 214), Precipath E (Lot No. 206 X). 3. Dade Division, American Hospital Supply Corporation (supplied by Canlab, Toronto, Ontario). Monitrol I (Lot No, LTD 112 B), Enzatrol (Lot No. ET 238 B), Electrophoresis Control (Lot No. EC 100). 4. Hyland, Division Travenol Laboratories Inc., Malton, Ontario. MultiEnzyme Sera A (Lot No, 3055TOOlAl), B (Lot No. 3056TOOlAl), C (Lot NO. 3057TOOlAl); Multi-Enzyme Reference Sera V (Lot No. 3045R004Al), VI (Lot No 3046TOOlAl); Control Sera Unassayed I (Lot No. 0750TOllAl), II (Lot No. 075OTOOTAl). 5. Lederle Diagnostics Cyanamid of Canada Ltd, Montreal, Quebec. LederZyme E (Lot No. 2911-645Hl), LederZyme (Lot No. 2910-64081). 6. Metrix Chemical and Diagnostics Division, Armour Pharmaceutical Company (supplied by Maynard Diagnostics, Downsview, Ontario). Met& Normal (Lot Nos 5446 and 6171), Metrix Abnormal (Lot No. 1020). 7. A.R. Smith Laboratories (supplied by Maynard Diagnostics) Normal Control (Lot No. 2021201). 8, Smith Kline and French Canada Ltd, Montreal, Quebec. Eskafab Normal Control (Lot No. 4058 A), Abnormal Control (Lot No. 4102). 9. Warner-Chilcott Laboratories, Scarborough, Ontario. Versatol-E (lot Validate-A (Lot No. No. 1763031), Versatol-E-N (Lot No. 2011061), 2466111), Validate (Lot Nos 2535111 and 0391042), CaIibrated Automa~d
55
Lock-In (Vial No. 1087112, Kit No. 1248122), Versatol Automated Hi (Lot No. 0863092), Versatol Automated Lo (Lot No. 2343101). 10. Wellcome Reagents Ltd (supplied by Warner-Chilcott Laboratories). Wellcomtrol I (Lot No. K5825), II (Lot No. K6483), III (Lot No. K3500). 11. Worthington Biochemical Corporation (supplied by Canlab). Statzyme (Lot No. 2KA). All the sera were used before their expiry dates, and were reconstituted instructions. Except where stated the sera according to the manufacturers’ were always used within a few hours of reconstitution. Determination of serum LDH isoenzymes LDH isoenzymes were separated by agarose thin film electrophoresis [9] and incubated with 7.5 mM NAD and 0.5 M L-lactate at 38” for 15 min using the Analytical Chemists Inc. (Palo Alto, Calif.) system. After drying, NADH formation was detected by fluorescence (excitation 356 nm, emission 405 nm) on a Densicomp Model 445-50 Fluorescent Recording Densitometer (Clifford Instruments Inc., Natick, Mass.). Peaks were quantitated by peak height measurements. The mobilities of the LDH isoenzyme bands of the quality control sera were standardised with those of a fresh normal human serum by simultaneously electrophoresing the two samples in adjacent parts of the agarose strip. The adjacent separations were then similarly aligned in the densitometer and the tracings were superimposed. This procedure was carried out at least twice for all sera examined. Quality control procedure Vials of certain human quality control sera were reconstituted according to the manufacturers’ instructions on Mondays, used immediately, but were thereafter stored at room temperature (20” ) and sampled on the Wednesday and Friday of that week and were then discarded. Within-batch precision was determined from vials freshly reconstituted, except where stated, and betweenbatch precision was obtained by pooling the series of all determinations, obtained as described above, over a 2-3 month period. Total LDH assay LDH activity was determined by the pyruvate/lactate assay of Henry et al. [lo] using a temperature of 37” instead of the original temperature of 32”. Purified animal lactate dehydrogenase preparations LDHl sources: Crystalline suspension in ammonium sulphate from bovine heart, LDH activity 5500 units/ml, and crystalline suspension in ammonium sulphate from porcine heart, LDH activity 4500 units/ml. LDHS source: Lyophilized preparation from leporine muscle dissolved in 1 ml of water, LDH activity 3700 units/ml. All preparations were obtained from Sigma Chemical Co., St. Louis, Missouri. Results The quality
control
sera which we used were classified
according
to their
56 TABLE
I
CLASSIFICATION OF QUALITY ENZYME ADDITIVES
CONTROL
SERA
BY SPECIES
ORIGIN
AN11 ‘iOL’RCE
Class 1: Human sera 1.1 With no LDH isoenzymr additives: Validate; Monitrol I; Dade Electrophoresls Normal: LederZyme; Eskalab Normal; A.R. Smith Normal. 1.2
With homologous
1.3
With heterologous LDH isoenzyme additives from: 1.3.1. Bovine Heart LDH: Validate-A; Versatol-E; VersatoI-E-N; Versatol Automated Hi; Versatol Automated Lo.
LDH ~soeruvmr
additives:
Muscle LDH,
Calibrate
1.3.2
Bovine Heart and Leporine
Porcine Heart LDH: Multi-EnLyme Sera A. B, and C; Multi-Enzyme VI; Control Sera Unassayed I and II.
1.3.4
Porcine Heart and Lepnrine ll0t7IlaI.
Wellcomtrol
Metrix
Muscle LDH:
Automated
Lock-In;
Enratrol.
Metrir
Abnormal;
Reference
Lederzyme
Sera V and
E; Eskalab Ab-
additives)
II: Seronorm
Class 3: Bovine Sera (With no LDH isoenzyme Wellcomtrol
Control;
StatLyme.
1.3.3
Class 2: Equine Sera (With no LDH isoenzyme
OF LDH lSO_
additives)
I and III.
Class 4: Other Bovine Albumin base with Porcine Heart LDH: __~~__..~__~_~_~~ ~~~
Precinorm
E; Precipath.
source (Table I). This information was primarily obtained from the technical literature available with each batch of serum but in nearly all instances further enquiry to the Technical Departments of each manufacturer was necessary. In all cases the manufacturers were extremely helpful. Fig. 1 shows the patterns obtained from pure human quality control sera without LDH isoenzyme additions (Table I: Class 1.1). These LDH isoenzymes have the same mobility and distribution of activity as the sample of fresh normal human serum shown in Fig. la, except for the sample of Minotrol I which has a series of curious ‘webs’ between each peak particularly between Bands 1 and 2, and 3 and 4. We are indebted to David Plaut (Dade Technical Services) who has pointed out that this ‘webbing’ is due to storage of serum for some time before freeze drying. Sera which are lyophilized immediately or soon after venesection will not show this effect. These findings suggest that all the sera tested, with the exception of Monitrol I, would be suitable as quality control materials for the LDH isoenzyme assay. This point will be returned to later. Fig. 2 shows the pattern obtained from a human serum pool (Statzyme, Class 1.2) with the addition of, presumably, purified human red cell LDH isoenzymes (Worthington Biochemical Corporation do not stipulate the exact source except to state that the materials are obtained entirely from normal human blood). Thus, the sample largely contains LDH isoenzymes 2 and 3 with very slight activities due to LDHi and LDH,, . Clearly, this type of material would prove less effective in controlling a process which has both a separative and enzymatic component to monitor. Fig, 3 is obtained from human sera with the addition of bovine heart LDH isoenzymes (Class 1.3.1). Bovine LDHl has a slightly greater negative charge,
57
b
d
c
t i
:
t
h
Fig, 1. LDH isoenayme patterns of human quality control sera with no LDH k&enzyme additions. I Normal fresh human serum (LDR activity 340 munits( (b) Validate (237 munitsjmf). (c) Metrix Nor mal (234 munits/ml). (d) Monitrol I (234 munits/ml). (e) Dade Ebctrophoresis Control (219 murdtsjml) 5 (f) A.% Smith Normal (222 munits/ml). (g) Eskalab Normal (198 munitslml). (h). LederZyme (21! :s munits/ml), The vertical numbered lines indicate the mobilities of the respective human LDH isoenzyme run at the same time, and under the same conditions, as the quality control sera.
58
Fig. 2. LDH isoenzyme pattern tions. Statzyme (730 munits/ml).
of human quality control sera with homologous LDH isoenzyme Refer to Fig. 1 for the explanation of the vertical lines.
addi-
b
C
Fig. 3. LDH isoenzyme patterns of human quality control sera with bovine heart LDH isoenzyme additions. (a) Validate-A (551 munits/mf). (b) Versatol-E (812 munitsfmi). (c) Versatol-E-N (155 munits/ ml). (d) Calibrate Automatic Lock-In (391 munits/ml). (e) Versatol Hi (590 munitslml). (f) Versatol Lo (246 munits/ml). Refer to Fig. 1 for the explanation of the vertical lines.
59
Fig. 4. LDH isoenzyme pattern of human quality control sera with bovine heart and leporine muscle LDH isoenzyme additions. Enzatrol (723 munits/ml). Refer to Fig. 1 for the explanation of the vertical lines.
and bovine LDHz a slightly lesser negative charge than human LDHz . The human LDH components are clearly present in Validate-A but much less so in Versatol-E and Versatol-E-N. The patterns obtained for Calibrate Automated Lock-In, Versatol Automated Hi and Automated Lo show wide variations in both the human LDH isoenzyme content and bovine heart LDH. They are therefore all unsuitable for control of the LDH isoenzyme assay as the peaks are neither symmetrical nor well defined. Fig. 4 shows a human serum (Enzatrol) with both bovine heart LDHl and LDHz and leporine muscle LDHS added (Class 1.3.2). The human component is clearly very minor and the peaks are asymmetric. This product is therefore not in our opinion suitable for LDH isoenzyme quality control. Porcine heart LDH additive is favoured by Hyland (Class 1.3.3) just as Warner-Chilcott are almost the only users of bovine heart LDH. From a study of Fig. 5 it is clear that the recently introduced Hyland Multi-Enzyme Sera A, B and C have had the human LDH isoenzymes removed by the preliminary treatment of the serum pool, and that the LDH component of these sera consists largely of porcine heart LDHl . The earlier Hyland range includes Multi-Enzyme Reference sera V and VI which show the addition of a less purified porcine heart LDH preparation. Control Sera Unassayed I and II show considerable human LDH isoenzyme activity (particularly Serum I) but the addition of the purified porcine heart LDH preparation spoils the symmetry of the peaks and the distribution of activity. In summary, therefore, none of the Hyland preparations tested appears to us suitable for the control of LDH isoenzyme assays. The Boehringer Mannheim products, Precinorm E and Precipath, also contain porcine heart LDHl isoenzyme with no other LDH enzyme source, as the protein base is not serum but bovine albumin (Class 4, Table I). The pattern obtained for both these sera is exactly similar to that of Multi-Enzyme Serum A (Fig. 5).
60
Fig. 5. LDH isoenzyme patterns of human quality control sera with porcine heart LDH isoenzyme additions. (a) Multi-Enzyme A (B and C have a similar pattern differing only in total activity, A is 232 munits/ml). (b) Multi-Enzyme Reference Sera V (activity not determined). (c) Control Sera I (activity not determined). (d) Multi-Enzyme Reference Sera VI (267 munits/ml). Refer to Fig. 1 for the explanation of the vertical lines.
Fig. 6 shows the Metrix Abnormal LDH isoenzyme pattern (Class 1.3.4). The human LDH, and LDH3 components are clearly shown but the LDHl is obscured by the more negatively charged porcine LDH, and human LDH, and LDHS are similarly overwhelmed by the very large quantity of leporine LDH, which has a negative charge very similar to, but slightly less than, human LDH4. Here again the shouldered asymmetrical peaks make this serum in our opinion unsuitable for use as a quality control serum for LDH isoenzyme assays. Similar conclusions can be drawn for the LDH isoenzyme patterns of Eskalab Abnormal and LederZyme E (Fig. 6). Fig. 7 shows two examples of equine sera (Class 2) from Wellcome (Wellcomtrol II) and BDH (Seronorm). Both these sera show 5 bands of LDH activity, the main isoenzyme being LDH3. Otherwise these patterns are not identical. Comparing these separations with Fig. la it is clear the equine sera would require a longer electrophoretic run in order to separate the LDH isoenzymes to the same extent as the human isoenzymes. Thus, this type of serum could not be directly compared with human serum for the purposes of LDH isoenzyme control. The final group of sera to be tested was the bovine sera (Class 3, Table I).
61
a
Fig. 6. LDH isoenzyme pattern of human quality control serum with porcine heart and leporine muscle LDH isoenzyme additions. (a) Metrix Abnormal (6’75 munitslml. (b) LederZyme E (650 mu&s/ml). (c) Eskalab Abnormal (709 mu&s/ml). Refer to Fig. 1 for explanation of the vertical lines.
Wellcome appears to be the only manufacturer of this type of sera. The pattern shown in Fig. 8 is exactly similar for both Wellcomtrol I and III. The objections applied to the use of equine sera for the control of human LDH isoenzyme assays apply, with more force, to the bovine sera. We were interested to learn whether the manufacturing processes applied to the quality control sera alter the mobilities of the heterologous LDH addi-
62
b
Fig. 7. LDH isoenzyme patterns of equine Seronorm (628 munits/ml). (b) Wellcomtrol tical lines.
quality control sera with no LDH isoenzyme additions. (a) (542 munits/ml). Refer to Fig. 1 for explanation of the ver-
2
Fig. 8. LDH isoenzyme patterns of bovine quality control sera with no Wellcomtrol I (1980 munits/ml) is shown. Wellcomtrol III (1964 munits/ml) isoenzyme pattern. Refer to Fig. 1 for explanation of the vertical lines.
LDH isoenzyme additions. had exactly the same LDH
63
a
d
Fig. 9. LDH isoenzyme patterns of fresh human leporine LDH5. (a) Fresh normal human serum (425 (1425 munitslml). (c) As for (a) with added Bovine leporine LDHs (1355 munitslml). Refer to Fig. 1 for
serum with added bovine LDHl, porcine LDHl or munitslml). (b) As for (a) with added porcine LDHl LDHl (1795 munitslml). (d) As for (a) with added explanation of the vertical lines.
tions and we therefore added samples of purified bovine or porcine LDHl and leporine LDH5 to normal human serum. Fig. 9 shows that the patterns obtained are similar to those from the quality control sera with these heterologous LDH additions. We also know that the manufacturing processes do not alter the human LDH mobilities (Fig. 1). In Table II we give the within batch precisions for a fresh normal human serum and for six quality control sera. In general a standard deviation of less than 1% of the total enzyme activity can be achieved for each peak. Elevitch [ll] has quoted similar but slightly greater, values. Between batch precisions using these sera over a period of many weeks are also given in Table II. Validate, Metrix Normal, LederZyme and Eskalab Normal sera can all be determined with standard deviations of less than 2%. A.R. Smith Normal and Dade Electrophoresis Control sera produced standard deviations of more than 2% but this disparity is due to our method of handling (see Materials and Methods) and it will be discussed later. The within- and between-batch precisions were determined for two separate lots of Validate and Metrix Normal sera. The results show that there is good agreement between both lots.
64
TABLE
II
PRECISION Mean
(x).
Sera
OF
LDH
Standard
used
ISOENZYME
Deviation LDH isoenzyme
(S.D.)
ASSAY and
Coefficient
Within-batch R
of Variation
(%)
(C.V.) Between-hatch
-.__
S.D.
c.v
n
S.D.
(%) C.V.
?i
-__human
Normal serum
Validate
control
serum Lot
No.
2535111
1
23.26
1.09
4.7
16
2
36.80
0.91
2.5
16
3
20.59
0.41
2.0
16
4
8.53
0.58
6.7
16
5
10.76
1.23
11.4
16
1
21.67
0.82
3.8
14
21.71
1.55
7.2
33
2
35.38
0.68
1.9
14
36.63
1.50
4.1
33
3
19.99
0.85
4.3
14
20.48
0.97
4.7
33
0.51
5.9
14
8.42
1.06
12.6
33
11.7
33
4
Lot
No.
0391042
8.65
5
14.30
0.64
4.5
14
12.83
1.50
1
23.3
0.64
2.7
15
22.6
1.7
7.4
28
2
37.6
0.78
2.0
15
38.7
1.6
4.1
28
3
22.1
0.80
3.6
15
21.9
0.8
3.6
28
4
6.8
0.58
8.5
15
6.6
1.0
14.9
28
5
10.0
0.66
6.6
15
9.9
1.2
12.1
28 24
Metrix
normal
1
23.3
1.11
4.8
24
21.4
1.33
6.2
control
serum
2
35.6
0.93
2.6
24
36.6
1.64
4.5
24
5446
3
22.4
0.51
2.3
24
23.2
0.78
3.4
24
Lot
Lot
No.
No.
6171
LederZyme
control
serum
Eskalab control
normal serum
4
8.2
0.61
7.4
24
8.4
1.26
1 5.1
24
5
10.4
0.14
7.1
24
10.6
1.72
1 6.3
24
1
22.3
0.87
3.9
16
23.9
1.32
5.5
30
2
37.7
0.86
2.2
16
39.1
1.93
4.9
30
3
22.2
0.63
2.8
16
21.7
1.00
4.6
30
4
7.1
0.37
5.1
16
6.3
1.12
1 1.7
30
1.9
21.8
30
5
10.5
1.36
12.9
16
8.9
1
20.7
0.43
2.0
15
22.5
1.14
5.0
2
35.9
0.34
0.9
15
37.9
1.27
3.3
18
3
22.3
0.48
2.1
15
21.4
0.83
3.8
18 18
4
9.1
0.36
3.9
15
7.8
0.80
11.4
5
11.8
0.46
3.8
15
10.3
0.97
9.3
18
1
19.7
0.75
3.8
16
20.4
1.31
6.4
18
2
34.9
0.52
1.4
16
36.3
1.92
5.3
18
3
21.7
0.63
2.8
16
21.7
0.66
3.0
18
0.54
5.8
16
8.8
1.04
11.7
18
1.85
14.6
18
4
A.R. control
Dade
Smith
normal
serum
Electrophore-
sis control
18
9.2
5
14.4
0.65
4.5
16
12.6
1
22.0
0.62
2.8
16
24.2
2.17
8.9
22
2
38.4
0.41
1.0
16
38.6
2.37
6.1
22
3
21.2
0.58
2.1
16
20.8
0.79
3.7
22
4
4.1
0.46
16
5.2
2.19
42.0
22
5
14.3
0.68
4.7
16
11.0
2.43
22.0
22
1
19.4
0.78
4.0
12
22.2
4.79
21.6
21
2
34.5
0.72
2.0
12
40.4
4.39
10.8
27
3
23.0
0.66
2.8
12
24.3
1.34
5.5
4
10.0
0.63
6.2
12
6.4
2.59
40.7
27
5
12.9
1.13
8.8
12
5.9
4.9
83.5
27
11.4
27
Discussion As suggested in the introduction, a serum containing the majority of its activity in the fast moving LDH isoenzymes would appear to be the best type of material to use for the quality control of human LDH isoenzyme separations. Our survey of LDH-containing quality control sera indicates that the
65
patterns obtained from pure human control sera with no LDH isoenzyme additions would best fit this requirement. Thus any of the sera listed in Class 1.1 of Table I could be used. However, it is clear from Fig. 1 that Monitrol I is not suitable because of the asymmetry of the peaks due apparently to the delay in lyophilizing this material after it was pooled and collected. We have considered how far the facts about any delay in lyophilizing are available to a user. An examination of the information available with each of the Class 1.1 (Table I) sera gave the following information. Dade Electrophoresis Control “ . . . . prepared from fresh human blood . ...“. Dade Monitrol I contains no statement on this point in the pack, but in back up literature from the manufacturer it is stated that “.... bleeding donor to freezing of minor pool must not exceed 6 hours”. It is not clear from the information contained in the Lederle pack how fresh the serum might be. The Metrix normal sera are “.... freshly drawn . . . (with) . . . rapid processing”; A.R. Smith state “.... the reconstituted serum parallels behaviour of freshly drawn serum . ...“. Smith, Kline and French (Eskalab Normal) “._. processing is completed as quickly as possible . ...“‘. General Diagnostics (Validate) “. . . . prepared from freshly drawn blood. The serum is rapidly freeze-dried . ...“. Dade thus appears to be the only manufacturer who gives a specific time limit as to processing, but the product about which they give this information is the only one of the nine sera tested (two lots each of Validate and Metrix Normal) which shows “webbing” (Fig. 1). We have therefore assumed that all the other sera are as “fresh” as normal serum on basis of our results but clearly in view of the almost complete absence of detailed information on the time limits associated with processing each batch, users should check the individual batches of serum which they wish to use for quality control purposes. The precisions associated with the use of the Class 1.1 sera (Table I) were investigated, with the exception of Monitrol I. From a consideration of Table II it is clear that the batches of Validate, Metrix Normal, LederZyme Control and Eskalab Normal which we tested would all be satisfactory for use as quality control sera for LDH isoenzyme separations. We have also observed satisfactory precision for different lots of Validate and Metrix Normal. It must be stressed that the conditions under which we tested the sera were very rigorous; they were left standing at room temperature for up to 4 days (see Materials and Methods) and each of the manufacturers specifically states (in their package leaflets) that serum, once reconstituted must not be handled in this manner. The reason for this treatment was, of course, the well-known cold lability of LDH5. Under this treatment neither Dade Electrophoresis Control nor A.R. Smith Normal Control did well (Table II). We noted that if only fresh samples of either serum were used, then between-batch precisions similar to those obtained with the other sera were observed. However, using fresh vials of either serum every day would clearly be considerably more expensive than the procedure which we adopted. We can conclude therefore that quality control of LDH isoenzyme separations is practicable and possible provided that the control serum is human, has no additives, and is lyophilized soon after venesection. In our experience any of
66
the following sera would be suitable (with the provisions noted above): Validate, Metrix Normal, LederZyme Normal, Eskalab Normal, A.R. Smith Normal and Dade Electrophoresis Control. References 1 S.B. Rosalki. Paper given at the Association of Clinical Biochemists Symposium: The Optimisation and Standardisation of Enzyme Assays. Manchester, England, 21 Feb. 1973. See also S.B. Rosalki. Clin. Biochem. (In Press). 2 D.B. Tanks, Clin. Chem.. 9 (1963) 127. 3 H.A. Zondag, Determination and Diagnostic Significance of Lactate Dehydrogenase Isoenzymes. Van Gorcum, Assen, 1962. 4 A.L Latner and A.W. Skillen. Isoenzymes in Biology and Medicine, Academic Press. London, 1968. 5 J.H. Wilkinson. Isoenzymes, Second Edition, Chapman & Hall, London, 1970. 6 G.J. Brewer, An Introduction to Isoenzyme Techniques, Academic Press, New York, 1970. 7 A.A. Dietz, T. Lubrano and H.M. Rubinstein in G.R. Cooper (Ed.) Standard Methods of Clinical Chemistry. Vol. 7, Academic Press, New York, 1972, P. 49. 8 W.R. Dito. Am. J. Clin. Pathol.. 59 (1973) 439. 9 F.R. Elevitch, S.B. Aronwn, T.V. Feichtmeir and M.L. Enterline. Am. J. Clin. Pathol.. 46 (1966) 692. 10 R.J. Henry. N. Chiamori, O.J. Golub and S. Berkman, Am. J. Clin. Pathol.. 34 (1960) 381. 11 F.R. Elevitch, Fluorometric Techniques in Clinical Chemistry, Little, Brown and Co., Boston. 1973. p. 233.
Clinica Chimica Acta, 51 (1974) 53-66 0 Elsevier Scientific Publishing Company,
Amsterdam
-- Printed
in The Netherlands
CCA 6213
THE QUALITY CONTROL OF SERUM LACTATE DEHYDROGENASE ISOENZYME SEPARATIONS USING COMMERCIAL QUALITY CONTROL SERA
DORIS MCKENZIE
and A.R. HENDERSON
Department of Clinical Biochemistry, London, Ontario N6G 2K3 (Canada) (Received
University Hospital, 339 Windermere Road,
July 2, 1973)
Summary
The lactate dehydrogenase (L-1actate:NAD oxidoreductase, EC 1.1. 1.27; LDH) isoenzyme content of 31 commercially available quality control sera was determined using thin layer agarose and fluorimetry. Only one of these sera was specifically intended for isoenzyme quality control purposes. The group of human sera, with no additives, proved to be the only one having an LDH isoenzyme distribution suitable for quality control use. Within-batch and between-batch precision has been determined over a 2-3 month period for LDH isoenzyme separations using several human quality control sera. The control of quality of this procedure has been shown to be feasible.
Introduction
At a recent Symposium on Clinical Enzymology [l] Rosalki commented on the diverse methodologies currently available for the assay of the lactate dehydrogenase (LDH) isoenzymes and advocated a standardised technique as a means of improving intra- and inter-laboratory control of this assay. It is remarkable that 10 years after Tonks’ paper [2] there are still procedures which are carried out in clinical laboratories with little or no regard to the control of quality. Typical examples of these procedures are the omission of quality control materials and methods from chemical estimations on cerebrospinal fluid and urine, and from all electrophoretic procedures. This aspect of LDH isoenzyme methodology receives no attention in monographs [3-S] on the subject and an article [7] in the latest volume of Standard Methods of Clinical Chemistry although laying great stress on methodology, contains no mention of the quality control of LDH isoenzyme assays. Clearly, methods of quality control should be applied, if possible, to LDH isoenzyme assays and we have therefore examined the LDH isoenzyme content
of most of the commercially available sera with this in mind. The sera had levels of total LDH activity in either the normal or above normal range. None of the samples were regarded, by their manufacturers, as being an LDH isoenzyme assay control serum, with one exception, but as these sera are generally available in most laboratories, we decided to examine them for LDH isoenzyme content. It would certainly be an added advantage for a commonly used serum to be also available for LDH isoenzyme control. An acceptable normal human LDH isoenzyme pattern displays, on densitometry, symmetrical peaks and good separation of each of the five bands. In our experience, and also of others [8], about 70% of aI1 LDH isoenzyme patterns have either a normal distribution, an isomorphic increase or an increased LDHi and LDH? . These patterns are basically similar, in that the majority of LDH activity resides in the fast moving isoenzymes, and it therefore seemed reasonable to select quality control sera with a similar isoenzyme pattern. We therefore examined samples of commercial quality control sera for LDH isoenzyme patterns with these properties. Our findings suggest that material is currently available for the introduction of a quality control programme for LDH isoenzyme assays. Materials and Methods
Quality control
sera
The source, supplier (if different) and the types and serial numbers of the sera used are as follows: 1. BDH Chemicals, Toronto, Ontario. Seronorm {Lot 113). 2. BMC (Boehringer Mannheim Corporation) Diagnostics & Biochemicals Ltd., St. Laurent, Quebec, Precinorm E (Lot No. 214), Precipath E (Lot No. 206 X). 3. Dade Division, American Hospital Supply Corporation (supplied by Canlab, Toronto, Ontario). Monitrol I (Lot No, LTD 112 B), Enzatrol (Lot No. ET 238 B), Electrophoresis Control (Lot No. EC 100). 4. Hyland, Division Travenol Laboratories Inc., Malton, Ontario. MultiEnzyme Sera A (Lot No, 3055TOOlAl), B (Lot No. 3056TOOlAl), C (Lot NO. 3057TOOlAl); Multi-Enzyme Reference Sera V (Lot No. 3045R004Al), VI (Lot No 3046TOOlAl); Control Sera Unassayed I (Lot No. 0750TOllAl), II (Lot No. 075OTOOTAl). 5. Lederle Diagnostics Cyanamid of Canada Ltd, Montreal, Quebec. LederZyme E (Lot No. 2911-645Hl), LederZyme (Lot No. 2910-64081). 6. Metrix Chemical and Diagnostics Division, Armour Pharmaceutical Company (supplied by Maynard Diagnostics, Downsview, Ontario). Met& Normal (Lot Nos 5446 and 6171), Metrix Abnormal (Lot No. 1020). 7. A.R. Smith Laboratories (supplied by Maynard Diagnostics) Normal Control (Lot No. 2021201). 8, Smith Kline and French Canada Ltd, Montreal, Quebec. Eskafab Normal Control (Lot No. 4058 A), Abnormal Control (Lot No. 4102). 9. Warner-Chilcott Laboratories, Scarborough, Ontario. Versatol-E (lot Validate-A (Lot No. No. 1763031), Versatol-E-N (Lot No. 2011061), 2466111), Validate (Lot Nos 2535111 and 0391042), CaIibrated Automa~d
55
Lock-In (Vial No. 1087112, Kit No. 1248122), Versatol Automated Hi (Lot No. 0863092), Versatol Automated Lo (Lot No. 2343101). 10. Wellcome Reagents Ltd (supplied by Warner-Chilcott Laboratories). Wellcomtrol I (Lot No. K5825), II (Lot No. K6483), III (Lot No. K3500). 11. Worthington Biochemical Corporation (supplied by Canlab). Statzyme (Lot No. 2KA). All the sera were used before their expiry dates, and were reconstituted instructions. Except where stated the sera according to the manufacturers’ were always used within a few hours of reconstitution. Determination of serum LDH isoenzymes LDH isoenzymes were separated by agarose thin film electrophoresis [9] and incubated with 7.5 mM NAD and 0.5 M L-lactate at 38” for 15 min using the Analytical Chemists Inc. (Palo Alto, Calif.) system. After drying, NADH formation was detected by fluorescence (excitation 356 nm, emission 405 nm) on a Densicomp Model 445-50 Fluorescent Recording Densitometer (Clifford Instruments Inc., Natick, Mass.). Peaks were quantitated by peak height measurements. The mobilities of the LDH isoenzyme bands of the quality control sera were standardised with those of a fresh normal human serum by simultaneously electrophoresing the two samples in adjacent parts of the agarose strip. The adjacent separations were then similarly aligned in the densitometer and the tracings were superimposed. This procedure was carried out at least twice for all sera examined. Quality control procedure Vials of certain human quality control sera were reconstituted according to the manufacturers’ instructions on Mondays, used immediately, but were thereafter stored at room temperature (20” ) and sampled on the Wednesday and Friday of that week and were then discarded. Within-batch precision was determined from vials freshly reconstituted, except where stated, and betweenbatch precision was obtained by pooling the series of all determinations, obtained as described above, over a 2-3 month period. Total LDH assay LDH activity was determined by the pyruvate/lactate assay of Henry et al. [lo] using a temperature of 37” instead of the original temperature of 32”. Purified animal lactate dehydrogenase preparations LDHl sources: Crystalline suspension in ammonium sulphate from bovine heart, LDH activity 5500 units/ml, and crystalline suspension in ammonium sulphate from porcine heart, LDH activity 4500 units/ml. LDHS source: Lyophilized preparation from leporine muscle dissolved in 1 ml of water, LDH activity 3700 units/ml. All preparations were obtained from Sigma Chemical Co., St. Louis, Missouri. Results The quality
control
sera which we used were classified
according
to their
56 TABLE
I
CLASSIFICATION OF QUALITY ENZYME ADDITIVES
CONTROL
SERA
BY SPECIES
ORIGIN
AN11 ‘iOL’RCE
Class 1: Human sera 1.1 With no LDH isoenzymr additives: Validate; Monitrol I; Dade Electrophoresls Normal: LederZyme; Eskalab Normal; A.R. Smith Normal. 1.2
With homologous
1.3
With heterologous LDH isoenzyme additives from: 1.3.1. Bovine Heart LDH: Validate-A; Versatol-E; VersatoI-E-N; Versatol Automated Hi; Versatol Automated Lo.
LDH ~soeruvmr
additives:
Muscle LDH,
Calibrate
1.3.2
Bovine Heart and Leporine
Porcine Heart LDH: Multi-EnLyme Sera A. B, and C; Multi-Enzyme VI; Control Sera Unassayed I and II.
1.3.4
Porcine Heart and Lepnrine ll0t7IlaI.
Wellcomtrol
Metrix
Muscle LDH:
Automated
Lock-In;
Enratrol.
Metrir
Abnormal;
Reference
Lederzyme
Sera V and
E; Eskalab Ab-
additives)
II: Seronorm
Class 3: Bovine Sera (With no LDH isoenzyme Wellcomtrol
Control;
StatLyme.
1.3.3
Class 2: Equine Sera (With no LDH isoenzyme
OF LDH lSO_
additives)
I and III.
Class 4: Other Bovine Albumin base with Porcine Heart LDH: __~~__..~__~_~_~~ ~~~
Precinorm
E; Precipath.
source (Table I). This information was primarily obtained from the technical literature available with each batch of serum but in nearly all instances further enquiry to the Technical Departments of each manufacturer was necessary. In all cases the manufacturers were extremely helpful. Fig. 1 shows the patterns obtained from pure human quality control sera without LDH isoenzyme additions (Table I: Class 1.1). These LDH isoenzymes have the same mobility and distribution of activity as the sample of fresh normal human serum shown in Fig. la, except for the sample of Minotrol I which has a series of curious ‘webs’ between each peak particularly between Bands 1 and 2, and 3 and 4. We are indebted to David Plaut (Dade Technical Services) who has pointed out that this ‘webbing’ is due to storage of serum for some time before freeze drying. Sera which are lyophilized immediately or soon after venesection will not show this effect. These findings suggest that all the sera tested, with the exception of Monitrol I, would be suitable as quality control materials for the LDH isoenzyme assay. This point will be returned to later. Fig. 2 shows the pattern obtained from a human serum pool (Statzyme, Class 1.2) with the addition of, presumably, purified human red cell LDH isoenzymes (Worthington Biochemical Corporation do not stipulate the exact source except to state that the materials are obtained entirely from normal human blood). Thus, the sample largely contains LDH isoenzymes 2 and 3 with very slight activities due to LDHi and LDH,, . Clearly, this type of material would prove less effective in controlling a process which has both a separative and enzymatic component to monitor. Fig, 3 is obtained from human sera with the addition of bovine heart LDH isoenzymes (Class 1.3.1). Bovine LDHl has a slightly greater negative charge,
57
b
d
c
t i
:
t
h
Fig, 1. LDH isoenayme patterns of human quality control sera with no LDH k&enzyme additions. I Normal fresh human serum (LDR activity 340 munits( (b) Validate (237 munitsjmf). (c) Metrix Nor mal (234 munits/ml). (d) Monitrol I (234 munits/ml). (e) Dade Ebctrophoresis Control (219 murdtsjml) 5 (f) A.% Smith Normal (222 munits/ml). (g) Eskalab Normal (198 munitslml). (h). LederZyme (21! :s munits/ml), The vertical numbered lines indicate the mobilities of the respective human LDH isoenzyme run at the same time, and under the same conditions, as the quality control sera.
58
Fig. 2. LDH isoenzyme pattern tions. Statzyme (730 munits/ml).
of human quality control sera with homologous LDH isoenzyme Refer to Fig. 1 for the explanation of the vertical lines.
addi-
b
C
Fig. 3. LDH isoenzyme patterns of human quality control sera with bovine heart LDH isoenzyme additions. (a) Validate-A (551 munits/mf). (b) Versatol-E (812 munitsfmi). (c) Versatol-E-N (155 munits/ ml). (d) Calibrate Automatic Lock-In (391 munits/ml). (e) Versatol Hi (590 munitslml). (f) Versatol Lo (246 munits/ml). Refer to Fig. 1 for the explanation of the vertical lines.
59
Fig. 4. LDH isoenzyme pattern of human quality control sera with bovine heart and leporine muscle LDH isoenzyme additions. Enzatrol (723 munits/ml). Refer to Fig. 1 for the explanation of the vertical lines.
and bovine LDHz a slightly lesser negative charge than human LDHz . The human LDH components are clearly present in Validate-A but much less so in Versatol-E and Versatol-E-N. The patterns obtained for Calibrate Automated Lock-In, Versatol Automated Hi and Automated Lo show wide variations in both the human LDH isoenzyme content and bovine heart LDH. They are therefore all unsuitable for control of the LDH isoenzyme assay as the peaks are neither symmetrical nor well defined. Fig. 4 shows a human serum (Enzatrol) with both bovine heart LDHl and LDHz and leporine muscle LDHS added (Class 1.3.2). The human component is clearly very minor and the peaks are asymmetric. This product is therefore not in our opinion suitable for LDH isoenzyme quality control. Porcine heart LDH additive is favoured by Hyland (Class 1.3.3) just as Warner-Chilcott are almost the only users of bovine heart LDH. From a study of Fig. 5 it is clear that the recently introduced Hyland Multi-Enzyme Sera A, B and C have had the human LDH isoenzymes removed by the preliminary treatment of the serum pool, and that the LDH component of these sera consists largely of porcine heart LDHl . The earlier Hyland range includes Multi-Enzyme Reference sera V and VI which show the addition of a less purified porcine heart LDH preparation. Control Sera Unassayed I and II show considerable human LDH isoenzyme activity (particularly Serum I) but the addition of the purified porcine heart LDH preparation spoils the symmetry of the peaks and the distribution of activity. In summary, therefore, none of the Hyland preparations tested appears to us suitable for the control of LDH isoenzyme assays. The Boehringer Mannheim products, Precinorm E and Precipath, also contain porcine heart LDHl isoenzyme with no other LDH enzyme source, as the protein base is not serum but bovine albumin (Class 4, Table I). The pattern obtained for both these sera is exactly similar to that of Multi-Enzyme Serum A (Fig. 5).
60
Fig. 5. LDH isoenzyme patterns of human quality control sera with porcine heart LDH isoenzyme additions. (a) Multi-Enzyme A (B and C have a similar pattern differing only in total activity, A is 232 munits/ml). (b) Multi-Enzyme Reference Sera V (activity not determined). (c) Control Sera I (activity not determined). (d) Multi-Enzyme Reference Sera VI (267 munits/ml). Refer to Fig. 1 for the explanation of the vertical lines.
Fig. 6 shows the Metrix Abnormal LDH isoenzyme pattern (Class 1.3.4). The human LDH, and LDH3 components are clearly shown but the LDHl is obscured by the more negatively charged porcine LDH, and human LDH, and LDHS are similarly overwhelmed by the very large quantity of leporine LDH, which has a negative charge very similar to, but slightly less than, human LDH4. Here again the shouldered asymmetrical peaks make this serum in our opinion unsuitable for use as a quality control serum for LDH isoenzyme assays. Similar conclusions can be drawn for the LDH isoenzyme patterns of Eskalab Abnormal and LederZyme E (Fig. 6). Fig. 7 shows two examples of equine sera (Class 2) from Wellcome (Wellcomtrol II) and BDH (Seronorm). Both these sera show 5 bands of LDH activity, the main isoenzyme being LDH3. Otherwise these patterns are not identical. Comparing these separations with Fig. la it is clear the equine sera would require a longer electrophoretic run in order to separate the LDH isoenzymes to the same extent as the human isoenzymes. Thus, this type of serum could not be directly compared with human serum for the purposes of LDH isoenzyme control. The final group of sera to be tested was the bovine sera (Class 3, Table I).
61
a
Fig. 6. LDH isoenzyme pattern of human quality control serum with porcine heart and leporine muscle LDH isoenzyme additions. (a) Metrix Abnormal (6’75 munitslml. (b) LederZyme E (650 mu&s/ml). (c) Eskalab Abnormal (709 mu&s/ml). Refer to Fig. 1 for explanation of the vertical lines.
Wellcome appears to be the only manufacturer of this type of sera. The pattern shown in Fig. 8 is exactly similar for both Wellcomtrol I and III. The objections applied to the use of equine sera for the control of human LDH isoenzyme assays apply, with more force, to the bovine sera. We were interested to learn whether the manufacturing processes applied to the quality control sera alter the mobilities of the heterologous LDH addi-
62
b
Fig. 7. LDH isoenzyme patterns of equine Seronorm (628 munits/ml). (b) Wellcomtrol tical lines.
quality control sera with no LDH isoenzyme additions. (a) (542 munits/ml). Refer to Fig. 1 for explanation of the ver-
2
Fig. 8. LDH isoenzyme patterns of bovine quality control sera with no Wellcomtrol I (1980 munits/ml) is shown. Wellcomtrol III (1964 munits/ml) isoenzyme pattern. Refer to Fig. 1 for explanation of the vertical lines.
LDH isoenzyme additions. had exactly the same LDH
63
a
d
Fig. 9. LDH isoenzyme patterns of fresh human leporine LDH5. (a) Fresh normal human serum (425 (1425 munitslml). (c) As for (a) with added Bovine leporine LDHs (1355 munitslml). Refer to Fig. 1 for
serum with added bovine LDHl, porcine LDHl or munitslml). (b) As for (a) with added porcine LDHl LDHl (1795 munitslml). (d) As for (a) with added explanation of the vertical lines.
tions and we therefore added samples of purified bovine or porcine LDHl and leporine LDH5 to normal human serum. Fig. 9 shows that the patterns obtained are similar to those from the quality control sera with these heterologous LDH additions. We also know that the manufacturing processes do not alter the human LDH mobilities (Fig. 1). In Table II we give the within batch precisions for a fresh normal human serum and for six quality control sera. In general a standard deviation of less than 1% of the total enzyme activity can be achieved for each peak. Elevitch [ll] has quoted similar but slightly greater, values. Between batch precisions using these sera over a period of many weeks are also given in Table II. Validate, Metrix Normal, LederZyme and Eskalab Normal sera can all be determined with standard deviations of less than 2%. A.R. Smith Normal and Dade Electrophoresis Control sera produced standard deviations of more than 2% but this disparity is due to our method of handling (see Materials and Methods) and it will be discussed later. The within- and between-batch precisions were determined for two separate lots of Validate and Metrix Normal sera. The results show that there is good agreement between both lots.
64
TABLE
II
PRECISION Mean
(x).
Sera
OF
LDH
Standard
used
ISOENZYME
Deviation LDH isoenzyme
(S.D.)
ASSAY and
Coefficient
Within-batch R
of Variation
(%)
(C.V.) Between-hatch
-.__
S.D.
c.v
n
S.D.
(%) C.V.
?i
-__human
Normal serum
Validate
control
serum Lot
No.
2535111
1
23.26
1.09
4.7
16
2
36.80
0.91
2.5
16
3
20.59
0.41
2.0
16
4
8.53
0.58
6.7
16
5
10.76
1.23
11.4
16
1
21.67
0.82
3.8
14
21.71
1.55
7.2
33
2
35.38
0.68
1.9
14
36.63
1.50
4.1
33
3
19.99
0.85
4.3
14
20.48
0.97
4.7
33
0.51
5.9
14
8.42
1.06
12.6
33
11.7
33
4
Lot
No.
0391042
8.65
5
14.30
0.64
4.5
14
12.83
1.50
1
23.3
0.64
2.7
15
22.6
1.7
7.4
28
2
37.6
0.78
2.0
15
38.7
1.6
4.1
28
3
22.1
0.80
3.6
15
21.9
0.8
3.6
28
4
6.8
0.58
8.5
15
6.6
1.0
14.9
28
5
10.0
0.66
6.6
15
9.9
1.2
12.1
28 24
Metrix
normal
1
23.3
1.11
4.8
24
21.4
1.33
6.2
control
serum
2
35.6
0.93
2.6
24
36.6
1.64
4.5
24
5446
3
22.4
0.51
2.3
24
23.2
0.78
3.4
24
Lot
Lot
No.
No.
6171
LederZyme
control
serum
Eskalab control
normal serum
4
8.2
0.61
7.4
24
8.4
1.26
1 5.1
24
5
10.4
0.14
7.1
24
10.6
1.72
1 6.3
24
1
22.3
0.87
3.9
16
23.9
1.32
5.5
30
2
37.7
0.86
2.2
16
39.1
1.93
4.9
30
3
22.2
0.63
2.8
16
21.7
1.00
4.6
30
4
7.1
0.37
5.1
16
6.3
1.12
1 1.7
30
1.9
21.8
30
5
10.5
1.36
12.9
16
8.9
1
20.7
0.43
2.0
15
22.5
1.14
5.0
2
35.9
0.34
0.9
15
37.9
1.27
3.3
18
3
22.3
0.48
2.1
15
21.4
0.83
3.8
18 18
4
9.1
0.36
3.9
15
7.8
0.80
11.4
5
11.8
0.46
3.8
15
10.3
0.97
9.3
18
1
19.7
0.75
3.8
16
20.4
1.31
6.4
18
2
34.9
0.52
1.4
16
36.3
1.92
5.3
18
3
21.7
0.63
2.8
16
21.7
0.66
3.0
18
0.54
5.8
16
8.8
1.04
11.7
18
1.85
14.6
18
4
A.R. control
Dade
Smith
normal
serum
Electrophore-
sis control
18
9.2
5
14.4
0.65
4.5
16
12.6
1
22.0
0.62
2.8
16
24.2
2.17
8.9
22
2
38.4
0.41
1.0
16
38.6
2.37
6.1
22
3
21.2
0.58
2.1
16
20.8
0.79
3.7
22
4
4.1
0.46
16
5.2
2.19
42.0
22
5
14.3
0.68
4.7
16
11.0
2.43
22.0
22
1
19.4
0.78
4.0
12
22.2
4.79
21.6
21
2
34.5
0.72
2.0
12
40.4
4.39
10.8
27
3
23.0
0.66
2.8
12
24.3
1.34
5.5
4
10.0
0.63
6.2
12
6.4
2.59
40.7
27
5
12.9
1.13
8.8
12
5.9
4.9
83.5
27
11.4
27
Discussion As suggested in the introduction, a serum containing the majority of its activity in the fast moving LDH isoenzymes would appear to be the best type of material to use for the quality control of human LDH isoenzyme separations. Our survey of LDH-containing quality control sera indicates that the
65
patterns obtained from pure human control sera with no LDH isoenzyme additions would best fit this requirement. Thus any of the sera listed in Class 1.1 of Table I could be used. However, it is clear from Fig. 1 that Monitrol I is not suitable because of the asymmetry of the peaks due apparently to the delay in lyophilizing this material after it was pooled and collected. We have considered how far the facts about any delay in lyophilizing are available to a user. An examination of the information available with each of the Class 1.1 (Table I) sera gave the following information. Dade Electrophoresis Control “ . . . . prepared from fresh human blood . ...“. Dade Monitrol I contains no statement on this point in the pack, but in back up literature from the manufacturer it is stated that “.... bleeding donor to freezing of minor pool must not exceed 6 hours”. It is not clear from the information contained in the Lederle pack how fresh the serum might be. The Metrix normal sera are “.... freshly drawn . . . (with) . . . rapid processing”; A.R. Smith state “.... the reconstituted serum parallels behaviour of freshly drawn serum . ...“. Smith, Kline and French (Eskalab Normal) “._. processing is completed as quickly as possible . ...“‘. General Diagnostics (Validate) “. . . . prepared from freshly drawn blood. The serum is rapidly freeze-dried . ...“. Dade thus appears to be the only manufacturer who gives a specific time limit as to processing, but the product about which they give this information is the only one of the nine sera tested (two lots each of Validate and Metrix Normal) which shows “webbing” (Fig. 1). We have therefore assumed that all the other sera are as “fresh” as normal serum on basis of our results but clearly in view of the almost complete absence of detailed information on the time limits associated with processing each batch, users should check the individual batches of serum which they wish to use for quality control purposes. The precisions associated with the use of the Class 1.1 sera (Table I) were investigated, with the exception of Monitrol I. From a consideration of Table II it is clear that the batches of Validate, Metrix Normal, LederZyme Control and Eskalab Normal which we tested would all be satisfactory for use as quality control sera for LDH isoenzyme separations. We have also observed satisfactory precision for different lots of Validate and Metrix Normal. It must be stressed that the conditions under which we tested the sera were very rigorous; they were left standing at room temperature for up to 4 days (see Materials and Methods) and each of the manufacturers specifically states (in their package leaflets) that serum, once reconstituted must not be handled in this manner. The reason for this treatment was, of course, the well-known cold lability of LDH5. Under this treatment neither Dade Electrophoresis Control nor A.R. Smith Normal Control did well (Table II). We noted that if only fresh samples of either serum were used, then between-batch precisions similar to those obtained with the other sera were observed. However, using fresh vials of either serum every day would clearly be considerably more expensive than the procedure which we adopted. We can conclude therefore that quality control of LDH isoenzyme separations is practicable and possible provided that the control serum is human, has no additives, and is lyophilized soon after venesection. In our experience any of
66
the following sera would be suitable (with the provisions noted above): Validate, Metrix Normal, LederZyme Normal, Eskalab Normal, A.R. Smith Normal and Dade Electrophoresis Control. References 1 S.B. Rosalki. Paper given at the Association of Clinical Biochemists Symposium: The Optimisation and Standardisation of Enzyme Assays. Manchester, England, 21 Feb. 1973. See also S.B. Rosalki. Clin. Biochem. (In Press). 2 D.B. Tanks, Clin. Chem.. 9 (1963) 127. 3 H.A. Zondag, Determination and Diagnostic Significance of Lactate Dehydrogenase Isoenzymes. Van Gorcum, Assen, 1962. 4 A.L Latner and A.W. Skillen. Isoenzymes in Biology and Medicine, Academic Press. London, 1968. 5 J.H. Wilkinson. Isoenzymes, Second Edition, Chapman & Hall, London, 1970. 6 G.J. Brewer, An Introduction to Isoenzyme Techniques, Academic Press, New York, 1970. 7 A.A. Dietz, T. Lubrano and H.M. Rubinstein in G.R. Cooper (Ed.) Standard Methods of Clinical Chemistry. Vol. 7, Academic Press, New York, 1972, P. 49. 8 W.R. Dito. Am. J. Clin. Pathol.. 59 (1973) 439. 9 F.R. Elevitch, S.B. Aronwn, T.V. Feichtmeir and M.L. Enterline. Am. J. Clin. Pathol.. 46 (1966) 692. 10 R.J. Henry. N. Chiamori, O.J. Golub and S. Berkman, Am. J. Clin. Pathol.. 34 (1960) 381. 11 F.R. Elevitch, Fluorometric Techniques in Clinical Chemistry, Little, Brown and Co., Boston. 1973. p. 233.