191
Clinica Chimica Acta, 59 (1975) 0 Elsevier
Scientific
Publishing
191-194 Company,
Amsterdam
-
Printed
in The NetherIands
CCA 6859
A COMPARISON OF RESULTS USING THREE COMMERCIALLY AVAILABLE VITAMIN B-12 RADIOASSAY KITS
INGO S. KAMPA
Department (Received
and JOY
M. HUNDERTMARK
of Pathology, The Valley Hospital, Ridgewood,
September
N.J. 07450
(U.S.A.)
27, 1974)
Introduction The use of radioassay for the determination of vitamin B-12 utilizing ’ 7 Co, has become popular as more clinical laboratories develop the techniques to detect gamma-emitting isotopes. Three manufacturers, Pharmacia, SchwarzMann, and Curtis Nuclear, have recently introduced kits for the determination of vitamin B-12. These kits contain all the reagents necessary for the performance of the test. The procedures are relatively simple, and the manufacturers claim that they provide a sensitive, accurate, and reproducible method for the determination of vitamin B-12 in serum. Of concern to the clinical chemist and the physician is the correlation between the results obtained from the assay and the actual quantity of vitamin B-12 in the patient’s serum. The reliability of these test results can only be established by assaying sera that contain a known quantity of vitamin B-12. Comparisons of data obtained by radioassay with data obtained by bioassay utilizing Euglena gracilis are of little value as the bioassays are non-specific, and are sensitive to the presence of antibiotics in the serum, varying organism growth rate, and other factors [l]. . A. R. Smith Laboratories recently introduced a dried control serum, VITEL (B-12) TM for vitamin B-12 assay. This control serum is made from human serum to which vitamin B-12 is added to achieve an elevated level. It is the purpose of this report to: 1. Present a critical comparison of results obtained through the use of these three commercial kits. 2. Examine VIT-EL (B-12) TM as a suitable control serum for vitamin B-l 2 radioassay. In each of the three vitamin B-12 assay procedures the cyanocobalamin bound to the intrinsic factor must be separated from the unbound cyanocobalamin once equilibrium has been reached. It is in the separation of the bound cyanocobalamin from the unbound that these kits essentially differ. The Phadehas B-12 kit uses a solid-phase technique utilizing intrinsic factor bound to
192
Sephadex [2,3]. Schwarz-Mann effects the separation by adsorption of the unbound cyanocobalamin on to albumin coated charcoal [4,5]. Curtis Nuclear removes the bound cyanocobalamin by precipitating it with DEAE cellulose
C6,71. Materials
and Methods
In this study, seventy serum samples were obtained from patients with a wide variety of pathology. Forty control samples, VIT-EL (B-12) TM *, were reconstituted according to the manufacturer’s directions and assayed along with the patient sera. The vitamin B-12 content of each patient sample was determined by using at least two commercial kits. All samples were coded and the statistical comparison was carried out after decoding. The Vitamin B-12 assay kits compared were obtained from SchwarzAll reagents were reconstiMann**, Pharmacia***, and Curtis Nuclear ****. tuted according to manufacturer’s directions. The analysis and calculation of results were performed according to manfacturer’s specifications. Results The vitamin B-12 serum concentration obtained with the Pharmacia kit was plotted against corresponding concentrations obtained when using the Schwarz-Mann and Curtis Nuclear kits. The comparison between the Pharmacia and Schwarz-Mann kits is shown in Fig. 1. The fitted regression line was y = 1.17 x + 69 (where y is the Pharmacia method, and x the Schwarz-Mann
Schwarz
- Mann
Curtis
Fig.
1. Comparison
of vitamin
B-12
values
of forty
serum
specimens.
Fig.
2. Comparison
of vitamin
B-12
values
of forty
serum
specimens.
Terminal
Annex,
* A.R. ** *** ****
Smith
Laboratories,
Schwarz-Mann, Pharmacia. Curtis
Mountain 800
Nuclear,
Centennial 1948
East
P.O. View
Box
54400,
Avenue,
Avenue, Fortysixth
Orangeburg,
Piscataway. Street,
Los
N.J.
N.Y. 08854,
Angeles,
Los
Angeles.
10962,
U.S.A.
Calif.
U.S.A. Calif.
90058.
U.S.A.
Nuclear
90054,
U.S.A.
193 TABLE
I
RELATIVE
ACTIVITY
RADIOASSAY
Curtis
Mean
(pg/dl)
OF
IN
VIT-EL(B-~~)~~
THREE
DIFFERENT
COMMERCIAL
VITAMIN
B-12
KITS
Nuclear
Pharmacia
Schwarz-Mann
LOW
Normat
Elevated
Low
Normal
Elevated
Low
Normal
Elevated
378
500
2202
320
480
1827
260
450
1633
S. D.
64.0
70.0
50.0
16.3
87.0
77.0
40.2
54.7
27.0
cv
16.9
14.1
2.3
5.1
18.1
4.2
15.5
12.2
3.5
methodology). The correlation coefficient was 0.957. The paired t test value was 2.26. The comparison between Pharmacia and Curtis Nuclear methodology is shown in Fig. 2. The fitted regression line was y =.786 x + 59 (where y is the Pharmacia method, and x is the Schwarz-Mann method). The correlation coefficient was 0.952. The paired t value was 2.36. Control samples, VIT-EL (B-12) TM, purchased from A.R. Smith Laboratories, were diluted in order to produce control samples in the low, normal, and elevated ranges. The results obtained by the three levels of control sera by the three methods are presented in Table I. The recovery of added exogenous crystalline vitamin B-12 was evaluated with each kit procedure. The ability to account for added vitamin B-12 is presented in Table II. All three kits produced satisfactory recovery of added crystalline vitamin B-12. Discussion The results indicate that all three commercial kits produce acceptable reproducibility and that the recovery of added crystalline B-12 is within acceptable limits. The normal range for the Schwarz-Mann kit is given as 200 to 900
TABLE
II
RECOVERY
Curtis
Nuclear
Schwarz-Mann
Pharmacia
OF
EXOGENOUS
VITAMIN
B-12
ADDED
TO
SERUM
Endogenous
Added
Predicted
B-12
B-12
total
(PglmI)
(p!z/mI)
(PglmI)
(PenI)
Measured
total
recovery %
500
125
625
623
99.7
500
250
750
780
104.0
500
500
1000
1025
102.5
480
125
605
620
102.5
480
250
730
753
103.2
480
500
980
887
90.5
450
125
575
550
95.7
450
250
700
730
104.3
450
500
950
930
97.9
194
pg/ml, for the Pharmacia kit as 300 to 1000 pg/ml, and for the Curtis Nuclear kit as 200 to 900 pg/ml. Vitamin B-12 assays are generally performed in order to identify vitamin B-12 deficient states. Patients with pernicious anemia fall below 100 pg/ml [ 81. Pernicious anemia should be confirmed by the Schilling test. Serum vitamin B-12 levels of above 1000 pg/ml are suggestive of liver disease and myeloproliferative disorders [9]. In such cases, vitamin B-12 levels are often in the 2000 to 9000 pg/ml range. Myeloproliferative disorders generally have values of above 4000 pg/ml [lo] . Therefore, the upper normal limit is difficult to define. The previously established normal range in our laboratory is 200 to 1200 pg/ml. A comparison between the Schwarz-Mann and Pharmacia kits indicates that the results of serum samples given by the Pharmacia kit were higher than those obtained with the Schwarz-Mann kit. However, twelve elevated and two low values were identified by both kits. Both patients with low vitamin B-12 levels had abnormal Schilling tests. Eight of the twelve patients with elevated levels had no history of liver disease or myeloproliferative disorders. No history was available on the four remaining patients. The comparison of the Pharmacia kit with the Curtis Nuclear kit showed good correlation, although the results obtained by the Curtis Nuclear kit were higher than those obtained by the Pharmacia kit. The upper normal range of 900 pg/ml stated by the manufacturer of the Curtis Nuclear kit is probably too low. Patients that exceeded the upper range had no clinical findings of conditions that are associated with vitamin B-12 elevations. The accuracy of these kits is difficult to establish. Results from all three kits exceeded the stated content of the VIT-EL (B-12) TM control sera. However, the recovery of added crystalline vitamin B-12 established the ability of each kit to measure added exogenous vitamin B-12 accurately. Any of the three kits is capable of providing comparative measurements of vitamin B-12. The major difference between the procedures is the ease of performing the test. Although the incubation time of the Schwarz-Mann kit is short, the procedure is more tedious because of the reagents that must be prepared and added to the tubes. The Pharmacia kit requires a long incubation time (three hours) and reagents must also be prepared. The Curtis Nuclear kit is easy to use, and all reagents, other than normal saline, are supplied with the kit. Pipetting is minimal, and incubation time is an acceptable two hours. This procedure is desirable for laboratories that can adjust to slightly higher serum values. References 1
S.H. Proc.
Hunter, Sm.
2
L. Wide
3
M. Ceska
4
K.-S.
5
V. Herbert,
L.
EXP.
and
A.
and
Lau.
Provasoli. Biol.
Med.,
Killander,
6
G. Tibbling,
7
E.P.
Frenkel,
8
J.L.
Raven,
L.R.
C. Gottlieb Clin. M.B.
Clin.
Acta, and
Robson,
M.S.
J. Clin.
Lab.
Chim.
Acta,
J.O.
Lau,
23
C.E.
and
(1969)
Invest.. 32
V.
Blood,
M.
Belt,
A.L.
Franklin
and
Herbert. 28
27
(1971)
(1971)
151.
339. Blood,
(1966)
26
(1965)
202.
130.
209.
MC Call, Morgan
Hoffmann.
118.
Wasserman
and K.-S.
Chim.
S. Keller
Stokstad,
(1949)
Stand.
U. Lundkuist.
C. Gottlieb,
E.L.R. 70
J. Lab.
and
A.V.
Clin.
Med.,
Hofbrand,
68
(1968)
510.
Br. J. Haematol.,
22
(1972)
21.
T.H.
Jukes,
Clinica Chimica Acta, 59 (1975) 0 Elsevier
Scientific
Publishing
191-194 Company,
Amsterdam
-
Printed
in The NetherIands
CCA 6859
A COMPARISON OF RESULTS USING THREE COMMERCIALLY AVAILABLE VITAMIN B-12 RADIOASSAY KITS
INGO S. KAMPA
Department (Received
and JOY
M. HUNDERTMARK
of Pathology, The Valley Hospital, Ridgewood,
September
N.J. 07450
(U.S.A.)
27, 1974)
Introduction The use of radioassay for the determination of vitamin B-12 utilizing ’ 7 Co, has become popular as more clinical laboratories develop the techniques to detect gamma-emitting isotopes. Three manufacturers, Pharmacia, SchwarzMann, and Curtis Nuclear, have recently introduced kits for the determination of vitamin B-12. These kits contain all the reagents necessary for the performance of the test. The procedures are relatively simple, and the manufacturers claim that they provide a sensitive, accurate, and reproducible method for the determination of vitamin B-12 in serum. Of concern to the clinical chemist and the physician is the correlation between the results obtained from the assay and the actual quantity of vitamin B-12 in the patient’s serum. The reliability of these test results can only be established by assaying sera that contain a known quantity of vitamin B-12. Comparisons of data obtained by radioassay with data obtained by bioassay utilizing Euglena gracilis are of little value as the bioassays are non-specific, and are sensitive to the presence of antibiotics in the serum, varying organism growth rate, and other factors [l]. . A. R. Smith Laboratories recently introduced a dried control serum, VITEL (B-12) TM for vitamin B-12 assay. This control serum is made from human serum to which vitamin B-12 is added to achieve an elevated level. It is the purpose of this report to: 1. Present a critical comparison of results obtained through the use of these three commercial kits. 2. Examine VIT-EL (B-12) TM as a suitable control serum for vitamin B-l 2 radioassay. In each of the three vitamin B-12 assay procedures the cyanocobalamin bound to the intrinsic factor must be separated from the unbound cyanocobalamin once equilibrium has been reached. It is in the separation of the bound cyanocobalamin from the unbound that these kits essentially differ. The Phadehas B-12 kit uses a solid-phase technique utilizing intrinsic factor bound to
192
Sephadex [2,3]. Schwarz-Mann effects the separation by adsorption of the unbound cyanocobalamin on to albumin coated charcoal [4,5]. Curtis Nuclear removes the bound cyanocobalamin by precipitating it with DEAE cellulose
C6,71. Materials
and Methods
In this study, seventy serum samples were obtained from patients with a wide variety of pathology. Forty control samples, VIT-EL (B-12) TM *, were reconstituted according to the manufacturer’s directions and assayed along with the patient sera. The vitamin B-12 content of each patient sample was determined by using at least two commercial kits. All samples were coded and the statistical comparison was carried out after decoding. The Vitamin B-12 assay kits compared were obtained from SchwarzAll reagents were reconstiMann**, Pharmacia***, and Curtis Nuclear ****. tuted according to manufacturer’s directions. The analysis and calculation of results were performed according to manfacturer’s specifications. Results The vitamin B-12 serum concentration obtained with the Pharmacia kit was plotted against corresponding concentrations obtained when using the Schwarz-Mann and Curtis Nuclear kits. The comparison between the Pharmacia and Schwarz-Mann kits is shown in Fig. 1. The fitted regression line was y = 1.17 x + 69 (where y is the Pharmacia method, and x the Schwarz-Mann
Schwarz
- Mann
Curtis
Fig.
1. Comparison
of vitamin
B-12
values
of forty
serum
specimens.
Fig.
2. Comparison
of vitamin
B-12
values
of forty
serum
specimens.
Terminal
Annex,
* A.R. ** *** ****
Smith
Laboratories,
Schwarz-Mann, Pharmacia. Curtis
Mountain 800
Nuclear,
Centennial 1948
East
P.O. View
Box
54400,
Avenue,
Avenue, Fortysixth
Orangeburg,
Piscataway. Street,
Los
N.J.
N.Y. 08854,
Angeles,
Los
Angeles.
10962,
U.S.A.
Calif.
U.S.A. Calif.
90058.
U.S.A.
Nuclear
90054,
U.S.A.
193 TABLE
I
RELATIVE
ACTIVITY
RADIOASSAY
Curtis
Mean
(pg/dl)
OF
IN
VIT-EL(B-~~)~~
THREE
DIFFERENT
COMMERCIAL
VITAMIN
B-12
KITS
Nuclear
Pharmacia
Schwarz-Mann
LOW
Normat
Elevated
Low
Normal
Elevated
Low
Normal
Elevated
378
500
2202
320
480
1827
260
450
1633
S. D.
64.0
70.0
50.0
16.3
87.0
77.0
40.2
54.7
27.0
cv
16.9
14.1
2.3
5.1
18.1
4.2
15.5
12.2
3.5
methodology). The correlation coefficient was 0.957. The paired t test value was 2.26. The comparison between Pharmacia and Curtis Nuclear methodology is shown in Fig. 2. The fitted regression line was y =.786 x + 59 (where y is the Pharmacia method, and x is the Schwarz-Mann method). The correlation coefficient was 0.952. The paired t value was 2.36. Control samples, VIT-EL (B-12) TM, purchased from A.R. Smith Laboratories, were diluted in order to produce control samples in the low, normal, and elevated ranges. The results obtained by the three levels of control sera by the three methods are presented in Table I. The recovery of added exogenous crystalline vitamin B-12 was evaluated with each kit procedure. The ability to account for added vitamin B-12 is presented in Table II. All three kits produced satisfactory recovery of added crystalline vitamin B-12. Discussion The results indicate that all three commercial kits produce acceptable reproducibility and that the recovery of added crystalline B-12 is within acceptable limits. The normal range for the Schwarz-Mann kit is given as 200 to 900
TABLE
II
RECOVERY
Curtis
Nuclear
Schwarz-Mann
Pharmacia
OF
EXOGENOUS
VITAMIN
B-12
ADDED
TO
SERUM
Endogenous
Added
Predicted
B-12
B-12
total
(PglmI)
(p!z/mI)
(PglmI)
(PenI)
Measured
total
recovery %
500
125
625
623
99.7
500
250
750
780
104.0
500
500
1000
1025
102.5
480
125
605
620
102.5
480
250
730
753
103.2
480
500
980
887
90.5
450
125
575
550
95.7
450
250
700
730
104.3
450
500
950
930
97.9
194
pg/ml, for the Pharmacia kit as 300 to 1000 pg/ml, and for the Curtis Nuclear kit as 200 to 900 pg/ml. Vitamin B-12 assays are generally performed in order to identify vitamin B-12 deficient states. Patients with pernicious anemia fall below 100 pg/ml [ 81. Pernicious anemia should be confirmed by the Schilling test. Serum vitamin B-12 levels of above 1000 pg/ml are suggestive of liver disease and myeloproliferative disorders [9]. In such cases, vitamin B-12 levels are often in the 2000 to 9000 pg/ml range. Myeloproliferative disorders generally have values of above 4000 pg/ml [lo] . Therefore, the upper normal limit is difficult to define. The previously established normal range in our laboratory is 200 to 1200 pg/ml. A comparison between the Schwarz-Mann and Pharmacia kits indicates that the results of serum samples given by the Pharmacia kit were higher than those obtained with the Schwarz-Mann kit. However, twelve elevated and two low values were identified by both kits. Both patients with low vitamin B-12 levels had abnormal Schilling tests. Eight of the twelve patients with elevated levels had no history of liver disease or myeloproliferative disorders. No history was available on the four remaining patients. The comparison of the Pharmacia kit with the Curtis Nuclear kit showed good correlation, although the results obtained by the Curtis Nuclear kit were higher than those obtained by the Pharmacia kit. The upper normal range of 900 pg/ml stated by the manufacturer of the Curtis Nuclear kit is probably too low. Patients that exceeded the upper range had no clinical findings of conditions that are associated with vitamin B-12 elevations. The accuracy of these kits is difficult to establish. Results from all three kits exceeded the stated content of the VIT-EL (B-12) TM control sera. However, the recovery of added crystalline vitamin B-12 established the ability of each kit to measure added exogenous vitamin B-12 accurately. Any of the three kits is capable of providing comparative measurements of vitamin B-12. The major difference between the procedures is the ease of performing the test. Although the incubation time of the Schwarz-Mann kit is short, the procedure is more tedious because of the reagents that must be prepared and added to the tubes. The Pharmacia kit requires a long incubation time (three hours) and reagents must also be prepared. The Curtis Nuclear kit is easy to use, and all reagents, other than normal saline, are supplied with the kit. Pipetting is minimal, and incubation time is an acceptable two hours. This procedure is desirable for laboratories that can adjust to slightly higher serum values. References 1
S.H. Proc.
Hunter, Sm.
2
L. Wide
3
M. Ceska
4
K.-S.
5
V. Herbert,
L.
EXP.
and
A.
and
Lau.
Provasoli. Biol.
Med.,
Killander,
6
G. Tibbling,
7
E.P.
Frenkel,
8
J.L.
Raven,
L.R.
C. Gottlieb Clin. M.B.
Clin.
Acta, and
Robson,
M.S.
J. Clin.
Lab.
Chim.
Acta,
J.O.
Lau,
23
C.E.
and
(1969)
Invest.. 32
V.
Blood,
M.
Belt,
A.L.
Franklin
and
Herbert. 28
27
(1971)
(1971)
151.
339. Blood,
(1966)
26
(1965)
202.
130.
209.
MC Call, Morgan
Hoffmann.
118.
Wasserman
and K.-S.
Chim.
S. Keller
Stokstad,
(1949)
Stand.
U. Lundkuist.
C. Gottlieb,
E.L.R. 70
J. Lab.
and
A.V.
Clin.
Med.,
Hofbrand,
68
(1968)
510.
Br. J. Haematol.,
22
(1972)
21.
T.H.
Jukes,
