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50 Urinalysis by automated nephelometry (AN); comparison with routine microscopic examination (ME)
P-11 (Subventionn~ par l'Instltut National du Cancer et par la Fondatlon Phoenix )
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49 I EVALUATION OF A BEHRING-LASER NEPHEMETER AUTOMATIC SYSTEM FOR THE MEASUREM E N T O F I g G , l g A , l g M A N D 0 3 c IN S E R U M . R . O a t ~ n e a u l t and F . O e s j a r l a i s , D~pt de B i o c h i m i e , H6pital Notre-Dame, Montreal, E)J~bec H2L 4M1 i
A Behr'ing-Laser Nephelometer Automatic system was e v a l u a t e d and u s e d i n o u r h o s p i t a l f o p the m e a s u r e m e n t o f IgG, IgA, IgM and C3c in serum. The patient sample dilut e d 101 f o l d w a s i n c u b a t e d f o r 3 0 P i n at r o o m t e m p e r a t u r e i n a d i s p o s a b l e p l a s t i c c u v e t t e w i t h the s p e c i f i c a n t i s e r u m diluted 5 fold (200 ul). The unknown concentrations were automatically computed by a Hewlett-Packamd 9815 A calc u l a t o r f r o m the i n t e n s i t y o f the l i g h t s c a t t e r e d b y t h e antigen-antibody complexes. The workirlg ranges were 170-5,435 mg/dl for lgG, 2 8 - 9 1 0 m g / d l f o p I g A , 4 3 - 6 8 0 m g / d l f o r I g M aP.d 1 5 - 4 7 0 m g / d l for" 0 3 c . T h e c o e f f i c i e n t o f v a r i a t i o n (n = 10) w a s 4 . 7 % f o r l g G at a c o n c e n t r " a t i o n o f 3 , 1 2 5 m g / d l ( s a m p | e s i z e : 10 u l ) and 4 . 2 % f o p C 3 c at 40 m g / d l ( s a m p l e s i z e : 100 u l ) . T h e p r e c i s i o n f i g u r e s wer"e i m p r o v e d w h e n c o m m e r c i a l l y available pre-diluted standards w e r e used. T h e results correlated well with radial immunodiffusion except for r m y e l o m a proteins at high concentrations. W e have me-used the disposable plastic cuvettes up to 5 times w h i c h d e c r e a s e s significantly the cost pep test.
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I 50 l URINALYSIS BY AUTOMATED NEPHELOMETRY (AN); COMPARISON WITH ROUTINE MICROSCOPIC EXAMINATION (ME). E.A. Stein, . P.M. Stelner, L.A. Kaplan, Dept. of Pathology (Clin.Chem.), University of Cincinnati Med. Center, Cincinnati, Ohio 45267. The benefit of routine ME of all urines has recently incurred increased attention. Also, technologlst's time, motivation, skill, and effort necessary to do an adequate ME in a busy hospital (100-150 urlnes/day) is a major problem. In an effort to reduce the number of unnecessary ME, a pre-sereenlng of unspun urine by nephelometry has been suggested. This study evaluates routine ME against AN on a Technicon AAI system (30 samples/hour) with and without the additional parameters given by automated Ames Multistlx (AM). The ME was considered the true value (positive) by the following criteria: RBC >i; WBC >5; Casts >11; Bacteria >15 (all parameters/HPF). A positive (Pos) result by AN was arbitrarily taken as >/18 peak height units. The results of AN vs. ME and AN + AM vs. ME + AM are shown in Table i. TABLE i. Pos ME Neg ME Positive Negative ME&AM ME&AM Pos AN 80 29 Pos AN & AM i01 26 Ne 8 AN 14 49 Ne~ AN & AM 7 38 85% Sensitivity 94% 63% Specificity 59% 73% Positive Predictive Value 80% 78% Negative Predictive Value 84% 75% Efficiency of AN/AN + AM 81% Almost 40% of false positives on AN were due to large amounts of precipitated phosphate crystals; these may be reduced by running the AN at 37 ° C. The false negatives (FN) were reduced considerably by the AM; of the remaining 7 FN, 5 were of doubtful clinical significance. In summary, AN together with AM offers a cost beneficial screening procedure for star and routine urinalysis, eliminating nearly 33% of samples from ME.
I
ILABELING SMALL MOLECULES FOR RIA WITH 2-1ODOHISTAMINE.
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W. Roy Slaunwhiter Jr., J. K. Tantchou (Depts. of Biochemistry and red., Sch. Med., SUNY/B, Buffalo, NY 14214). We propose a simple, 2-step procedure: treatment of 12512-1odohlstamine with the activated ester of a small molecule followed by direct TLC to remove unlabeled ligand. Only one radioactive substance, 1251-2-iodohlstamine, need be stoc~-d. The principle is illustrated by the use of testosterone (T). N-HydroxTsucclnlmldyl esters of testosterone hemisuccinate and of testosterone-3-(O-carboxymethyl)oxlme (T-3-CMO) are coupled to 1251-2-iodohlstamine. Optimum conditions require mixing a 20-50 fold molar excess of ester in 75 ~i of tetrahydrofuran with iodohistamine in 75 ul of buffer at pH 8.5 for 30 mln at 4°. The reaction mixture applied dlrect&y to pre-absorbent TLC plates (benzene:ethanol:acetlc acid, 75:24:1) allowed isolation of labeled ligand in 85% yield in 2-3 hours. The affinity of such ligands for antibodies was slightly greater than that of 3N-testosterone when T was the displacing steroid and 4 times greater when T-3-CMO was the displacing steroid. Unexpectedly the 1251-2,5-dilodohlstaminyl ligand was decidedly inferior to the monolodo analog, being chemically more unstable (decomposition rate, 0.00]6 day -! or t½, 182 days) and exhibiting less affinity for antibody than 3H-T. The specificity of the antibody was greater when the 125I-2-iodohistamlnyl llgand was used than when 3H-T was employed. Thus, the activated ester approach to the "custom" synthesis of radlollgands appears practical and promising; it offers increased ease and simplicity and probably increased yield over the customary mixed anhydride synthesis. Its general applicability awaits extension to other small molecules.
I
53 I MONTHLY VALIDATION OF THE REFERENCE VALUES AND OF THE CALIBRATION OF A SMAC SYSTEM USING DATA FROM AN APPARENTLY HEALTHY POPULATION. G. Letellier, D@ptde Biochimie, H6pital Notre-Dame, Montreal. O,-~bec H 2 L 4M1 T h e e m p l o y e e s at H h p i t a l N o t r e - D a m e have a SMAC profile as part ortheir annual medical checkup. Sixteen hundred of these profiles were used to establish reference v a l u e s a c c o r d i n g to age a n d s e x ( O a i g n e a u l t . R . , Gagn@, U. and Letellier, G. (1977)Advances in Automated Analysis, Medtad Inc., Tarrytown, Vol. I, 219). The results o f 50 o r m o p e p r o f i l e s o b t a i n e d e a c h m o n t h For w o m e n o f a g e g r o u p 3 0 - 3 9 w e r e k e y p u n c h e d on I B M 80 c o l u m n c a r d s a n d p r o c e s s e d o n a V a r i a n 75 c o m p u t e r ( L e t e l l i e r , G . , et a t . ( 1 9 7 5 ) C o m p t e s r e n d u s du 3 i ~ m e C o l l o q u e I n t e r n a t i o n a l , O r ' g a n i s a t i o n d e s l a b o r a t o i P e s et B i o l o g i e P r o s p e c t i v e , L'Expansion Scientifique Franqaise, p. 3). The mean and S.D, were calculated for each parameter. This group w a s c h o s e n f o r t w o r e a s o n s : the l a r g e p o p u l a t i o n s i z e and t h e n e g l i g e a b l e v a r i a t i o n s o b s e r v e d a c c o r d i n g to a g e . T h e d a t a w e r e c o m p a r e d to the m e a n s o f p r e v i o u s m o n t h s and t h e o r i g i n a l m e a n u s e d i n 1975 w h e n w e e s t a b l i s h e d o,J r r e f e r e n c e v a l u e s . The results show minimal v a r i a t i o n s f r o m m o n t h to m o n t h , t h u s v a l i d a t i n g the r e f e r e n c e v a l u e s a n d d e m o n s t r a t i n g the s t a b i l i t y i n t h e c a l i b r a t i o n o f the i n s t r u m e n t f o p a l l 19 p a r a m e t e r s o v e r a four year period.
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51 I LA FERRITINE CO~94E MARQUEUR BIOLOGIQUE DE LA NEOPLASIE. M. P a ~ , L. Th~riault et M. Caron, Centre de recherche, HStel-Dieu de Quebec, Quebec, ClR 2J6. L'enzymoimmunoessai en phase solide que nous avons d~velopp~, a montr~ que le taux de ferritine s~rique s'@levait chez les patients leuc~miques. On a trouv~ dans la leuc~mie my~lolde aigue (AML) et chronique (CML) respectivement 9/9 et 13/17 patients avec des taux ~lev~s de ferritine s~rique. Lee concentrations moyennes ~taient de 503 ng/ml dans I'AML, 287 ng/ml dans la C M L e t 426 ng/ml dans la leuc~mle my~loide aigue (ALL) et ce, pour un taux normal moyen de 58 ng/ml. On a aussl effeetu~ les dosages de ferritine s~rlque chez un groupe de patientes non s~lectlonn~es, porteuses d'un carcihome mammalre (n:129) & t o u s l e s stades de la maladie. L'antig~ne carcinoembryonnalre (CEA) ~tait ~lev~ chez 26/129 de ces patlentes tandls que 35/129 de ees patientes avaient un taux anormal de ferritine s~rlque; 47/129 desdites patientes avalent un des deux marqueurs s~riques anormal et seulement 13 patlentes montraient des taux anormaux pour les deux marqueure. Des ~tudes in vitro ont montr~ que la eellule leue~mlque synth~tlse de la ferritlne mais l'origlne de l'~l~vation de cette prot~ine dans le cancer du sein est encore Inconnue.
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54 |MEASUREMENT OF GLYCOSYLATED HEMOGLOBINS BY MINICOLUMN CHROMATOGRAPHY. M.C.Wans, M.B.Sonawanee, and P.R.Wals~ Department of Chemistry, Laboratory Procedures East, Inc., King of Prussia, PA, 19406, U.S.A. The measurement of glycosylated hemoglobins in erythrocytes has been shown to be a useful index for monitoring long-term diabetic control. We describe a simplified chromatographic procedure for the measurement using the Pasteur pipette as a column. It improves reagent cost and assay time. Bio-Rex 70 is suspended in Developer 06 (0.bM phosphate buffer, pH 6.70 containing O.01M KCN) with pH adjusted to 6.70. The resin is packed into a Pasteur pipette 8 cm high, and the column was equilibrated with 150 ml Developer 06 to pH 6.70 - 6.73. Erythroeyte hemolysate containing 2.1 - 2.5 mg hemoglobin (in 25 ~i) was applied to the column. Separation of glycosylated hemoglobin A-, . . . . from the rest of the hemoglobins L~a~D~C was achieved by e l u t l o n wlt~ 20 ml Developer 06 c o n t i n u o u s l y u s i n g a Tygon t u b i n g (O.D. 5 n~) to c o n n e c t t h e column and t h e
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49 I EVALUATION OF A BEHRING-LASER NEPHEMETER AUTOMATIC SYSTEM FOR THE MEASUREM E N T O F I g G , l g A , l g M A N D 0 3 c IN S E R U M . R . O a t ~ n e a u l t and F . O e s j a r l a i s , D~pt de B i o c h i m i e , H6pital Notre-Dame, Montreal, E)J~bec H2L 4M1 i
A Behr'ing-Laser Nephelometer Automatic system was e v a l u a t e d and u s e d i n o u r h o s p i t a l f o p the m e a s u r e m e n t o f IgG, IgA, IgM and C3c in serum. The patient sample dilut e d 101 f o l d w a s i n c u b a t e d f o r 3 0 P i n at r o o m t e m p e r a t u r e i n a d i s p o s a b l e p l a s t i c c u v e t t e w i t h the s p e c i f i c a n t i s e r u m diluted 5 fold (200 ul). The unknown concentrations were automatically computed by a Hewlett-Packamd 9815 A calc u l a t o r f r o m the i n t e n s i t y o f the l i g h t s c a t t e r e d b y t h e antigen-antibody complexes. The workirlg ranges were 170-5,435 mg/dl for lgG, 2 8 - 9 1 0 m g / d l f o p I g A , 4 3 - 6 8 0 m g / d l f o r I g M aP.d 1 5 - 4 7 0 m g / d l for" 0 3 c . T h e c o e f f i c i e n t o f v a r i a t i o n (n = 10) w a s 4 . 7 % f o r l g G at a c o n c e n t r " a t i o n o f 3 , 1 2 5 m g / d l ( s a m p | e s i z e : 10 u l ) and 4 . 2 % f o p C 3 c at 40 m g / d l ( s a m p l e s i z e : 100 u l ) . T h e p r e c i s i o n f i g u r e s wer"e i m p r o v e d w h e n c o m m e r c i a l l y available pre-diluted standards w e r e used. T h e results correlated well with radial immunodiffusion except for r m y e l o m a proteins at high concentrations. W e have me-used the disposable plastic cuvettes up to 5 times w h i c h d e c r e a s e s significantly the cost pep test.
!
I 50 l URINALYSIS BY AUTOMATED NEPHELOMETRY (AN); COMPARISON WITH ROUTINE MICROSCOPIC EXAMINATION (ME). E.A. Stein, . P.M. Stelner, L.A. Kaplan, Dept. of Pathology (Clin.Chem.), University of Cincinnati Med. Center, Cincinnati, Ohio 45267. The benefit of routine ME of all urines has recently incurred increased attention. Also, technologlst's time, motivation, skill, and effort necessary to do an adequate ME in a busy hospital (100-150 urlnes/day) is a major problem. In an effort to reduce the number of unnecessary ME, a pre-sereenlng of unspun urine by nephelometry has been suggested. This study evaluates routine ME against AN on a Technicon AAI system (30 samples/hour) with and without the additional parameters given by automated Ames Multistlx (AM). The ME was considered the true value (positive) by the following criteria: RBC >i; WBC >5; Casts >11; Bacteria >15 (all parameters/HPF). A positive (Pos) result by AN was arbitrarily taken as >/18 peak height units. The results of AN vs. ME and AN + AM vs. ME + AM are shown in Table i. TABLE i. Pos ME Neg ME Positive Negative ME&AM ME&AM Pos AN 80 29 Pos AN & AM i01 26 Ne 8 AN 14 49 Ne~ AN & AM 7 38 85% Sensitivity 94% 63% Specificity 59% 73% Positive Predictive Value 80% 78% Negative Predictive Value 84% 75% Efficiency of AN/AN + AM 81% Almost 40% of false positives on AN were due to large amounts of precipitated phosphate crystals; these may be reduced by running the AN at 37 ° C. The false negatives (FN) were reduced considerably by the AM; of the remaining 7 FN, 5 were of doubtful clinical significance. In summary, AN together with AM offers a cost beneficial screening procedure for star and routine urinalysis, eliminating nearly 33% of samples from ME.
I
ILABELING SMALL MOLECULES FOR RIA WITH 2-1ODOHISTAMINE.
i
W. Roy Slaunwhiter Jr., J. K. Tantchou (Depts. of Biochemistry and red., Sch. Med., SUNY/B, Buffalo, NY 14214). We propose a simple, 2-step procedure: treatment of 12512-1odohlstamine with the activated ester of a small molecule followed by direct TLC to remove unlabeled ligand. Only one radioactive substance, 1251-2-iodohlstamine, need be stoc~-d. The principle is illustrated by the use of testosterone (T). N-HydroxTsucclnlmldyl esters of testosterone hemisuccinate and of testosterone-3-(O-carboxymethyl)oxlme (T-3-CMO) are coupled to 1251-2-iodohlstamine. Optimum conditions require mixing a 20-50 fold molar excess of ester in 75 ~i of tetrahydrofuran with iodohistamine in 75 ul of buffer at pH 8.5 for 30 mln at 4°. The reaction mixture applied dlrect&y to pre-absorbent TLC plates (benzene:ethanol:acetlc acid, 75:24:1) allowed isolation of labeled ligand in 85% yield in 2-3 hours. The affinity of such ligands for antibodies was slightly greater than that of 3N-testosterone when T was the displacing steroid and 4 times greater when T-3-CMO was the displacing steroid. Unexpectedly the 1251-2,5-dilodohlstaminyl ligand was decidedly inferior to the monolodo analog, being chemically more unstable (decomposition rate, 0.00]6 day -! or t½, 182 days) and exhibiting less affinity for antibody than 3H-T. The specificity of the antibody was greater when the 125I-2-iodohistamlnyl llgand was used than when 3H-T was employed. Thus, the activated ester approach to the "custom" synthesis of radlollgands appears practical and promising; it offers increased ease and simplicity and probably increased yield over the customary mixed anhydride synthesis. Its general applicability awaits extension to other small molecules.
I
53 I MONTHLY VALIDATION OF THE REFERENCE VALUES AND OF THE CALIBRATION OF A SMAC SYSTEM USING DATA FROM AN APPARENTLY HEALTHY POPULATION. G. Letellier, D@ptde Biochimie, H6pital Notre-Dame, Montreal. O,-~bec H 2 L 4M1 T h e e m p l o y e e s at H h p i t a l N o t r e - D a m e have a SMAC profile as part ortheir annual medical checkup. Sixteen hundred of these profiles were used to establish reference v a l u e s a c c o r d i n g to age a n d s e x ( O a i g n e a u l t . R . , Gagn@, U. and Letellier, G. (1977)Advances in Automated Analysis, Medtad Inc., Tarrytown, Vol. I, 219). The results o f 50 o r m o p e p r o f i l e s o b t a i n e d e a c h m o n t h For w o m e n o f a g e g r o u p 3 0 - 3 9 w e r e k e y p u n c h e d on I B M 80 c o l u m n c a r d s a n d p r o c e s s e d o n a V a r i a n 75 c o m p u t e r ( L e t e l l i e r , G . , et a t . ( 1 9 7 5 ) C o m p t e s r e n d u s du 3 i ~ m e C o l l o q u e I n t e r n a t i o n a l , O r ' g a n i s a t i o n d e s l a b o r a t o i P e s et B i o l o g i e P r o s p e c t i v e , L'Expansion Scientifique Franqaise, p. 3). The mean and S.D, were calculated for each parameter. This group w a s c h o s e n f o r t w o r e a s o n s : the l a r g e p o p u l a t i o n s i z e and t h e n e g l i g e a b l e v a r i a t i o n s o b s e r v e d a c c o r d i n g to a g e . T h e d a t a w e r e c o m p a r e d to the m e a n s o f p r e v i o u s m o n t h s and t h e o r i g i n a l m e a n u s e d i n 1975 w h e n w e e s t a b l i s h e d o,J r r e f e r e n c e v a l u e s . The results show minimal v a r i a t i o n s f r o m m o n t h to m o n t h , t h u s v a l i d a t i n g the r e f e r e n c e v a l u e s a n d d e m o n s t r a t i n g the s t a b i l i t y i n t h e c a l i b r a t i o n o f the i n s t r u m e n t f o p a l l 19 p a r a m e t e r s o v e r a four year period.
I
51 I LA FERRITINE CO~94E MARQUEUR BIOLOGIQUE DE LA NEOPLASIE. M. P a ~ , L. Th~riault et M. Caron, Centre de recherche, HStel-Dieu de Quebec, Quebec, ClR 2J6. L'enzymoimmunoessai en phase solide que nous avons d~velopp~, a montr~ que le taux de ferritine s~rique s'@levait chez les patients leuc~miques. On a trouv~ dans la leuc~mie my~lolde aigue (AML) et chronique (CML) respectivement 9/9 et 13/17 patients avec des taux ~lev~s de ferritine s~rique. Lee concentrations moyennes ~taient de 503 ng/ml dans I'AML, 287 ng/ml dans la C M L e t 426 ng/ml dans la leuc~mle my~loide aigue (ALL) et ce, pour un taux normal moyen de 58 ng/ml. On a aussl effeetu~ les dosages de ferritine s~rlque chez un groupe de patientes non s~lectlonn~es, porteuses d'un carcihome mammalre (n:129) & t o u s l e s stades de la maladie. L'antig~ne carcinoembryonnalre (CEA) ~tait ~lev~ chez 26/129 de ces patlentes tandls que 35/129 de ees patientes avaient un taux anormal de ferritine s~rlque; 47/129 desdites patientes avalent un des deux marqueurs s~riques anormal et seulement 13 patlentes montraient des taux anormaux pour les deux marqueure. Des ~tudes in vitro ont montr~ que la eellule leue~mlque synth~tlse de la ferritlne mais l'origlne de l'~l~vation de cette prot~ine dans le cancer du sein est encore Inconnue.
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54 |MEASUREMENT OF GLYCOSYLATED HEMOGLOBINS BY MINICOLUMN CHROMATOGRAPHY. M.C.Wans, M.B.Sonawanee, and P.R.Wals~ Department of Chemistry, Laboratory Procedures East, Inc., King of Prussia, PA, 19406, U.S.A. The measurement of glycosylated hemoglobins in erythrocytes has been shown to be a useful index for monitoring long-term diabetic control. We describe a simplified chromatographic procedure for the measurement using the Pasteur pipette as a column. It improves reagent cost and assay time. Bio-Rex 70 is suspended in Developer 06 (0.bM phosphate buffer, pH 6.70 containing O.01M KCN) with pH adjusted to 6.70. The resin is packed into a Pasteur pipette 8 cm high, and the column was equilibrated with 150 ml Developer 06 to pH 6.70 - 6.73. Erythroeyte hemolysate containing 2.1 - 2.5 mg hemoglobin (in 25 ~i) was applied to the column. Separation of glycosylated hemoglobin A-, . . . . from the rest of the hemoglobins L~a~D~C was achieved by e l u t l o n wlt~ 20 ml Developer 06 c o n t i n u o u s l y u s i n g a Tygon t u b i n g (O.D. 5 n~) to c o n n e c t t h e column and t h e