Journal of Clinical Laboratory Analysis 6:176-181 (1992)
Development of a Rapid Microparticle-Enhanced Nephelometric lmmunoassay for Serum Myoglobin in Acute Myocardial Infarction F. Cliquet,’ t? Montagne,’ M.L. Cuilliere,’ I?Varcin,* and J. Duheille’ ‘Immunology Laboratoty Faculty of Medicine, Nancy, France; Sanofi Research, Montpellier, France A microparticle-enhanced nephelometric immunoassay was developed for myoglobin quantitation in human serum. It uses rabbit antimyoglobin serum and hydrophilic polyacrylic microparticles covalently coated with baboon myoglobin in a competitive immunoagglutination system. The level of microparticle agglutinationis assessed with a specially designed nephelometer. This sensitive (45 ng/ml of myoglobin detected in serum) and accurate (coefficients of variation from 3.0% to 8.2% in precision study and linear recov-
Key words:
s s ,
microparticle agglutination, immunonephelometry,AM1
INTRODUCTION Microparticle-enhanced nephelometric immunoassay has been previously described as a new easy-to-perform and sensitive method to assay various serum proteins (1-3), milk proteins (4-7), and haptens (8). These tests are based on the inhibition, by free antigen, of the agglutination of antigencoated microspheres (MS) by specific antiserum. Quantification of free antigen levels is performed by the measurement with an adapted nephelometer of the light scattered by antigen-MS conjugate clusters during this inhibition. The measurement of serum myoglobin (Mb) levels is used to rule out acute myocardial infarction (AMI). In spite of its relative nonspecificity, it could also contribute to the AM1 diagnosis in emergency units, allowing rapid instauration of reperfusion and evaluation of its efficiency (9,lO). The most recently reported methods for Mb measurement (1 1) are usually inadequate for emergency conditions (constraint linked to the use of radioisotopes, long reaction times, necessity of numerous washings or phase separation). Latex-slide tests ( 12) are rapid, unsophisticated, and well adapted to emergency conditions, but they are only semiquantitative and the detection of latex agglutination is subjective. An automated nephelometric immunoassay using latex agglutination has been described recently for Mb quantitation (13). It uses antibodycoated latex with a calibration range limited to 400 ng/ml and thus presents a risk of false-negative results through underevaluation of the highest Mb levels (hook effect in antigen excess conditions). 0 1992 Wiley-Liss, Inc.
ery of myoglobin in overloaded sera) immunoassay was evaluated with human sera from patients suffering from acute myocardial infarction. Myoglobin levels in patient‘s serum on admission appeared to be correlated with clinical and biological parameters assessed in emergency wards and later. This new, rapid, and easy microparticle-enhanced nephelometric immunoassay could thus be useful in emergency conditions for the early quantitationof serum myoglobin. o ~ g ~ ~ i ~ e y Inc. - ~ i
A rapid and easy-to-perform assay, capable of providing an early and accurate quantitation of Mb levels in serum from patients with AM1 will be very beneficial. We report here an improved competitive microparticle-enhanced nephelometric immunoassay using Mb-MS conjugate and its evaluation as a method to be used in emergency conditions for the quantitation of Mb in serum from patients with AMI.
MATERIALS AND METHODS Reagents Acrolein, 2-hydroxyethylmethacrylate,methacrylic acid, and polyethylene glycol 6000 were obtained from Merck (Darmstadt, Federal Republic of Germany). N,N’-methylenebisacrylamide was from Eastman Kodak Co. (Rochester, NY). Sodium dodecyl sulfate, hydroquinone, 2-aminoethanol, sucrose, sodium azide, potassium and sodium chlorides, potassium and sodium dihydrogenophosphates, and disodium hydrogenophosphate were purchased from Prolabo Rh6ne Poulenc (Paris, France). Immunonephelometric assays were carried out with the nephelometry buffer supplied by Diagnostics Pasteur (Marnes, France). Human Mb (hMb), cardiac and skeletal horse Mb, and sperm whale Mb were purchased from Dakopatts (Glostrup,
Received November7, 1991; accepted February 18, 1992. Address reprint requests to P. Montagne, Immunology Laboratory, Faculty of Medicine, B.P.184, F-54505 Vandoeuvre les Nancy Cedex, France.
Microparticle-Based Assay of Myoglobin
Denmark), Calbiochem Corporation (San Diego, CA), and Sigma Chemical Co. (St. Louis, MO), respectively. Baboon Mb (bMb) was extracted as metmyoglobin from skeletal muscle according to the method described by Malin et a]. (14) and characterized by its absorbance profile (15). Anti-hMb rabbit immunoglobulins were obtained from Dakopatts.
Human Serum Samples Human sera were collected in the University Hospital of Nancy by centrifugation of blood samples (3,OOOg, 10 min) and stored frozen at - 20°C until use. Forty sera were obtained from subjects without known heart disorders. A pool of 10 of these sera, without measurable Mb concentration and rheumatoid factors, was used to perform the calibration curve of the nephelometric assay of Mb. Thirty-seven sera were obtained from 21 patients (five women and 16 men, 37-89 years old) with AM1 and from two men (62 and 73 years old) with angina pectoris. Ten of the AM1 patients presented large, mostly anterior, myocardial necrosis. Eleven patients presented a more limited necrosis. Diagnosis and extent of the myocardial necrosis were defined by cardiologists according to a combination of clinical, biochemical (total released amount of diagnostic enzymes), electrocardiographic, and radiological analyses. The microparticle-enhanced nephelometric immunoassay of Mb has been performed without knowledge of this clinical and biochemical information. Serum levels of glutamic oxalacetic transaminase (GOT), total creatine kinase (CK), and CK-MB isoenzyme were determined on admission (from 1 to 24 hr, mean 5.5 hr after first distress signs), after 6 and 12 hr, and then every day for 6 days. GOT (normal range 5-25 IUiliter, 30"C), total CK (normal range 15- 130 IU/liter, 30"C), and CK-MB (normal concentration
5
10
1
1000
1OD
M b Concentration (ng/rnl) Fig. 2. Inhibitions, by various free Mb, of agglutinations of Mb-MS conhMb-MS (0.2 mg1rnP); jugates in the presence of anti-hMh antiserum: W, hMh-MS (0.2mg1mP) with anti-hMh(111600) hy hMh. hMb-MS(0.125 hMh; 0 ,hMb; V and A , cardiac and mg1ml) with anti-hMb (113200) by skeletal horse Mb; *, sperm whale Mb.
A,
v,
concentration yielding 50% inhibition 14 and 7 ng/ml, respectively) and diverged only for the highest Mb concentrations (>30 ng/ml). Cardiac and skeletal horse Mb and sperm whale Mb did not interfere (cross reaction 45 ng/ml were observed in nephelometry in 33 of these sera (mean 305 ng/ml, SD 289 ng/ml), while Mb was detected only by slide test (detection threshold 100 ng/ml) in 14 of them. The concentrations of Mb measured by nephelometry in these 14 sera ranged from 149 to 1,628 ng/ml (mean 470 ng/ml, SD 390 ng/ml) and were significantly higher than those obtained for the 19 other (from
Microparticle-BasedAssay of Myoglobin h
> E 500 x
'rF 350 t (u
c,
cn
1
L)
1
Ll
0 o 300
Mb Serum Concentration (pg/ml) Fig. 3. Calibration curve of assay for hMb in serum sample, bMb-MS conjugate, 0.6 mg/ml, anti-hMb antiserum 400-fold diluted, and serum sample tenfold diluted in the reaction mixture. Reaction time 15 min.
76 to 251 ngiml (mean 184 ng/ml, SD 48 nglml). Mb was detected in nephelometry in two of 30 control serum samples (590 and 51 ng/ml, respectively). The latex slide test was positive in the first only. A correlation was observed between results obtained by nephelometry and RIA (r = 0.85, nephelometry = 116.02 0.46 RIA) for the 26 sera where Mb concentration was measurable at the same time by both methods. Mb concentrations measured by microparticle-enhanced nephelometric immunoassay in the earliest serum collected for each patient (n = 23, mean 354 ng/ml, SD 335 ng/ml) were significantly higher than those obtained in later blood samples (n = 11, mean 152 ng/ml, SD = 82 ng/ml). Serum Mb levels measured by microparticle-enhanced nephelometric immunoassay on admission correlated with GOT (n = 12, r = 0.50) and CK-MB (n = 9, r = 0.57) maximum levels, observed later during the following 6 days of hospitalization. For 71% of patients, the level of these enzymes was not yet increased on admission. Furthermore, as for the maximum levels of GOT, CK, and CK-MB observed later, the mean of Mb concentrations measured in the first serum of each patient appeared higher for patients with AM1 and a large myocardial necrosis (n = 10, mean 497 ng/ml, SD 456 ng/ml) than for patients with a more limited necrosis (n = 13, mean 245 ng/ml, SD 142 ng/ml). However, this difference was not significant because of the great disparity in Mb levels in patients with large myocardial necrosis.
+
DISCUSSION Polyacrylic MS, especially synthesized as nephelometric marker of the antigen-antibody reaction (2), were coated with hMb and bMb. The Mb-MS conjugates thus produced were agglutinated by anti-hMb immunoglobulins. The light scattered by Mb-MS clusters could be quantified using an adapted
179
nephelometer. Changes in the scattered light during these agglutinations were large enough for accurate measurements after only 15 min of reaction. Total inhibition by free pure hMb and bMb, respectively, of the hMb-MS and bMb-MS agglutinations occurring in the presence of specific anti-hMb antibodies confirmed the immunological specificity of agglutinations. It also showed that baboon Mb, but not Mb from horse and sperm whale, could be used to replace hMb for a sensitive immunoassay of hMb in human serum. This cross reaction allowed a simplification in the preparation of the conjugate and a decrease in its cost (constraint linked to the use of human tissu,e). The microparticle-enhanced nephelometric immunoassay for hMb in serum, as described in this report, used a pretreatment of human serum samples by control uncoated MS before the assay. A preliminary study had indeed shown the possible unstability of bMb-MS in some highly concentrated human sera, a drawback readily eliminated by pretreatment of the samples with control, uncoated MS. With the intent to evaluate the microparticle-enhanced nephelometric immunoassay of Mb in serum from patients with AM1 under the conditions of most reliability, the serum samples were systematically pretreated, although this procedure increased the complexity of the assay. The instability of the dispersed solid phase in some highly concentrated human sera was not peculiar to this Mb assay and can be compared with the nonspecific phenomena encountered in other solid-phase immunoassays currently used. Study of the serum components responsible for these phenomena is in progress. It shows a parallelism between the microparticle instability and the ability of these serum components to be adsorbed on the solidphase surface. Their identification may allow us to decrease the constraints brought about by this sample pretreatment (report in progress). The calibration range obtained for hMb quantitation by microparticle-enhanced nephelometric immunoassay (45 ng to 11.5 p,g/ml) largely covered with a sufficient accuracy (precision and recovery) pathological Mb concentrations in sera of patients with AM1 observed using other methods. The method presented here is easier to perform and faster than ELISA or RIA (1 1) and is totally accurate for the highest Mb concentrations (antigen-coated MS and competitive mode). The use of antibody-coated latex in immunonephelometry (13) may, conversely, carry a risk of underevaluation in antigenexcess conditions (calibration range limited to 400 ng/ml and possible hook effect). The slope of the regression curve in the correlation study with RIA suggested that there was a difference in standardization between the RIA used and the new assay described in this report. Only an available international reference preparation for hMb would allow us to solve this standardization problem. Evaluation of the microparticle-enhanced nephelometric immunoassay for hMb confirmed the great interest of Mb assessment in serum samples to rule out AM1 and, in spite
Cliquet et al.
180
TABLE 1. Clinical and Biological Picture of Patients With AM1 and Angina Pectoris
Patient Sex, age
GOT
CK
CK-MB
Extent of necrosisb
M,59
ND
2,921
ND
++
Enzymes (1UIliter)a
1
2 M.37
509
2,748
148
3 M.73 4 M,38 5 F, 77
58 160 72
79 1,630 92
ND ND ND
++ -
+ +
+ ++
6 F, 88
103
37 1
ND
7
M.64
274
2,200
213
8 M,62
35
200
ND
-
9 M.41 10 M,58
I98 339
2,405 3,800
243 193
+ ++
I1
M,57
170
1,628
119
12 F, 71 13 M,42 14 M.47 15 M,44 16 M,57
182 176 64 490 209
1,406 1,180 682 3,870 2,500
ND 104 79 199 127
17 M,51
89
1,031
108
18 M,47
I80
1,355
123
19 F, 89 20 F, 84 21 M.49
ND 117 54
898 749 609
132 93 61
22 M,75
92
612
74
23 M,75
188
1,481
42
”Maximum level. b + + , Large; + , limited; - , without necrosis. , Agglutination and last agglutinating dilution; dND. not determined.
+
-
++ ++ + + ++ ++
+ + ++ + + + ++
Time (hr) after admission
Mb slide test‘
RIA
72 96 120 0 96 0 96 0 120 0 144 0 ? 0 48 144 0 96 24 120 96 0 96 0 12 144 0 120 0 120 0 24 24 120 0 72 72
-
102 71 89 > 1,000 53 23 I 55 165 207 206 164 > 1,000 238 > 1,000 69 78 327 48 816 81 1,000
Development of a Rapid Microparticle-Enhanced Nephelometric lmmunoassay for Serum Myoglobin in Acute Myocardial Infarction F. Cliquet,’ t? Montagne,’ M.L. Cuilliere,’ I?Varcin,* and J. Duheille’ ‘Immunology Laboratoty Faculty of Medicine, Nancy, France; Sanofi Research, Montpellier, France A microparticle-enhanced nephelometric immunoassay was developed for myoglobin quantitation in human serum. It uses rabbit antimyoglobin serum and hydrophilic polyacrylic microparticles covalently coated with baboon myoglobin in a competitive immunoagglutination system. The level of microparticle agglutinationis assessed with a specially designed nephelometer. This sensitive (45 ng/ml of myoglobin detected in serum) and accurate (coefficients of variation from 3.0% to 8.2% in precision study and linear recov-
Key words:
s s ,
microparticle agglutination, immunonephelometry,AM1
INTRODUCTION Microparticle-enhanced nephelometric immunoassay has been previously described as a new easy-to-perform and sensitive method to assay various serum proteins (1-3), milk proteins (4-7), and haptens (8). These tests are based on the inhibition, by free antigen, of the agglutination of antigencoated microspheres (MS) by specific antiserum. Quantification of free antigen levels is performed by the measurement with an adapted nephelometer of the light scattered by antigen-MS conjugate clusters during this inhibition. The measurement of serum myoglobin (Mb) levels is used to rule out acute myocardial infarction (AMI). In spite of its relative nonspecificity, it could also contribute to the AM1 diagnosis in emergency units, allowing rapid instauration of reperfusion and evaluation of its efficiency (9,lO). The most recently reported methods for Mb measurement (1 1) are usually inadequate for emergency conditions (constraint linked to the use of radioisotopes, long reaction times, necessity of numerous washings or phase separation). Latex-slide tests ( 12) are rapid, unsophisticated, and well adapted to emergency conditions, but they are only semiquantitative and the detection of latex agglutination is subjective. An automated nephelometric immunoassay using latex agglutination has been described recently for Mb quantitation (13). It uses antibodycoated latex with a calibration range limited to 400 ng/ml and thus presents a risk of false-negative results through underevaluation of the highest Mb levels (hook effect in antigen excess conditions). 0 1992 Wiley-Liss, Inc.
ery of myoglobin in overloaded sera) immunoassay was evaluated with human sera from patients suffering from acute myocardial infarction. Myoglobin levels in patient‘s serum on admission appeared to be correlated with clinical and biological parameters assessed in emergency wards and later. This new, rapid, and easy microparticle-enhanced nephelometric immunoassay could thus be useful in emergency conditions for the early quantitationof serum myoglobin. o ~ g ~ ~ i ~ e y Inc. - ~ i
A rapid and easy-to-perform assay, capable of providing an early and accurate quantitation of Mb levels in serum from patients with AM1 will be very beneficial. We report here an improved competitive microparticle-enhanced nephelometric immunoassay using Mb-MS conjugate and its evaluation as a method to be used in emergency conditions for the quantitation of Mb in serum from patients with AMI.
MATERIALS AND METHODS Reagents Acrolein, 2-hydroxyethylmethacrylate,methacrylic acid, and polyethylene glycol 6000 were obtained from Merck (Darmstadt, Federal Republic of Germany). N,N’-methylenebisacrylamide was from Eastman Kodak Co. (Rochester, NY). Sodium dodecyl sulfate, hydroquinone, 2-aminoethanol, sucrose, sodium azide, potassium and sodium chlorides, potassium and sodium dihydrogenophosphates, and disodium hydrogenophosphate were purchased from Prolabo Rh6ne Poulenc (Paris, France). Immunonephelometric assays were carried out with the nephelometry buffer supplied by Diagnostics Pasteur (Marnes, France). Human Mb (hMb), cardiac and skeletal horse Mb, and sperm whale Mb were purchased from Dakopatts (Glostrup,
Received November7, 1991; accepted February 18, 1992. Address reprint requests to P. Montagne, Immunology Laboratory, Faculty of Medicine, B.P.184, F-54505 Vandoeuvre les Nancy Cedex, France.
Microparticle-Based Assay of Myoglobin
Denmark), Calbiochem Corporation (San Diego, CA), and Sigma Chemical Co. (St. Louis, MO), respectively. Baboon Mb (bMb) was extracted as metmyoglobin from skeletal muscle according to the method described by Malin et a]. (14) and characterized by its absorbance profile (15). Anti-hMb rabbit immunoglobulins were obtained from Dakopatts.
Human Serum Samples Human sera were collected in the University Hospital of Nancy by centrifugation of blood samples (3,OOOg, 10 min) and stored frozen at - 20°C until use. Forty sera were obtained from subjects without known heart disorders. A pool of 10 of these sera, without measurable Mb concentration and rheumatoid factors, was used to perform the calibration curve of the nephelometric assay of Mb. Thirty-seven sera were obtained from 21 patients (five women and 16 men, 37-89 years old) with AM1 and from two men (62 and 73 years old) with angina pectoris. Ten of the AM1 patients presented large, mostly anterior, myocardial necrosis. Eleven patients presented a more limited necrosis. Diagnosis and extent of the myocardial necrosis were defined by cardiologists according to a combination of clinical, biochemical (total released amount of diagnostic enzymes), electrocardiographic, and radiological analyses. The microparticle-enhanced nephelometric immunoassay of Mb has been performed without knowledge of this clinical and biochemical information. Serum levels of glutamic oxalacetic transaminase (GOT), total creatine kinase (CK), and CK-MB isoenzyme were determined on admission (from 1 to 24 hr, mean 5.5 hr after first distress signs), after 6 and 12 hr, and then every day for 6 days. GOT (normal range 5-25 IUiliter, 30"C), total CK (normal range 15- 130 IU/liter, 30"C), and CK-MB (normal concentration
5
10
1
1000
1OD
M b Concentration (ng/rnl) Fig. 2. Inhibitions, by various free Mb, of agglutinations of Mb-MS conhMb-MS (0.2 mg1rnP); jugates in the presence of anti-hMh antiserum: W, hMh-MS (0.2mg1mP) with anti-hMh(111600) hy hMh. hMb-MS(0.125 hMh; 0 ,hMb; V and A , cardiac and mg1ml) with anti-hMb (113200) by skeletal horse Mb; *, sperm whale Mb.
A,
v,
concentration yielding 50% inhibition 14 and 7 ng/ml, respectively) and diverged only for the highest Mb concentrations (>30 ng/ml). Cardiac and skeletal horse Mb and sperm whale Mb did not interfere (cross reaction 45 ng/ml were observed in nephelometry in 33 of these sera (mean 305 ng/ml, SD 289 ng/ml), while Mb was detected only by slide test (detection threshold 100 ng/ml) in 14 of them. The concentrations of Mb measured by nephelometry in these 14 sera ranged from 149 to 1,628 ng/ml (mean 470 ng/ml, SD 390 ng/ml) and were significantly higher than those obtained for the 19 other (from
Microparticle-BasedAssay of Myoglobin h
> E 500 x
'rF 350 t (u
c,
cn
1
L)
1
Ll
0 o 300
Mb Serum Concentration (pg/ml) Fig. 3. Calibration curve of assay for hMb in serum sample, bMb-MS conjugate, 0.6 mg/ml, anti-hMb antiserum 400-fold diluted, and serum sample tenfold diluted in the reaction mixture. Reaction time 15 min.
76 to 251 ngiml (mean 184 ng/ml, SD 48 nglml). Mb was detected in nephelometry in two of 30 control serum samples (590 and 51 ng/ml, respectively). The latex slide test was positive in the first only. A correlation was observed between results obtained by nephelometry and RIA (r = 0.85, nephelometry = 116.02 0.46 RIA) for the 26 sera where Mb concentration was measurable at the same time by both methods. Mb concentrations measured by microparticle-enhanced nephelometric immunoassay in the earliest serum collected for each patient (n = 23, mean 354 ng/ml, SD 335 ng/ml) were significantly higher than those obtained in later blood samples (n = 11, mean 152 ng/ml, SD = 82 ng/ml). Serum Mb levels measured by microparticle-enhanced nephelometric immunoassay on admission correlated with GOT (n = 12, r = 0.50) and CK-MB (n = 9, r = 0.57) maximum levels, observed later during the following 6 days of hospitalization. For 71% of patients, the level of these enzymes was not yet increased on admission. Furthermore, as for the maximum levels of GOT, CK, and CK-MB observed later, the mean of Mb concentrations measured in the first serum of each patient appeared higher for patients with AM1 and a large myocardial necrosis (n = 10, mean 497 ng/ml, SD 456 ng/ml) than for patients with a more limited necrosis (n = 13, mean 245 ng/ml, SD 142 ng/ml). However, this difference was not significant because of the great disparity in Mb levels in patients with large myocardial necrosis.
+
DISCUSSION Polyacrylic MS, especially synthesized as nephelometric marker of the antigen-antibody reaction (2), were coated with hMb and bMb. The Mb-MS conjugates thus produced were agglutinated by anti-hMb immunoglobulins. The light scattered by Mb-MS clusters could be quantified using an adapted
179
nephelometer. Changes in the scattered light during these agglutinations were large enough for accurate measurements after only 15 min of reaction. Total inhibition by free pure hMb and bMb, respectively, of the hMb-MS and bMb-MS agglutinations occurring in the presence of specific anti-hMb antibodies confirmed the immunological specificity of agglutinations. It also showed that baboon Mb, but not Mb from horse and sperm whale, could be used to replace hMb for a sensitive immunoassay of hMb in human serum. This cross reaction allowed a simplification in the preparation of the conjugate and a decrease in its cost (constraint linked to the use of human tissu,e). The microparticle-enhanced nephelometric immunoassay for hMb in serum, as described in this report, used a pretreatment of human serum samples by control uncoated MS before the assay. A preliminary study had indeed shown the possible unstability of bMb-MS in some highly concentrated human sera, a drawback readily eliminated by pretreatment of the samples with control, uncoated MS. With the intent to evaluate the microparticle-enhanced nephelometric immunoassay of Mb in serum from patients with AM1 under the conditions of most reliability, the serum samples were systematically pretreated, although this procedure increased the complexity of the assay. The instability of the dispersed solid phase in some highly concentrated human sera was not peculiar to this Mb assay and can be compared with the nonspecific phenomena encountered in other solid-phase immunoassays currently used. Study of the serum components responsible for these phenomena is in progress. It shows a parallelism between the microparticle instability and the ability of these serum components to be adsorbed on the solidphase surface. Their identification may allow us to decrease the constraints brought about by this sample pretreatment (report in progress). The calibration range obtained for hMb quantitation by microparticle-enhanced nephelometric immunoassay (45 ng to 11.5 p,g/ml) largely covered with a sufficient accuracy (precision and recovery) pathological Mb concentrations in sera of patients with AM1 observed using other methods. The method presented here is easier to perform and faster than ELISA or RIA (1 1) and is totally accurate for the highest Mb concentrations (antigen-coated MS and competitive mode). The use of antibody-coated latex in immunonephelometry (13) may, conversely, carry a risk of underevaluation in antigenexcess conditions (calibration range limited to 400 ng/ml and possible hook effect). The slope of the regression curve in the correlation study with RIA suggested that there was a difference in standardization between the RIA used and the new assay described in this report. Only an available international reference preparation for hMb would allow us to solve this standardization problem. Evaluation of the microparticle-enhanced nephelometric immunoassay for hMb confirmed the great interest of Mb assessment in serum samples to rule out AM1 and, in spite
Cliquet et al.
180
TABLE 1. Clinical and Biological Picture of Patients With AM1 and Angina Pectoris
Patient Sex, age
GOT
CK
CK-MB
Extent of necrosisb
M,59
ND
2,921
ND
++
Enzymes (1UIliter)a
1
2 M.37
509
2,748
148
3 M.73 4 M,38 5 F, 77
58 160 72
79 1,630 92
ND ND ND
++ -
+ +
+ ++
6 F, 88
103
37 1
ND
7
M.64
274
2,200
213
8 M,62
35
200
ND
-
9 M.41 10 M,58
I98 339
2,405 3,800
243 193
+ ++
I1
M,57
170
1,628
119
12 F, 71 13 M,42 14 M.47 15 M,44 16 M,57
182 176 64 490 209
1,406 1,180 682 3,870 2,500
ND 104 79 199 127
17 M,51
89
1,031
108
18 M,47
I80
1,355
123
19 F, 89 20 F, 84 21 M.49
ND 117 54
898 749 609
132 93 61
22 M,75
92
612
74
23 M,75
188
1,481
42
”Maximum level. b + + , Large; + , limited; - , without necrosis. , Agglutination and last agglutinating dilution; dND. not determined.
+
-
++ ++ + + ++ ++
+ + ++ + + + ++
Time (hr) after admission
Mb slide test‘
RIA
72 96 120 0 96 0 96 0 120 0 144 0 ? 0 48 144 0 96 24 120 96 0 96 0 12 144 0 120 0 120 0 24 24 120 0 72 72
-
102 71 89 > 1,000 53 23 I 55 165 207 206 164 > 1,000 238 > 1,000 69 78 327 48 816 81 1,000
