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Neonatal screening for familial hypercholesterolaemia by immunoturbidimetric assay of apolipoprotein B in dried blood spots
Clinica Chimica Acta, 213 (1992) 95-101 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009~8981/92/$05.00
95
CCA 05435
Short Communication
Neonatal screening for familial hypercholesterolaemia by immunoturbidimetric assay of apolipoprotein B in dried blood spots SK. Bangerta, P.H. Eldridgeb and T.J. Petersa “Department of Clinical Biochemistry, King’s College School of Medicine and Dentistry, Bessemer Road, London, SE5 9PJ and bChemical Pathology Department, Lewisham Hospital, Lewisham. London SE13 6LH (UK)
(Received 20 February 1992; revision received 25 September 1992; accepted 29 September 1992) Key work
Neonatal screening; Familial hypercholesterolaemia;
Apolipoprotein B; Blood spots
Introduction Homozygotes and heterozygotes with familial hypercholesterolaemia have raised LDL cholesterol concentrations in serum and an increased risk of premature coronary atherosclerosis [ 11. Early treatment may improve the prognosis [2] and so early detection is desirable. However, assay of cholesterol in cord blood does not appear to be useful in screening for hypercholesterolaemia [3,4]. It would be particularly convenient to be able to screen using the dried blood spots already collected from infants at about 6 days of age for hypothyroidism, phenylketonuria, etc. screening. Measurement of total cholesterol in dried blood spots has been reported [5], but involves a solvent extraction step and has been criticized [6] as a screening test since it is not straightforward or reliable. Van Biervliet et al., using a manual immunonephelometric method, showed that it was possible to assay apo B, a good marker for familial hypercholesterolaemia, in dried blood spots 171. This is a reasonable approach to screening, since apo B levels rise rapidly during the first few days of life but then remain constant for up to 30 days [4]. In an investigation of methods suitable for large scale screening, Dudman et al. [8] chose radial immunodiffusion as an assay procedure, although this is a lengthy, labour intensive procedure. We have developed a rapid, mechanised, immunoturbidimetric method for measurement of apo B in dried blood spots and applied it to screening for hyperlipidaemias. Correspondence lo: Professor T.J. Peters, Department of Clinical Biochemistry, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK.
Materials and Methods
Assays were performed on a Cobas Bio centrifugal analyser (Roche Products Ltd., Welwyn Garden City, UK). Serum apo B measurements were made with goat antihuman ApoB, Immunoneph Reference Standard, polyethylene glycol (PEG) buffer concentrate (40%) and detergent solution (Immuno Ltd, Sevenoaks, Kent) in the protocol of Mount et al. [9]. In the dried blood spot assay, goat anti-human ApoB and PEG buffer 40% (Immuno Ltd) were also used. Phosphate buffered saline (PBS, pH 7.4) was prepared according to Mount et al. [9], with the addition of bovine serum albumin at a concentration of 10 g/l. Blood spot standards were prepared by first selecting a serum sample with a high apoB concentration (around 2 g/l). This was assayed repeatedly and assigned a value. It was then diluted with PBS to give six standards over the approximate range 0.15-2.0 g/l (exact values depending on the value assigned to the serum). Washed red blood cells were prepared by collecting blood into lithium heparin, washing 3 times with 0.15 mol/l saline, and centrifuging (1,000 x g for 10 min) to remove as much saline as possible. Each dilution of serum was then mixed with an equal volume of packed washed red cells, mixed thoroughly and spotted (20 ~1) on to filter paper blood collection cards. These were allowed to dry overnight at room temperature and then stored at -20°C. Splitting batches into small sets of standards minimized the number of times standards were removed from the freezer. Quality control material was prepared by spotting blood from normal and hyperlipidaemic subjects on to filter paper cards and drying and storing in a similar manner.
TABLE I Assay variables for blood spot apo B Variable
Setting
1 2 3 4 5 6 I 8 9 IO 11 12 13 14 15 16 17 18 19
DA
Units Calculation factor Standard 1 Standard 2 Standard 3 Limit Temperature (“C) Type of analysis Wavelength (nm) Sample volume (~1) Diluent volume (~1) Reagent volume (~1) Incubation time (min) Start reagent volume (~1) Time of first reading (s) Time interval (s) No of readings Blanking mode Printout mode
1,000 0 0 0 0 25 5 310 80 2 200 0 0 5 600 2
I I
97
Assay procedure
Discs (3 mm) were punched from standard, control and unknown blood spots, using an automated punch (Delfia, Pharmacia-Wallac (UK) Ltd., Milton Keynes, UK) which dropped the discs into consecutive wells of a 96-well micro-titre plate (Dynatech Labs Ltd., Billingshurst, UK). Neonatal blood spot cards were stored dry at 4°C and harvested once each week. The Screening Laboratory computer system was used to generate worklists, so that no additional record of demographic data was required. A suitable eluant was prepared (see later) and 190 ~1 added to each well. The plate was shaken for 2 h at room temperature on a mechanical plate shaker. The eluates were then transferred to sample cups for analysis on the Cobas Bio analyser. PEG stock reagent (40%) was diluted to 50 g/l and used to dilute the anti-apoB antiserum 1:21. The diluted antibody was placed in the reagent boat of the Cobas Bio and the assay run using the variables listed in Table I. A standard curve was plotted from which values for controls and unknowns were derived. Results Choice of wavelength
Monitoring the change in absorbance at the recommended wavelength of 340 nm was complicated by high background absorbances, due to the haemoglobin which 1.10 1 .oo 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 280
320
360
400
440
480
520
560
Wavelength
600
640
680
720
760
800
(nm)
Fig. 1. Spectrophotometric scans of the immune complex in buffer (-) and the pigment eluted from dried blood spots (--- ). 310 nm was selected for use in the assay to minimize the background absorbance.
80 70 60 50 40 30 20 10 0 2
1
0
Apo
B
concentratron
3
(g/l)
Fig. 2. Comparison of detergent (e) and PEG (0) eluants on the standard curve of the blood spot assay. Values are mean f S.D.
30
20
10
0
o-.02
21-.4
.41-.6
Apo
.61-.8
B
.81-1.0
1.01-1.2
concentration
121-1.4
1.41-1.6
1.61-1.8
(g/l)
Fig. 3. Histogram of blood spot apo B values for randomly selected neonatal samples.
99
was also eluted from the blood spots. Scans of the blood spot eluate and the immune reaction mixture (Fig. 1) indicated that reading at 310 nm would minimize the background readings. Choice of eluant
The first eluant tested was the detergent solution supplied by Immuno Ltd. for the dilution of serum samples. This initially appeared to be satisfactory, but when used with samples from patients led to a significant proportion of values being less than the limit of detection. Eluting with 50 g/l PEG solution moved the standard curve (Fig. 2) so that it covered the whole neonatal range (Fig. 3). Optimal PEG concentration
The optimal PEG concentration was confirmed to be 50 g/l, as in the serum apoB assay. Accuracy
Twenty seven pairs of serum and blood spot samples were collected from hyperlipidaemic patients and from volunteer controls. The results are shown in Fig. 4, the line representing a linear regression analysis of the data, with a correlation coefficient of 6.79 and i < O.OOl.3
I
0 0
1
1 2 Serum
Apo
6
I
I
3
4
(g/l)
Fig. 4. Relationship between serum and blood spot apo B results for 27 paired samples.
5
100
Imprecision Intra-batch variation was assessed by assaying pairs of blood spots over a range of concentrations and calcuIating the coefficients of variation (C.V.) in three groups. At a mean level of 1.39 g/l the C.V. was 7.6%, (n = 7), at a mean level of 1.58 g/l the C.V. was 9.5% (n = 8) and at a mean level of 2.02 g/l the C.V. was 6.2% (n = 7). Inter-batch variation was assessed by assaying single blood spots at 2 levels in repeat assays. At a level of I .72 g/l the C.V. was 12.7% (n = 11) and at 2.2 1 g.0 12.2% (n = 10). Stability Blood spots were stable at -20°C for at least 3 months. However, in any large scale screening programme, storage at -20°C from sample collection until analysis may not be possible, so other storage conditions were investigated. Samples kept dry at 4°C were relatively stable for at least 2 weeks: storage at room temperature led to 50% loss of apo B after 2 weeks. Similar conclusions were reached by Wang et al. [lo], with respect to apo Al concentrations in blood spots. Reference interval The trial batch of 105 neonatal samples shown in Fig. 3 had a mean concentration of 1.04 g/l and S.D. of 0.34 g/l. Thus the reference interval based on the mean f 2 SD. is 0.36-1.71 g/l. Discussion Familial hypercholesterolaemia (FH) satisfies the requirements for disease screening in the neonatal population [ 111, but an automated method for screening large numbers of samples has not previously been available. Blood spot assays cannot be expected to be as good as the corresponding serum assays, but should still be adequate for screening purposes, This assay has precision comparable to other blood spot assays and the correlation between serum and blood spot values shouid enable high values to be readily identified. Standardization is a particular problem with apo B assays [12], and a secondary standard had to be used in the blood spot assay due to the lack of calibration material with a sufficiently high value. The serum assay used has a commercially prepared calibrant, but this has a relatively low value which is increased in the assay by using larger volumes of calibrant than sample. This approach could not be used in the blood spot assay because of the elution step, but in practice the use of a secondary standard should not present problems, as ‘positives’ on initial blood spot screening would be defined in terms of centiles (e.g. top 1%). The assay has been used for regular screening of all neonates in Camberwell over the past nine months. Approximately 100 samples are analysed weekly and the entire procedure takes approx. 2 h. Good consistency and reliability of the assay procedure has been achieved.
101
References 1 Goldstein JL, Brown MS: Familial hypercholesterolaemia. In: Striver CL, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1989. 2 Glueck CJ, Mellies MJ, Drue M, Perry T, Laskarzewski P: Safety and efficacy of long-term diet and diet plus bile acid-binding resin cholesterol-lowering therapy in 73 children heterozygous for familial hypercholesterolaemia. Paediatrics 1986;78:338-348. 3 Damady JM, Fosbrooke AS, Lloyd JK: Prospective study of serum cholesterol levels during first year of life. Br Med J 1972;2:685-688. 4 Lane DM, McConathy WJ: Changes in serum lipids and apolipoproteins in the first four weeks of life. Pediatr Res 1986;20:332-337. 5 Asami T: Screening for hypercholesterolaemia on blood-spotted filter paper (letter). Lancet 1983;11:229-230. 6 Bradbury WH, Forrest ARW: Screening for hypercholesterolaemia by use of blood spotted on filter paper (letter). Clin Chem 1985;31:648-649. 7 Van Biervliet JP, Vinaimmont N, Caster H, Rosseneu M, Belpaire F: A screening procedure for dyslipoproteinaemia in the newborn. Apoprotein quantitation on dried blood spots. Clin Chim Acta 1982;120:191-200. 8 Dudman NPD, Blades BL, Wilcken DEL, Aitken JM: Radial immunodiffusion assay of apolipoprotein B in blood dried on filter paper - a potential screening method for familial type 11 hypercholesterolaemia. Clin Chim Acta 1985;149:117-127. 9 Mount JN, Kearney EM, Rosseneu M, Salvin BM: Immunoturbidimetric assays for serum apolipoproteins Al and B using Cobas Bio centrifugal analyser. J Clin Path01 1988;41:471-474. 10 Wang XL, Dudman NPB, Blases BL, Wilcken DEL: Changes in the immunoreactivity of apo A-l during storage. Clin Chim Acta 1989;179:285-294. 11 Frankenburg WK: Slection of diseases and tests in pediatric screening. Pediatrics 1974;54:612-616. 12 Albers JJ, Marcovina SM: Standardization of apolipoprotein B and A-l measurements. Clin Chem 1989:35:1357-1361.
95
CCA 05435
Short Communication
Neonatal screening for familial hypercholesterolaemia by immunoturbidimetric assay of apolipoprotein B in dried blood spots SK. Bangerta, P.H. Eldridgeb and T.J. Petersa “Department of Clinical Biochemistry, King’s College School of Medicine and Dentistry, Bessemer Road, London, SE5 9PJ and bChemical Pathology Department, Lewisham Hospital, Lewisham. London SE13 6LH (UK)
(Received 20 February 1992; revision received 25 September 1992; accepted 29 September 1992) Key work
Neonatal screening; Familial hypercholesterolaemia;
Apolipoprotein B; Blood spots
Introduction Homozygotes and heterozygotes with familial hypercholesterolaemia have raised LDL cholesterol concentrations in serum and an increased risk of premature coronary atherosclerosis [ 11. Early treatment may improve the prognosis [2] and so early detection is desirable. However, assay of cholesterol in cord blood does not appear to be useful in screening for hypercholesterolaemia [3,4]. It would be particularly convenient to be able to screen using the dried blood spots already collected from infants at about 6 days of age for hypothyroidism, phenylketonuria, etc. screening. Measurement of total cholesterol in dried blood spots has been reported [5], but involves a solvent extraction step and has been criticized [6] as a screening test since it is not straightforward or reliable. Van Biervliet et al., using a manual immunonephelometric method, showed that it was possible to assay apo B, a good marker for familial hypercholesterolaemia, in dried blood spots 171. This is a reasonable approach to screening, since apo B levels rise rapidly during the first few days of life but then remain constant for up to 30 days [4]. In an investigation of methods suitable for large scale screening, Dudman et al. [8] chose radial immunodiffusion as an assay procedure, although this is a lengthy, labour intensive procedure. We have developed a rapid, mechanised, immunoturbidimetric method for measurement of apo B in dried blood spots and applied it to screening for hyperlipidaemias. Correspondence lo: Professor T.J. Peters, Department of Clinical Biochemistry, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK.
Materials and Methods
Assays were performed on a Cobas Bio centrifugal analyser (Roche Products Ltd., Welwyn Garden City, UK). Serum apo B measurements were made with goat antihuman ApoB, Immunoneph Reference Standard, polyethylene glycol (PEG) buffer concentrate (40%) and detergent solution (Immuno Ltd, Sevenoaks, Kent) in the protocol of Mount et al. [9]. In the dried blood spot assay, goat anti-human ApoB and PEG buffer 40% (Immuno Ltd) were also used. Phosphate buffered saline (PBS, pH 7.4) was prepared according to Mount et al. [9], with the addition of bovine serum albumin at a concentration of 10 g/l. Blood spot standards were prepared by first selecting a serum sample with a high apoB concentration (around 2 g/l). This was assayed repeatedly and assigned a value. It was then diluted with PBS to give six standards over the approximate range 0.15-2.0 g/l (exact values depending on the value assigned to the serum). Washed red blood cells were prepared by collecting blood into lithium heparin, washing 3 times with 0.15 mol/l saline, and centrifuging (1,000 x g for 10 min) to remove as much saline as possible. Each dilution of serum was then mixed with an equal volume of packed washed red cells, mixed thoroughly and spotted (20 ~1) on to filter paper blood collection cards. These were allowed to dry overnight at room temperature and then stored at -20°C. Splitting batches into small sets of standards minimized the number of times standards were removed from the freezer. Quality control material was prepared by spotting blood from normal and hyperlipidaemic subjects on to filter paper cards and drying and storing in a similar manner.
TABLE I Assay variables for blood spot apo B Variable
Setting
1 2 3 4 5 6 I 8 9 IO 11 12 13 14 15 16 17 18 19
DA
Units Calculation factor Standard 1 Standard 2 Standard 3 Limit Temperature (“C) Type of analysis Wavelength (nm) Sample volume (~1) Diluent volume (~1) Reagent volume (~1) Incubation time (min) Start reagent volume (~1) Time of first reading (s) Time interval (s) No of readings Blanking mode Printout mode
1,000 0 0 0 0 25 5 310 80 2 200 0 0 5 600 2
I I
97
Assay procedure
Discs (3 mm) were punched from standard, control and unknown blood spots, using an automated punch (Delfia, Pharmacia-Wallac (UK) Ltd., Milton Keynes, UK) which dropped the discs into consecutive wells of a 96-well micro-titre plate (Dynatech Labs Ltd., Billingshurst, UK). Neonatal blood spot cards were stored dry at 4°C and harvested once each week. The Screening Laboratory computer system was used to generate worklists, so that no additional record of demographic data was required. A suitable eluant was prepared (see later) and 190 ~1 added to each well. The plate was shaken for 2 h at room temperature on a mechanical plate shaker. The eluates were then transferred to sample cups for analysis on the Cobas Bio analyser. PEG stock reagent (40%) was diluted to 50 g/l and used to dilute the anti-apoB antiserum 1:21. The diluted antibody was placed in the reagent boat of the Cobas Bio and the assay run using the variables listed in Table I. A standard curve was plotted from which values for controls and unknowns were derived. Results Choice of wavelength
Monitoring the change in absorbance at the recommended wavelength of 340 nm was complicated by high background absorbances, due to the haemoglobin which 1.10 1 .oo 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 280
320
360
400
440
480
520
560
Wavelength
600
640
680
720
760
800
(nm)
Fig. 1. Spectrophotometric scans of the immune complex in buffer (-) and the pigment eluted from dried blood spots (--- ). 310 nm was selected for use in the assay to minimize the background absorbance.
80 70 60 50 40 30 20 10 0 2
1
0
Apo
B
concentratron
3
(g/l)
Fig. 2. Comparison of detergent (e) and PEG (0) eluants on the standard curve of the blood spot assay. Values are mean f S.D.
30
20
10
0
o-.02
21-.4
.41-.6
Apo
.61-.8
B
.81-1.0
1.01-1.2
concentration
121-1.4
1.41-1.6
1.61-1.8
(g/l)
Fig. 3. Histogram of blood spot apo B values for randomly selected neonatal samples.
99
was also eluted from the blood spots. Scans of the blood spot eluate and the immune reaction mixture (Fig. 1) indicated that reading at 310 nm would minimize the background readings. Choice of eluant
The first eluant tested was the detergent solution supplied by Immuno Ltd. for the dilution of serum samples. This initially appeared to be satisfactory, but when used with samples from patients led to a significant proportion of values being less than the limit of detection. Eluting with 50 g/l PEG solution moved the standard curve (Fig. 2) so that it covered the whole neonatal range (Fig. 3). Optimal PEG concentration
The optimal PEG concentration was confirmed to be 50 g/l, as in the serum apoB assay. Accuracy
Twenty seven pairs of serum and blood spot samples were collected from hyperlipidaemic patients and from volunteer controls. The results are shown in Fig. 4, the line representing a linear regression analysis of the data, with a correlation coefficient of 6.79 and i < O.OOl.3
I
0 0
1
1 2 Serum
Apo
6
I
I
3
4
(g/l)
Fig. 4. Relationship between serum and blood spot apo B results for 27 paired samples.
5
100
Imprecision Intra-batch variation was assessed by assaying pairs of blood spots over a range of concentrations and calcuIating the coefficients of variation (C.V.) in three groups. At a mean level of 1.39 g/l the C.V. was 7.6%, (n = 7), at a mean level of 1.58 g/l the C.V. was 9.5% (n = 8) and at a mean level of 2.02 g/l the C.V. was 6.2% (n = 7). Inter-batch variation was assessed by assaying single blood spots at 2 levels in repeat assays. At a level of I .72 g/l the C.V. was 12.7% (n = 11) and at 2.2 1 g.0 12.2% (n = 10). Stability Blood spots were stable at -20°C for at least 3 months. However, in any large scale screening programme, storage at -20°C from sample collection until analysis may not be possible, so other storage conditions were investigated. Samples kept dry at 4°C were relatively stable for at least 2 weeks: storage at room temperature led to 50% loss of apo B after 2 weeks. Similar conclusions were reached by Wang et al. [lo], with respect to apo Al concentrations in blood spots. Reference interval The trial batch of 105 neonatal samples shown in Fig. 3 had a mean concentration of 1.04 g/l and S.D. of 0.34 g/l. Thus the reference interval based on the mean f 2 SD. is 0.36-1.71 g/l. Discussion Familial hypercholesterolaemia (FH) satisfies the requirements for disease screening in the neonatal population [ 111, but an automated method for screening large numbers of samples has not previously been available. Blood spot assays cannot be expected to be as good as the corresponding serum assays, but should still be adequate for screening purposes, This assay has precision comparable to other blood spot assays and the correlation between serum and blood spot values shouid enable high values to be readily identified. Standardization is a particular problem with apo B assays [12], and a secondary standard had to be used in the blood spot assay due to the lack of calibration material with a sufficiently high value. The serum assay used has a commercially prepared calibrant, but this has a relatively low value which is increased in the assay by using larger volumes of calibrant than sample. This approach could not be used in the blood spot assay because of the elution step, but in practice the use of a secondary standard should not present problems, as ‘positives’ on initial blood spot screening would be defined in terms of centiles (e.g. top 1%). The assay has been used for regular screening of all neonates in Camberwell over the past nine months. Approximately 100 samples are analysed weekly and the entire procedure takes approx. 2 h. Good consistency and reliability of the assay procedure has been achieved.
101
References 1 Goldstein JL, Brown MS: Familial hypercholesterolaemia. In: Striver CL, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1989. 2 Glueck CJ, Mellies MJ, Drue M, Perry T, Laskarzewski P: Safety and efficacy of long-term diet and diet plus bile acid-binding resin cholesterol-lowering therapy in 73 children heterozygous for familial hypercholesterolaemia. Paediatrics 1986;78:338-348. 3 Damady JM, Fosbrooke AS, Lloyd JK: Prospective study of serum cholesterol levels during first year of life. Br Med J 1972;2:685-688. 4 Lane DM, McConathy WJ: Changes in serum lipids and apolipoproteins in the first four weeks of life. Pediatr Res 1986;20:332-337. 5 Asami T: Screening for hypercholesterolaemia on blood-spotted filter paper (letter). Lancet 1983;11:229-230. 6 Bradbury WH, Forrest ARW: Screening for hypercholesterolaemia by use of blood spotted on filter paper (letter). Clin Chem 1985;31:648-649. 7 Van Biervliet JP, Vinaimmont N, Caster H, Rosseneu M, Belpaire F: A screening procedure for dyslipoproteinaemia in the newborn. Apoprotein quantitation on dried blood spots. Clin Chim Acta 1982;120:191-200. 8 Dudman NPD, Blades BL, Wilcken DEL, Aitken JM: Radial immunodiffusion assay of apolipoprotein B in blood dried on filter paper - a potential screening method for familial type 11 hypercholesterolaemia. Clin Chim Acta 1985;149:117-127. 9 Mount JN, Kearney EM, Rosseneu M, Salvin BM: Immunoturbidimetric assays for serum apolipoproteins Al and B using Cobas Bio centrifugal analyser. J Clin Path01 1988;41:471-474. 10 Wang XL, Dudman NPB, Blases BL, Wilcken DEL: Changes in the immunoreactivity of apo A-l during storage. Clin Chim Acta 1989;179:285-294. 11 Frankenburg WK: Slection of diseases and tests in pediatric screening. Pediatrics 1974;54:612-616. 12 Albers JJ, Marcovina SM: Standardization of apolipoprotein B and A-l measurements. Clin Chem 1989:35:1357-1361.