Blood spots

There are also RMs which are prepared for a specific application and are used for validation of relevant methods. Cobbaert et al. (1999) made use of Ion Selective Electrode (ISE)-protein-based materials when evaluating a procedure which used an electrode with an enzyme-linked biosensor to determine glucose and lactate in blood. Chance et al. (1999) are involved with the diagnosis of inherited disorders in newborn children and they prepared a series of reference materials consisting of blood spotted onto filter paper and dried, from which amino-acids can be eluted and... 

Chance DH, Adam BW, Smith SJ, Alexander JR, Hillman SL, Hannon WH (1999) Validation of accuracy-based amino acid reference materials in dried-blood spots by tandem mass spectrometry for newborn screening assays. Clin Chem 45 1269-1277. 

RASHED, M.S., BUCKNALL, M.P., LITTLE, D., AWAD, A., JACOB, M., ALAMOUDI, M., ALWATTAR, M., OZAND, P.T., Screening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles, Clin. Chem., 1997,43,1129-1141. 

This in fact is what happened. Tandem MS has clearly been shown to be the only technology to screen for disorders of fatty acid oxidation and could also detect many disorders of organic acid metabolism. Tandem MS has the ability to detect both compound classes (amino acids and acylcarnitines) and after demonstrating that both classes could be prepared in the same manner, the MS/MS analysis of blood spots for newborn screening applications was born. 

Figure 14.1. Tandem mass spectra of amino acids. Top left panel shows the product ion spectra of the buylester of phenylalanine. Fragmentation that explains the neutral loss of 102 Da is shown in the top left panel of the figure. The bottom panel is a neutral loss scan from m/z 125 to 270 which includes many common amino acids. The profile is obtained by an analysis of a blood spot from a patient with PKU.

Figure 14.2. Acylcarnitine profile obtained using a precursor ion scan of 85 Da. The profile is from the blood spot of a normal patient.

A competitive ELISA assay for Lp(a) was recently described (Y4) in which the microtiter plate was coated with Lp(a) purified from a pool of donors. The method is simple and easy to perform, with satisfactory analytical parameters. A good stability and a reproducible coating of plates with the large Lp(a) lipoprotein is, however, critical in this type of assay. Wang et al. (W6) described an indirect sandwich assay for the measurement of Lp(a) in plasma and in dried blood spots, which can be applied to screening elevated Lp(a) levels in newborns (V3, V4). 

V4. Van Biervliet, J. P., Michiels, G., and Rosseneu, M., Quantification of lipoprotein(a) in dried blood spots and screening for above-normal lipoprotein(a) concentrations in newborns. Clin. Chem. (Winston-Salem. NC) 37, 706-708 (1991). 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

Koal, T., Burhenne, H., Romling, R., Svoboda, M., Resch, K., and Kaever, V., Quantification of antiretroviral drugs in dried blood spot samples by means of liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 19(21), 2995-3001, 2005. 

Rapid diagnosis of MCAD deficiency quantitative analysis of octanoylcamitine and other acylcamitines in newborn blood spots by tandem mass spectrometry. 

Thyroid status can be assessed using dried blood spot specimens for epidemiological surveys. Since TSH levels (as a marker of hypothyroidism) are stable in dried blood spot specimens for months this is the preferred assay for monitoring purposes. TSH monitoring is used in this way in India, China, Zaire, Thailand and Indonesia. 

Al-Dirbashi OY, Rashed MS, Brink HJ, Jakobs C, Filimban N, Al-Ahaidib LY, Jacob M, Al-Sayed MM, Al-Hassnan Z, Faqeih E (2006) Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry. J Chromatogr 831 274-280... 

A variety of body fluids can be used for acylcarnitine analysis. While initially the favored specimen, urine acylcarnitine analysis is the least appropriate when an FAO disorder is under diagnostic consideration. Heparinized plasma or whole blood spotted on filter paper are preferred in this context. 

Acylcarnitine analysis of dried blood or bile spots is very similar to the analysis of plasma. A small disk (diameter typically 5 mm or less) is punched out of the blood spot and the acylcarnitines extracted by the addition of methanol and known concentrations of isotopically labeled acylcarnitines, which function as internal standards. The extract is dried under a stream of nitrogen, and derivatized by the addition of either n-butanol HC1 or n-methanol HC1. The acylcarnitines are measured as their butyl or methyl esters by MS-MS. The concentrations of the analytes are established by computerized comparison of ion intensities of these analytes to that of the internal standards. 

Acylcarnitine species Blood spots (n = 1480) Bile spots (n = 1590)... 

Schmidt-Sommerfeld E, Penn D, Duran M, et al (1992) Detection and quantitation of acylcarnitines in plasma and blood spots from patients with inborn errors of fatty acid oxidation. Prog Clin Biol Res 375 355-362... 

Matern D, Strauss AW, Hillman SL, Mayatepek E, Millington DS, Trefz FK (1999) Diagnosis of mitochondrial trifunctional protein deficiency in a blood spot from the newborn screening card by tandem mass spectrometry and DNA analysis. Pediatr Res 46 45-49... 

Van Hove JL, Kahler SG, Feezor MD, et al (2000) Acylcarnitines in plasma and blood spots of patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency. J Inherit Metab Dis 23 571-582... 

Gibson KM, Bennett MJ, Naylor EW, Morton DH (1998) 3-Methylcrotonyl-coenzyme a carboxylase deficiency in amish/mennonite adults identified by detection of increased acylcarnitines in blood spots of their children. J Pediatr 132 519-523... 

Severe plasma turbidity due to hyperlipidaemia, as found in lipoprotein lipase deficiency, was shown to result in false-positive newborn screening results when dried blood spots on filter paper are used, but does not usually affect the quantitative colorimetric assay employing plasma samples (reference [21] and our own unpublished experience). 

A calibration curve is recorded for each plate (Fig. 4.1.10). 4-MU standards at absolute concentrations of 0.25 pmol (5 pi), 0.5 pmol (10 pi), 1.0 pmol (20 pi), 2.5 pmol (50 pi) and 5.0 pmol (100 pi) are measured in duplicates. The volume for each well is adjusted with demineralized water to the volume used in the assay. Before measurement, 200 pi stop solution are added and the plate is shaken for 5 min on a plate shaker. Fluorescence is read with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. For leukocyte and dried blood measurements blanks that contain substrate and buffer solution without leukocyte homogenate or dried blood spots are prepared. After incubation and addition of the stop solution, the plate is read immediately for all assays based on leukocytes, while one dried blood spot from an arbitrary sample is added to each blank in case of dried blood assays. Hence, hemoglobin is eluted from blood spots for 30 min and these spots are removed again before measurement. It is crucial to match the age of these specimens to the age of the patient samples. If large variations (several weeks) are evident concerning the age of the samples on the same plate then a separate blank for each patient sample has to be prepared. 

The preparation of a large batch of dried blood spot samples (e.g., ten filter paper cards) from one source requires about 5 ml whole blood. These samples can serve as quality control for at least 150 separate runs. Positive controls from patients may also be prepared in that manner and patients are far more likely to consent to this use of their blood due to the small amount necessary. 

Dried blood spots are prepared from native blood on standardized filter paper (Whatman 903, Whatman). The paper has to be fully soaked with blood and dried at room temperature. Blood has to be applied from one side only, to ensure even distribution throughout the filter paper. Do not use any plastic wrapping before the paper is completely dry. For further details, consult the guidelines from your closest newborn screening facility or metabolic laboratory processing dried blood specimens. 

---