Dried filter paper blood spot

Screening for congenital hypothyroidism is mandatory throughout the United States. Most locales initially test dried filter paper blood spots for T4 and follow-up with TSH for results in the lowest decile. It is estimated that as many as 10% of cases may be missed initially, because of either absent abnormafities at birth or errors in sample processing (Rhead and Irons, 2004). 

Specimen collection for amino acids is comprehensively covered in Chap. B. Filter paper blood spots (PKU cards) are increasingly used for blood collection. Urine specimens for the testing of purines and pyrimidines, and for acylglycines are typically frozen and shipped on dry ice. They can also be applied to filter paper strips. For further details, refer to Chap. 21 and refs. 7 and 8. 

Serum proteins are fractionated by electrophoresis on hlter paper in a buffer solution, using a wet chamber. We use filter paper Whatman No. 1, measuring 4.5 X 37 cm, impregnated with a buffer solution, the excess of which is removed by placing the strip upon dry filter paper for several minutes. Spot 25-30 pi (cubic millimeters) of fresh serum obtained from blood sampled on the same day serum remaining from the previous day cannot be used anymore. If the protein level is low, greater volumes are spotted. Perform the protein fractionation at room temperature, 110 V, about 1 mA per strip, for 16 hr. Then place the filter paper strips hori-... 

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... 

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. 

All sample types, except for dried blood and bile spots (which can be sent at room temperature), should be kept frozen until analysis. Reliable results, particularly for short-chain acylcarnitine species, for any sample, liquid or dried on filter paper, can not be achieved following long-term storage at ambient temperatures [55]. 

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). 

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. 

Dried blood spots (3 mm) are punched from filter paper cards (Whatman 903 paper, Whatman) and distributed in a microtiter plate. Each patient sample is run in duplicates. Subsequently, 100 pi of substrate buffer and 100 pi of citrate buffer are added to each specimen. The plate is shaken for 45 min, sealed with tape, covered with aluminum wrapping and incubated at 37°C for 21 h. The reaction is terminated by the addition of 200 pi stop solution and the fluorescence is read with an excitation wavelength of 365 nm and an emission wavelength of 450 nm. 

Chamoles NA, Blanco MB, Gaggioli D, Casentini C (2001) Hurler-like Phenotype enzymatic diagnosis in dried blood spots on filter paper. Clin Chem 47 2098-2102... 

FIGURE 3 (A) Photograph of dried blood spots on filter paper (white portion) attached to a... 

As newborn screening by MS/MS is discussed in detail, one of the primary lessons learned has been that the method of sample collection is critical in directing the methods used in analysis and interpretation of results. In the case of dried blood spots, it was apparent that the blood volume indeed varies substantially from sample to sample and has a significant analytical impact on quantification and interpretation. The quantity of blood contained with each disk, as observed in Figure 3A and B, will vary by hematocrit, for example. Higher hematocrit increases the viscosity of blood and it will therefore spread less when applied to the filter paper. The net result is a high blood volume per area of filter paper. Lower hematocrit specimens, which spread more readily, have a larger area and a lower blood volume per area. Plasma, which has no red cells, spreads to nearly twice the area of blood... 

Maeda M, Arakawa H, Tsuji A, Yamagami Y, Isozaki A, Takahashi T. et al. Enzyme-linked immunosorbent assay for 17a-hydroxyprogesterone in dried blood spotted on filter paper. Clin Chem 1987 33 761-4. 

Figure 55-3 shows a flow chart for the postmortem evaluation of sudden and unexpected death that is centered on the analysis of acylcarnitines in blood and bile spots. Blood and bile could be conveniently collected on a single filter paper card, one identical to those used for newborn screening, which can be shipped at room temperature once properly dried. Both specimens should be collected to detect patients who may have only mild blood abnormalities when carnitine is depleted (Figure 55-4). In cases with a higher level of suspicion, an effort should be made to collect and freeze a specimen of liver and a skin biopsy, which could be analyzed only if needed to reduce cell culture workload. ...

Methods for the assay of hexosaminidase A and total hexosaminidase activities in dried blood spots on filter paper offer considerable advantages for screening (Chamoles et al., 2002). The deficient activity of the lysosomal enzymes hexosaminidase A and total hexosaminidase (hexosaminidase A plus B) are usually measured in plasma or extracts of leukocytes. To tubes containing a 3-mm-diameter blood spot, elution liquid and substrate solution were added. After incubation at 37°C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. The method was proven reliable even after storage for up to 38 months at room temperature. For total hexosaminidase, the substrate was 4-methyl-umbelliferyl-2-acetamido-... 

Bogden and Joselow (B7) also spotted a drop of blood onto filter paper, dried it, and then burned a standard-sized disc punched from the filter paper in an atomic absorption spectrophotometer. Blood lead analysis by this method was reported to compare favorably with macromethods, and specimen collection was much easier. 

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