Prevention is better than Treatment
ISNS Meeting, June 26-30, Genoa, Italy
George J. Reclos1, Evangelos D. Papaconstantinou2, Claudio Sampaio Jr.3, Nancy Mack Fadden3, Kleopatra H. Schulpis2 & Kenneth A. Pass4
1R&D Diagnostics Ltd., Holargos, Greece, 2Institute of Child Health, Athens, Greece, 3Intercientifica Ltda, Sao Jose dos Campos, Brazil, and 4NY Dept. of Health, Wadsworth Center, Albany, New York, USA
Abstract
Since the introduction of colorimetric kits for the determination of phenylalanine (Phe) levels it has been a common practice to use the “blank” as the “zero” point of the standard curve against which all samples were calculated. The blank doesn’t contain any sample at all therefore we used our new kit (PKUMMR2000) to investigate whether the use of a real “zero - Phe” blood spot would make a difference. The kit was used with both the Millipore and the transfer protocols and yielded comparable results in all aspects. Due to the higher cost of the Millipore plate protocol the normal “transfer protocol” was used for massive screening. Results presented in this report compare the statistics obtained from 200.000 neonates screened with the “blank” used as the “zero” point of the standard curve (data from n=17.120 standard curves) versus the statistics from 20.000 neonates (data from n=1.698 standard curves) obtained with a “zero - Phe” dried blood spot. Production of the “zero” blood was made by repeated washing of red blood cells from a normal donor with normal saline and the Phe value was verified by 10 independent runs in HPLC using aquatic Phe solutions for comparison. Results show that the use of the “zero-Phe” dried blood spot leads to a lower slope (0,0078 vs. 0,0095) but improved accuracy and reproducibility especially in the low end (0-8 mg/dL Phe) while the SD at higher values is not affected. In contrast, the values and SD of the mid range controls was significantly better using the “zero-Phe” blood spot (6,102+0,85 vs. 7,05+0,97; target value: 6,0 mg/dl and 10,26+0,91 vs. 11,04+1,1; target value: 10,0 mg/dl). The new method employing the “zero-Phe” blood spot has been found to offer significant advantages especially in the low end values therefore is being normally used for routine neonatal screening for PKU in the Institute of Child Health in Greece.
George J. Reclos1, Evangelos D. Papaconstantinou2, Claudio Sampaio Jr.3, Nancy Mack Fadden3, Kleopatra H. Schulpis2 and Kenneth A. Pass4
1R&D Diagnostics Ltd., Holargos, Greece, 2Institute of Child Health, Athens, Greece, 3Intercientifica Ltda, Sao Jose dos Campos, Brazil, and 4NY Dept. of Health, Wadsworth Center, Albany, New York, USA
Abstract
We have developed a colorimetric assay for the determination of Phenylalanine (Phe) in neonates which utilizes a new tetrazolium salt (Nitro Blue Tetrazolium; NBT) and diaphorase. This salt results in a much higher slope and better discrimination of Phe levels in the 0-20 mg/dl range, improved accuracy and excellent reproducibility. The new kit (PKUMMR2000/550nm) has been used with the Millipore assay and the “transfer” protocol yielding comparable results in every aspect. In the literature NBT is reported to be particularly unstable and leading to an increased auto-NADH+ production which results in increased background levels and low accuracy especially in the low end values (0-5 mg/dl) which are critical for the correct classification and diagnosis. We have used a combination of buffers which increased stability to well over 12 months while auto NADH+ production is kept to an absolute minimum. Studies comparing this kit to other PKU screening kits in the market (including our own “standard” 570 nm kit utilizing an MTT / Phenzainium color reagent; PKUMMR2000/570nm) revealed that the “blank” of our kit is 60-85% lower, while the slope is on average 100% higher. Statistics from more than 200.000 neonates screened at the Institute of Child Health in Athens employing the “transfer” protocol reveal a slope of 0,0095+0,0008 while the mean Phe value was 1,45+0,92 mg/dl (cut off point 3.5 mg/dl). Both PKUMMR2000 kits offer an accurate determination of Phe levels in neonatal specimens while the one using the NBT-diaphorase combination may be especially useful for laboratories using the “transfer” protocol which usually yields lower ODs and slopes. The entire procedure with the new kit takes approximately 70 minutes.
George J. Reclos1, Christine J. Hatzidakis1, Eurico Camargo Neto2, Claudio Sampaio Jr.3 and Kenneth A. Pass4
1R&D Diagnostics Ltd., Holargos, Greece, 2Laboratório Nobel RIE. Porto Alegre, Brazil, 3Intercientifica Ltda, Sao Jose dos Campos, Brazil, and 4NY Dept. of Health, Wadsworth Center, Albany, New York, USA
Abstract
We have developed an one step assay (OSMMR2000) for the determination of Glucose-6-Phosphate Dehydrogenase (G-6-PD) activity in neonates, which includes our “Hemoglobin Normalization” invention. This invention allows the direct expression of results in U/g Hb while it normalizes the blood content of all samples against the control. The assay is performed in microtiter plates using dried blood spots of any size or a quantity of whole blood or red blood cells (usually 5 microliters). It should be noted that dried blood spots and whole blood samples can be tested in the same microplate with the same control. After the elution a portion of the eluate is transferred to a clean microplate well and the reagent mixture (containing a substrate, coenzyme and buffer) is added. G-6-PD present in the specimen converts NADP to NADPH which is detected by a kinetic reading at 340 nm. G-6-PD activity in U/g Hb is calculated from the rate of a control. After termination of the kinetic readings the microplate is read at a suitable wavelength where haemoglobin (Hb) is measured. We strongly recommend 405 nm as the best wavelength (highest peak for Hb) although alternative wavelengths (e.g. 570 nm) can be used. Correlation between the Sigma assay and OSMMR2000 results was very good. Results from 14.192 neonates screened in Porto Allegre, Brazil and 12.500 neonates screened in Athens, Greece showed remarkably similar results (range : 0.4 – 18 U/g Hb; mean value: 11.5 U/g Hb). A cut-off point of 2,5 U/g Hb was selected for total deficiency whereas samples with an activity between 2,5 and 6,5 U/g Hb were classified as being partially deficient. In contrast 5.115 neonates screened in a pilot study in the New York State revealed higher activity (mean value 20,5+5,0) while the cut-off points was set to 4,1 and 12,2 U/g Hb for the totally deficient and partially deficient populations respectively. The prevalence of this disorder was 1.5, 1.6 and 1.8% for total deficiency (USA, Brazil and Greece respectively) and 4.2, 5.0 and 5.7 % for the two forms combined (Brazil, Greece and USA respectively). The new kit proved to be quick, easy to perform and reliable with a good reproducibility. The entire procedure (Hb normalization and elution time included) takes less than 40 minutes and uses standard laboratory equipment.
Claudio Sampaio Jr.1, George J. Reclos2, Nancy Mack Fadden1 and Kenneth A. Pass3
1Intercientifica Ltda, Rua Paschoal Moreira 485, Sao Jose dos Campos, Brazil, 2R&D Diagnostics Ltd., 41 El. Venizelou str., Holargos, Greece and 3NY Dept. of Health, Wadsworth Center, Albany, New York, USA
Abstract
Since the introduction by Bob Guthrie of filter paper for the collection and analysis of human blood in the early 1960s, the direct spotting method by heel pick has been by far the most widely used collection method. This method is characterized by ease of sample transport and use as well as very good stability and reproducibility. One of the drawbacks of this method as compared to alternative collection methods (vacuum tubes and capillary pipettes) is the relatively high number of false positive results which can be attributed to inaccuracies in estimating the amount of blood in the spot, due either to multiple applications or incomplete saturation of the paper. This can result in an increased number of recalls which in turn is time and labour consuming for any lab and thus increases the cost, a key factor in routine neonatal screening. Normalization of samples for their haemoglobin content would largely solve this problem. We have determined phenylalanine, galactose and leucine levels by colorimetric means in dried blood spot samples on which varying amounts of blood were spotted. Our results show that the ratio of the analyte concentration to the haemoglobin content (as determined by a separate reading at 405 nm) is stable for the range of 0.8 – 4 drops / filter paper disk although it differs from donor to donor as expected. Since current standard and control dried blood spots supplied by most companies are calibrated to 55% hematocrit, determination of haemoglobin content in them can be used as the basis for comparison for samples to discriminate between true and false positives. We propose a rapid, inexpensive and easy method for this haemoglobin normalization process. Results of work now in progress in a screening center to evaluate the usefulness and ease of implementation of this technique in routine screening will be presented. While its implementation in automated protocols is relatively easy, this method is not limited to enzymatic colorimetric assays and can be easily adapted to other methods (e.g. Luminex LabMAP system, fluorimetric assays, HPLC, enzymatic kinetic assays etc.).
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