Elucidation of the complex metabolic profile of cerebrospinal fluid using an untargeted biochemical profiling assay.

We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.

Lessons from 30 years of selective screening for tetrahydrobiopterin deficiency.

In addition to being hyperphenylalaninemic, patients lacking tetrahydrobiopterin (BH4) are deficient in the neurotransmitters whose synthesis depends on the normal activity of tetrahydrobiopterin-dependent tyrosine and tryptophan hydroxylases. Consequently, these patients have to be rapidly recognized among hyperphenylalaninemic babies, since they need specific and early substitutive therapy. Since 1980, BH4 metabolism has been investigated in 2,186 hyperphenylalaninemic babies, using HPLC measurement of pteridines in urine to recognize tetrahydrobiopterin synthesis deficiency (GTP cyclohydrolase and PTPS deficiency) and direct DHPR assay in dried blood samples to recognize DHPR deficiency. A total of 73 tetrahydrobiopterin deficient patients have been detected. Considering the group of neonates born in France (1,342), out of the 32 BH4 deficient patients which have been detected, only 8 were from caucasian families. The lessons from that experience are: (1) tests on blood and urine collected on filter paper cards commend itself by their convenience and simplicity, and samples can be collected on the first visit of the screened infants to the out-patient clinic; and (2) the preconceaved idea that newborns with moderate elevation of blood phenylalanine are false positives of the screening or mild forms of hyperphenylalaninemia explains that a significant number of cases were investigated after 1 month of age; however, in half of BH4-deficient babies, blood phenylalanine was below 10 mg/dl (0.6 mmol/l).

Serum prolactin as a tool for the follow-up of treated DHPR-deficient patients.

Deficiency of dihydropteridine reductase causes a variant form of phenylketonuria associated with a devastating neurological disease characterized by mental retardation, hypokinesis and other features relating to basal ganglia disorder. Hyperphenylalaninaemias with tetrahydrobiopterin deficiency make up about 1-3% of all hyperphenylalaninaemias. We describe three patients from Calabria, a southern region of Italy, who have a dihydropteridine reductase deficiency, caused by the same mutation (p.L14P) also found in the nearby region of Sicily. We report the evolution of clinical and biochemical data during the treatment of these patients where we used prolactin serum determination to adapt the specific therapy. This report suggests that serum prolactin levels can be a good biomarker for optimal dosage of hydroxylated precursors in long-term treatment monitoring.

Outcome and long-term follow-up of 36 patients with tetrahydrobiopterin deficiency.

We describe the treatment, the clinical, and biochemical findings and the outcome of 26 patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency and 10 patients with dihydropteridine reductase (DHPR) deficiency. These are the two most common forms of the autosomal-recessively inherited tetrahydrobiopterin (BH4) deficiency. Time of diagnosis, dosage of BH4 and neurotransmitter precursors, folinic acid substitution, and levels of 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in cerebrospinal fluid (CSF) are essential parameters in the follow-up of patients. Unfortunately, treatment protocols vary greatly among patients and clinical centers, and CSF investigations and outcome assessments are not always available. Seventeen patients with PTPS deficiency and four patients with DHPR deficiency were diagnosed within 2 months after birth. In 14 patients with PTPS deficiency (54%; 9 early and 5 late diagnosed) and 2 patients with DHPR deficiency (20%; all early diagnosed) no developmental delay is observed, while in 10 patients with PTPS deficiency (38%; 6 early and 4 late diagnosed) and 8 patients with DHPR deficiency (80%; 2 early and 6 late diagnosed) development was delayed. Two PTPS-deficient patients died in the newborn period. DHPR deficiency seems to be more severe than PTPS deficiency and it is clearly the onset of treatment that determines the outcome. Our data suggest that diagnosis within the first month of life is essential for a good outcome and that low CSF5 HIAA and HVA values in CSF could be an indicator for the ongoing developmental impairment

[Clinical study of tetrahydrobiopterin responsive phenylalanine hydroxylase deficiency in southern and northern Chinese patients].

OBJECTIVE: To analyze characteristics of different hyperphenylalaninemia (HPA) and to discuss the clinical difference between southern and northern Chinese patients with tetrahydrobiopterin (BH4) responsive phenylalanine hydroxylase (PAH) deficiency. METHODS: (1)BH4 (20 mg/kg) loading test was performed in all 108 HPA patients. These patients, 63 males and 45 females, were at a mean age of 7.05 months. A combined phenylalanine (Phe) and BH4 loading test was carried out in the patients who had a basic blood Phe concentration less than 600 micromol/L. The urine pterine profile analysis and the dihydropteridine reductase (DHPR) activity in dry blood filter spot were analyzed simultaneously. (2)BH4 responsive patients were divided to southern and northern groups by their parent's native place and geographic boundary determined by Changjiang River. The change of Phe concentration after BH4 loading test was compared between the two groups. RESULTS: (1)Among the 108 HPA cases, 36 patients (33.3%) were BH4 responsive PAH deficiency, 49 (45.4%) were non-BH4 no responsive phenylketonuria (PKU)and 23(21.3%)were BH4 deficiency (BH4D). The Phe concentration of patients with BH4 responsive PAH deficiency decreased by 49.24% and 65.35% at 8 h and 24 h after oral BH4, 23 in southern group and 13 in northern group among 36 patients. (2)The mean Phe concentration at 24 h after loading test in southern and northern groups were (217.02+/-189.03) micromol/L and 458.75+/-342.54 micromol/L respectively (P<0.05), although the decrease percent of plasma Phe concentration at 2 h, 4 h, 8 h, 24 h was no distinct difference between southern and northern groups (P>0.05). CONCLUSION: Most of mild and moderate HPA patients affected by PAH deficiency show plasma Phe concentration decrease >30% in 24 h after oral BH4 20 mg/kg, few are classic PKU. The responsiveness to BH4 is no difference between southern and northern Chinese patients with BH4 responsive PAH deficiency according to the decrease percent of plasma Phe concentration, although the Phe concentration is lower in southern patients than that in northern patients.

Evaluation of pteridine metabolism in battery workers chronically exposed to lead.

Occupationally-exposed lead affects the neuromuscular junction and might cause disturbances in the locomotor activity. This study was undertaken to evaluate pteridine metabolism, in which neurotransmitters are synthesized in battery workers. Urinary neopterin, biopterin and creatinine were measured using high performance liquid chromatography. Serum neopterin concentrations were detected by enzyme-linked immunoassay. Blood dihydropteridine reductase (DHPR) activities and deltaaminolevulinic acid (delta-ALA) were measured spectrophotometrically. Blood and urinary lead were detected by atomic absorption spectroscopy. Significantly increased blood and urinary lead levels, urinary neopterin, biopterin and delta-ALA were found in workers, while DHPR activities were indifferent compared to control group. Urinary creatinine decreased. This is the first study to demonstrate that increased activity of the pteridine pathway results in the accumulation of the neurotransmitters that may be responsible for the neurological disorders.

[Study on tetrahydrobiopterin deficiency in Northern Chinese population].

OBJECTIVE: To emphasize early differential diagnosis from patients with hyperphenylalaninemia (HPA) and to evaluate the treatment and long-term outcome of patients with tetrahydrobiopterin synthase (BH4) deficiency in Northern Chinese population. METHODS: From 1992 to 2005, a total of 618 patients with HPA were diagnosed and/or cared for in our outpatient clinic. Urinary pterin analysis, detection of dihydropteridine reductase (DHPR) activity in blood, and then BH4 loading tests were carried out to differentiate BH4 deficiency in these patients from classical phenylketonuria. BH4 deficient patients were treated with BH4, levodopa and 5-hydroxytryptophane (5-HTP) immediately while the diagnosis was done to disease. Patientso blood phenylalanine levels, psychomotor and intelligence development were followed up. RESULTS: A total of 38 cases were diagnosed as BH4 deficiency, all of them were revealed as 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency from the extremely decreased urine biopterin, normal DHPR activities and drop down of blood phenylalanine level to normal range within 4 to 8 hours after BH4 loading. The most common manifestations were progressively psychomotor and mental retardation to patients even after taking early dietary treatment. The patients were diagnosed and treated with drugs at the ages of 2.1 months to 13 years. With 4 patients died of pneumonia, 7 patients refused to treatment, only 27 patients were under treatment and followed up. The average full scale development or intelligence quotient (DQ/IQ) of patients who were treated within and after 6 months were 86+/- 10 or 66+/- 7 respectively. Development was not even in different aspects. A significant negative correlation was observed between the level of the DQ and the age of treatment commenced (r was -0.714, P< 0.01). Eleven patients experienced the extrapyramidal movement disorders, 3 of them combined with epilepsy. The extrapyramidal disorders were controlled by administration of levodopa. CONCLUSION: The differential diagnosis for BH4 deficiency should be carried out in all patients with HPA. PTPS deficiency is the most common form of BH4 deficiency in Northern Chinese population. The long-term outcome of these patients benefits from diagnosis and treatment with BH4, levodopa and 5-HTP as early as possible.

[Tetrahydrobiopterin loading test in differential diagnosis among hyperphenylalaninemia patients].

OBJECTIVE: To perform tetrahydrobiopterin (BH(4)) loading test and to further understand its usefulness in differential diagnosis among hyperphenylalaninemia(HPA) patients. METHODS: BH(4) loading test was carried out in 73 HPA patients, including the positive cases unveiled by neonatal screening and the clinically suspected cases. These patients, 47 males and 26 females, were at a mean age of 1.93 months. BH(4) (20 mg/kg) loading test was performed in all patients, and a combined phenylalanine (Phe)(100 mg/kg) and BH(4) loading test was performed among the patient who had a basic blood Phe concentration less than 600 micro mol/L. The urine pterine profile analysis and the dihydropteridine reductase activity in dry blood filter spot were tested simultaneously. RESULTS: During BH(4) loading test or combined Phe and BH(4) loading test, the patients with classic phenylketonuria showed no response to BH(4), the patients with moderate HPA caused by Phe hydroxylase deficiency decreased 32.8% of blood Phe level and the patients with BH(4) deficiency showed a prompt reduction in blood Phe level and it decreased to normal level at 4 h and lasted until 24 h. Twenty-two cases were diagnosed as classic phenylketonuria, 39 were moderate phenylketonuria and 12 were BH(4) deficiency. CONCLUSION: Hyperphenylalaninemia may be caused by deficiency of Phe hydroxylase or by deficiency of co-factor BH(4). Early diagnosis is important. BH(4) loading test is a safe and fast test in vivo. It is sensitive, easy-to-do, and is highly useful in differential diagnosis for suspected cases of HPA.

Effects of the metals on dihydropteridine reductase activity.

Metals are the oldest toxins known to human. Particularly, occupational and environmental exposure to aluminium, lead, mercury, cadmium, and manganese cause serious health problems by interaction with biological systems. Cellular targets of these metals are mostly specific biochemical processes (enzymes) and/or membranes of cells and organelles. To prevent and/or reduce the untoward or irreversible toxic effects of the metals by using biomarkers are as important as to know and to understand of their toxicity mechanisms. Dihydropteridine reductase (DHPR), which possessed essential thiol groups at the activity site, plays a crucial role in the maintenance of tetrahydrobiopterin (BH4). BH4 is the cofactor in the synthesis and regulation of neurotransmitters. A limited number of the evidences have shown that DHPR may be a target for the metals. Therefore, the present study was designed to assess possible in vitro effects of the commonly exposed metals on the enzyme activity. It was found that aluminium, cadmium, mercury, di-phenyl mercury, lead, diethyl lead, in chloride forms, and manganese, in sulphate form, led to statistically significant decreases in DHPR activity, in a concentration-dependent manner, in vitro.

Coronary endothelial dysfunction in the insulin-resistant state is linked to abnormal pteridine metabolism and vascular oxidative stress.

OBJECTIVES: We investigated whether abnormal pteridine metabolism is related to coronary endothelial dysfunction in insulin-resistant subjects. BACKGROUND: Depletion of tetrahydrobiopterin (BH(4)) and elevation of the 7,8-dihydrobiopterin (BH(2)) (activating and inactivating cofactors of nitric oxide synthase [NOS], respectively) contribute to impairment of NO-dependent vasodilation through reduction of NOS activity as well as increased superoxide anion generation in insulin-resistant rats. METHODS: Thirty-six consecutive nondiabetic, normotensive and nonobese subjects with angiographically normal coronary vessels were studied. Traditional coronary risk factors, plasma pteridine levels, activities of erythrocyte dihydropteridine reductase (DHPR), the recycling enzyme that converts BH(2) to BH(4) and lipid peroxide (LPO) levels were measured and coronary endothelial function was assessed with graded infusions of acetylcholine (ACh). RESULTS: When we divided patients into tertiles based on insulin sensitivity, we observed stepwise decreases in the maximal ACh-induced vasodilation and plasma BH(4)/7,8-BH(2) ratio, and increases in coronary LPO production as insulin sensitivity decreased. The ACh-induced vasodilation was positively correlated with insulin sensitivity, BH(4)/7,8-BH(2) ratio and DHPR activity. Furthermore, BH(4)/7,8-BH(2) was inversely correlated with DHPR activity and insulin sensitivity. In multiple stepwise regression analysis, BH(4)/BH(2) was independently related to ACh-induced vasodilation and accounted for 39% of the variance. However, no significant correlation existed between other traditional risk factors and BH(4)/7,8-BH(2). CONCLUSIONS: These results indicate that both abnormal pteridine metabolism and vascular oxidative stress are linked to coronary endothelial dysfunction in the insulin-resistant subjects.

Dihydropteridine reductase activity and neopterin levels in leukemias and lymphomas: is there any correlation between these two parameters?

Urinary neopterin levels, blood dihydropteridine reductase activity as well as other frequently used clinical parameters were evaluated in 110 patients suffering from various types of lymphomas and leukemias. Among them neopterin was detected as the most sensitive marker representing the severity of malignancy (p<0.00001). All patients with active diseases had significantly raised urinary neopterin levels compared to those in remission and healthy controls. Of 69 patients with active disease 66 (96%) were above the upper limit seen in healthy subjects. In addition, the highest neopterin excretion was found in patients with active chronic myeloid leukemia (1469+/-479 micromol/mol creatinine n=16). In contrast, only 1 of 41 patients in stable responsive disease and remission (2.4%) had increased urinary neopterin levels above the upper limit. Dihydropteridine reductase (DHPR) activities were also detected in all patients and control groups. In active disease slightly reduced (DHPR) activities were evident (3.42+/-0.37 for controls, 2.92+/-0.39 in active disease and 3.28+/-0.42 nmol red cytochrome C/min/5 mm diameter disc in remission patients). However in patients under medication this was strengthened. This data also suggest that DHPR activity can be effected by chemotherapy. The results of the present study support the fact that urinary neopterin levels may be an useful and reliable early prognostic marker for neoplasia when used together with other prognostic indicators. Our data also suggest that reductions in DHPR activities may also be an underlying cause for the neurological disorders that are commonly seen in patients with haematological malignancies.

Urinary neopterin excretion and dihydropteridine reductase activity in rheumatoid arthritis.

Neopterin and biopterin are two products of the pteridine pathway. Even though their roles and interrelationships have not been exactly clarified, neopterin is known as a biomarker of cell-mediated immunity. In this case, the highly elevated neopterin levels are parallel to the slightly elevated biopterin levels. On the other hand, the reduced form of biopterin-tetrahydrobiopterin is an essential cofactor of aromatic monoxygenases that leads to synthesis of tyrosine, tryptophan and dopamine neurotransmitters and its concentration in body fluids and tissues is maintained by the enzyme dihydropteridine reductase (DHPR). Increased numbers of activated lymphocytes can be found in peripheral blood, in the synovial fluid and synovial membranes or patients with rheumatoid arthritis (RA). Since the present study was undertaken to evaluate the role of the pteridine pathway in RA, we measured urine neopterin levels and dried blood DHPR activities in 36 patients with RA and in 20 healthy volunteers, in parallel with other clinical parameters. We found that neopterin excretion was significantly increased in RA patients compared with controls. The means were 433 +/- 216, 153 +/- 43 and 111 +/- 34 mumol/mol creatinine for patients in active stage, in remission and controls, respectively. Our results suggest that urine neopterin levels were strongly dependent on the stage and activity of RA. Either as an effect of the disease itself or of drug administration, slightly reduced DHPR activities were detected (3.484 +/- 0.304 for control, 2.974 +/- 0.255 in active stage RA, and 3.048 +/- 0.302 red cytochrome C/min/5 mm disc in remission).

Tetrahydrobiopterin and inherited hyperphenylalaninemias.

Tetrahydrobiopterin deficiency, a variant of hyperphenylalaninemia, may be caused by deficiency of one of the following enzymes: guanosine triphosphate cyclohydrolase 1,6-pyruvoyltetrahydropterin synthase, dihydropteridin reductase and pterin-4a-carbinolamine dehydratase. The first two enzymes are involved in the biosynthesis of tetrahydrobiopterin, the last two in its regeneration. Although these diseases are rare, early detection by selective screening is essential for the treatment and outcome. Tetrahydrobiopterin deficiencies are very heterogenous ranging from mild forms requiring only marginal if any treatment to severe forms which are in some cases very difficult to treat. All variants of tetrahydrobiopterin deficiency can be differentiated from the classical phenylketonuria (PKU) by measurement of pterin metabolites in patients' urine, tetrahydrobiopterin loading test, and by dihydropteridine reductase activity in erythrocytes from the Guthrie card.

Atypical (mild) forms of dihydropteridine reductase deficiency: neurochemical evaluation and mutation detection.

We investigated two patients with an atypical (mild) form of dihydropteridine reductase (DHPR) deficiency. Both responded to the loading test with tetrahydrobiopterin; their plasma phenylalanine levels were lowered from 278 mumol/L to 85 and 48 mumol/L and from 460 mumol/L to 97 and 36 mumol/L after 4 and 8 h, respectively. In one of the patients, a combined loading test with phenylalanine followed by tetrahydrobiopterin was also carried out and showed a profile typical for DHPR deficiency. The phenylalanine hydroxylation rate was calculated to be 43 and 87%, 4 and 8 h after cofactor administration, respectively. Diagnosis was confirmed by the absence of DHPR activity in the patient's erythrocytes. In cultured fibroblasts, residual activity of 4 and 10%, respectively, was found. Excretion of urinary pterins was essentially normal, and the biopterin to neopterin ratio in cerebrospinal fluid was increased. Although in both patients cerebrospinal fluid homovanillic acid was found to be normal, and 5-hydroxyindoleacetic acid was substantially reduced, there was no sign of neurologic alterations until the age of 2 y. However, one of the patients recently developed deceleration of head growth, whereas psychomotor development continued to be normal for age. Using the chemical cleavage method on the amplified cDNA, mismatches of T to G at nucleotide 659 and of G to A at nucleotide 475, respectively, were identified. These results also demonstrate that screening for tetrahydrobiopterin deficiency by urinary pterin analysis alone can miss some newborns with mild DHPR deficiency and that all children with tetrahydrobiopterin defects need full neurochemical evaluation together with analysis of the enzyme activity.

[Measurement of dihydropteridine reductase activity in dried blood eluates: physiological and pathological implications]. / Mesure de l'activité dihydroptéridine réductase sur éluat de sang séché: implications physiologiques et pathologiques.

Tetrahydrobiopterin deficiency in hyperphenylalaninemic babies has to be rapidly recognized since the disease requires a specific treatment. Although the measurement of pteridines in urine can detect most of tetrahydrobiopterin deficiencies, DHPR measurement has to be performed to circumvent the risk of missing DHPR deficiency, especially at the neonatal period. The possibility to measure DHPR activity in dried blood samples justifies itself by its convenience and simplicity. Expression of activity per mg of hemoglobin improves the precision of the assay by removing variation in the elution step (insufficient blood being loaded onto the filter paper, hematocrit variation, thickness of filter paper used). The distribution of DHPR activities, corrected from the progressive decrease with age, showed that 2.5% of the hyperphenylalaninemic or normal population have low levels of activity (below 50% of normal). This observation suggests that genetic variations may exist, as illustrated by the analysis of 11 families. Although blood DHPR measurement seems efficient for screening DHPR deficiency, the finding in a family of clinically normal subjects with zero activity illustrates the risk of false positive results of the enzymatic test.

Dihydropteridine reductase activity: lack of association with serum aluminum levels and cognitive functioning in patients with end-stage renal disease.

Although increased levels of aluminum (Al) are present in patients with dialysis encephalopathy (DE), it is unclear if the association is causal. The enzyme dihydropteridine reductase (DHPR) plays a critical role in neurotransmitter formation and its activity. Elevated levels of Al are reported to decrease DHPR activity, which would alter neurotransmitter metabolism, thus producing DE. We examined the association between erythrocyte DHPR activity and Al levels, attention/psychomotor skills, and depression in a group of 21 patients with end-stage renal disease. DHPR activity was not related to Al level, mental status, psychomotor ability, or depression score. After administration of deferoxamine (an Al chelating agent), Al level increased significantly but DHPR activity remained the same. Our results suggest that the mechanism for the development for DE does not involve alterations of neurotransmitter metabolism caused by Al-mediated reductions in DHPR activity.

Strategy for the screening of tetrahydrobiopterin deficiency among hyperphenylalaninaemic patients: 15-years experience.

Tetrahydrobiopterin deficiency in hyperphenylalaninaemic babies has to be rapidly recognized since the disease requires a specific treatment. Based on 15 years experience, we report on the evolution of a strategy for the detection of such patients. A total of 913 hyperphenylalaninaemic patients have been studied and 15 tetrahydrobiopterin deficiencies have been detected or confirmed. DHPR assay in dried blood samples and pteridine measurement in urine collected on filter paper combine convenient sampling and reliable tests for systematic investigation of hyperphenylalaninaemic patients for cofactor deficiency.

DHPR activity decrease in dried blood spots stored at 4 degrees C.

Dihydropteridine reductase (DHPR) activity has been measured in dried blood spots by the Arai method in 286 subjects divided into 4 groups: 45 newborns aged between 4 and 7 days; 40 subjects with hyperphenylalainaemia; 199 apparently normal subjects, and 2 patients with DHPR deficiency. Furthermore, a study of the persistence of enzymatic activity on the spots stored at 4 degrees C revealed that after 1 year it decreased to one third of the original value, corresponding to the mean value of controls minus 2 SD.

The estimation of dihydropteridine reductase in human blood cells.

Significant homology between dihydropteridine reductase (DHPR) from rat and human sources has been established by the ability of polyclonal antibodies raised to the rat-liver enzyme to detect the human protein in Western blots. The antibody also reacted with a single protein in bovine, dog and porcine kidney extracts, however, only trace reactivity was detected in rabbit. Quantitation of Western blots by soft laser densitometry showed that the response was proportional to total protein present in analyses of both pure rat-liver enzyme samples and crude extracts of rat and human liver. The DHPR contents of human blood cells were analysed by this method and the results compared to levels determined in enzymatic assays. Extracts of platelets and lymphocytes showed good correlation between these two methods, however, granulocytes exhibited high apparent enzyme activity but no DHPR protein detectable in blots. Erythrocyte extracts showed approximately 50% lower DHPR protein levels than predicted by activity measurements. These results are discussed in relation to the accuracy of detecting DHPR deficiencies in humans by enzymatic assay of whole blood samples.