Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry

doi:10.1016/j.cca.2006.04.013

Clinica Chimica Acta

Volume 373, Issues 1–2, November 2006, Pages 27-31

https://doi.org/10.1016/j.cca.2006.04.013 Get rights and content

Abstract

Background

Detection of amino acids (AA), acylcarnitines (AC), and guanidinoacetate (GAA) in dried blood spots by tandem mass spectrometry has made it possible to detect different inborn errors of metabolism in neonatal screening programs. Despite its proven sensitivity many issues related to sample preparation remain unsolved. Hematocrit has a profound effect on blood viscosity, and may thereby influence flux and diffusion properties of the blood. As newborn infants show a considerable interindividual variability of hematocrit levels, we investigated its effect on levels of AA and AC in dried blood spots.

Methods

Blood samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were produced by diluting blood cells with plasma from a single donor. Forty dried blood spots were made for each hematocrit level and a central as well as a peripheral 3 mm disk was punched and analysed for AA, AC, and GAA, respectively.

Results

Levels of most AA and GAA increased significantly with increasing hematocrit (p < 0.001), while the effect of hematocrit on some AA was less pronounced. Total AC, free carnitine, some long, medium and short chain AC correlated positively with hematocrit levels (p < 0.001). In samples with low hematocrit, levels of most AA and free carnitine were higher in the peripheral than in the central disk (p < 0.0001).

Conclusions

Both hematocrit and position of the disk within the dried blood spot have a significant and sometimes additive effect on levels of AA, AC and GAA in dried blood spots. Theoretically, diagnoses may be missed depending on hematocrit and position of the disk.

Introduction

The integration of electrospray injection tandem mass spectrometry (ESI-MS/MS) into newborn screening programs allows the detection of numerous metabolites that are relevant for the diagnosis of many inborn errors of metabolism (IEM) [1], [2], [3], [4], [5]. It has become the mainstay for screening of IEM, including amino acidopathies, defects of fatty acid oxidation and organic acidopathies [1], [2], [3], [4], [5].

Newborn screening by ESI-MS/MS is based on the semi-quantitative analysis of amino acids (AA) and acylcarnitine ester (AC) using stable isotopically labelled internal standards in whole blood dried on filter cards. Recently, analysis of GAA, a characteristic metabolite for guanidinoacetate-methyl-transferase deficiency, which represents an inborn error in creatine synthesis, was included in our newborn screening program. Diagnosis of IEM is suspected when certain AA, GAA or AC concentrations exceed population dependent cut-off limits. These limits typically reflect the 99th to 99.5th percentile [2], [3].

Most IEM detected by ESI-MS/MS in newborn screening programs show characteristic metabolite profiles that unequivocally exceed the respective cut-off limits. While this may be true for some disorders, diagnosis may be difficult for others. In addition metabolite levels depend on the metabolic status, dietary intake and maturity of the infants which may pose a diagnostic challenge requiring confirmatory tests such as further biochemical testing, measurement of enzyme activity in fibroblasts or mutation analysis [1], [2], [4].

There may be additional factors that have a profound effect on the analysis of metabolites. Hematocrit, for example, has a considerable effect on blood viscosity, and thereby may affect flux and diffusion properties of the blood put on filter paper used for newborn screening. In addition there may be a significant difference of metabolite concentrations between central and peripheral areas within the dried blood spot, due to chromatographic effects. Neonates screened on the third day of life show considerable interindividual variability of hematocrit levels and may therefore be particularly amenable to changes of metabolite concentrations [6], [7], [8]. This may be particularly true for preterm infants and those who are critically ill on the day of screening [8].

Consequently, we devised a study to investigate whether hematocrit or the location of the punch within the dried blood spot has any effect on concentrations of AA, free carnitine (FC), total carnitine (TC), AC and guanidinoacetate (GAA) measured by ESI-MS/MS.

Section snippets

Samples

Heparinised blood (50 ml) was taken from a healthy adult volunteer following written informed consent. Blood was spun down immediately and the plasma was separated. Plasma was then added in respective amounts to the corpuscular elements so that samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were obtained. Forty dried blood spots were made for each hematocrit level using standard filter cards (Schleicher, SS 2992). Blood spots were generated by dispensing blood into the centre

Results

Levels of most AA and GAA increased significantly with increasing hematocrit (p < 0.001), while the effect of hematocrit on aspartate and tyrosine was less pronounced and not statistically significant (Table 1).

Total carnitine, FC, some long chain AC (C16, C18, C18:1, C18:2) as well as some medium and short chain AC (C2, C3, C4, C4DC, C5OH) correlated with hematocrit (p < 0.001) (Table 1, Table 2). For other AC species (C6, C8, C10, C12, C14) a significant correlation was not observed (data not

Discussion

Newborn screening programs employing tandem mass spectrometry rely on population based cut-off values for different metabolites in dried blood filter cards that are typically sampled on day of life three [3], [4]. In addition, this analytical set-up is used for selective metabolic diagnosis and retrospective analysis of stored filter cards [11], [12], [13]. For analysis a 3 mm disk is punched from the filter card, the metabolites eluted and typically derivatised to yield corresponding butyl

References (18)

T.H. Zytkovicz et al.
Tandem mass spectrometric analysis of amino, organic, and fatty acid disorders in newborn dried blood spots: a two-year summary from the New England Newborn Screening Program
Clin Chem
(2001)
D.H. Chace et al.
Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns
Clin Chem
(2003)
A. Schulze et al.
Expanded newborn screening for inborn errors of metabolism by electrospray ionization-tandem mass spectrometry: results, outcome, and implications
Pediatrics
(2003)
B. Wilcken et al.
Screening newborns for inborn errors of metabolism by tandem mass spectrometry
N Engl J Med
(2003)
M.S. Rashed et al.
Application of electrospray tandem mass spectrometry to neonatal screening
Semin Perinatol
(1999)
S.M. Kayiran et al.
Significant differences between capillary and venous complete blood counts in the neonatal period
Clin Lab Haematol
(2003)
J.A. Leipala et al.
Blood volume assessment with hemoglobin subtype analysis in preterm infants
Biol Neonate
(2003)
M.J. Bizzarro et al.
Differential diagnosis and management of anemia in the newborn
Pediatr Clin North Am
(2004)
O.A. Bodamer et al.
Analysis of acylcarnitine ester for neonatal screening of inborn errors of metabolism using tandem mass-spectrometry
Chem Mon
(2005)

There are more references available in the full text version of this article.

Cited by (115)

Internal standard metabolites for estimating origin blood volume of bloodstains
2023, Forensic Science International
The volume of blood leaked from blood vessels may change due to evaporation of water under the natural influence of the external environment. Bloodstains and dried blood spots (DBS), which describes blood dried in the external environment, are similar in their production and their metabolite quantification profiles. In both bloodstain metabolite analysis in the forensic science field and DBS metabolite analysis in the clinical field, it is important to determine the volume of the origin blood as this affects metabolite quantification results. Therefore, the purpose of this study is to discover the internal standard metabolites that have quantitatively proportional relationships with origin blood volume and maintain constant concentrations even as the age of the bloodstain increases. As a result, the concentrations of L-isoleucine and L-phenylalanine increased in proportion to the origin blood volume of the bloodstain. The differences in concentration of L-isoleucine were significant in all volume comparisons except in the comparison between 65 μL and 85 μL. The differences in concentration of L-phenylalanine were significant in all volume comparisons except between 65 μL and 45 μL and between 65 μL and 85 μL. In addition, it was confirmed that both metabolites tended to maintain constant concentrations without being affected by bloodstain age as the volume became smaller. These internal standard metabolites can be used for estimating the origin blood volume of bloodstains during metabolite analysis of bloodstains and DBS and could provide a volume criterion for standardization when comparing metabolite quantification between samples.
Evaluation of volumetric blood collection devices for the measurement of phenylalanine and tyrosine to monitor patients with phenylketonuria
2022, Clinica Chimica Acta
Measurement of dried blood spot (DBS) phenylalanine (Phe) is central to the monitoring of patients with phenylketonuria. However, the volume and hematocrit (Hct) of the blood applied to conventional DBS cards significantly affects analytical results. Volumetric blood collection devices are reported to be more accurate, precise and less prone to Hct effects.
Accuracy, imprecision, effect of blood volume and Hct were evaluated for measurement of Phe and tyrosine using three volumetric devices and compared with the conventional PerkinElmer-226 filter-paper collection devices. i.e. conventional DBS cards. Applicability for use in a clinical laboratory was assessed qualitatively.
Blood volume did not impact on the performance of the volumetric devices; however, significant biases were observed with the conventional DBS card. A higher Hct introduced unacceptable bias for Neoteryx-Mitra and conventional DBS card. All devices had a mean relative standard deviation (RSD) ≤ 4.1 %, except for the Neoteryx-Mitra (≤ 6.2 %). Relative to liquid blood, the mean biases of Phe for the various devices were −5.1 (HemaXis-DB10), −7.8 (Capitainer-qDBS), −12.0 (Neoteryx-Mitra) and −32.6 % (conventional DBS card).
Introducing volumetric collection devices will overcome the significant pre-analytical issues associated with conventional DBS collection and improve the biochemical monitoring of patients with PKU.
Determination of selected α-keto acids in dried blood samples using HPLC with fluorescence detection
2022, Journal of Pharmaceutical and Biomedical Analysis
The determination of α-keto acids derived from amino acids is currently the most reliable approach for the diagnosis of some congenital metabolic diseases. An HPLC method for the simultaneous measurement of selected α-keto acids in dried blood samples has been developed and evaluated. Blood spot samples from a group of healthy blood donors were collected onto #903 Specimen Collection Paper. Prior the separation, the α-keto acids were derivatized with 1,2-diamino-4,5-dimethoxybenzene to the corresponding 3-substituted-6,7-dimethoxy-2(1 H)-quinoxalinol derivatives. For the separation, a reverse-phase column LichroCart 125–4, Purospher RP-18e, 5 µm, was used. The mixture of 25% ACN in deionized water (mobile phase A) and 100% ACN (mobile phase B) were used for a gradient elution of α-keto acids derivatives. Analytical performance of this method is satisfactory for all α-keto acids. The intra-assay and inter-assay coefficients were below 10% and recoveries were close to 100%. We have developed relatively simple, rapid, selective and sufficiently sensitive HPLC method with fluorescence detection for the determination of selected α-keto acids in dried blood samples. The presented method is suitable for clinical testing purposes.
Methodological aspects of dried blood spot sampling for the determination of isoprostanoids and prostanoids
2022, Microchemical Journal
Dried Blood Spot (DBS) analysis is a well-established practice for monitoring newborns in neonatal units because of the non-invasive collection of a very limited blood volume. Unfortunately, the haematocrit level and the drop volume can adversely affect the quality of the analytical data. For this reason, we performed an in-depth comparison of non-volumetric and volumetric sampling (e.g. Minivette® POCT microcapillary, and the microfluidic-based systems HemaXis^TM DB and Capitainer-B) for the DBS analysis of the isoprostanoids 8-iso prostaglandin F_2α and 8-iso prostaglandin E_2, and prostaglandin E₂, which represent well recognized indexes of oxidative stress and inflammation.
Results highlighted a significant impact of drop volume on the analytical performances for the non-volumetric approach due to the inhomogeneous distribution of the analytes across the DBS. The innovative addition of labelled internal standards (ISs) on the filter paper before sampling did not circumvent the problem, as ISs did not accurately mimic the spreading of analytes in the DBS. Differently from HemaXis^TM DB and Capitainer-B, the Minivette® POCT device showed an almost quantitative recovery with an overall variability of about 15%, and resulted the best option for an easy and reliable DBS collection in the clinic. Our procedure was used for the determination of the three metabolites in preterm newborns suffering from Patent Ductus Arteriosus (PDA). During our longitudinal monitoring, most of the preterm newborns with PDA showed a marked decrease (40–60%) of target analytes upon ibuprofen therapy, suggesting the potential involvement of the isoprostanoids and prostanoids in promoting drug responsiveness and ductal closure.
Quantitative and qualitative application of a novel capillary microsampling device, Microsampling Wing™ (MSW), using antiepileptic drugs in rats
2021, Journal of Pharmaceutical and Biomedical Analysis
A novel microsampling device, namely, the Microsampling Wing™ (MSW), was evaluated using three anti-epileptic drugs (AEDs): carbamazepine, lamotrigine, and phenytoin. A simultaneous assay method of the three AEDs was developed and qualified via liquid chromatography with tandem mass spectrometry. Using 2.8 μL plasma, the three AEDs were quantifiable from 1 or 2 ng/mL. According to the intra-assay reproducibility assessment and additional validation parameters, the established method is reproducible. To apply the device to a pharmacokinetic (PK) study in rats, a cocktail of the three AEDs was orally administered to rats. Whole blood samples were serially collected using the MSW device and a glass capillary from the tail vein, and plasma samples (each 2.8 μL) from each device were assayed to compare PK parameters. The PK parameters of the three AEDs were similar between the two devices. A metabolite identification study was also conducted after oral administration of carbamazepine to rats. At least seven metabolites were detected in plasma, and the major metabolite was carbamazepine 10,11-epoxide, which is in accordance with the reported results. These findings suggest that the MSW device is a useful microsampling device for PK and metabolite identification studies.
A novel functional C1 inhibitor activity assay in dried blood spot for diagnosis of Hereditary angioedema
2020, Clinica Chimica Acta
Citation Excerpt :
As shown in Table 1, the intra-day precision and accuracy ranged from 4.0% to 11.6%, and −11.1% to −2.1%, and inter-day precision and accuracy were 8.1–13.1%, and −10.3% to 0.9%, respectively. The effect of hematocrit on assay performance [14,18,20,23,24] was investigated at four hematocrit levels, 25%, 45%, 60% and 75%, where 45% hematocrit was used as a control. The fC1-INH levels showed a linear correlation with hematocrit levels (Fig. 4).
Hereditary angioedema (HAE) is a rare genetic disease caused by deficiency or dysfunction of C1 esterase inhibitor (C1-INH). Timely and accurate diagnosis is an ongoing challenge. Measurement of plasma C1-INH activity is currently the critical standard test. We describe a novel and highly robust point-of-care assay to quantify C1-INH activity in dried blood spot (DBS).
C1-INH was extracted from 3 mm punches of DBS samples and incubated with excess amount of C1 esterase (C1s). The mixture was subsequentially incubated with C1s substrate, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitation of the enzyme reaction product.
The assay was validated within a quantification range from 100 to 1500 mU/mL. The intra-day precision and accuracy ranged from 4.0% to 11.6% and −11.1% to −2.1%, and the inter-day precision and accuracy were 8.1–13.1% and −10.3% to 0.9%, respectively. Normal C1-INH activity (n = 103) ranged from 311 to 1090 mU/mL, whereas 23 out of 24 HAE patients exhibited C1-INH activity lower than 100 mU/mL.
DBS specimen collection for measurement of functional C1-INH activity in a physician’s office is straightforward and not limited by logistic considerations and therefore, appropriate for the diagnosis of HAE in high throughput diagnostic laboratories.

View all citing articles on Scopus

View full text

Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Samples

Results

Discussion

Tandem mass spectrometric analysis of amino, organic, and fatty acid disorders in newborn dried blood spots: a two-year summary from the New England Newborn Screening Program

Clin Chem

Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns

Clin Chem

Expanded newborn screening for inborn errors of metabolism by electrospray ionization-tandem mass spectrometry: results, outcome, and implications

Pediatrics

Screening newborns for inborn errors of metabolism by tandem mass spectrometry

N Engl J Med

Application of electrospray tandem mass spectrometry to neonatal screening

Semin Perinatol

Significant differences between capillary and venous complete blood counts in the neonatal period

Clin Lab Haematol

Blood volume assessment with hemoglobin subtype analysis in preterm infants

Biol Neonate

Differential diagnosis and management of anemia in the newborn

Pediatr Clin North Am

Analysis of acylcarnitine ester for neonatal screening of inborn errors of metabolism using tandem mass-spectrometry

Chem Mon