- Email: [email protected]
L arabinitol ratios by mass spectrometry
207
Journal o f .'~.'fi,.'r.obi'd"e;"qi.~., .~,, M~,,t(,~,.~ l_, (1992i 20"; 2i4
!992Else~ierSciencePublishcrsB.V. All rights reser~,ed 0167
7012/92/$ 5.00
MIMET 00495
Filter paper sampling of whole blood and urine for the determination of D/L arabinitol ratios by mass spectrometry J. Roboz, Q. Yu and J.F. Holland T..I. Martell Laboratories for Leukemia, Cancer and AIDS Research, Derald H. Ruttenberg Cancer Center, Mount Sinai School o f Medicine, New York, N Y 10029, U.S.A.
(Received 31 October 1991; re,,ision received 20 January 1992; accepted 30 January 1992)
Summary The methodology described permits the determination of the ratios of the o and L enantiomers of arabinitol in a small dorp of venous (arm) or finger-puncture blood or of urine placed as a spot on conventional filter paper. Replicate analyses using samples from healthy subjects and patients with confirmed disseminated candidiasis gave results that are statistically indistinguishable from those obtained from serum or urine using techniques previously developed by the authors. Replicate analyses in = 8) on days 1, 3, 7, and 14, and on samples after roundtrip long distance airmail transportation revealed that the filter paper spots have storage stability for at least two weeks at room temperature and that samples can be safely transported. Advantages include the initiation of pediatric applications using finger or heel punctures, frequent samplings from adults using drops of blood from samples taken for unrelated routine tests, and the design of inter-institutional studies to evaluate the clinical utility of using D/L arabinitol ratios for the diagnosis and monitoring of disseminated candidiasis.
Key words: Arabinitol enantiomer; D/L arabinitol ratio; Cand!diasis; Filter paper sampling
Introduction Disseminated candidiasis is an opportunistic infection that continues to be the cause of high morbidity and mortality in immunocompromised patients. Because of the lack of distinctive clinical manifestations and the dearth of reliable microbiological, biochemical, or immunological diagnostic techniques, antifungal chemotherapy is often started when the infection is already at an advanced stage [1]; there is a recent tre,d for empirical antifungal therapy in persistently febrile neutropenic patients [2]. Correspondence to: J. Roboz, Mount Sinai School of Medicine, Box 1247, One Gustave Levy Place, New York, NY 10029, U S.A.
208 Based on the identification of D-arabinitol as a major metabolite of several pathologic Candida species, a number of gas chromatographic and gas chromatographi c - m a s s spectrometric techniques have been developed for the quantification of arabinltol in serum [3]. Initial enthusiasm for the 'arabinitol technique' has considerably eroded because of poor specificity in the presence of renal dysfunction. This problem has been solved by new methodologies which separate the o and L enantiomers of arabinito! with the aid of chiral columns prior to detection by gas chromatography [4] or positive [5] or negative [6] ionization mass spectrometry. The use of serum D/L arabinitol ratios, instead of concentrations, has led to considerable improvement in both diagnostic specificity and sensitivity [5, 6]. A much needed inter-institutional prospective evaluation of the clinical utility of the D/L arabinitol technique would require convenient transportation of samples to laboratories equipped with the reqoired instrumentation. The well known restrictions and limitations in taking blood from infants and children ~equire acceptable sampli~lg techniques for the extension of the arabinitol technique to pediatrics. Accordingly, the objective of the present work was to further improve our methodology for the determination of O/L arabinitol ratios in blood (also in urine) by using a small drop of whole blood instead of serum or urine and collecting samples on conventional filter paper. Materials and Methods
Chemicals 2HT-labeled D-arabinitol (96 atom %0 deuterium), was purchased from Merck Sharp & Dohme, Isotopes Div. (Montreal, Canada). Methylene chloride and methanol (HPLC grade), toluene (certified ACS grade) and acetone (Optima grade) were obtained from Fisher Scientific (Fair Lawn, NJ, U.S.A.) and trifluoroacetic anhydride from Supelco (Bellefonte, PA, U.S.A.).
Study subjects Three types of blood samples, designated as 'serum', 'venous blood', and 'finger blood' (see below) were taken from two healthy adults and from a cancer patient with disseminated visceral candidiasis. Urine samples were from a healthy adult and from another cancer patient with disseminated candidiasis. Candidiasis was confirmed in both cases using conventional blood cultures as well as increased serum O/L arabinitol ratios.
Types of samples and method of sample taking 'Serum' samples were obtained conventionally, i.e., venous blood was collected in 'red top' glass tubes, and serum separated by centrifugation from clotted (20 min) blood which was stored frozen. 'Venous blood' samples were obtained by placing, with a calibrated micropipette, 20/zl of freshly drawn whole blood on a filter paper normally used for phenylketonuria screening of newborn infants (#903, Schleicher & Schuell Inc., Keene, NH, U.S.A.). 'Finger blood' samples were obtained by touching a filter paper to a drop of blood appearing after puncturing fingertips (or infant .heels) with a lance until the ring area on the paper was filled. The filter papers were allowed
209 to dry at room temperature (about 30 min) and stored for 1, 3, 7, or 14 days at room temperature. They were also used for transportation studies. The spots were cut from the paper pior to analysis. For the comparison studies, i.e., whole venous blood and finger blood against serum.. 88 samoles, were_ . .taken . . from . . . n .ho~lth,,,,z ....~.,, ,.,j;.,... ,..,. as r..,,,,.,l,UW~,. ..... Whole blood, taken by venipuncture, was divided into two parts. From one part serum was obtained and eight aliquets were taken to serve as standard. From the other part, eight drops of whole blood were placed separately on filter paper in quintuplicate for storage and transportation studies. At the same time, 40 filter paper samples were taken from finger blood for similar studies. In a similar manner, 72 samples were taken from a second healthy subject (no transportation studies), and 12 samples (four of each type) from a patient with confirmed dissemi~mted candidiasis. Urine samples were collected in glass tubes and were stored frozen without preservative. Ten microliter aliqiJots wcrc p,,,c,.,., on filter paper and treated like whole blood. Transportation stability
A set of eight each of venous-blood and finger-blood samples, dried on filter papers, was sealed in a plastic bag and sent airmail to East Lansing, MI, and back to this laboratory in New York City. Similarly, a set of urine samples dried on filter paper also made the round trip by air to Michigan. Sample preparation
A filter paper spot, containing venous blood, finger blood, or urine, was cut from the filter papers and extracted with one ml methanol for 15 min in an ultrasonic bath (conventional, low power, no cooling necessary). The solution was transferred to a 2-ml reaction vial which contained 10 ~! intornnl etnncl~rci (O 0 , - , , , / . . ! ~ ~'.-.n . . . . . .4 u . . evaporation to dryness under nitrogen at 45 °C in a waterbath. To the dried samples, 200 tzl methylene chloride and 100 #1 trifluoroacetic anhydride were added and, after vortexing for 30 s, derivatization was accomplished at 37 °C (metal heating block) for 45 min. After cooling to room temperature, the solvent and excessive reagent were evaporated to a final volume of approximately 5 ul (almost dryness) with a gentle stream of nitrogen at room temperature. The residues were dissolved in 50 ~1 toluene, and 1.0-#1 aliquots were analyzed. Serum (20-#1 aliquots) and urine (10-#1 aliquots) were prepared as follows: after adding 10 #1 internal standard, proteins were removed with one ml acetone, the mixture was vortexed for 30 s, centrifuged at 3000 g for 10 min, the supernatant was transferred into a small vial and evaporated to dryness. Derivatization followed as described above. Gas c h r o m a t o g r a p h y - mass spectrometry
The arabinitol enantiomers were separated using a 30 m long, 0.25 mm i.d. fused silica capillary column, coated with permethylated/3-cyclodextrin in a polysiloxane gum phase (B-Dex 120, Supelco, Inc., Bellefonte, PA), kept isothermally at 106 °C. A metal on-column injector (Model 16715, Chrompack, Bridgewater, N J), operated at room temperature, was connected to the analytical column via a one meter long
210 deactivated uncoated fused silica capillary precolumn. The precolumn, which traps a number of involatile sample constituents, was changed after every 30 analyses. This resulted in a considerable extension of the life of the analytical column, permitting the analysis of as many as 500 samples. The end of the analytical column was inserted into the ion source of the mass spectrometer; the transfer line was kept at 175 °C. The carrier gas was helium, no separator was used. A single quadrupole mass spectrometer (Model 3300, Finnigan Corp., San Jose, CA) combined with a Vector-One (Teknivent Co., Maryland Heights, MO) data system was used. The mass spectrometer was operated in negative chemical ionization mode with isobutane as reagent gas. D-, t.-arabinitol and the internal standard (2H 7labelled o-arabinitol) were monitored at m/z = 5!8 and m/z = 525, respectively, by selected ion monitoring, and O/k ratios were determined from peak arcas [6]. Results and Discussion
Rationale Some of the potential advantages of collecting whole blood samples on conventional filter paper, instead of serum, are obvious: extension of the use of the arabinitol method to include infants and children and overall sampling convenience. Easy and safe transportation is coi~.sidered of major importance. It will permit large-scale inter!nstitutional prospective studies which are essential to establish the clinical utility of the arabinitol enantiomer technique. The absence of the necessary instrumentation in most laboratories and the rarity of confirmed cases of visceral candidiasis makes some convenient transportation method essential. We have developed a technique that can be used for whole blood collected directly on filter paper by touching it to a tiny drop of blood obtained by puncturing a finger (or heel) with a lance, or by expressing a drop of blood from the needle of a syringe used for venipuncture. The first method is the obvious choice |or intended applications with infants and children, but may also be of considerable convenience for adults where samples may be taken at the time of finger stick blood work. The second method permits the study of a drop of blood from samples routinely taken for clinical chemistry or microbiology, thereby eliminating the need for separate sampling for this purpose. More frequent sampling will permit the evaluation of the utility of the arabinitol technique for the monitoring of antifungal chemotherapy.
Methodology Our previous methodology [5, 6] started with serum obtained from whole blood and used acetone to remove proteins. The present technique starts with whole blood which is collected on filter paper; the protein removal step is eliminated. Although not essential, we still use a known qu~mtity of internal standard because its presence provides additional analytical spec:,ficity for the D-arabinitol peak, its size reveals possible errors in sample preparatioJ~ (although this does not affect the ratio measurement), izs shape provides information on column performance and, when needed, its height cr area permits quantification of the individual enantiomers [6]. The method of derivatization, the chiral chromatographic separation, and the analysis by negative chemJca! ionization and selected ion monitoring mass spectrometry remained essen-
211 WHOLE BLOO0 Serum
Finger
Venous
2.6
2.6
2.6
5.6
5.6
5.7
HOR~£
m / z = 5~8 D/L
CAHDTDIASI$
m / z = 518
D/L
Fig. 1. Selected ion monitoring (at m / z = 518) profiles of D- and ~-zrzbinitol from a healthy subject (normalj and a cancer patient with disseminated candidiasis (candidiasis). ?rofiles labeled as 'serum' were from serum analyzed by our previous technique [6]. The label 'finger' refers to sample taken by finger stick expressing a drop of whole blood onto a filter paper. The label 'venous' refers to a drop of whole blood taken by venipuncture and placed onto a filter paper.
TABLE 1 R E P R O D U C I B I L I T Y , STORAGE ,aND T R A N S P O R T A T I O N STABILITY OF D / L A R A B I N I T O L RATIOS OF A H E A L T H Y S U B J E C T IN W H O L E BLOOD ON FILTER P A P E R , TAKEN BY V E N I P U N C T U R E (VENOUS) OR F;,NGER STICK (FINGER), C O M P A R E D TO SERUM AS STANDARD Type
Serum
Days on filter paper
0
D/I. ratio
Meanbiood/Meanserum (n = 8)
Mean (n = 8)
S.D.
Range
2.63
0.22
2.31 - 2.88
Venous
1 3 7 14 Transport
2.62 2.65 2.58 2.58 2.62
0.12 0.22 0.22 0.16 0.14
2.41 2.392.31 2.44 2.38 -
2.80 3.00 2.90 2.94 2.82
0.996 1.008 0.981 0.981 0.996
Finger
1 3 7 14 Transport
2.68 2.62 2.63 2.57 2.64
0.08 0.19 0.06 0.i2 0.14
2.57 - 2.83 2.35 - 2.88 2.55 - 2.73 2.41 - 2 . 7 9 2.39 - 2.79
1.019 0.996 1.000 0.977 1.004
~1"~ tiaii,,' unchar, ged, except for replacing the previously used Lipodex A type glass capillary co!~mns [5] with the ¢3-Dex-120 fused silica type. It is noted that the two column L, pes gave comparable performance. The efficie~acy of the methanol extraction of arabinitol from the filter papers was determined by comparing the recovery of known quantities of added D-arabinito! using our previous technique with the present one. Recovery was 9 5 - 105% using added D-arabinitol in six samples in a concentration range from 2 . 8 - 9 2 ng. To test t,h,.x, new sampling and ~,.,,.,~,.,~,.~,;,-,n . . . . . . . v:.- cananl~c by , , - v . . . . . . . . . . . . . . . m,-h,-,,~,-,1,-,~,, ........... o: ,~,e , - , h , ~ ; n , ~ A m!!!t;ni~ both techniques and compared results with those for serum samples taken and analyzed by our previous techniques which was considered to be the standard. D/L Arabinitol ratios in whole blood
Our objective was to prove that the results using venous or finger stick whole blood samples on filter paper are indistinguishable from those using serum with our previous vechnique. Fig. 1 shows the selected ion monitoring of o- and L-arabinitol in the three types of samples from a healthy subject (normal) and a patient with confirmed disseminated candidiasis, together with the corresponding D/L arabinitol rat'os determined from the respective peak areas. The separation of the enantiomers was nearly baseline. The profiles for serum are similar to those we have previously reported [5]. The profiles for finger and venous blood samples appear nearly identical to serum (Fig. 1). Table 1 summarizes reproducibility, storage and transportation stability data for D/L arabinitol ratios in whole blood on filter paper, both venous and fingerstick, and serum for the 88 samples taken from a healthy subject. Each mean value refers to n = 8, thus these and the corresponding standard deviations (and ranges) may be used to evaluate the reproducibility of the analyses for a given type of sample for a given period of storage. The data in Table 1 may be expressed in four ways. (1) The standard deviations of all groups of filter paper samples are equal to or smaller than that of serum. (2) The means of the venous- and finger-blood samples are statistically indistinguishable from that of serum (t-test, independent samples) for all storage days as well as for transportation. (3) M! ratios of the means of whole blood (venous as well as finger) over the mean of serum (taken as the standard) closely converge around unity (Ta-
E
2 J 0
o
1.10 1.05
A
®o
~
1.00 A
A
f
_.e 0.95 0 0.90
Day I
Day 3
Day 7
Day 14
Fig. 2. Testing the stability of blood samples placed on filter paper on days 1, 3, 7, and 14. The ratios of the mean (n = 8) values of whole blood samples analyzed by the present technique and serum analyzed by our previous technique [6] and taken as standard. Filled symbols refer to healthy subject 1, open symbols to healthy subject 2. Circles refer to finger blood, triangles to venous blood.
213 TABLE 2 C O M P A R I S O N OF D / L A R A B I N I T O L RATIOS IN W H O L E B L O O D ON F I L T E R P A P E R , TAKEN BY V E N I P U N C T U R F (VENOUS) A N D F I N G E R STICK (FINGER), W I T H S E R U M AS S T A N D A R D ; S A M P L E F R O M PA FIENT W I T H C O N F I R M E D D I S S E M I N A T E D C A N D I D I A S I S Type
Serum Venous Finger
Days on filter paper
0 1 1
D / L ratio
Meant, lood/Meanser,, m (n = 4)
Mean (n = 4)
S.D.
Range
5.42 5.40 5.32
0.13 0.20 0. ! 7
5.23 - 5.59 5.22 - 5.74 5.15 - 5.58
0.996 0.982
ble 1, last column). (4) The range of mean values of both venous and finger blood is between 2.57 and 2.68, i.e., within meanserum ___0.3 standard deviation (2.56- 2.70). Similar data were obtained for the samples from the second healthy subject. Fig. 2 shows ratios of the mean values of whole blood to serum for venous and fingerstick blood for all storage studies for both healthy subjects; values are well within the 0 . 9 5 - 1.05 range. We also tested the technique using samples from a patient with confirmed disseminated candidiasis. The D/L arabinitol ratios were greater than 3.3, which was designated in our previous studies [6] as the upper limit of normal. The data shown in Table 2 confirm that the technique is applicable in situations where the D/L ratio is abnormal. n / I A r a h l n i t o i in
,,";"~
The analysis of eight replicates of urine from a healthy subject gave the mean (_+ S.D.) D/L arabinitol ratio as 1.67 ( _+0.07) with a range of ! . 5 2 - 1.73. This is in agreement with our previous finding that urinary D/L arabinitol ratios in normals are in the same range as in normal serum (0.77 - 2.76) in spite of the fact that the absolute amount of arabinitol is approx. 60 times larger in urine than in serum [5, 6]. The corresponding data for filter paper analysis and storage stability are, mean + S.D. (range), day 1" 1.64+0.07 (1.53-1.72); day 3 : 1 . 6 5 + 0 . 0 5 (1.59-1.73); day 7: 1.62+C.07 (1.51 - 1.74); day 14:1.67 +0.10 (1.51 - 1.80). The results for the urine samples transported on filter paper to Michigan and back are: 1.65+0.07 (1.53- 1.75). The small standard deviation and the narrow range indicate essentially identical, and very good, reproducibility for both the original and the fi!ter paper techniques. Similar to the results with whole blood, comparison of the storage and transportation stability samples of urine analyzed by the present and previous techniques reveal that they are statistically indistinguishable (t-test, independent samples). Urine from a patient with confirmed disseminated candidiasis was analyzed by our previous method and the filter paper sampling technique, both after one day standing and after round trip transportation to Michigan ( n = 8 for each). The respective results for D/L arabinitol ratios are: 3.90_+0.12 (3.67-4.09); 3.87+_0.21
214 (3.47-4.10); 3.83_+0.18 (3.51 -4.04). The data are statistically indistinguishable. Determination of D/L arabinitol ratios in both blood and urine in a patient may further increase the reliability of the arabinitol technique. Experiments are in progress to establish the clinical utility of this approach, and the possibility of using urine alone.
Conclusions Based on the data presented we conclude that: (1) The technique developed permits the determination of the D and L enantiomers of arabinitol in a small drop of venous or finger stick blood or of urine placed as a spot on conventional filter paper with results that are statistically indistinguishable from those obtained from serum or urine using our previous technique. (2) The filter paper spots have storage stability for at least two weeks at room temperature. (3) Blood or urine samples can be safely air transported as filter paper spots. The method offers the following advantages: (1) B~ood samples can be directly collected on conventional filter paper either from finger stick or heel stick which permits applications in infants and children or from venous blood, which permits taking a one-drop aliquot from samples taken for other routine diagnostic purposes. (2) Only one small drop of blood is needed. (3) The filter paper spots can be stored for up to two weeks at room temperature and can be air transported to laboratories equipped to perform D/L arabinitol analyses, thereby offering the potential for largescale inter-institutional studies. (4) Whole blood is analyzed directly, the serum protein removal step is eliminated. (5) The technique is applicable to urine.
Acknowledgements This work was supported by a grant from the Department of Health and Human Services (CA40050) and by the T.J. Martell Memorial Foundation for Leukemia, Cancer and AIDS Research.
References 1 Sobel, J. and Vazquez, J. (1990) Candidemia and systemic candidiasis. Sem. Respir. Infect. 5, 123- 137. 2 Hughes, W., Armstrong, D., Bodey, G., et al. (1990) Guidelines for the use of antimicrobial agents in neutropenic patients with unexplained fever. J. Infect. Dis. 161,381 - 396. 3 Roboz, J. (1990) Gas chromatographic and mass spectrometric techniques for the diagnosis of disseminated candidiasis. In: Analytical Microbiology Methods: Chromatography and Mass Spectrometry (Fox, A., Morgan, S., Larsson, L. and Odham, G., eds.), pp. 2 3 9 - 258, Plenum, New York. 4 Wong, B. and Castellanos, M. (1989) Enantioselective measurement of the Candida metabolite Darabinitol in human serum using multidimensional gas chromatography and a new chiral phase. J. Chromatogr. 495, 21 - 3 0 . 5 Roboz, J., Nieves, E. and Holland, J.F. (1990) Separation and quantification by gas chromatographymass spectrometry of arabinitol enantiomers to aid the differential diagnosis of disseminated candidiasis. J. Chromatogr, 500, 4 1 3 - 426. 6 Roboz, J. and Katz, R. (1992) Diagnosis of disseminated candidiasis based on serum D/L arabinitol ratios using negative chemical ionization mass spectrometry. J. Chromatogr. (in press).
Journal o f .'~.'fi,.'r.obi'd"e;"qi.~., .~,, M~,,t(,~,.~ l_, (1992i 20"; 2i4
!992Else~ierSciencePublishcrsB.V. All rights reser~,ed 0167
7012/92/$ 5.00
MIMET 00495
Filter paper sampling of whole blood and urine for the determination of D/L arabinitol ratios by mass spectrometry J. Roboz, Q. Yu and J.F. Holland T..I. Martell Laboratories for Leukemia, Cancer and AIDS Research, Derald H. Ruttenberg Cancer Center, Mount Sinai School o f Medicine, New York, N Y 10029, U.S.A.
(Received 31 October 1991; re,,ision received 20 January 1992; accepted 30 January 1992)
Summary The methodology described permits the determination of the ratios of the o and L enantiomers of arabinitol in a small dorp of venous (arm) or finger-puncture blood or of urine placed as a spot on conventional filter paper. Replicate analyses using samples from healthy subjects and patients with confirmed disseminated candidiasis gave results that are statistically indistinguishable from those obtained from serum or urine using techniques previously developed by the authors. Replicate analyses in = 8) on days 1, 3, 7, and 14, and on samples after roundtrip long distance airmail transportation revealed that the filter paper spots have storage stability for at least two weeks at room temperature and that samples can be safely transported. Advantages include the initiation of pediatric applications using finger or heel punctures, frequent samplings from adults using drops of blood from samples taken for unrelated routine tests, and the design of inter-institutional studies to evaluate the clinical utility of using D/L arabinitol ratios for the diagnosis and monitoring of disseminated candidiasis.
Key words: Arabinitol enantiomer; D/L arabinitol ratio; Cand!diasis; Filter paper sampling
Introduction Disseminated candidiasis is an opportunistic infection that continues to be the cause of high morbidity and mortality in immunocompromised patients. Because of the lack of distinctive clinical manifestations and the dearth of reliable microbiological, biochemical, or immunological diagnostic techniques, antifungal chemotherapy is often started when the infection is already at an advanced stage [1]; there is a recent tre,d for empirical antifungal therapy in persistently febrile neutropenic patients [2]. Correspondence to: J. Roboz, Mount Sinai School of Medicine, Box 1247, One Gustave Levy Place, New York, NY 10029, U S.A.
208 Based on the identification of D-arabinitol as a major metabolite of several pathologic Candida species, a number of gas chromatographic and gas chromatographi c - m a s s spectrometric techniques have been developed for the quantification of arabinltol in serum [3]. Initial enthusiasm for the 'arabinitol technique' has considerably eroded because of poor specificity in the presence of renal dysfunction. This problem has been solved by new methodologies which separate the o and L enantiomers of arabinito! with the aid of chiral columns prior to detection by gas chromatography [4] or positive [5] or negative [6] ionization mass spectrometry. The use of serum D/L arabinitol ratios, instead of concentrations, has led to considerable improvement in both diagnostic specificity and sensitivity [5, 6]. A much needed inter-institutional prospective evaluation of the clinical utility of the D/L arabinitol technique would require convenient transportation of samples to laboratories equipped with the reqoired instrumentation. The well known restrictions and limitations in taking blood from infants and children ~equire acceptable sampli~lg techniques for the extension of the arabinitol technique to pediatrics. Accordingly, the objective of the present work was to further improve our methodology for the determination of O/L arabinitol ratios in blood (also in urine) by using a small drop of whole blood instead of serum or urine and collecting samples on conventional filter paper. Materials and Methods
Chemicals 2HT-labeled D-arabinitol (96 atom %0 deuterium), was purchased from Merck Sharp & Dohme, Isotopes Div. (Montreal, Canada). Methylene chloride and methanol (HPLC grade), toluene (certified ACS grade) and acetone (Optima grade) were obtained from Fisher Scientific (Fair Lawn, NJ, U.S.A.) and trifluoroacetic anhydride from Supelco (Bellefonte, PA, U.S.A.).
Study subjects Three types of blood samples, designated as 'serum', 'venous blood', and 'finger blood' (see below) were taken from two healthy adults and from a cancer patient with disseminated visceral candidiasis. Urine samples were from a healthy adult and from another cancer patient with disseminated candidiasis. Candidiasis was confirmed in both cases using conventional blood cultures as well as increased serum O/L arabinitol ratios.
Types of samples and method of sample taking 'Serum' samples were obtained conventionally, i.e., venous blood was collected in 'red top' glass tubes, and serum separated by centrifugation from clotted (20 min) blood which was stored frozen. 'Venous blood' samples were obtained by placing, with a calibrated micropipette, 20/zl of freshly drawn whole blood on a filter paper normally used for phenylketonuria screening of newborn infants (#903, Schleicher & Schuell Inc., Keene, NH, U.S.A.). 'Finger blood' samples were obtained by touching a filter paper to a drop of blood appearing after puncturing fingertips (or infant .heels) with a lance until the ring area on the paper was filled. The filter papers were allowed
209 to dry at room temperature (about 30 min) and stored for 1, 3, 7, or 14 days at room temperature. They were also used for transportation studies. The spots were cut from the paper pior to analysis. For the comparison studies, i.e., whole venous blood and finger blood against serum.. 88 samoles, were_ . .taken . . from . . . n .ho~lth,,,,z ....~.,, ,.,j;.,... ,..,. as r..,,,,.,l,UW~,. ..... Whole blood, taken by venipuncture, was divided into two parts. From one part serum was obtained and eight aliquets were taken to serve as standard. From the other part, eight drops of whole blood were placed separately on filter paper in quintuplicate for storage and transportation studies. At the same time, 40 filter paper samples were taken from finger blood for similar studies. In a similar manner, 72 samples were taken from a second healthy subject (no transportation studies), and 12 samples (four of each type) from a patient with confirmed dissemi~mted candidiasis. Urine samples were collected in glass tubes and were stored frozen without preservative. Ten microliter aliqiJots wcrc p,,,c,.,., on filter paper and treated like whole blood. Transportation stability
A set of eight each of venous-blood and finger-blood samples, dried on filter papers, was sealed in a plastic bag and sent airmail to East Lansing, MI, and back to this laboratory in New York City. Similarly, a set of urine samples dried on filter paper also made the round trip by air to Michigan. Sample preparation
A filter paper spot, containing venous blood, finger blood, or urine, was cut from the filter papers and extracted with one ml methanol for 15 min in an ultrasonic bath (conventional, low power, no cooling necessary). The solution was transferred to a 2-ml reaction vial which contained 10 ~! intornnl etnncl~rci (O 0 , - , , , / . . ! ~ ~'.-.n . . . . . .4 u . . evaporation to dryness under nitrogen at 45 °C in a waterbath. To the dried samples, 200 tzl methylene chloride and 100 #1 trifluoroacetic anhydride were added and, after vortexing for 30 s, derivatization was accomplished at 37 °C (metal heating block) for 45 min. After cooling to room temperature, the solvent and excessive reagent were evaporated to a final volume of approximately 5 ul (almost dryness) with a gentle stream of nitrogen at room temperature. The residues were dissolved in 50 ~1 toluene, and 1.0-#1 aliquots were analyzed. Serum (20-#1 aliquots) and urine (10-#1 aliquots) were prepared as follows: after adding 10 #1 internal standard, proteins were removed with one ml acetone, the mixture was vortexed for 30 s, centrifuged at 3000 g for 10 min, the supernatant was transferred into a small vial and evaporated to dryness. Derivatization followed as described above. Gas c h r o m a t o g r a p h y - mass spectrometry
The arabinitol enantiomers were separated using a 30 m long, 0.25 mm i.d. fused silica capillary column, coated with permethylated/3-cyclodextrin in a polysiloxane gum phase (B-Dex 120, Supelco, Inc., Bellefonte, PA), kept isothermally at 106 °C. A metal on-column injector (Model 16715, Chrompack, Bridgewater, N J), operated at room temperature, was connected to the analytical column via a one meter long
210 deactivated uncoated fused silica capillary precolumn. The precolumn, which traps a number of involatile sample constituents, was changed after every 30 analyses. This resulted in a considerable extension of the life of the analytical column, permitting the analysis of as many as 500 samples. The end of the analytical column was inserted into the ion source of the mass spectrometer; the transfer line was kept at 175 °C. The carrier gas was helium, no separator was used. A single quadrupole mass spectrometer (Model 3300, Finnigan Corp., San Jose, CA) combined with a Vector-One (Teknivent Co., Maryland Heights, MO) data system was used. The mass spectrometer was operated in negative chemical ionization mode with isobutane as reagent gas. D-, t.-arabinitol and the internal standard (2H 7labelled o-arabinitol) were monitored at m/z = 5!8 and m/z = 525, respectively, by selected ion monitoring, and O/k ratios were determined from peak arcas [6]. Results and Discussion
Rationale Some of the potential advantages of collecting whole blood samples on conventional filter paper, instead of serum, are obvious: extension of the use of the arabinitol method to include infants and children and overall sampling convenience. Easy and safe transportation is coi~.sidered of major importance. It will permit large-scale inter!nstitutional prospective studies which are essential to establish the clinical utility of the arabinitol enantiomer technique. The absence of the necessary instrumentation in most laboratories and the rarity of confirmed cases of visceral candidiasis makes some convenient transportation method essential. We have developed a technique that can be used for whole blood collected directly on filter paper by touching it to a tiny drop of blood obtained by puncturing a finger (or heel) with a lance, or by expressing a drop of blood from the needle of a syringe used for venipuncture. The first method is the obvious choice |or intended applications with infants and children, but may also be of considerable convenience for adults where samples may be taken at the time of finger stick blood work. The second method permits the study of a drop of blood from samples routinely taken for clinical chemistry or microbiology, thereby eliminating the need for separate sampling for this purpose. More frequent sampling will permit the evaluation of the utility of the arabinitol technique for the monitoring of antifungal chemotherapy.
Methodology Our previous methodology [5, 6] started with serum obtained from whole blood and used acetone to remove proteins. The present technique starts with whole blood which is collected on filter paper; the protein removal step is eliminated. Although not essential, we still use a known qu~mtity of internal standard because its presence provides additional analytical spec:,ficity for the D-arabinitol peak, its size reveals possible errors in sample preparatioJ~ (although this does not affect the ratio measurement), izs shape provides information on column performance and, when needed, its height cr area permits quantification of the individual enantiomers [6]. The method of derivatization, the chiral chromatographic separation, and the analysis by negative chemJca! ionization and selected ion monitoring mass spectrometry remained essen-
211 WHOLE BLOO0 Serum
Finger
Venous
2.6
2.6
2.6
5.6
5.6
5.7
HOR~£
m / z = 5~8 D/L
CAHDTDIASI$
m / z = 518
D/L
Fig. 1. Selected ion monitoring (at m / z = 518) profiles of D- and ~-zrzbinitol from a healthy subject (normalj and a cancer patient with disseminated candidiasis (candidiasis). ?rofiles labeled as 'serum' were from serum analyzed by our previous technique [6]. The label 'finger' refers to sample taken by finger stick expressing a drop of whole blood onto a filter paper. The label 'venous' refers to a drop of whole blood taken by venipuncture and placed onto a filter paper.
TABLE 1 R E P R O D U C I B I L I T Y , STORAGE ,aND T R A N S P O R T A T I O N STABILITY OF D / L A R A B I N I T O L RATIOS OF A H E A L T H Y S U B J E C T IN W H O L E BLOOD ON FILTER P A P E R , TAKEN BY V E N I P U N C T U R E (VENOUS) OR F;,NGER STICK (FINGER), C O M P A R E D TO SERUM AS STANDARD Type
Serum
Days on filter paper
0
D/I. ratio
Meanbiood/Meanserum (n = 8)
Mean (n = 8)
S.D.
Range
2.63
0.22
2.31 - 2.88
Venous
1 3 7 14 Transport
2.62 2.65 2.58 2.58 2.62
0.12 0.22 0.22 0.16 0.14
2.41 2.392.31 2.44 2.38 -
2.80 3.00 2.90 2.94 2.82
0.996 1.008 0.981 0.981 0.996
Finger
1 3 7 14 Transport
2.68 2.62 2.63 2.57 2.64
0.08 0.19 0.06 0.i2 0.14
2.57 - 2.83 2.35 - 2.88 2.55 - 2.73 2.41 - 2 . 7 9 2.39 - 2.79
1.019 0.996 1.000 0.977 1.004
~1"~ tiaii,,' unchar, ged, except for replacing the previously used Lipodex A type glass capillary co!~mns [5] with the ¢3-Dex-120 fused silica type. It is noted that the two column L, pes gave comparable performance. The efficie~acy of the methanol extraction of arabinitol from the filter papers was determined by comparing the recovery of known quantities of added D-arabinito! using our previous technique with the present one. Recovery was 9 5 - 105% using added D-arabinitol in six samples in a concentration range from 2 . 8 - 9 2 ng. To test t,h,.x, new sampling and ~,.,,.,~,.,~,.~,;,-,n . . . . . . . v:.- cananl~c by , , - v . . . . . . . . . . . . . . . m,-h,-,,~,-,1,-,~,, ........... o: ,~,e , - , h , ~ ; n , ~ A m!!!t;ni~ both techniques and compared results with those for serum samples taken and analyzed by our previous techniques which was considered to be the standard. D/L Arabinitol ratios in whole blood
Our objective was to prove that the results using venous or finger stick whole blood samples on filter paper are indistinguishable from those using serum with our previous vechnique. Fig. 1 shows the selected ion monitoring of o- and L-arabinitol in the three types of samples from a healthy subject (normal) and a patient with confirmed disseminated candidiasis, together with the corresponding D/L arabinitol rat'os determined from the respective peak areas. The separation of the enantiomers was nearly baseline. The profiles for serum are similar to those we have previously reported [5]. The profiles for finger and venous blood samples appear nearly identical to serum (Fig. 1). Table 1 summarizes reproducibility, storage and transportation stability data for D/L arabinitol ratios in whole blood on filter paper, both venous and fingerstick, and serum for the 88 samples taken from a healthy subject. Each mean value refers to n = 8, thus these and the corresponding standard deviations (and ranges) may be used to evaluate the reproducibility of the analyses for a given type of sample for a given period of storage. The data in Table 1 may be expressed in four ways. (1) The standard deviations of all groups of filter paper samples are equal to or smaller than that of serum. (2) The means of the venous- and finger-blood samples are statistically indistinguishable from that of serum (t-test, independent samples) for all storage days as well as for transportation. (3) M! ratios of the means of whole blood (venous as well as finger) over the mean of serum (taken as the standard) closely converge around unity (Ta-
E
2 J 0
o
1.10 1.05
A
®o
~
1.00 A
A
f
_.e 0.95 0 0.90
Day I
Day 3
Day 7
Day 14
Fig. 2. Testing the stability of blood samples placed on filter paper on days 1, 3, 7, and 14. The ratios of the mean (n = 8) values of whole blood samples analyzed by the present technique and serum analyzed by our previous technique [6] and taken as standard. Filled symbols refer to healthy subject 1, open symbols to healthy subject 2. Circles refer to finger blood, triangles to venous blood.
213 TABLE 2 C O M P A R I S O N OF D / L A R A B I N I T O L RATIOS IN W H O L E B L O O D ON F I L T E R P A P E R , TAKEN BY V E N I P U N C T U R F (VENOUS) A N D F I N G E R STICK (FINGER), W I T H S E R U M AS S T A N D A R D ; S A M P L E F R O M PA FIENT W I T H C O N F I R M E D D I S S E M I N A T E D C A N D I D I A S I S Type
Serum Venous Finger
Days on filter paper
0 1 1
D / L ratio
Meant, lood/Meanser,, m (n = 4)
Mean (n = 4)
S.D.
Range
5.42 5.40 5.32
0.13 0.20 0. ! 7
5.23 - 5.59 5.22 - 5.74 5.15 - 5.58
0.996 0.982
ble 1, last column). (4) The range of mean values of both venous and finger blood is between 2.57 and 2.68, i.e., within meanserum ___0.3 standard deviation (2.56- 2.70). Similar data were obtained for the samples from the second healthy subject. Fig. 2 shows ratios of the mean values of whole blood to serum for venous and fingerstick blood for all storage studies for both healthy subjects; values are well within the 0 . 9 5 - 1.05 range. We also tested the technique using samples from a patient with confirmed disseminated candidiasis. The D/L arabinitol ratios were greater than 3.3, which was designated in our previous studies [6] as the upper limit of normal. The data shown in Table 2 confirm that the technique is applicable in situations where the D/L ratio is abnormal. n / I A r a h l n i t o i in
,,";"~
The analysis of eight replicates of urine from a healthy subject gave the mean (_+ S.D.) D/L arabinitol ratio as 1.67 ( _+0.07) with a range of ! . 5 2 - 1.73. This is in agreement with our previous finding that urinary D/L arabinitol ratios in normals are in the same range as in normal serum (0.77 - 2.76) in spite of the fact that the absolute amount of arabinitol is approx. 60 times larger in urine than in serum [5, 6]. The corresponding data for filter paper analysis and storage stability are, mean + S.D. (range), day 1" 1.64+0.07 (1.53-1.72); day 3 : 1 . 6 5 + 0 . 0 5 (1.59-1.73); day 7: 1.62+C.07 (1.51 - 1.74); day 14:1.67 +0.10 (1.51 - 1.80). The results for the urine samples transported on filter paper to Michigan and back are: 1.65+0.07 (1.53- 1.75). The small standard deviation and the narrow range indicate essentially identical, and very good, reproducibility for both the original and the fi!ter paper techniques. Similar to the results with whole blood, comparison of the storage and transportation stability samples of urine analyzed by the present and previous techniques reveal that they are statistically indistinguishable (t-test, independent samples). Urine from a patient with confirmed disseminated candidiasis was analyzed by our previous method and the filter paper sampling technique, both after one day standing and after round trip transportation to Michigan ( n = 8 for each). The respective results for D/L arabinitol ratios are: 3.90_+0.12 (3.67-4.09); 3.87+_0.21
214 (3.47-4.10); 3.83_+0.18 (3.51 -4.04). The data are statistically indistinguishable. Determination of D/L arabinitol ratios in both blood and urine in a patient may further increase the reliability of the arabinitol technique. Experiments are in progress to establish the clinical utility of this approach, and the possibility of using urine alone.
Conclusions Based on the data presented we conclude that: (1) The technique developed permits the determination of the D and L enantiomers of arabinitol in a small drop of venous or finger stick blood or of urine placed as a spot on conventional filter paper with results that are statistically indistinguishable from those obtained from serum or urine using our previous technique. (2) The filter paper spots have storage stability for at least two weeks at room temperature. (3) Blood or urine samples can be safely air transported as filter paper spots. The method offers the following advantages: (1) B~ood samples can be directly collected on conventional filter paper either from finger stick or heel stick which permits applications in infants and children or from venous blood, which permits taking a one-drop aliquot from samples taken for other routine diagnostic purposes. (2) Only one small drop of blood is needed. (3) The filter paper spots can be stored for up to two weeks at room temperature and can be air transported to laboratories equipped to perform D/L arabinitol analyses, thereby offering the potential for largescale inter-institutional studies. (4) Whole blood is analyzed directly, the serum protein removal step is eliminated. (5) The technique is applicable to urine.
Acknowledgements This work was supported by a grant from the Department of Health and Human Services (CA40050) and by the T.J. Martell Memorial Foundation for Leukemia, Cancer and AIDS Research.
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