field-study screening of blood folate concentrations - american

1398 Am J C/in Nutr 1997:66:1398-405. Printed in USA. © 1997 American Society for Clinical Nutrition

Field-study screening of blood folate concentrations:specimen stability and finger-stick sampling1�

Sean D 0 ‘Broin, Brian P Kelleher, Ann Davoren, and Elaine W Gunter

ABSTRACT We describe optimized procedures for field

studies of blood folate concentrations by using finger-stick blood

sampling and include relevant studies on blood folate stability. We

introduce whole-blood folate adjustment using sample hemoglobin

(folate/hemoglobin, nmollg) as a novel and practical tool yielding

accurate and precise results when blood volume or dilution is

unknown. Red cell folate concentrations (nmollL) of 1 1 887

Americans correlated well with hemoglobin-corrected whole-

blood folate concentrations (r� = 0.993; red cell folate = 0.347 X

hemoglobin folate + I nmol/L), which supports the approach of

using the mean cell hemoglobin concentration (g/L) to interconvert

red cell and hemoglobin folate data. Folate concentrations in

capillary (finger stick) and venous blood samples from 28 normal

donors were similar (P > 0.87), correlating closely (r = 0.98, P <

0.001). Whole-blood samples (collected into K2-EDTA-contain-

ing evacuated tubes) in field studies are best stored intact at 4 #{176}C

until they can be processed and frozen (-20 #{176}C).Specific knowl-

edge of blood folate stability is essential in planning and designing

field studies. Am J Gum Nutr l997;66: I 398-405.

KEY WORDS Red cell folate, hemoglobin folate, driedblood spot, specimen stability, finger-stick blood sampling,

field studies

INTRODUCTION

Recent focus on folate status as a serious clinical and public

health issue has led to an increased demand for nutritionalsurveys of blood folate concentrations. Diminished folate sta-

tus may be associated with cervical (1) and colonic (2) carci-

nogenesis and is a primary determinant for homocysteinemia,

which may be an independent risk factor for atherosclerotic

vascular disease (3). The relations of these new clinical corre-

lates with folate status have yet to be evaluated fully. Because

data exist (4) that confirm the role of folate in the prevention ofspina bifida and anencephaly, extensive clinical and epidemi-

ologic studies are needed to support the development of effec-

tive primary prevention strategies for these birth defects (5).

To control costs in nutritional surveys, it is desirable tosimplify both blood collection and assay procedures when

possible. Red cell folate concentrations are unstable (6) andspecial care must be taken when deviating from standard pro-

tocols of collection and handling. For field studies in remote

areas, additional factors relating to climate, local facilities,

transportation, and storage can compromise blood folate sta-bility and must be evaluated in advance.

This study was initiated to simplify blood folate screening in

general and to develop effective logistics for conducting

screening in remote areas. The approach of analyzing folate

concentrations in capillary blood collected by finger-stick sam-

pling was selected because this method of sample collection

has proved to be both practical and economical compared with

venipuncture in field studies for other analytes (7).

MATERIALS AND METHODS

Materials

Type 903 filter paper cards (1 5 X 10 cm) for preparing dried

blood spots (DBS) were obtained from Schleicher and Schuell

(Keene, NH). Microvettes, cryovials, and microtiter plateswere from Sarstedt (Wexford, Ireland) and EDTA-anticoagu-

lated Microtainers (500 p.L) were from Becton Dickinson(Orangeburg, NY). EDTA-anticoagulated whole blood

(EDTA-WB) for complete blood counts (CBCs) was collected

into K2-EDTA-containing Vacutainers (Becton Dickinson) in

Ireland and into K3-EDTA elsewhere. Unistik 2 lancets were

from Owen Mumford Inc (Atlanta). Sodium ascorbate, ascor-

bic acid, Tween 80, Triton X-lOO, and sodium lauryl sulfate

were from Sigma Chemical Co (St Louis). A Decon FS-lOO

sonication bath was from Ultrasonics Ltd (Sussex, United

Kingdom). Serum femtin concentrations were determined with

the Abbott IMX system (Abbott Laboratories, Abbott Park,

IL). Serum vitamin B- 12 concentrations were measured by

microbiological assay on microtiter plates (8). Standard finger-

stick protocols were followed (9).

Folate assays

Red cell and serum folate concentrations were measuredwith the Bio-Rad Quantaphase II radioassay kit (Bio-Rad Lab-

I From the Department of Haematology, St James’s Hospital, Dublin;The Blood Transfusion Service Board, Dublin; and the Centers for DiseaseControl and Prevention, Atlanta.

2 Use of trade names is for the purpose of identification only and does

not constitute endorsement by St James’s Hospital, the US Public Health

Service, or the US Department of Health and Human Services.

3 Supported in part by the National Center for Environmental Health,

Centers for Disease Control and Prevention, Atlanta.

4 Address reprint requests to SD O’Broin, Department of Haematology,St James’s Hospital, Dublin 8, Ireland.

Received April 14, 1997.Accepted for publication July 17, 1997.

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FIELD STUDIES OF BLOOD FOLATE CONCENTRATIONS I 399

oratories, Hercules, CA) and also with a microbiological assay

using Lactobacillus casei (NCIB 10463) on microtiter plates

(10). We compared the red cell folate concentrations in 1 18EDTA-WB samples using both assays. The samples repre-sented results of sequential diagnostic folate assays and in-

cluded never-thawed hemolysates from donors with poor folatestatus (n = 30), which had been stored at -70 #{176}C.Erythrocyte

5-methyltetrahydrofolate polyglutamates must be deconjugated

to the monoglutamate form before being assayed (1 1). Hydro-

lysis is achieved by lysing cells in ascorbic acid at a pH suitable

for the activity of endogenous plasma conjugase enzyme (‘y-glutamyl hydrolase, EC 3.4.12.10). Microbiological assay of

lysates of EDTA-WB, prepared by diluting the EDTA-WB 1:9

with 10 g ascorbic acid/L (eg, 100 p.L EDTA-WB + 900 �tL

of 10 g ascorbic acidlL), mixing, and then incubating theresulting hemolysate at room temperature for 30 mm, was used

as the 100% control whole-blood folate concentrations

throughout the study. In some stability studies low-folate se-

rum from folate-deficient patients was incubated with hemo-lysates as an additional conjugase source to establish endoge-

nous plasma conjugase sufficiency. In these cases, a volume of

low-folate serum was added that was equivalent to the endog-enous plasma volumes of the hemolysates.

Measurement of red cell indexes

CBCs were performed electronically on a Coulter STKScalibrated by using SC hematology reference controls (Coulter

Diagnostics, Hialeah, FL). Hemoglobin concentrations of

EDTA-WB samples from patient donors were measured by

using the reference cyanmethemoglobin method (12), and ly-

sates of these samples prepared as described were then used as

hemoglobin controls in later assays. Hemoglobin concentra-

tions in EDTA-WB in ascorbic acid (as prepared for red blood

cell folate determination) were quantified spectrophotometri-

cally (535 nm) after 1:20 dilution with a solution of sodium

lauryl sulfate (2.42 mmol/L) and Triton X- 100 (1 mLJL) in

0.03 mol sodium phosphate bufferlL (pH 7.3) mixed, and

allowed to stand for 3 mm. Hemoglobin concentrations weremeasured directly after precalibrating the spectrophotometerwith lysates of the hemoglobin control samples (n = 3).

Whole-blood hemoglobin samples (n = 83) measured with the

Coulter STKS correlated well with hemoglobin measured incorresponding hemolysates in 10 g ascorbic acidlL (r� = 0.97,

y = 1.03x - 0.29) and there was no significant difference

between the results (P > 0.7). Also, there was no significant

difference between hemoglobin concentrations in WB-EDTAsamples (n = 10) diluted 1:20 in water and those in WB-EDTAsamples diluted in 2.5, 5.0, 10, or 20 g ascorbic acid/L (P >

0.8). Sodium lauryl sulfate is used widely for conventionalhemoglobin measurements because of its comparatively lowtoxicity and because excellent comparisons have been obtainedwith the reference cyanmethemoglobin method (13, 14).

Dried blood spots

Blood specimens were stored as DBS on filter paper cards

(15 X 10 cm), each with 15 preprinted 1.25-cm circles. DBSwere prepared by pipetting SO p.L EDTA-WB onto the circles

on the cards, then placing the cards horizontally on a rack andallowing them to air-dry overnight at ambient temperature.Recovery studies indicated that > 90% of the original folate

concentration and > 96% of the original hemoglobin concen-tration were eluted from the spots prepared in this manner, after

the entire spot had been cut out with scissors and sonicated in

10 g ascorbic acidlL containing 1% (by vol) Tween 80 for 60mm. Some filter paper was preimpregnated with ascorbic acid

as an antioxidant in an effort to improve the stability of folate

in the DBS. This was done by immersing the paper card flat in10 mL 10-20 g ascorbic acid/L for 60 s, followed by air-dryingthe card horizontally on a rack.

Blood folate correction

We evaluated two approaches for adjusting whole-bloodfolate concentrations: the use of hemoglobin and the use of

traditional hematocrit correction. The close relation that exists

between these hematologic indexes is expressed as the mean

corpuscular (cell) hemoglobin concentration (MCHC) or he-

moglobin/hematocrit. The MCHC, as calculated by automated

analyzers as part of a CBC, has shown a remarkable constancy

across a range of ages in cord blood and in children and adults

(15, 16) of different ethnic groups, both males and females

(17).

Hematologic data, including serum folate and red cell folateconcentrations measured with the Bio-Rad radioimmunoassay

as well as CBCs from a nutritional survey of 1 1 887 healthyAmericans, were examined (EW Gunter, unpublished observa-

tions, 1994). These data were collected by using methods of

analyses described previously (I 8). Red cell folate concentra-

tions were derived from whole-blood folate and serum folate

concentrations by using the traditional hematocrit correction of

Hoffbrand et al ( 1 1) as follows:

Red cell folate (nmolIL)

- whole-blood folate - [serum folate (1 - hematocrit)] (1)

- hematocrit

Hemoglobin folate concentrations were determined by divid-

ing the whole-blood folate concentration by the concentration

of hemoglobin in the sample:

Hemoglobin folate (nmol/g)

- whole-blood folate_(nmollL) (2)

- hemoglobin (g/L)

We also analyzed the results of sequential diagnostic CBCrequests (n 2049) and nutritional screen requests (n = 868)

in the Department of Haematology of St James’s Hospital to

determine the constancy of the MCHC ratio in such samples.CBCs on EDTA-WB samples were estimated by using theCoulter STKS, and serum and red cell folate concentrations bymicrobiological assay.

Red cell and hemoglobin folate concentrations compared

Blood samples for red cell folate assay (n = 187) wereprepared by accurate volumetric dilution of fresh EDTA-WB

as described above. The same blood samples (n = 187) werediluted nonvolurnetrically by an independent laboratory tech-

nician who was supplied with tubes containing variable vol-umes of 10 g ascorbic acid/L and instructed to prepare hemo-

lysates by adding a few drops of blood to each tube. The

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1400 O’BROIN ET AL

dilution factor of these blood lysates was thus unknown and

variable. Lysate folate concentrations were assayed by micro-

biological assay as described previously and hemoglobin con-

centrations determined from the lysates described previously

were used to correct the results of nonvolumetrically preparedsamples. Red cell folate concentrations of conventionally di-

luted blood samples were calculated from the hematocrit. The

folate concentrations of nonvolumetrically prepared lysates

were expressed as the folate concentration per unit of hemo-

globin (folate/hemoglobin, pmol/g); a dilution factor thus be-

comes irrelevant because it is common to both the numerator

and denominator.

Blood folate stability

Preliminary blood folate stability screens helped to approx-

imate rates of loss of microbiological assay activity and to

identify conditions suitable for further study. One such study

was made by using DBS stored at temperatures of -20, 8, 22,and 37 #{176}C.DBS were prepared by pipetting S0-pL aliquotsfrom three fresh EDTA-WB samples onto both plain filter

paper and paper that had been pretreated with 10 or 20 gascorbic acidlL as described. Blood spots were air-dried over-

night at ambient temperature and then stored in plain polyeth-

ylene bags without desiccant on day 1 . On subsequent days, up

to the completion of the study, DBS were removed fromstorage and placed at - 20 #{176}Cuntil extracted and assayed as

described previously.

Similar folate stability studies were made with EDTA-WB

samples (n = 14) stored as unlysed whole blood (K2-EDTA)

and as hemolysates in 10 g ascorbic acidlL at temperatures of

-20, 4, 22, and 37 #{176}C.Controls prepared as described were

stored at - 20 #{176}C.EDTA-WB samples were mixed and dis-

pensed in 50-pt aliquots into 1-mL cryovials and ascorbated

hemolysates of these samples were stored under the same

conditions. On days 1, 2, 4, 5, and 7, an aliquot of whole blood

was removed from storage, diluted with 10 g ascorbic acidlL

(1 : 10), mixed thoroughly, incubated at room temperature for 30mm, and stored at -20 #{176}Cuntil assayed. Blood lysates were

placed at - 20 #{176}Cat the same times and the folate concentra-

tions of all lysates and controls were measured in quadruplicate

in a single microbiological assay run.

Freeze-thaw stability of EDTA-WB folate was studied. In atypical experiment, control hemolysates were prepared and

l00-�L aliquots from 10 fresh EDTA-WB samples were fro-

zen overnight at -20 #{176}C,placed at room temperature (20 #{176}C),

and diluted by adding 10 g ascorbic acidlL immediately and

after 1, 6, and 24 h. After dilution, each lysate was mixedextensively by vortex, incubated at 20 #{176}Cfor 60 mm, and

frozen at - 20 #{176}C.Finally, all aliquots were thawed and the

folate concentration was measured by microbiological assay.

The stability of whole-blood folate at -20 #{176}Cwas observedas a between-assay reproducibility study. Aliquots (200 pL) ofEDTA-WB from three donors were frozen at -20 #{176}C.They

were removed, extracted as above, and assayed for folateweekly over 10 wk.

Factors influencing plasma conjugase activity

Stability

A study of EDTA-WB folate recovery included an evalua-

tion of plasma conjugase activity. Aliquots from six

EDTA-WB samples in cryovials were stored in batches at both4 and 22 #{176}C.On days 1, 4, 5, 6, and 7, an aliquot from eachbatch was stored at -20 #{176}C.Finally, all aliquots were diluted

1: 10 with 10 g ascorbic acidlL, incubated at 20 #{176}Cfor 30 mm,

and assayed. A portion of each aliquot was also incubated with

supplemental fresh low-folate plasma, as described previously.

Ascorbic acid concentration

Blood samples (n = 9) were diluted 10-fold in solutions of

20, 15, 12.5, 10, 7.5 and 5.0 g ascorbic acidlL. The dilutedsamples were incubated at 20 #{176}Cfor 30 mm, assayed for folate

concentrations, and the relative folate recoveries comparedwith those of the hemolysate in 10 g ascorbic acid/L. Blood

samples diluted in 10 or 5.0 g ascorbic acid/L containing 1%Tween 80 or 0.1% Triton X-100 were also compared with

controls.

Dilution factor

The relation between the degree of initial dilution of wholeblood in ascorbic acid and blood folate recovery was also

studied. Fresh blood samples (n 10) were diluted 1 : 10, 1:20,1:40, and 1:50 in 10 g ascorbic acid/L, then each dilution was

divided and supplementary fresh plasma conjugase was added

to one portion as a control before the samples were mixed and

incubated at 20 #{176}Cfor 30 mm. In a separate experiment, therelative folate recoveries of whole blood (n = 10) diluted 1:10,1:20, 1:50, and 1:100 in both 10 and 5.0 g ascorbic acid/L

containing 0.1% Triton X-100 were determined.

Finger-stick and venous blood folate concentrations

compared

Venous EDTA-WB samples were obtained from 28 labora-tory staff along with concurrent capillary blood finger-sticksamples collected into EDTA-containing cryovials after eachdonor’s hand was prewarmed as recommended (9). Lysates ofboth sets of blood samples were prepared by standard dilution

in 10 g ascorbic acidlL and assayed for folate. Finger-sticksamples in EDTA-containing cryovials were also obtained

from blood bank donors (n = 45). These finger-stick sampleswere taken from unwarmed finger-stick sites by donor atten-dants secondary to a primary sampling from the identical site

for hemoglobin screening. Blood was washed nonvolumetri-cally from these cryovials with a solution of 5.0 g ascorbicacid/L containing 0.1% Triton X-100, and venous control he-molysates were diluted 1 :20 in the same solution for compar-

ison. Similar fmger-stick samples from 56 blood bank donorswere collected directly into 1 mL of sterile 5.0 g ascorbic

acid/L and 0.1% Triton X-100 in cryovials. Donor attendantswere instructed to add a drop of blood directly from thefinger-stick site into the ascorbic acid, cap the vial, and mix thesolution immediately and thoroughly by inversion six times.Venous control sample lysates were prepared as described and

all samples were assayed together for folate either on the sameday, or else the samples were frozen at -20 #{176}Cwithin 3 h ofsampling and assayed later.

Statistical analysis

Results were compared by using the t test for paired obser-vations and by simple-regression analysis and are reported as

means ± SDs. The statistical package STATGRAPHICS (ver-

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7000

8000

0.

0

I:0

r = 0.99

y=3.06x+ 118.4n = 187

2000ire

Red cell folate (nmoVL)ire

�2000

0

E

F500c8to(5� moo

0

0

�51xD

. 8,.#{149}

r = 0.84y = 1.8x + 107.8n = 218

0 200 400 800 800 000 200

Bio-Rad Quantaphase II (nmol/L)

FIGURE 2. Comparison of red cell folate concentrations estimated by

using a microbiological assay and by radioassay with Bio-Rad’s Quant-

aphase II (Bio-Rad Laboratories, Hercules, CA).

FIELD STUDIES OF BLOOD FOLATE CONCENTRATIONS 1401

TABLE 1

Comparison of red cell folate and hemoglobin folate concentrations in

healthy US subjects’

Males Females

Red cell folate (nmollL) 608.6 ± 278.9 [6045] 620.2 ± 313.2 [61 191

Hemoglobin folate 1744.8 ± 800 [5913] 1786. 1 ± 899.6 [59741(pmollg)

, I ± SD; n in brackets. Folate concentrations were assayed by using

Bio-Rad Quantaphase II; Bio-Rad Laboratories, Hercules, CA.

sion 1 .2; Statistical Graphics Corp. Rockville, MD) was used

for the analyses.

RESULTS

Distribution of MCHC in normal and disease states

In almost 12 000 healthy Americans, red cell folate andhemoglobin folate concentrations correlated well (r� 0.993,

P � 0.001; n = 11887) and were similarly distributed acrossthe full range of values (Table 1), thus reflecting the constancy

of the MCHC. This supports the use of hemoglobin for cor-

recting data and of MCHC for interconverting blood folatedata. The regression equations were as follows:

Red cell folate = 0.347 hemoglobin folate + 1.0 nmolIL (3)

Hemoglobin folate = 2.86 red cell folate + 10 pmollg (4)

Data from Ireland on 2049 sequential, diagnostic, full bloodcount requests supported this finding (MCHC = 343 ± 8 g/L)

but slightly lower MCHCs were noted in patients with lowserum ferritin concentrations (< 20 �g/L). In these patients(n 2 16) MCHCs (320.6 ± 1 7 g/L) correlated poorly with

serum ferritin concentrations (r = 0.2, P = < 0.01) but thiscorrelation improved with the degree of iron deficiency

(Table 2).

In the Irish data set there was no relation between severity of

red cell folate deficiency and MCHC (33 1 .6 ± 1 1 .6 g/L) in 457sequential patients selected on the basis of having simulta-

neously low serum ferritin (< 5.7 nmol/L) and red cell folate(< 340 nmolJL) concentrations. Also, MCHCs (335.5 ± 1 1.7gIL) of 481 sequential patients with a low serum vitamin B-l2concentration (< 1 10 pmol/L) correlated negatively with theserum vitamin B- 12 concentration when patients with low

serum femtin concentrations were excluded (r = -0. 1 16, P =

< 0.05).

TABLE 2

Mean cell hemoglobin concentrations of patients with iron deficiency,

correlated with serum ferritin concentrations

Serum femtin I ± SD r

gIL< 20 �tg/L (n = 216) 321 ± 17 0.2

< 15 p�g/L (n = 188) 320 ± 17 0.235

< 10 �g/L (n = 145) 319 ± 17 0.324

< 5 ,.Lg/L (n = 82) 316 ± 19 0.346

< 3 ;ig/L (n = 23) 305 ± 19 -

FIGURE 1. Comparison of traditionally prepared red cell and hemo-

globin folate concentrations from nonvolumetrically prepared lysates esti-

mated by using a microbiological assay.

Folate assays

Volumetrically prepared red cell folate concentrations (n

187) correlated well (r� 0.98, P < 0.001) with theirequivalent hemoglobin folate concentrations from lysates pre-pared by using dilutions of unknown proportions (Figure 1).Random nonvolumetric dilution of lysates for hemoglobin fo-

late was confirmed by comparing the whole-blood folate con-centrations from both data sets. Red cell folate concentrations

(n 2 1 8) determined by microbiological assay (85 1 .6 ± 465nmol/L) were substantially higher than those obtained by using

the Bio-Rad Quantaphase II radioassay (417 ± 219 nmol/L;

Figure 2.

Blood folate stability

Storage temperature was identified as a major influence onstability in all studies. In initial screens of blood folate stability,

lysates in 10 g ascorbic acid/L incurred an overnight folate loss

of > 50% at 37 #{176}C,thus this condition was not included in

subsequent studies. Comparatively good stability was achieved

for folate concentrations in DBS on plain filter paper but thesewere progressively less stable on filter paper that was pre-

treated with 10 or 20 g ascorbic acidlL and with increasing

2500

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acid.

-�. 120

0

� 100

SCO

0

11)

>

02

0)C

C

CO

E0)

0)

CO0

LI- 0

FIGURE 3. Effect of temperature on folate stability when blood sam-

ples (n = 14) were stored as anticoagulated (with EDTA) whole blood (A)and lysates (B) in ascorbic acid. I ± 1 SD. Values < 10% are expressed

as 10%.

temperature (Table 3). A folate recovery > 90% was obtainedfrom 10 DBS on plain paper after 4 d at 4 #{176}C(90.5 ± 9.5%).

The addition of supplemental plasma conjugase to extracts

from DBS as described did not improve folate recovery.In general, folate recovery was related to storage tempera-

ture, whether blood was stored as EDTA-WB (Figure 3A), oras lysates in 10 g ascorbic acid/L (Figure 3B), or as DBS on

filter paper (Table 3). Maximum folate stability at 4 and 22 #{176}C

was achieved with intact whole blood (n = 14), with 73%recovery at 22 #{176}Cafter 5 d. Folate stability was poorest withlysed blood specimens. Results varied considerably in individ-

TABLE 3

80

60

40

20

ual samples stored as hemolysates in 10 g ascorbic acid/L, with

recoveries ranging from < 10% to 100% after 4 d at 22 #{176}C

(Figure 3B).Frozen and thawed EDTA-WB samples (n = 20) had 12%

lower folate concentrations than controls, which was signifi-cant (t = 3.79, P < 0.005). Folate concentrations ofEDTA-WB samples frozen at -20 #{176}C(n = 10) diminishedrapidly at 20 #{176}Cafter being thawed, with recoveries of 90%,

77.8%, and 51.2% after 1, 6, and 24 h, respectively. However,the folate concentrations of three intact EDTA-WB samples

that had been frozen in aliquots at -20 #{176}Cremained stable,with between-assay CVs of 12.0%, 8.6%, and 12.6%, after 10consecutive weekly assays.

Factors influencing plasma conjugase activity

Folate recovery from six EDTA-WB samples after storagefor 5, 6, and 7 d was 91%, 90%, and 92%, respectively, at 4 #{176}C

and 79%, 72%, and 66%, respectively, at 22 #{176}C.Folate recov-

ery was not improved by incubating hemolysates with supple-

mental fresh plasma, confirming the retention of sufficient

endogenous conjugase enzyme activity after such storage.Whole-blood folate concentrations were not significantly dif-

ferent when ascorbic acid at concentrations of 5.0, 7.5, 10,12.5, and 15 gIL were used to lyse blood samples (n = 9);when blood samples (n 10) were diluted 1 : 10 (controls),1 :20, 1:40, or 1 :50 in ascorbic acid; or when supplementary

plasma conjugase was added to these lysates. The folate recov-eries from blood samples (n = 10) diluted 1:10, 1:20, 1:50, or1:100, both in 10 g ascorbic acid/L and in 5.0 g ascorbic acid/L

containing 0.1% Triton X-l00, were virtually identical and the

inclusion of 1 % Tween 80 in the ascorbic acid solutions did not

influence recovery.

Finger-stick and venous blood folate concentrationscompared

Hemoglobin folate concentrations of volumetrically pre-pared finger-stick (2060 ± 663 pmollg) and of venous (2087 ±

657 pmoL/g) EDTA-WB samples from 28 normal volunteers

correlated well (,2 0.98) and were not significantly different(t = 0.155, P > 0.87; Figure 4). Folate concentrations of

finger-stick samples taken as second samples from unwarmed

sites (2205 ± 680 pmol/g) and diluted nonvolumetrically also

correlated significantly (r� = 0.9, P < 0.001) with venouscontrols (2178 ± 672 pmol/g) and were not significantly dif-

ferent (t = 0.86, P > 0.39). Estimation of hemoglobin folate

Mean percentage recovery of folate remaining in dried blood spots when stored for 1 wk at three different temperatures on both plain and ascorbated

filter paper’

Plain paper Paper A Paper B

8 #{176}C 22 #{176}C 37 #{176}C 8 #{176}C 22 #{176}C 37 #{176}C8 #{176}C 22 #{176}C 37 #{176}C

%

Day 1 87 83 59 99 75 44 86 62 21

Day 4 85 7 1 56 79 47 18 60 39 11

Day 5 80 75 58 72 47 18 62 28 11

Day6 77 71 51 71 44 18 60 29 10

Day 7 81 71 57 76 44 16 64 32 13

‘ Results are from three separate blood samples compared with a control. Paper A was pretreated with 1% ascorbic acid and paper B with 2% ascorbic

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0) 3500

0

E.9’ 3000

0)

CO.2 2500

03

12000

sire

0)0)

�ire

r = 0.98

y = 0.98x + 73.5n = 28

moo ire 2000 2500 3000 3500 4000

Venous control sample folate (pmol/g)

B7000

0) 6000

0

E.9��ooo

03

CO.90)

53000

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�ire

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r = 0.92y = 0.94x + 236n = 56

0 ire 2000 3000 4000 5000 6000

Venous control sample folate (pmoVg)7000


FIGURE 4. Comparison by microbiological assay of folate concentra-

tions in finger-stick blood samples collected into K3-EDTA (A) or 5.0 g

ascorbic acidlL (B) and venous blood samples.

overcomes the problem of accurately diluting such very smallsamples (< 300 p.L). Initially, 80% of small samples (< 100p.L) taken into EDTA showed small clots on microscopic

examination; this problem was eliminated by ensuring ade-quate mixing by shaking and inverting at least six times. Thefolate concentrations of finger-stick samples collected directlyinto sterile ascorbic acid solution (Figure 4) also compared wellwith venous controls (r� = 0.85, P < 0.001), giving similar

results (P > 0.3).

DISCUSSION

These studies were undertaken to support population screen-

ing of blood folate status in conventional settings as well as inremote areas with limited facilities, to simplify blood collectionand reduce costs without unduly sacrificing analytic accuracyand precision. Folate concentrations are unstable and studies ofblood folate recoveries under different conditions were consid-

ered important because few of the available studies were rel-evant and some were contradictory. Endogenous folate plasmaconjugase stability was a concern but did not limit folaterecovery under any conditions studied here; an inhibition of itsactivity was noted previously in sera from uremic patients (19)

The temperature of blood storage had a major influence onthe recovery of folate assayed microbiologically from all spec-

imen types, including intact whole-blood samples (Figure 3A),whole blood lysates (Figure 3B), and DBS (Table 3). Also,

folate concentrations in all unfrozen blood samples were morestable in the intact red cell than as a hemolysate. The generally

decreased stability of blood folate concentrations as lysates orDBS in the presence of ascorbic acid was disconcerting. Alikely explanation is that ascorbic acid rapidly lyses the red

cells and that in its absence the intact cell affords greaterprotection to labile folate concentrations.

Storage as unfrozen intact EDTA-WB offered the best over-all folate stability, yielding recoveries of 86% on storage at

4 #{176}Cfor 7 d (Figure 3A) and 92% at 4 #{176}Cin a follow-up study(n 6). Previous studies at 4 #{176}Cnoted either no deterioration

in the folate concentrations of similar samples after storage for7-10 d (I 1) or for 14 d (20), or a 20% loss after 3 d when

shaken (6). Blood samples that cannot be processed immedi-ately should be stored in this manner at a temperature > 0 #{176}C(eg, 4 #{176}C),a temperature at which folate degradation is slow.

The relative instability and erratic recoveries of folate from

ascorbate-treated blood hemolysates (Figure 3B) confirm thefindings of previous studies (6, 1 1), despite one report to the

contrary (21). These problems limit the potential of preparing

such hemolysates in some field-study settings because the

samples need to be either assayed immediately or frozen at

-20 #{176}C(or -70 #{176}C).These hemolysate folate concentrations

will remain stable when frozen at -20 #{176}Cand have served wellas long-term quality control materials (22, 23).

Contrary to the findings of a previous study (1 1), we foundthat folate concentrations in EDTA-WB samples at - 20 #{176}C

remained stable for � 10 wk despite poor between-assay re-

producibility (CV: > 10%) relative to that of conventional

microbiological assay controls (22). Difficulties in resuspend-

ing these viscous frozen and thawed samples may account for

the sporadic errors that occurred; a systematic folate loss of� 12% was noted as a result of the freezing and thawing. Also,

when thawed, the folate concentrations of frozen EDTA-WBsamples degrade rapidly with almost a 50% loss of activityoccurring at 20 #{176}Cover 24 h; therefore, blood samples thatthaw during transportation should not be used.

Traditional whole-blood folate correction by using hemato-crit was introduced because red cells can contain SO times the

amount of folate that serum contains (24) and red cell volumesvary. The usefulness of correcting whole-blood folate concen-trations by using the sample hemoglobin became apparent early

in the present study; previous work had suggested the use of aderived hematocrit value to estimate red cell folate concentra-tions (25). Blood folate concentrations may be adjusted by

using the hemoglobin concentration because the MCHC ratio is

physiologically controlled within relatively narrow limits (26).

This relation has been well established both in clinical hema-tology (15, 16) and in the establishment of hematologic refer-

ence data for normal populations ( 17). Also, an internationalhematologic study of consecutive patients admitted to teachinghospitals in Wales, California, and Japan (n = 3500) noted nosignificant geographic difference in mean MCHC values (26).

The potential of using population mean MCHCs to manipulate

blood folate data was validated in our analysis of data fromalmost 12 000 free-living American subjects (Table 1) becausered cell folate concentrations and hemoglobin folate concen-

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trations correlated well (r� = 0.993). Little variation in MCHCwas noted in an analysis of 2049 consecutive diagnostic CBC

requests: MCHC 343 ± 8.2 gIL, comparing well with mean

hematologic reference data (17) established for males (343 ±

12.5 g/L; n = 8449) and females (338 ± 12.9 g/L; n 8829)

aged 3-74 y. It is apparent that the CVs obtained for MCHCsin these hematologic screens (CV: < 5%) can be less than

those obtained for within-assay reproducibilities of many ana-

lytes. Falsely elevated MCHCs (> 360 g/L) occasionally arise

in our diagnostic CBC screens when the turbidity of hyperli-

pemic plasma causes falsely elevated hemoglobin concentra-tions (27) or when hematocrit is calculated incorrectly because

of the presence of cold agglutinins (28) and needs adjustment.Moderately altered MCHCs, which may be seen in iron defi-

ciency (Table 2), may result in small distortions in the inter-conversion of blood folate data, but this effect is difficult toquantitate. Studies of MCHC in disease are hampered by thetechnical difficulties associated with hematocrit measurement,which have been reported extensively (29-35), and by the

absence of an international standard for hematocrit (30). Nosuch technical shortcomings have been associated with the

estimation of hemoglobin, which is a simple colorimetric assay

whether measured manually or by automated methods. Inter-national reference preparations exist for hemoglobin assay

calibration (1 1), and blood folate data adjusted by using thesample hemoglobin will have less interlaboratory variation.

The minimum objective of these studies was to develop amethod for evaluating screening of compliance with folatesupplementation. A substantial lack of agreement exists cur-rently between the results of readily available conventional

serum and red cell folate assays (36) and is noted here in a

comparison of results between the radioassay and microbiolog-

ical assay of red cell folate concentrations (Figure 2). Because

the results of these analyses are method specific (36), we

considered that any method that was practical and optimized

for folate stability in the field would have potential for devel-

opment if it gave reproducible results in the laboratory. A

nonvolumetric folate screening assay with DBS has such po-tential; studies of folate stability with use of DBS are a pre-

requisite to further development and show some promise (Ta-

ble 3). A report on folate analysis in DBS is forthcoming.Simple finger-stick blood sampling emerged as being suit-

able even for conventional volumetric analysis of blood folate

concentrations (Figure 4A), and is useful in any setting. Accu-rate blood folate concentrations can also be obtained when the

blood volume or dilution factor is unknown (Figure 1), thus

allowing folate analysis of small finger-stick samples or those

collected directly into ascorbic acid solution to form hemoly-sates (Figure 4B); hematocrit measurements and dilution fac-

tors can now be ignored. This flexibility should be particularlyadvantageous under field conditions, where accurate dilution ofblood can be difficult. Red cell folate and hemoglobin folate

data become interconvertible later when applying either spe-

cific or population mean MCHCs. In certain studies, ideally, a

plasma or serum folate concentration is needed to calculate the

red cell folate concentration (37); however, this is not possiblewith very small or lysed finger-stick samples; these small

specimens may be suitable, however, for evaluating compli-ance in folate-intervention trials.

In field surveys, standard techniques often have to be mod-ified to suit local conditions and to meet financial resources. It

is important to identify the variables associated with these

changes and to compensate for them. For blood folate screens,

the suitability and economy of finger-stick peripheral bloodsampling coupled with the flexibility and stability of correction

by using sample hemoglobin should prove useful. However, an

awareness of the instability of blood folate concentrationsremains crucial in the design of field-study protocols. U

We thank Emer Lawlor and Joan 0’ Riordan of the Blood Transfusion

Service Board and the staff of the Centers for Disease Control and Pre-

vention (Atlanta) and St James’s Hospital (Dublin) for their help in

providing blood samples.

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