Eur J Clin Chem Clin Biochem 1997; 35(10):781 -785 © 1997 by Walter de Gruyter · Berlin · New York

Whole Blood Folate Concentrations: Comparison between Stratus Folate(DADE) and Radioassay (DPC) Methods

Fabrizia Bamonti-Catena1, Antonella Porcella1, Mauro Pomati1, Carlo Franzini2, Mariele Rosina5,Viviana Cavalca4 and Anna Teresa Maiolo1

1 Istituto di Scienze Mediche, Universita di Milano, Ospedale Maggiore-IRCCS, Milano, Italy2 Istituto Scienze Biomediche, Universita di Milano, Ospedale L. Sacco, Milano, Italy3 Istituto di Chirurgia Generale ed Oncologia Chirurgica, Universita di Milano, Ospedale Maggiore-IRCCS,

Milano, Italy4 Istituto di Cardiologia, Universita di Milano, Milano, Italy

Summary: The analytical performance of the Stratus Folate assay for intra-erythrocyte folate determination innormal subjects and in patients affected by folate-related diseases was compared with that of the radioassay (DPC)routinely employed by us. Folate concentrations were measured in freshly obtained EDTA whole blood from 100subjects. Haemolysis was performed with the appropriate lysis reagent. In addition, to compare two differenthaemolysis procedures folate determination was carried out in 51 samples haemolysed according to the two pro-cedures in parallel. Data were analyzed using Wilcoxon's test and standardized principal component analysis. StratusFolate assay and radioassay performances were comparable in terms of analytical characteristics as well as inindividual intraerythrocyte folate values across the range of whole blood concentrations examined in the survey.Significant differences were detected between the two different haemolysis procedures only for the radioassay. Inconclusion, we observed no significant differences between the two folate determination methods despite theirdifferent analytical principles, which indicates the suitability of routine use of the automated non-isotopic StratusFolate assay for clinical purposes. Moreover, with the latter assay the laboratory staff could choose the moreconvenient haemolysis procedure.

Introduction

Folic acid is a water-soluble vitamin whose derivativesplay an essential role in cell proliferation and differenti-ation (1, 2). As folate deficiency induces severe alter-ations especially in proliferating cells, an adequate andconstant dietary supply of folates is required. Supple-mentation of this nutrient during pregnancy can reducethe incidence of neural tube defects such as spina bifidaand anencephaly in developing fetuses, and in adults lifemay be beneficial in preventing development and pro-gression to cardiovascular disease (4—7). Evaluation offolate body stores is therefore useful in many clinicalsituations (6, 7).

Several studies have identified normal pathways of fo-late supply and utilization, and have clarified the mecha-nisms that maintain folate homeostasis (3). Red bloodcell folate concentrations have been shown to be the bestindicator of folate stores, not immediately influenced bydiet, alcohol or pharmacological treatments.

Although folate determination is becoming more andmore important, owing to its increasingly recognizedmedical and epidemiological significance (4—7), thereis still no validated reference method to perform this

measurement (8). For some time radioassay has beenused in our laboratory to measure both serum and redblood cell folate concentrations for clinical purposes.Subsequently, for practical reasons, we became inter-ested in employing a non-radioactive automated method,and we investigated the performance of a commerciallyavailable, automated immunoenzymatic assay (Stratussystem) in comparison with our routine radioassay inthe measurement of serum and whole blood folate (9).

In this study we compared these two methods in wholeblood sample determination, and in addition, we com-pared two different haemolysis procedures for bothmethods.

Materials and MethodsApparatusA Stratus II radial partition fluorometric immunoassay analyser(DADE International, Inc., Deerfield IL, USA) and γ-counterKontron MDA 312 (Kontron Instruments S. A., Montigny Le Bret-onneux, France) were used.

Kits and reagents

The diagnostic kits used for radioassay and Stratus determinationswere respectively Solid Phase No Boil Dual Count DPC (Diagnos-

782 Bamonti-Catena et al.: Red blood cell folate levels: comparison of methods

tic Product Corporation, Los Angeles, CA, USA) for the simulta-neous measurement of serum vitamin B12 and folic acid concentra-tions and red blood cell folic acid concentrations, and Stratus FolateFluorometric Enzyme-Linked Assay (DADE International, Inc.,Deerfield IL, USA) for the measurement of serum and red bloodcell folate concentrations.

The haemolysis reagents were ascorbic acid solutions as suggestedby the manufacturers (10, 11). For the Stratus Folate assay, a buf-fered ready-to-use solution (containing folate free bovine serumalbumin, 2% detergent and 0.1% sodium azide) was used for fur-ther dilution according to the manufacturer's instructions (12).

Principles of assays

The radial partition immunoassay for folate was performed auto-matically by the Stratus analyzer using pteroylglutamic acid, a sta-ble form of folate, as calibrator and as enzyme-linked conjugate(11). For folate radioassay determination the same form of the folicacid was used for standardization and its 125I-labelled analogue astracer (10). In both kits, endogenous proteins underwent an alkalinedenaturation to avoid the boiling step. The following steps were:sequential binding to a folate binding indicator protein immobi-lized on a square piece of glass fiber paper retained in plastic carri-ers (tabs), and radial elution of unbound enzyme and applicationof substrate wash solution for Stratus and for radioassay, competi-tion for purified binder a pH 9.3, binder immobilization on micro-crystalline cellulose particles and solid phase separation (10, 11).

Patients and procedures

Blood samples were obtained from 100 fasting subjects who hadall signed informed consent forms, 80 with haematological, gastro-intestinal or neurological disease and 20 healthy individuals. Forthis study, differences in sex and age were not considered.

Two blood specimens from each subject were collected into pre-evacuated and light-protected tubes, either plain for serum deter-minations or containing EDTA as anticoagulant for haemolysatepreparations. Serum and haemolysate aliquots were frozen immedi-ately and stored at —20 °C until analysis (from a few days to 12months later). Alternative aliquots of EDTA-whole blood from 51subjects were frozen and stored at —20 °C; lysis reagent was addedimmediately before assay.

Haemolysis was performed by diluting whole blood samples 1/21with a freshly prepared ascorbic acid solution (10 g/1) for radioas-say. For Stratus assay whole blood samples were diluted 1/11 withan ascorbic acid solution (4 g/1), left for 90 ± 5 min, and thenfurther diluted 1/2 (final dilution 1/22) with the system's specificdiluent.

Each sample was assayed in duplicate for radioassay and singlyfor Stratus determinations.

Both methods were calibrated using pteroylglutamic acid as cali-brator. The concentrations of the calibrators ranged from 0 to20 μg/l (corresponding to 0-45.3 nmol/1 of folate) for the Stratusassay and from 0 to 24 μ^ (corresponding to 0—54.4 nmol/1 offolate) for radioassay. Analytical results with both methods wereexpressed as nanomoles of folate per litre of whole blood, takinginto account the respective dilution factor and the relative molecu-lar mass (Mr = 441.42) of folate.

Red blood cell folate concentrations are usually calculated accord-ing to the following equation:

folate = (Z X R - (S Χ (100 - H)/100)) Χ 100/ΗwhereΖ = dilution factor (21 for radioassay and 22 for Stratus),R = whole blood folate concentration,S = serum folate concentration, andH = haematocrit.

In this study we did not apply such a calculation, but simply reportwhole blood folate concentration values multiplied by the dilu-tion factor.

Samples with concentrations exceeding the value of the higher cali-brator were remeasured after proper dilution, but were not consid-ered in the method comparison in order to exclude any additionalsource of error.

Each characteristic was first tested in serum samples and then, ifpossible, in whole blood haemolysate samples.

Performance characteristics

ImprecisionIntra- and inter-assay imprecisions were determined as coefficientsof variation (CV) from 12 replicate measurements in a single run(three haemolysates at different concentrations) and from repeatedmeasurements in five different runs (three haemolysates at differentconcentrations).

RecoveryRecovery was determined in 10 haemolysate samples containingdifferent folate levels, spiked with a whole blood haemolysate withknown folate concentration and calculated as the percentage of theobserved/expected concentration.

Detection limitThe zero standard was measured ten times along with the set ofstandards and controls and the lowest detectable serum or haemoly-sate folate quantity was defined as the concentration obtained attwo standard deviations from the mean signal (counts for radioas-say and mV/min for Stratus) by assaying 12 replicates in threeseparate runs.

LinearityAssay linearity was determined with eight dilutions obtained bymixing aliquots of whole blood haemolysate pools with high andlow folate concentrations (27.6 and 5.7 nmol/1).

Statistical analysis

Data were analysed using Wilcoxon 's test and standardized princi-pal component analysis (13). The measuring agreement betweenthe two methods was performed according to Bland and Altman 'smethod (14).

Results

Performance characteristics

Imprecision

Table 1 shows intra- and inter-assay imprecision of thetwo methods.

Tab. 1 Radioassay (DPC) and Stratus Folate assay (DADE) intra-and inter-assay imprecision measured in haemolysates at low, me-dium and high concentrations of folate, respectively. Means arewhole blood folate concentration values.

Imprecision Radioassay Stratus

Intra-assayIntra-assayIntra-assayInter-assayInter-assayInter-assay

Mean(nmol/1)

5.515.233.1

8.119.927.9

CV(%)

5.24.62.46.86.45.8

Mean(nmol/1)

5.915.133.97.5

15.327.4

CV(%)

7.34.93.88.26.93.5

Bamonti-Catena et al.: Red blood cell folate levels: comparison of methods 783

Recovery

The range of recoveries was 97.2% to 105% for radioas-say and 96.4% to 104.9% for Stratus, with a mean re-covery of 103% and 102%, respectively.

Analytical sensitivity

The detection limits of whole blood folate were0.57 nmol/1 in radioassay and 1.81 nmol/1 in Stratus as-say.

Linearity

Red blood cell folate linearity was good for both theradioassay and Stratus methods (data not shown).

Comparison of methods

Median values of whole blood folate levels of 100 sub-jects determined in parallel with radioassay and Stratusmethods were 329.5 and 331.5 nmol/1, respectively(p = 0.064, Wilcoxon's test). Biometrie analysis showeda linear correlation between the two assays (fig. la; y= 26.2 + 0.96x, Syx = 76.8 nmol/1, r = 0.889). Themeasuring agreement between the two methods is repre-sented in figure Ib.

Haemolysis procedures

Median values of red blood cell folate levels obtainedby Stratus assay using two different haemolysis pro-cedures on 51 blood samples in parallel were304.1 nmol/1 for frozen whole blood samples and324 nmol/1 for frozen haemolysates (p = 0.300, Wilcox-on 's test). Regression analysis showed a linear correla-tion between the two procedures (fig. 2a; y = —2.8 +1.03x, Syx = 55.4 nmol/1, r = 0.943); the measuringagreement between the procedures is shown in figure2b. On the other hand, the same haemolysis procedurestested on the same samples by radioassay yielded dif-ferent folate values (305.5 nmol/1 in frozen whole bloodsamples vs 333.1 nmol/1 in frozen haemolysates;p = 0.018) whereas regression analysis showed a linearcorrelation between the two procedures (y = 0.67 +1.18x, Syx = 96.8 nmol/1, r = 0.904).

Discussion

The importance of correct folate measurements isincreasingly evident as the role of this nutrient becomesclearer. No "gold standard" method or material is yetavailable, and we therefore compared radioassay (DPC)

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784 Bamonti-Catena et al.: Red blood cell folate levels: comparison of methods

with the Stratus Folate assay (DADE) in determiningwhole blood folate concentrations, which reflects intra-erythrocyte folate content, the latter being a better indi-cator of long-term folate status in tissues than serumfolate measurements.

Some time ago we chose the Dual Count No Boil (DPC)for routine determinations of serum and red blood cellfolate concentrations on the basis of a previous compari-son with a radioassay boil method (Quantaphase II B12-Folate Radioassay, Bio-Rad Diagnostic Group, Hercules,CA, USA ), which revealed a good correlation of serumlevels between the two methods (data not shown).

We found the analytical performances of the Stratus Fo-late assay and radioassay DPC satisfactory in serumsamples, as in our previous study (9), and in wholeblood samples. Adequate preparation of the haemolysatesamples and the first step of both methods, alkaline de-naturation, should ensure the release of the folate form(pteroyl glutamic acid) that is detected by the two meth-ods. It should also avoid some drawbacks of existingassays for red blood cell folate determination as for ex-ample binding to folate-binding proteins. The perfor-mance characteristics of the two methods were compar-able, except the detection limit, which proved lower forthe radioassay. Our data, previously presented in abstractform (15), are in agreement with another study (16)which compared the new immunofluoroenzymatic Stra-tus assay for determination of red blood cell folate levelswith another radioassay method (Magic No Boil CibaCorning).

Although complete agreement between different meth-ods is most unlikely, the two methods compared by usyielded very similar mean folate values. We also foundthat high and low red blood cell folate values agreed inboth assays despite the differences in analytical pro-cedures. As the aim of our study was to evaluate thepossibility of replacing the comparison method by a newmeasurement technique, we considered a population af-fected by the most important folate-related diseases. Sta-tistical analysis, which revealed good linear correlationand measuring agreement, demonstrated the inter-changeability of the two methods for clinical purposes.In the absence of a reference method for folate measure-ment, our findings seem interesting even though in ap-parent contrast with the recent international interlabora-

tory comparison study performed by Gunter et al. (8) toassess differences in folate concentrations among somemethods now available; however none of the 20 partici-pating laboratories used the Stratus Folate assay(DADE).

In our study sample stability (—20 °C) was good for10-12 months with both radioassay and Stratus Folateassay, provided that specimen processing was accurate.As inadequate sample preparation is a source of error indetermining red blood cell folate concentration (17), wecompared two different haemolysis techniques in sam-ples tested by the two methods. The Stratus Folate assayhad an advantage over radioassay in that the two hae-molysis procedures were interchangable, allowing thetechnician to choose the more convenient one as occa-sion required.

The cost of a sample analysis is similar with both meth-ods as radioassay determines samples in duplicate andneeds a calibration curve for each assay kit used,whereas the Stratus Folate assay tests single samples andthe analyser automatically produces an internal calibra-tion curve, performed every 3 to 4 weeks in accordancewith the laboratory protocol and validated only by twolevels of controls in each run.

In conclusion, our findings show that the automatedStratus Folate assay is suitable for routine application,thus avoiding the problems related to the use of radioac-tive materials and saving laboratory staff time. All theanalytical performance characteristics studied by us in-dicate that the new Stratus Folate assay could replaceradioassay in clinical laboratories, even though in thisstudy we considered neither our own reference intervalsnor clinical aspects. Despite our satisfactory results,there remains an urgent need for developing and validat-ing a reference method and material for serum and redblood cell folate determinations.

AcknowledgementsThis study was in part supported by MPI 60% no.12.01.025.00001 grant.We thank Mr. Oronzo Morabito for his technical assistance in pre-paring and storing samples, Dr. Alessandro Ortisi and his staff(DADE Division, Milano) for consultation, Ms. Alix Green for lin-guistic consultation, and Dr. Concetto Panioto and Miss ConcettoToscano for secretarial assistance.

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Received April 23/ July 25, 1997Corresponding author: Dott. Fabrizia Bamonti-Catena, IstitutoScienze Mediche, Pad. Granelli, Ospedale Maggiore-IRCCS, viaF. Sforza 35,1-20122 Milano, ItalyFax: +39255012111,e-mail: Filobus @IMIUCCA. CSI. UNIMI. IT