Maternal folate status and neural tube defects in Ireland: the need for a national food fortification program.

To determine the present risk of a Neural Tube Defect [NTD] pregnancy in the caucasian primigravid population in Dublin by comparing the serum folate and red cell folate status of primigravid patients attending the first prenatal booking clinic with data from the late 80's. This Cross-sectional population study looking at blood folate status of over 400 sequential primigravid caucasian women with a singleton pregnancy, booking at less than or equal to 20 weeks gestation. All patients were attending a prenatal booking clinic at the Rotunda Hospital in Dublin during 2003-2004. Comparing serum and red cell folate values in 454 primigravid patients in 2003-4 to values to in a large case-control study based on over 56,000 women attending maternity hospitals in Dublin from 1986 to 1990. Just 13.9% of our patients took periconceptual folate, 33.5% of patients took folate in the first 20 weeks of pregnancy and 58.8% of mothers were taking no folate supplement. Overall, 30% of mothers had RCF levels below 400 ug/L--a level recommended as the minimum value required for protection. NTD risk occurred most frequently amongst patients with RCF levels between 300 and 400 ng/mL.

Evaluation of serum transferrin receptor assay in a centralized iron screening service.

This study assesses the impact of permitting unrestricted access to requests for soluble transferrin receptor (sTfR) analysis in screening for iron deficiency (ID). Biochemical data including sTfR, serum ferritin (sFn), transferrin saturation, zinc protoporphyrins (ZPP) and also erythrocyte indices are used to highlight the differences between hospital (H) and general practitioner (GP) patient groups. A significantly higher number of abnormal sFn values (40%) over abnormal sTfR values (25%) occurred in GP patients. This trend was reversed in the H patient group where high sTfR values predominated. Consequently, screening with sFn, exclusively, missed ID (sTfR > 28.1 nmol/l) in 5% of GP patients and in 20% of H patients. Some 40% of H patients had elevated CRP values (CRP > 10 mg/l) indicating inflammatory disease, however, ZPP was more efficient than CRP at screening the validity of normal sFn values in the group. Unrestricted access to sTfR, sFn and ZPP analyses should expedite diagnosis in all patients, particularly H patients, but may be costly. The high specificity (>90%) of the mean cell haemoglobin for ID may be under-utilized diagnostically.

High serum folates and the simplification of red cell folate analysis.

Recent substantial increases in clinical blood folate concentrations are noted. Since red cell folates (RCF) are calculated from whole blood folates (WBF) by subtraction of the endogenous serum folate (SF) component, the reporting of clinical RCF results may be delayed because an ever increasing proportion (15%) of diagnostic SF levels are high (> 20 ng/ml) and need a repeat analysis. We evaluated 'plasma replacement' as a simple preanalytical procedure in which endogenous blood plasma is removed from red cells by washing and substituted with 'low-folate' plasma (serum) as an alternative conjugase (gamma-glutamyl carboxypeptidase) source for folate polyglutamate hydrolysis. Washed and conventional RCF assays compared well after both manual (n = 115, r = 0.98, y = 1x + 1.26) and automated washing of red cells (n = 170, r = 0.96, y = 0.96x - 0.73 ng/ml) and were not significantly different. The interassay reproducibility of folate results from washed blood samples was good (CV = < 6%). This novel 'plasma replacement' step halves the cost of a valid RCF assay by eliminating the need for endogenous SF analysis, and it expedites the reporting of clinical results.

Optimization of routine plasma homocysteine monitoring.

An elevated plasma homocysteine (Hcy) level is now considered to be an important risk factor in arterial and venous thromboembolic events. As a result of this relatively recent finding, there has been a dramatic increase in the number of requests for Hcy measurement. In our laboratory, this demand has been met by employing an automated immunoassay and improving the pre-analytical handling of blood samples. An automated fluorescent polarization immunoassay (FPIA) gave similar results to a reference high-pressure chromatographic (HPLC) method (r2 = 0.98, enzyme immunoassay = 0.998 HPLC - 0.3) and excellent between-run reproducibility (coefficient of variation <3%). The new assay also required less specialized technical input, and improved the sample throughput two-fold. Pre-analytical stability of plasma Hcy concentrations in blood samples is crucial to the accuracy of Hcy monitoring. This stability was improved 10-fold by adopting the anticoagulant acidic citrate instead of ethylenediamine tetraacetic acid for Hcy screening by FPIA. Acidic citrate dramatically inhibits time-related plasma contamination by red-cell Hcy, resulting in improved accuracy and a reduced number of 'spoiled' specimen discards.

Screening of folate status with use of dried blood spots on filter paper.

BACKGROUND: Dried blood spots (DBS) on filter paper have been a successful and economical matrix for neonatal screening. OBJECTIVE: Our objective was to develop and evaluate an optimized method for DBS folate analysis and to assess DBS folate stability. DESIGN: DBS were eluted from paper by sonication in 5 g ascorbic acid/L containing 0.1% (by vol) Triton X-100 and hemoglobin folate values (HF; as pmol/g) were calculated from DBS eluate folate and hemoglobin concentrations. RESULTS: Over 95% of DBS folate was eluted during a standardized sonication cycle and DBS folate assay reproducibility was acceptable both within (CV: <8%) and between (CV: <9%) runs. HF means (+/-1 SD) from finger-stick DBS and conventional venous methods were 2513 +/- 1144 and 2607 +/- 1195 pmol/g, respectively, in blood samples taken concurrently from 80 donors, and they correlated well (r = 0.97, P < 0.001). HF values and erythrocyte folate measures may be interconverted by using the mean cell hemoglobin concentration. CONCLUSION: The DBS matrix has potential as an inexpensive and practical option for folate screening studies.

Use of folic acid-fortified milk in the elderly population.

Folic acid deficiency is common in the elderly population, resulting in anaemia, dementia, many neurological sequelae and an indirect role in atheromatous disease. An increase in natural food folate is relatively ineffective at increasing folate status and the use of folate fortification of foodstuffs is recommended. The aim of our study was to assess the benefits of folic acid-fortified milk to the folate status of an elderly institutionalised population. 49 subjects received fortified milk as part of their daily diet for at least 6 months (active group) and 40 subjects received unfortified milk (control group). Our results showed a mean serum folate level in the active group of 5.81 (1.1-17.6) microgram/l compared to the control group mean of 2.16 (0.5-9.4) microgram/l (p < 0.001; normal range for serum folate 2.7-20 microgram/l). Similarly the mean red cell folate level in the active group of 316.5 (130-905) microgram/l was significantly higher than the control group mean of 196.1 (95-490) microgram/l (p < 0.001; normal range for red cell folate 150-1,000 microgram/l). Our results suggest that folic acid-fortified milk is an efficacious and acceptable method of administration of folic acid in the elderly population and we recommend the use of folic acid-fortified milk in the regular daily diet of the elderly population.

Field-study screening of blood folate concentrations: specimen stability and finger-stick sampling.

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, nmol/g) as a novel and practical tool yielding accurate and precise results when blood volume or dilution is unknown. Red cell folate concentrations (nmol/L) of 11,887 Americans correlated well with hemoglobin-corrected whole-blood folate concentrations (r2 = 0.993; red cell folate = 0.347 x hemoglobin folate + 1 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-containing evacuated tubes) in field studies are best stored intact at 4 degrees C until they can be processed and frozen (-20 degrees C). Specific knowledge of blood folate stability is essential in planning and designing field studies.

Evaluation of factors influencing precision in the analysis of samples taken from blood spots on filter paper.

Dried blood spots (DBS) on filter paper have potential as a collection method in screening for haematinic deficiencies. Factors influencing the volumetric precision of uniform 'centre' punches (6.35 mm diameter) removed from dried blood spots have been evaluated. The volume of blood in each DBS punch (n = 234) was greatly influenced by both sample haematocrit (r = 0.63) and haemoglobin concentration (r = 0.63). The volume in identical punches (n = 57) also differed significantly when measured independently using either 125I human serum albumin or haemoglobin relative to the original blood sample. DBS punch volumes should be predetermined for individual batches of filter paper using the specific analyte of interest.

Microbiological assay on microtitre plates of folate in serum and red cells.

AIMS: To develop a simple microbiological assay for serum and red cell folates on 96-well microtitre plates, suitable for use in routine clinical diagnosis. METHODS: Use of a chloramphenicol resistant organism (NCIB 10463) saved time by avoiding aseptic precautions. Use of plate sealers facilitated mixing. Evaluation of assay performance included estimations of folate recovery, assay reproducibility, and response to reduced folate. Results obtained on sera (193) and red cell folates (150) were compared with those obtained using a traditional microbiological assay. RESULTS: Good recovery of folic acid added to serum and also good interassay and intra-assay precision were obtained with both serum (CV% of less than 5) and red cell folate pools (CV% of less than 5). Equimolar assay responses were obtained with folic acid, 5-formyltetrahydrofolate (L-form), and 5-methyltetrahydrofolate (L-form). The microassay correlated well with a traditional assay for estimation of folate in both serum (n = 193, r = 0.975) and red cells (n = 150, r = 0.96). CONCLUSIONS: This assay is more compact and less time consuming than the traditional assay. It is extremely economical and is easy to perform in a routine clinical laboratory.

The management of heart transplant recipients treated with cyclosporine in Ireland: monitoring of cyclosporine concentrations in blood.

Thirty seven cardiac transplants have taken place at the National Cardiac Centre in Ireland since 1985. Data is presented on three still-surviving male patients aged 19 to 42 who received cardiac transplants in 1985 and 1986. Circulating levels of blood cyclosporine were measured by high pressure liquid chromatography and radioimmunoassay; plasma creatinine and bilirubin were also measured. In one of these patients the distribution of cyclosporine in blood was measured by high pressure liquid chromatography in a long term study. For all three patients cyclosporine levels in blood were compared with the daily dose of cyclosporine and biochemical and histopathological parameters.

Cryo-preservation of Lactobacillus leichmannii for vitamin B12 microbiological assay.

An optimised method for preparing cryo-preserved cultures of Lactobacillus leichmannii for vitamin B12 microbiological assay is described. Cultures are mixed with an equal volume of 800 ml/l sterile glycerol before freezing. The percentage recovery of viable cells after thawing is highest when cultures are enclosed in polystyrene insulation and cooled to -70 degrees C. Cells cryo-preserved in the stationary phase of growth have a higher percentage recovery after thawing, but the same numbers of log phase cells give a larger growth response to cyanocobalamin under assay conditions. Cryo-preserved cultures are stable for many months, giving consistency of standard curve shape. Results obtained for control sera are also more consistent over time, with a range of CV values from 2.58% to 4.82%, as compared to a range of 4.63% to 6.11% for control sera in assays using conventionally prepared fresh cultures. The use of large inocula also allows for assay completion after overnight incubation.

Use of beta-lactamase to hydrolyse interfering antibiotics in vitamin B12 microbiological assay using Lactobacillus leichmannii.

The presence of certain antibiotics in patients' sera can invalidate microbiological assays for vitamin B12 using Lactobacillus leichmannii by inhibiting organism growth. Analysis of 3291 consecutive serum vitamin B12 assays in a large general hospital service showed inhibition of growth in 303 samples (9.2%). In nearly all cases the interfering substance was a beta-lactam antibiotic. The value of using a beta-lactamase preparation to eliminate antibiotic effects was studied using antibiotic-containing patient sera and also normal serum to which antibiotics were added. In-vitro additions of penicillins, cephalosporins, and other antibiotics to normal serum resulted in varying levels of organism inhibition, but excellent recovery of a control value after treatment with beta-lactamase (greater than 98%). Patient sera showing inhibition were reassayed in duplicate following treatment with beta-lactamase (n = 200), the overall effectiveness of the treatment being verified by the excellent recovery (greater than 98%) of a cyanocobalamin 'spike' added to a control. In only 0.2% of cases (6 of 3291 samples) was this treatment ineffective and it has thus proved an efficient method of eliminating the problem of antibiotic interference in the vitamin B12 microbiological assay.