The transferrin/log(ferritin) ratio: a new tool for the diagnosis of iron deficiency anemia.

BACKGROUND: Serum ferritin is the best single laboratory test to diagnose iron deficiency anemia (IDA). Ferritin levels <20 µg/L are highly specific for IDA, and ferritin levels >100 µg/L usually exclude IDA. However, ferritin concentrations between 20 and 100 µg/L are often inconclusive. The objective of this study was to improve the diagnosis of IDA when ferritin levels are inconclusive. METHODS: We evaluated the predictive performance of classic (ferritin, mean corpuscular volume, transferrin and serum iron) and modern [reticulocyte hemoglobin content, serum transferrin receptor and soluble transferrin receptor (sTfR)/log(ferr)] iron status parameters to diagnose IDA in 2084 anemic, non-hospitalized patients. The results were validated in an independent cohort of 274 anemic patients. RESULTS: In our study population, 29% (595 patients) of the patients had a ferritin level between 20 and 100 µg/L, hampering diagnosis of IDA. None of the classic or modern parameters was capable of completely separating the IDA population from the non-IDA population. However, using a new parameter, the transferrin/log(ferritin) ratio, the IDA and non-IDA populations can be completely separated. At a cut-off value of 1.70, the transferrin/log(ferritin) ratio indicates IDA in 29% of the patients with inconclusive ferritin levels. CONCLUSIONS: The transferrin/log(ferritin) ratio is a practical new tool that improves diagnosis of iron deficiency when ferritin levels are inconclusive.

Screening for gastrointestinal malignancy in patients with iron deficiency anemia by general practitioners: an observational study.

BACKGROUND: The prevalence of iron deficiency anemia (IDA) is 2-5% in men and postmenopausal women in the developed world. IDA is commonly caused by chronic gastrointestinal blood loss, and a thorough examination of the gastrointestinal tract must be standard practice. OBJECTIVE: To retrospectively study endoscopic evaluations of patients from general practitioners diagnosed with IDA in a peripheral hospital laboratory in order to determine the cause of IDA and the number of gastrointestinal malignancies. MATERIAL AND METHODS: We retrospectively evaluated all patients with IDA diagnosed in a peripheral hospital laboratory by the general practitioner in the region of our hospital from 1 January 2004 until 31 December 2005. We included women older than 50 and men 18 years and older without a history of IDA in the previous 2 years. RESULTS: In 2 years, 287 patients were newly diagnosed with IDA in our hospital laboratory. Only 90 (31%) patients were endoscopically evaluated within 4 months. Gastrointestinal endoscopy revealed at least one lesion potentially responsible for blood loss in 41 of 90 (46%) patients. The most common lesions identified by gastroduodenal endoscopy were erosive esophagitis, gastritis and duodenitis (14%). Cancer was the most commonly detected lesion in the colon, accounting for 17 of 21 colonic lesions explaining IDA. In total, gastrointestinal malignancy was diagnosed in 2% of screened patients. Factors determining the decision for endoscopic screening were lower hemoglobin level, lower ferritin level and male gender. CONCLUSION: In our retrospective study of patients with IDA, only 31% received any form of endoscopic evaluation. In general practice, IDA is investigated suboptimally, and interventions other than the issuing of guidelines are needed to change practice.

Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma.

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDpsi mothers and that RHDpsi fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made. STUDY DESIGN AND METHODS: Real-time quantitative (RQ)-polymerase chain reaction (PCR) assays were developed for prenatal diagnostic use with cell-free fetal DNA from maternal plasma. An RQ-PCR assay on RHD exon 5 (amplicon 361 bp), negative on RHDpsi, was developed with genomic DNA and evaluated with cell-free fetal DNA. A previously published RHD exon 5 RQ-PCR (amplicon 82 bp) was duplexed with an in-house developed RHD exon 7 RQ-PCR and evaluated with cell-free fetal DNA from pregnant D-RHDpsi+ women. RESULTS: The RHD exon 5 361 bp assay showed on cell-free plasma DNA from D- women carrying a D+ fetus, low amplification levels, resulting in high Ct values and false-negative results. Owing to fragmentation of cell-free plasma DNA, too few DNA stretches of sufficient length (> 360 bp) are present. The RHD exon 5 82 bp and exon 7 RQ-PCR duplex was evaluated with RHDpsi+ cell-free plasma DNA and showed complete specificity and maximal sensitivity. CONCLUSION: Assays designed for prenatal genotyping should be developed and evaluated on cell-free plasma DNA. Prenatal RHD typing is accurate with the RHD exon 5 82 bp and exon 7 duplex strategy.

RHD(T201R, F223V) cluster analysis in five different ethnic groups and serologic characterization of a new Ethiopian variant DARE, the DIII type 6, and the RHD(F223V).

BACKGROUND: The RHD phylogeny in humans shows four main clusters of which three are predominantly observed in (African) black persons. Each of the African clusters is characterized by specific amino acid substitutions relative to the Eurasian RHD allele. RH phylogeny defines the framework for identification of clinically relevant aberrant alleles. This study focuses on the weak D type 4 cluster (characterized by RHD(T201R, F223V) (602C>G 667T>G)) in five ethnic groups. STUDY DESIGN AND METHODS: A total of 1702 samples were screened for the presence of 602C>G and 667T>G by sequence-specific polymerase chain reaction (PCR-SSP). Eighty samples were assigned to the weak D type 4 cluster and were molecularly characterized by PCR-SSP and RHD sequencing. Antigens of aberrant alleles were characterized with monoclonal anti-D according to the 37-epitope model when possible. RESULTS: Five new aberrant alleles, DIII type 6, DIII type 7, DARE, RHD(T201R, F223V) (without 819G>A), and RHD(F223V), were identified and DIII type 6, DARE, and RHD(F223V) were serologically characterized with monoclonal anti-D. Both the DARE and RHD(F223V) showed epitope loss. It is postulated that the 1136C>T nucleotide substitution (characteristic for the DAU allele cluster) is present on the DVa(KOU) allele. CONCLUSION: Identification of the new variant alleles refines the phylogeny of RHD in humans. The proposed DVa(KOU) allele with 1136C>T (DVa(KOU)T379M) is probably caused by conversion of the DAU0 allele and the DVa(KOU) allele, forming a phylogenetic link between the DV allele and the DAU cluster. By describing the RHD(F223V) (602C>G) and RHD(T201R, F223V) (602C>G and 667T>G) alleles formal proof is given for the origin of the non-Eurasian cluster.

The highly variable RH locus in nonwhite persons hampers RHD zygosity determination but yields more insight into RH-related evolutionary events.

BACKGROUND: Knowledge about paternal RHD hemi- or homozygosity is of clinical interest in alloimmunized pregnant women. D negativity in white persons is usually caused by deletion of the RHD gene. Recently, the physical structure of the RH locus and the mechanism causing the deletion of the RHD gene have been explored, enabling RHD zygosity determination in white persons by specific detection of a hybrid Rhesus box characteristic for the RHD- locus. STUDY DESIGN AND METHODS: RHD zygosity was determined in 402 samples from five different ethnic groups by polymerase chain reaction (PCR)-restriction fragment length polymorphism and by a newly developed real-time quantitative PCR. The Rhesus boxes of samples showing discrepancies between both tests were cycle sequenced. RESULTS: In nonwhite persons, several mutated Rhesus boxes exist that hamper zygosity determination by detection of the RHD- locus. Such mutated Rhesus boxes in D+RHD homozygous black persons have a frequency of 0.22. In white persons, no mutated Rhesus boxes were encountered so far. CONCLUSIONS: Owing to the high frequency of the mutated Rhesus boxes, zygosity determination by detection of the RHD- locus is not feasible in nonwhite persons. The cosegregation of variant RHD genes (RHDpsi and (C)cdes) with specific mutated Rhesus boxes yields more insight into the evolutionary events concerning variant RHD genes and mutated Rhesus boxes.

RHC and RHc genotyping in different ethnic groups.

BACKGROUND: RH genotyping assays are mainly based on research in whites. These assays may not be reliable in a multiracial society because of the genetic variation in RH among ethnic groups. STUDY DESIGN AND METHODS: Five groups from different ethnic backgrounds were serologically typed for C and c and were genotyped on nucleotide C48 and intron 2 for RHC and RHc on nucleotides C178 and C307. RESULTS: RHc genotyping with both methods proved to be reliable. RHC genotyping on C48 is not reliable because of a 48G>C mutation in the RHce allele (false-positive prediction of C). This mutation was found in every ethnic group and does not affect c or e expression. RHC genotyping on intron 2 is unreliable because of r's (Cdes) alleles (a false-negative prediction of C). This allele was found in whites and blacks from Curaçao and South Africa. Reactions of r's cells with anti-C are weaker, but no negative reactions with various MoAbs were found. A new method (RHC/c/hex3-intron 4/exon 7 multiplex PCRs) was developed based on intron 2 and r's hybrid exon 3 characteristics (RHC) and C307 (RHc). CONCLUSIONS: Reliable RHC and RHc genotyping is possible in different ethnic groups with the RHC/c/hex3-intron 4/exon 7 multiplex PCR approach.