The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on 'subvital' staining. The specialist 'subvital' stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsa's stain (commonly used microbiology and parasitology) in a 'subvital' manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.
Peripheral blood (PB) CD34+ cells enumeration is currently the most reliable method to guide the timing of stem cell harvest. However, its usage is restricted by being technically challenging, costly, and time-consuming. Immature reticulocyte fraction (IRF) determination, which is simpler and cheaper and has a faster turn-around time, has been proposed for a similar purpose. The purpose of this study is to evaluate the value of IRF in guiding stem cell harvest and examine the correlation between IRF and PB CD34+ cells count. Daily pre-harvest tests, i.e. PB CD34+ cells and IRF from 21 patients scheduled for autologous PBSC transplant were assessed. Stem cells harvests were commenced when the PB CD34+ cell count were more than 10 cell/ul. A total of 205 pre-harvest tests were analysed. Following stem cell mobilisations, both the IRF and PB CD 34+ cell counts rose with a variable pattern. In this study, we observed that the IRF peaks preceded the PB CD34+ count by 2 days. On the day of stem cell harvest, all the peak IRF values were >0.3. The PB CD34+ cell counts correlated with the harvested stem cell yield, whereby r2 = 0.77, p < 0.021. In autologous stem cell mobilisation, we believe that IRF is a useful screening tool to predict the rise of the PB CD34+ cell counts as it is a simple, fast and less costly. An IRF of > 0.3 may be used as a cut-off value for the initiation of PB CD34+ quantification prior to stem cell harvest.
Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152-625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157-560 aa.) and PvRBP1a-C (606-962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.
Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P vivax These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.
A 40-year-old Malay woman presented with increasing lethargy, palpitation and shortness of breath, 17 years after a mitral and aortic valve replacement. A Starr-Edwards prosthetic valve replaced the mitral valve, and a Bjork-Shiley prosthetic valve replaced the aortic valve. Biochemical parameters demonstrated intravascular haemolysis, as evidenced by haemoglobin 7.8 g/dL, reticulocyte count 8.4%, lactate dehydrogenase 2,057 IU/L and low haptoglobulin levels (less than 6 mg/dL). Transoesophageal echocardiography revealed a paravalvular leakage over the mitral valve. The haemoglobin levels remained persistently low despite frequent blood transfusions. She successfully underwent a second mitral valve replacement. Her anaemia resolved subsequently.
To establish the benefits of immature reticulocyte fraction (IRF) measurement using an automated hematology cells analyzer over absolute neutrophil count (ANC) in predicting bone marrow recovery post induction chemotherapy.
Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.
Iron deficiency anaemia (IDA) frequently occurs in haemodialysis
(HD) patients undergoing recombinant human erythropoietin (rHuEPO)
therapy and is commonly associated with rHuEPO hypo-responsiveness.
However, the conventional iron indices are inadequate to exhibit the status or
utilisation of iron during erythropoiesis. The aim of this study was to elucidate
the accuracy and usefulness of the reticulocyte haemoglobin (RET-He) test
for diagnosing IDA in HD patients undergoing rHuEPO therapy. Methods: In
this cross-sectional study, fifty-five blood samples of HD patients on rHuEPO
therapy were collected and analysed for haematological and biochemical
parameters. A receiver operating characteristics curve was also plotted for
sensitivity and specificity analysis. IDA detection rates by RET-He, soluble
transferrin receptor (sTfR) and serum ferritin were 63.64%, 3.64% and 0%,
respectively. RET-He level was significantly correlated with sTfR level, mean
cell volume, mean cell haemoglobin level and the transferrin receptor-ferritin
index. The sensitivity and specificity of RET-He in detecting IDA were 78.3%
and 92.0%, respectively, with an area under the curve of 0.864. IDA was more
frequently detected by RET-He than by ferritin or sTfR in HD patients
undergoing rHuEPO therapy. The RET-He level also showed higher sensitivity
and specificity for the iron status in these patients. Therefore, RET-He is a
useful biomarker for the detection of IDA in HD patients undergoing rHuEPO
therapy.
Haemoglobin E complicates 22.2°/o of pregnancy in Malaysian aborigines, the prevalence of variants associated with pregnancy being, 15.8% with Hb E trait abnormality, 3.9% with Hb E homozygous disease, and 2.5% with Hb E thalassaemia disease. Minor haematological abnormalities occur with the trait and homozygous conditions, though a more unfavourable response is expected with Hb E thalassaemia. Haemolysis is not a prominent feature and it is suggested that factors other than the haemoglobinopathic state
probably accounts for any unfavourable response in pregnancy.
Key Words: Haemoglobin E; Haemoglobinopathies; Haemolytic anaemias; Hb E thalassaemia; Malaysia; Pregnancy
Study site: Hospital Orang Asli, Gombak, Selangor, Malaysia
The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes.