Additional value of red blood cell parameters in predicting uncommon α-thalassemia; experience from 10 years of α-globin gene sequencing and copy number variation analysis.

INTRODUCTION: The diagnosis of rare forms of α-thalassemia requires laborious genetic analyses. Accurate sample selection for such evaluation is therefore essential. The main objectives of this study were to investigate the predictive power of red blood cell parameters to detect rare forms of α-thalassemia (substudy 1), and to explore the frequency of rare versus common forms of α-thalassemia in our sample population (substudy 2). METHODS: In substudy 1, we reviewed all blood samples selected for extended α-hemoglobinopathy evaluation at our laboratory during 2011-2020 (n = 1217), which included DNA sequencing and/or copy number variation analysis. We assessed α-thalassemia positive samples at different levels of mean corpuscular hemoglobin (MCH) alone and in combination with results for red blood cell count (RBC) or red cell distribution width (RDW). In substudy 2, we examined the distribution of α-thalassemia genotypes for all samples submitted to a first-tier hemoglobinopathy evaluation at our laboratory during 2014-2020 (n = 6495). RESULTS: In substudy 1, both RBC and RDW added predictive value in detecting rare forms of α-thalassemia in samples from adults and children. In adult samples with MCH ≤ 23 pg, the presence of erythrocytosis increased the detection rate from 27% to 74% as compared to non-erythrocytosis, while normal RDW increased the detection rate from 36% to 86% as compared to elevated RDW. In substudy 2, rare forms of α-thalassemia were detected in 12% of α-thalassemia positive samples. CONCLUSION: Initial assessment of MCH, RBC, and RDW provided valuable predictive information about the presence of rare forms of α-thalassemia during hemoglobinopathy evaluation.

Correction to: Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR.

[This corrects the article DOI: 10.1186/2052-1839-14-4.].

Hb Oslo [ß42(CD1)Phe→Ile; HBB: c.127T>A]: A Novel Unstable Hemoglobin Variant Found in a Norwegian Patient.

Unstable hemoglobin (Hb) variants are the result of sequence variants in the globin genes causing precipitation of Hb molecules in red blood cells (RBCs). Intracellular inclusions derived from the unstable Hb reduce the life-span of the red cells and may cause hemolytic anemia. Here we describe a patient with a history of hemolytic anemia and low oxygen saturation. She was found to be carrier of a novel unstable Hb variant, Hb Oslo [ß42(CD1)Phe→Ile (TTT>ATT), HBB: c.127T>A] located in the heme pocket of the ß-globin chain. Three-dimensional modeling suggested that isoleucine at position 42 creates weaker interactions with distal histidine and with the heme itself, which may lead to altered stability and decreased oxygen affinity. At steady state, the patient was in good clinical condition with a Hb concentration of 8.0-9.0 g/dL. During virus infections, the Hb concentration fell and on six occasions during 4 years, the patient needed a blood transfusion.

Aggregation of monocytes and platelets interferes in measurement of monocyte viability with phosphatidylserine expression but not with Mitochondrial membrane potential in whole blood.

BACKGROUND: Bacterial lipopolysaccharides (LPS) induce cell death and procoagulant tissue factor (TF) in purified cultured human monocytes. Phosphatidylserine (PS)-expression on the outer membrane of monocytes following cell death contributes to increased procoagulant activity. LPS also induce TF on monocytes in whole blood (WB), but it is unknown whether LPS induce cell death. Activated platelets that aggregate with monocytes in non-EDTA anticoagulated WB also express PS and may potentially interfere in viability measurement on monocytes in WB. METHODS: Viability of monocytes was estimated in purified monocytes and unlysed citrated WB incubated without and with LPS using two markers of early cell death; Lactadherin (PS-exposure) and Mitoprobe™ DilC (Mitochondrial Membrane Potential, MMP), and one late marker Sytox® Blue (membrane impermeant DNA stain). To study the interfering effect of monocyte-platelet aggregates (MPAs) on estimated monocyte viability, addition of EDTA or anti-CD62P was used to inhibit monocyte-platelet binding. RESULTS: In WB, the median percentage of viable monocytes without and with EDTA was 21% and 93% with PS-exposure and 89% and 99% with MMP, respectively. Addition of EDTA reduced the percentage of MPAs (81%-8.2%). Addition of anti-CD62P also reduced the percentage of MPAs (97-42%) and PS-expression on monocytes (MFI 28.9-3.9). LPS induced death of monocytes after 3 h in purified monocytes, but not in WB. CONCLUSIONS: MPAs interfered in monocyte viability measurement in citrated WB with PS-exposure, but not with MMP. LPS induced death of monocytes in purified culture, but not in WB. © 2015 International Clinical Cytometry Society.

Six-day stability of erythrocyte and reticulocyte parameters in-vitro: a comparison of blood samples from healthy, iron-deficient, and thalassemic individuals.

INTRODUCTION: Stability for up to 6 days' storage of erythrocyte and reticulocyte parameters in samples from iron-deficient and thalassemic individuals has not yet been reported. This lack of knowledge challenges evaluation of the full blood count in referral samples for hemoglobinopathy evaluation. We therefore hereby present such sample stability data. METHODS: We included fresh (less than 4 hours old) blood samples from eight healthy, eight iron-deficient, and 11 thalassemic individuals. A full blood count, including reticulocyte parameters, was performed on a Sysmex XE-2100 once daily during a 6-day storage period at room temperature. For healthy individuals, we also studied stability of refrigerated samples and investigated analytical and biological variation. RESULTS: Hemoglobin concentration, erythrocyte count, and mean corpuscular hemoglobin were stable for 6 days in all diagnostic groups. Mean corpuscular volume increased less in samples from iron-deficient individuals while the number of reticulocytes increased more in samples from thalassemic, as compared to healthy individuals. Ret-He stability depended on its baseline value. Within-person biological variation in samples from healthy individuals was low both for erythrocyte parameters and for reticulocyte hemoglobin, while higher for reticulocyte counts. CONCLUSION: Results for hemoglobin concentration, erythrocyte count, and mean corpuscular hemoglobin are reliable in hemoglobinopathy investigation of referred samples for up to 6 days. Storage time-dependent changes of other erythrocyte and reticulocyte parameters in blood samples from iron-deficient and thalassemic individuals differ from those of healthy individuals.

Aggregation of monocytes and platelets interferes in measurement of monocyte viability with phosphatidylserine expression but not with mitochondrial membrane potential in whole blood.

Background: Bacterial lipopolysaccharides (LPS) induce cell death and procoagulant tissue factor (TF) in purified cultured human monocytes. Phosphatidylserine (PS)-expression on the outer membrane of monocytes following cell death contributes to increased procoagulant activity. LPS also induce TF on monocytes in whole blood (WB), but it is unknown whether LPS induce cell death. Activated platelets that aggregate with monocytes in non-EDTA anticoagulated WB also express PS and may potentially interfere in viability measurement on monocytes in WB. Methods: Viability of monocytes was estimated in purified monocytes and unlysed citrated WB incubated without and with LPS using two markers of early cell death; Lactadherin (PS-exposure) and Mitoprobe™ DilC (Mitochondrial Membrane Potential, MMP), and one late marker; Sytox® Blue (membrane impermeant DNA stain). To study the interfering effect of monocyte-platelet aggregates (MPAs) on estimated monocyte viability, addition of EDTA or anti-CD62P was used to inhibit monocyte-platelet binding. Results: In WB, the median percentage of viable monocytes without and with EDTA was 21% and 93% with PS-exposure and 89% and 99% with MMP, respectively. Addition of EDTA reduced the percentage of MPAs (81% to 8.2%). Addition of anti-CD62P also reduced the percentage of MPAs (97% to 42%) and PS-expression on monocytes (MFI 28.9 to 3.9). LPS induced death of monocytes after 3 hours in purified monocytes, but not in WB. Conclusions: MPAs interfered in monocyte viability measurement in citrated WB with PS-exposure, but not with MMP. LPS induced death of monocytes in purified culture, but not in WB. © 2014 Clinical Cytometry Society.

Automated alarm to detect antigen excess in serum free immunoglobulin light chain kappa and lambda assays.

BACKGROUND: Antigen excess causing a falsely low concentration result may occur when measuring serum free immunoglobulin light chains (SFLC). Automated antigen excess detection methods are available only with some analyzers. We have now developed and verified such a method. METHODS: Residuals of sera with known SFLC-κ and -λ concentrations were analyzed using Binding Site reagents and methods adapted to the Roche Cobas® c.501 analyzer. RESULTS: We analyzed 117 sera for SFLC-κ and -λ and examined how the absorbance increased with time during the 7 minutes of reaction (absorbance reading points 12-70). From this an antigen excess alarm factor (ratio of absorbance increases between reading points 68-60 and 20-12, multiplied by 100) was defined. Upon our request, Roche added to our two SFLC assays a program which calculated this antigen excess alarm factor and triggered an alarm when the factor was below a defined value. We verified this antigen excess alarm function by analyzing serum from 325 persons of whom 143 were multiple myeloma patients. All samples with a known concentration above 30 mg/L triggered either an antigen excess alarm, an 'above test' alarm or both. Also, all samples above 200 mg/L (SFLC-λ) and 300 mg/L (SFLC-κ) triggered the antigen excess alarm and all but one triggered the above test alarm. CONCLUSIONS: The antigen excess alarm function presented here detected all known antigen excess samples at no increased time of analysis, a reduced workload and reduced reagent cost.

Serum bilirubin concentration in healthy adult North-Europeans is strictly controlled by the UGT1A1 TA-repeat variants.

The major enzyme responsible for the glucuronidation of bilirubin is the uridine 5'-diphosphoglucose glucuronosyltransferase A1 (UGT1A1) enzyme, and genetic variation in the UGT1A1 gene is reported to influence the bilirubin concentration in the blood. In this study, we have investigated which gene-/haplotype variants may be useful for genetic testing of Gilbert's syndrome. Two groups of samples based on serum bilirubin concentrations were obtained from the Nordic Reference Interval Project Bio-bank and Database (NOBIDA): the 150 individuals with the highest bilirubin (>17.5 µmol/L) and the 150 individuals with normal bilirubin concentrations (<17.5 µmol/L). The individuals were examined for the TA6>TA7 variant in the UGT1A1 promoter and 7 tag-SNPs in an extended promoter region of UGT1A1 (haplotype analysis) and in selected SNPs in candidate genes (SLCO1B3, ABCC2 and NUP153). We found significant odds ratios for high bilirubin level for all the selected UGT1A1 variants. However, in stepwise multivariate logistic regression analysis of all genetic variants together with age, sex, country of origin and fasting time, the repeat variants of UGT1A1 TA6>TA7 and SLCO1B3 rs2117032 T>C were the only variants significantly associated with higher bilirubin concentrations. Most individuals with high bilirubin levels were homozygous for the TA7-repeat (74%) while only 3% were homozygous for the TA7-repeat in individuals with normal bilirubin levels. Among individuals heterozygous for the TA7-repeat, a low frequent UGT1A1-diplotype harboring the rs7564935 G-variant was associated with higher bilirubin levels. In conclusion, our results demonstrate that in testing for Gilbert's syndrome, analyzing for the homozygous TA7/TA7-genotype would be appropriate.

Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR.

BACKGROUND: Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in ß-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). RESULTS: Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the -α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/µL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. CONCLUSIONS: HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results.

Hb Ullevaal [ß78(EF2)Leu→Val; HBB: c.235C>G], a new hemoglobin variant interfering with Hb A1c measurement using a cation exchange high performance liquid chromatography method.

A new hemoglobin (Hb) variant was detected during Hb A1c measurement. DNA sequencing showed heterozygosity for the single nucleotide substitution (C > G) on the ß-globin gene causing an amino acid change [ß78(EF2)Leu→Val; HBB: c.235C > G]. The new Hb variant was designated Hb Ullevaal after the hospital at which it was discovered. Heterozygosity for Hb Ullevaal appears to have no clinical significance. However, it interferes with Hb A1c measurement by cation exchange high performance liquid chromatography (HPLC), causing falsely low Hb A1c concentration when using the Tosoh G7 apparatus in variant mode.

Absolute neutrophil counts from automated hematology instruments are accurate and precise even at very low levels.

Using 106 samples from patients with an absolute neutrophil count (ANC) less than 2.0 × 10(9)/L, two 5-part differential hematology instruments (Sysmex XE-2100, Sysmex, Kobe, Japan, and Advia 2120i, Siemens Healthcare Diagnostics, Deerfield, IL), two 3-part differential hematology instruments (Sysmex K4500, Sysmex, and Advia 60, Siemens Healthcare Diagnostics), and an automated system for examination of microscopic slides (CellaVision DM96, CellaVision, Lund, Sweden) were compared with a flow cytometric (FCM) neutrophil count using monoclonal antibodies for cell classification. The precision and accuracy of the 5-part differential instrument ANC was very good at more than 0.1 × 10(9)/L, although a small systematic difference (10.3%) was found between the 2 instruments. The ANC of the 3-part differential instruments was less reliable, but the WBC count correlated very well with the WBC count from the 5-part differential instruments. Also, the neutrophil count from the CellaVision DM96 compared very well with FCM. When used in the correct laboratory setting, all of the evaluated instruments provide ANCs and WBCs with adequate accuracy and precision.

Is the blood basophil count sufficiently precise, accurate, and specific?: three automated hematology instruments and flow cytometry compared.

We compared the performance of the basophil count of 3 hematology instruments with a flow cytometric method (FCM) in which CD123 and CD193 were used as basophil markers. By analyzing 112 patient samples, we found the ADVIA 120 (Siemens Healthcare Diagnostics, Deerfield, IL) and CELL-DYN Sapphire (Abbott Diagnostics, Santa Clara, CA) to underestimate the number of basophils by approximately 50% and the Sysmex XE-2100 (Sysmex, Kobe, Japan) and ADVIA to overestimate the basophil count in some samples with pathologic leukocytes. All 3 instruments had large (25%-50%) analytic within-run coefficients of variation. Compared with the FCM, we found a relatively good correlation for the CELL-DYN basophil count (r = 0.81), an intermediate correlation for the Sysmex (r = 0.64), and a poor correlation for the ADVIA (r = 0.24). When excluding the 52 samples flagged for the presence of pathologic leukocytes, these correlations were found to be 0.84, 0.90, and 0.57, respectively. The basophil count of the 3 instruments is, at least presently, of unsatisfactory quality.

Quantitation of serum free light chains in combination with protein electrophoresis and clinical information for diagnosing multiple myeloma in a general hospital population.

BACKGROUND: Serum free light chain (SFLC) measurements have recently come into use as an aid for diagnosing monoclonal gammopathy. We evaluated SFLC measurements in combination with serum protein electrophoresis (SPE) and clinical information for diagnosing multiple myeloma (MM) in a hospital population. METHODS: We measured SFLCs in 3818 sera received for SPE over a 1-year period when patient symptoms or biochemical findings suggested myeloma-related tissue damage (n = 1067). We reviewed SPE and SFLC results from 489 patients together with their final diagnoses obtained from the hospital information technology department. RESULTS: SFLC measurement, combined with SPE and clinical information, allowed identification of 95% of patients (38 of 40) with previously undiagnosed MM, macroglobulinemia, or primary amyloidosis. Additionally, we identified 45 patients with monoclonal gammopathy of undetermined significance (MGUS) and 4 with plasmacytoma. Of patients followed at our hospital in whom SFLCs were not measured, only 1 patient was diagnosed with MM. This patient had anemia and was mistakenly not tested for SFLCs. An abnormal kappa/lambda ratio was found in 26 of 29 patients with MM but also in 36 of 203 patients with renal impairment, polyclonal immunoresponse, or other nonhematological diagnoses. None of the 203 patients with nonhematological disease had a kappa/lambda ratio <0.05 or >10. CONCLUSIONS: The combined use of SPE, SFLC measurements, and clinical criteria allows MM to be efficiently diagnosed or excluded based on serum measurements only.

Effects of long-term exercise and diet intervention on plasma adipokine concentrations.

BACKGROUND: In a randomized, controlled, 2 x 2 factorial trial on the effect of long-term changes in diet and exercise, a significant reduction in body weight and fat mass was observed. Alterations in leptin and plasminogen activator inhibitor-1 concentrations were previously reported from this study. OBJECTIVE: We examined the separate and combined effects of a 1-y exercise and diet intervention on several adipokines; adiponectin, interleukin-6 and -8, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, hepatocyte growth factor, nerve growth factor, C-reactive protein, and resistin. DESIGN: One hundred eighty-eight men with several risk factors for diabetes and cardiovascular disease were randomly allocated to 4 groups: diet, exercise, combined diet and exercise, and control. RESULTS: Plasma adiponectin concentrations remained unchanged, whereas body mass index and fat mass decreased after dietary changes and an increase in physical activity. In the control group, adiponectin concentrations were reduced. Analyzed according to the factorial design, only diet intervention had a significant (P = 0.03) positive effect on plasma adiponectin relative to control, and this effect was largely explained by changes in fat mass. After adjustment for change in percentage body fat, there were significant positive effects on tumor necrosis factor-alpha in all 3 intervention groups (P = 0.01 for the diet group, 0.03 for the exercise group, and 0.05 for the combined diet and exercise group). Minor changes were observed for the other adipokines. Neither baseline concentrations of nor changes in adiponectin and plasminogen activator inhibitor-1 were significantly correlated to the other adipokines, whereas concentrations of and changes in the other adipokines were significantly correlated. CONCLUSION: Diet intervention had a significant positive effect on adiponectin concentrations, which is largely explained by a reduction in fat mass.

Apolipoprotein E genotypes and their relation to lipid levels in a rural South African population.

AIMS: Genetic variation at the apolipoprotein E (apoE) locus is an important determinant of plasma lipids. The aim of the present study was to evaluate the association between apolipoprotein E genotype and plasma lipid levels among a rural black population in South Africa. METHODS: Lipid levels and apoE genotypes were studied in 505 volunteer subjects (363 women, 142 men) resident in the Dikgale demographic surveillance site. RESULTS: Allele frequencies were found to be 0.190 for epsilon2, 0.518 for epsilon3, and 0.293 for epsilon4, indicating a relatively low frequency of the epsilon3 allele and a high frequency of the epsilon4 allele. To determine the effect of apoE polymorphism on lipid levels three groups were formed: namely epsilon2-, epsilon3-, and epsilon4-expressing groups. A significant effect of the apoE genotype on total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C)/Total cholesterol (TC) ratio, and triglycerides was observed. LDL-C was significantly lower and the HDL-C/TC ratio was significantly higher in the epsilon2 group compared with the epsilon3 and epsilon4 groups. Triglyceride levels were significantly higher in the epsilon2 group than in the epsilon3 group. CONCLUSIONS: With the unfavourable apoE allele distribution, and the lifestyle changes taking place in rural South African populations, preventive strategies need to be developed to limit a potential epidemic of cardiovascular disease in the black population of South Africa.

Extremely slow formate elimination in severe methanol poisoning: a fatal case report.

UNLABELLED: Methanol poisoning is a potentially fatal medical emergency because of its metabolism to formic acid. The half-life of formate has been reported in the range of 2.5-12.5 hours, but the degree of inter-individual variation is not known. We studied methanol and formate kinetics in a case of late diagnosed methanol poisoning with persisting metabolic acidosis and circulatory failure. CASE REPORT: A 63-year-old man was referred to our hospital with a tentative diagnosis of stroke. He was awake on admission, but he soon deteriorated in the emergency department and a metabolic acidosis was revealed. Methanol poisoning was then suspected approximately five hours after admission but in spite of intensive treatment he died after six days. RESULTS: The S-methanol half-lives during treatment with fomepizole before and during hemodialysis were 49.5 and 4.1 hours, respectively, while the similar half-lives of S-formate were 77.0 and 2.9 hours. S-fomepizole was measured and found to be within the therapeutic range during treatment. DISCUSSION: The patient was treated with the established dosing regimen for fomepizole and the measured S-fomepizole levels throughout the treatment were adequate; the S-methanol elimination also suggests that methanol metabolism was blocked. Hence, other explanations for this exceptionally long formate half-life include slow formate metabolism, due to small hepatic folate stores or to genetic deficiencies in formate-metabolizing enzymes, or slow formate excretion, due to renal tubular acidosis, to a non-oliguric renal failure, or to genetic deficiencies in the renal formate transporters. CONCLUSION: This case report indicates that the half-life of S-formate may have greater inter-individual variation than earlier expected, being by far the longest half-life reported in the medical literature. These results support the use of hemodialysis in the treatment of such patients.

Early detection of breast cancer based on gene-expression patterns in peripheral blood cells.

INTRODUCTION: Existing methods to detect breast cancer in asymptomatic patients have limitations, and there is a need to develop more accurate and convenient methods. In this study, we investigated whether early detection of breast cancer is possible by analyzing gene-expression patterns in peripheral blood cells. METHODS: Using macroarrays and nearest-shrunken-centroid method, we analyzed the expression pattern of 1,368 genes in peripheral blood cells of 24 women with breast cancer and 32 women with no signs of this disease. The results were validated using a standard leave-one-out cross-validation approach. RESULTS: We identified a set of 37 genes that correctly predicted the diagnostic class in at least 82% of the samples. The majority of these genes had a decreased expression in samples from breast cancer patients, and predominantly encoded proteins implicated in ribosome production and translation control. In contrast, the expression of some defense-related genes was increased in samples from breast cancer patients. CONCLUSION: The results show that a blood-based gene-expression test can be developed to detect breast cancer early in asymptomatic patients. Additional studies with a large sample size, from women both with and without the disease, are warranted to confirm or refute this finding.

Increased serum formate in the diagnosis of methanol poisoning.

Early diagnosis is essential for successful treatment in methanol poisoning. Methanol detection by gas chromatography is not available in most hospitals. Methanol increases the osmolal gap in serum and its metabolite formate increases the anion gap. The sensitivity of these indirect diagnostic methods is not good at low concentrations of methanol or formate. We therefore studied the usefulness of formate measurement in diagnosing methanol poisoning. In 15 patients poisoned with methanol, serum formate was measured enzymatically on a Cobas Mira analyzer using formate dehydrogenase and nicotinamid adenine dinucleotid. Day-to-day coefficient of variation was 5%, and the upper reference limit was 2 mg/dL (0.4 mmol/L). Methanol was detected in all 15 patients of whom 14 had elevated serum formate concentrations. Anion gap was increased in 11 of 11, and osmolal gap in 11 patients of 15 examined. Metabolic acidosis was present in 12 of 15 patients, but pH was below 7.30 in only 9 of them. Four patients with no symptoms had formate concentrations in the range 2-38 mg/dL (0.5-8.3 mmol/L), indicating that increased serum formate was a sensitive indicator of methanol poisoning. Our results proved formate analyzes to be a simple, sensitive, and specific way of diagnosing methanol poisoning. Confounders are patients admitted early, or concomitant ethanol ingestion, and therefore no acidosis. This problem may, however, be omitted by repeated formate analysis in patients developing metabolic acidosis.