[Sickle cell disease ­ clinical manifestations and diagnostics]. / Sigdcellesykdom ­ kliniske manifestasjoner og diagnostikk.

Sickle cell disease is a group of diseases presenting with a set of characteristic acute and chronic manifestations. Sickle cell disease has traditionally been uncommon in the Northern European population; however, due to demographic changes, it is increasingly also something that Norwegian clinicians should be cognisant of. In this clinical review article we wish to present a brief introduction to sickle cell disease, with an emphasis on its aetiology, pathophysiology, clinical manifestation and how the diagnosis is established based on laboratory testing.

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.

Exploring cardiac effects after oxytocin 2.5 IU or carbetocin 100 µg: A randomised controlled trial in women undergoing planned caesarean delivery.

BACKGROUND: Oxytocin can stimulate release of myocardial biomarkers troponin I and T, prolong QTc and induce ST-depression. OBJECTIVE: To explore cardiac changes after either intravenous carbetocin or oxytocin. STUDY DESIGN: Exploratory phase 4 randomised controlled trial. SETTING: Obstetrics units of Oslo University Hospital, Norway between September 2015 and May 2018. PARTICIPANTS: Forty healthy, singleton pregnant women aged 18 to 50 years at gestational age at least 36 weeks with a planned caesarean delivery. INTERVENTIONS: Participants were randomised to receive either oxytocin 2.5 IU or carbetocin 100 µg immediately after delivery. MAIN OUTCOME MEASURES: The primary endpoint was the assessment of troponin I within 48 h of study drug administration. Troponin I and T, and creatine kinase myocardial band assessments were measured before spinal anaesthesia (baseline), and again at 4, 10 and 24 h after delivery. QTc, ST-depression and relative increase in heart rate were recorded from start of study drug administration to 10 min after delivery. All adverse events were monitored. RESULTS: Compared with the carbetocin group, higher troponin I levels were observed in the oxytocin group at 4 h and 10 h after delivery. For both treatment groups, an increase from baseline in troponin I and T was most pronounced at 10 h after delivery, and it had begun to decline by 24 h. QTc increased with time after administration of both study drugs, with a mean maximum increase of 10.4 ms observed at 9 min (P   <  0.001). No statistical differences were observed in QTc ( P  = 0.13) or ST-depression ( P  = 0.11) between the treatment groups. CONCLUSIONS: Oxytocin 2.5 IU and carbetocin 100 µg caused a similar increase in QTc. The trial was underpowered with regards to ST-depression and the release of myocardial biomarkers and these warrant further investigation. Data from this trial will inform a larger phase 4 trial to determine potential drug differences in troponin release. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02528136.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) and cystatin C are early biomarkers of acute kidney injury associated with cardiac surgery.

Acute kidney injury (AKI) is a serious complication in as much as half of the patients undergoing cardiac surgery, and early diagnosis and treatment are of the utmost importance. There is a need for robust biomarkers that can detect cardiac surgery-associated AKI (CSA-AKI) prior to rise in plasma creatinine, which typically occurs at least 48 h postoperatively. We compared pre- and 4, 12 and 48 h postoperative plasma (P) neutrophil gelatinase-associated lipocalin (NGAL), cystatin C, urea and creatinine, and urine (U) NGAL, as markers of AKI, in 49 patients (67% men, median age 65 years) scheduled for elective cardiac surgery (e.g. coronary artery bypass graft and/or valve replacement surgery) with the use of extracorporeal circulation. Patients with preoperative sepsis, renal replacement therapy, or estimated glomerular filtration rate <30 mL/min/1.73m2 were excluded. P- and U-NGAL were measured using the Roche Modular P (Roche Diagnostics®) NGAL immunoassay. According to AKIN/KDIGO criteria, nine patients (18%) were diagnosed with CSA-AKI. Compared to patients without CSA-AKI, these patients had significantly higher P-NGAL and P-cystatin C values 4 h (p-values .002 and <.001) and 12 h (p-values <.001 and <.001) postoperatively. The same differences were not observed for U-NGAL. Patients with AKI also had significantly higher P-creatinine 4 and 12 h postoperatively (p-values .001 and <.001), however the rise in P-creatinine was just above the upper reference limit. In conclusion, plasma NGAL and cystatin C seem to detect CSA-AKI earlier than the more commonly used biomarkers creatinine and urea.

Hemoglobinopathy gone astray-three novel forms of α-thalassemia in Norwegian patients characterized by quantitative real-time PCR and DNA sequencing.

α-thalassemia is one of the most common monogenic diseases worldwide and is caused by reduced or absent synthesis of α-globin chains, most commonly due to deletions of one or more of the α-globin genes. α-thalassemia occurs with high frequency in tropical and subtropical regions of the world and are very rarely found in the indigenous Scandinavian population. Here, we describe four rare forms of α-thalassemia out of which three are novel, found in together 20 patients of Norwegian origin. The study patients were diagnosed during routine hemoglobinopathy evaluation carried out at the Department of Medical Biochemistry, Oslo University Hospital, Norway. The patients were selected for their thalassemic phenotype, despite Norway as country of origin. All samples went through standard hemoglobinopathy evaluation. DNA sequencing and copy number variation (CNV) analysis using quantitative real-time polymerase chain reaction (qPCR) was applied to detect sequence variants and uncommon deletions in the α-globin gene cluster, respectively. Deletion breakpoints were characterized using gap-PCR and DNA sequencing. DNA sequencing revealed a single nucleotide deletion in exon 3 of the HBA2 gene (NM_000517.4(HBA2):c.345del) and a novel deletion of 20 nucleotides in exon 2 of the HBA2 gene (NM_000517.4(HBA2):c.142_161del). qPCR CNV analysis detected two novel large deletions in the α-globin gene cluster, -(NOR) deletion covering both α-globin genes and (αα)Aurora Borealis affecting the regulatory region, leaving the downstream α-globin genes intact. Even though inherited globin gene disorders are extremely rare in indigenous Scandinavians, the possibility of a carrier state should not be ignored.

Hb Calgary (HBB: c.194G>T): A Highly Unstable Hemoglobin Variant with a ß-Thalassemia Major Phenotype.

We describe two unrelated patients, both heterozygous for an unstable hemoglobin (Hb) variant named Hb Calgary (HBB: c.194G>T) that causes severe hemolytic anemia and dyserythorpoietic, resulting in transfusion dependence and iron overload. The molecular pathogenesis is a missense variation on the ß-globin gene, presumed to lead to an unstable Hb. The phenotype of Hb Calgary is particularly severe presenting as transfusion-dependent anemia in early infancy, precluding phenotypic diagnosis and highlighting the importance of early genetic testing in order to make an accurate diagnosis.

Prognostic Value of Nucleated RBCs for Patients With Suspected Sepsis in the Emergency Department: A Single-Center Prospective Cohort Study.

OBJECTIVES: Increase of nucleated RBCs in peripheral blood has been shown to be predictive of mortality in ICU patients. The aim of this study was to explore the prognostic value of nucleated RBCs in the first blood sample taken at admission to the emergency department from patients with suspected sepsis. DESIGN: Single-center prospective cohort study. SETTING: Emergency department. PATIENTS: One-thousand two-hundred thirty-one consecutive adult patients with suspected sepsis were included in a prospective quality register-based cohort study. Inclusion criteria were as follows: patients received in rapid response team with blood cultures taken and immediate antibiotics given in the emergency department. INTERVENTION: Not applicable. MEASUREMENT AND MAIN RESULTS: Nucleated RBCs, Sequential Organ Failure Assessment score, Quick Sequential Organ Failure Assessment, Charlson Comorbidity Index, and commonly used laboratory tests measured in the emergency department were compared with 30-day mortality. Nvaucleated RBC counts were divided into five groups, called "Nucleated RBC score," according to nucleated RBC count levels and analyzed with logistic regression together with the Sequential Organ Failure Assessment score and Charlson Comorbidity Index. Of the 262 patients with nucleated RBCs equal to or higher than the detection limit (0.01 × 109/L), 26% died within 30 days, compared with 12% of the 969 patients with nucleated RBCs below the detection limit (p < 0.0001). Mortality was significantly higher for each increase in Nucleated RBC score, except from score 2 to 3, and was 62% in the highest group. In multivariate logistic regression, odds ratios for 30-day mortality were as follows: Nucleated RBC score: 1.33 (95% CI, 1.13-1.56), Sequential Organ Failure Assessment score: 1.32 (1.29-1.56), and Charlson Comorbidity Index: 1.17 (1.09-1.25). CONCLUSIONS: Most patients with suspected sepsis in emergency department had undetectable nucleated RBCs at admission to the emergency department. However, increased nucleated RBCs significantly predicted 30-day mortality. Nucleated RBCs may provide additional prognostic information to Sequential Organ Failure Assessment score and other laboratory tests.

A study protocol for the cardiac effects of a single dose of either oxytocin 2.5 IU or carbetocin 100 µg after caesarean delivery: a prospective randomized controlled multi-centre trial in Norway.

Background: Both oxytocin and carbetocin are used to prevent uterine atony and post-partum haemorrhage after caesarean delivery in many countries, including Norway. Oxytocin causes dose-dependent ST-depression, troponin release, prolongation of QT-time and arrythmia, but little is known about myocardial effects of carbetocin. We have previously demonstrated comparable vasodilatory effects of oxytocin and carbetocin and are now undertaking a Phase 4 trial to investigate whether carbetocin causes similar changes to myocardial markers compared with oxytocin. Methods: Our randomized controlled trial will be conducted at three obstetrics units at Oslo University Hospital and Akershus University Hospital, Norway. Planned enrolment will be of 240 healthy, singleton pregnant women aged 18 to 50 years undergoing planned caesarean delivery. Based on pilot study data, each participant will receive a one-minute intravenous injection of either oxytocin 2.5 IU or carbetocin 100 µg during caesarean delivery. The prespecified primary outcome is the change from baseline in high-sensitive troponin I plasma concentrations at 6-10 hours after study drug administration. Secondary outcomes include uterine tone grade at 2.5 and five minutes after study drug administration, adverse events for up to 48 hours after study drug administration, estimated blood loss within eight hours of delivery, need for rescue treatment and direct/indirect costs. Enrolment and primary analysis are expected to be completed by the end of 2021. Discussion: Women undergoing caesarean delivery should be assessed for cardiovascular risk particularly as women with an obstetric history of pregnancy induced hypertension, gestational diabetes mellitus, preterm birth, placental abruption, and stillbirth are at increased risk of future cardiovascular disease. Any additional ischaemic myocardial risk from uterotonic agents will need to be balanced with the benefit of reducing the risk of postpartum haemorrhage. Any potential cardiotoxicity difference between oxytocin and carbetocin will help inform treatment decisions for pregnant women. Registration: Clinicaltrials.gov NCT03899961 (02/04/2019).

Hypogammaglobulinemia and Risk of Exacerbation and Mortality in Patients with COPD.

Introduction: Chronic obstructive pulmonary disease (COPD) may, in some patients, be characterized by recurring acute exacerbations. Often these exacerbations are associated with airway infections. As immunoglobulins (Ig) are important parts of the immune defence against airway infections, the aim of this study was to relate the levels of circulating immunoglobulins to clinical features in unselected patients with COPD included in a Norwegian multicenter study. Methods: Clinical and biological data, including circulating levels of immunoglobulins, were assessed in 262 prospectively included patients with COPD GOLD stage II-IV at five hospitals in south-eastern Norway. A revisit was done after one year, and survival was assessed after five years. Clinical features and survival of those with immunoglobulin levels below reference values were compared to those with normal levels. Results: In total, 11.5% of all COPD patients and 18.5% of those with GOLD stage IV had IgG concentrations below reference values. These patients were more likely to use inhaled or oral steroids, had lower BMI, and lower FEV1%. Moreover, they had significantly more COPD-related hospital admissions (2.8 vs 0.6), number of prednisolone courses (3.9 vs 1.2), and antibiotic treatments (3.7 vs 1.5) in the preceding year. Importantly, hypogammaglobulinemia was significantly associated with reduced survival in a log-rank analysis. In multivariate regression analysis, we found that the higher risk for acute exacerbations in these patients was independent of other risk factors and was associated with impaired survival. Conclusion: In conclusion, our study suggests that hypogammaglobulinemia may be involved in poor outcome in COPD and may thus be a feasible therapeutic target for interventional studies in COPD.

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 Aalesund (HBA2: c.400_406delAGCACCG), an Unstable α-Globin Variant Found in a Norwegian Patient Causing Moderate Hemolytic Anemia and Falsely High Hb A1c Using Ion Exchange High Performance Liquid Chromatography.

A new unstable hemoglobin (Hb) variant, named Hb Aalesund, was detected during Hb A1c measurement in a patient with a nearly compensated hemolytic anemia. Sequencing of the α-globin genes revealed a 7 bp deletion in exon 3 of the HBA2 gene (HBA2: c.400_406delAGCACCG) (NM_000517.4) causing a frameshift and a premature termination codon (PTC) two positions downstream. Apparently, the transcript bypassed nonsense-mediated decay (NMD), and a truncated protein was translated. The unstable Hb variant presumably underwent rapid denaturation, as heterozygosity of Hb Aalesund was associated with mild hemolytic anemia. In addition, the Hb variant interfered with Hb A1c measurement by cation exchange high performance liquid chromatography (HPLC), causing a falsely high Hb A1c result when using the Bio-Rad D10™ Hemoglobin Analyzer fast Hb A1c Program.

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.

Characterization of Two Deep Intronic Variants on the ß-Globin Gene with Inconsistent Interpretations of Clinical Significance.

Sequence variants located in the introns of the ß-globin gene may affect the mRNA processing and cause ß-thalassemia (ß-thal). Sequence variants that change one of the invariant dinucleotides at the exon-intron boundaries may have fatal consequences for normal mRNA splicing. Intronic variants located far from obvious regulatory sequences can be more difficult to evaluate. There is a potential for misinterpretation of such sequence variants. Hence, thorough evaluation of patient data together with critical use of databases and in silico prediction tools are important. Here, we describe two rare sequence variants in the second intron of the ß-globin gene, HBB: c.316-70C>G and HBB: c.316-125A>G (NM_000518.4), both previously reported as variants causing ß-thal, and later as benign sequence variants. Due to the limited number of published cases and inconsistent interpretations, the significance of these sequence variants has been unclear. We have identified these two sequence variants in multiple individuals, alone and in a variety of combinations with other δ- and ß-globin defects, and we find no influence of the sequence variants on the phenotype.

ß-Thromboglobulin may not reflect platelet activation during haemodialysis with the HeprAN membrane.

BACKGROUND: When blood passes through the extracorporeal circuit during haemodialysis (HD) undesirable effects including platelet degranulation and coagulation activation take place. ß-thromboglobulin (ß-TG) is a sensitive marker of platelet activation. The aim of this study was to investigate platelet degranulation and coagulation activation during HD with the heparin-coated dialysis membrane HeprAN. METHODS: Four HD sessions were evaluated in each of 12 chronic HD patients. None of the patients used oral warfarin, other anticoagulants or antiplatelet drugs. In the first session the HeprAN membrane or a conventional polyflux membrane was used in a randomized manner and thereafter alternately in a cross-over design, and 50% of the conventional dalteparin dose was given at start of HD. Prothrombin fragment 1 + 2 (PF1 + 2), ß-TG and anti-factor Xa activity were measured repeatedly. RESULTS: No dialysis sessions were terminated early due to clotting of the extracorporeal system. Activation of intravascular coagulation as assessed by change in PF1 + 2 during 4 hours of HD was the same with the two membranes. ß-TG concentration decreased significantly during 4 hours of HD with the HeprAN membrane but remained stable with the polyflux membrane. CONCLUSION: There were no differences in clotting scores or coagulation activation with the two membranes. The decrease in ß-TG during HD with the HeprAN membrane suggests ß-TG to be an inferior marker of platelet degranulation when using a heparin-coated dialysis membrane. A possible mechanism for the decline in ß-TG concentration may be adherence of this heparin-binding protein to the heparin-coated dialysis membrane.

HbA1c analysis by capillary electrophoresis - comparison with chromatography and an immunological method.

BACKGROUND: Haemoglobin A1c (HbA1c) has become an even more important analyte for clinical laboratories during recent years with the introduction of its diagnostic use for diabetes mellitus. Several different analytical principles can be used, each with their advantages and disadvantages. AIM: We wanted to compare Sebia Capillarys 2 Flex Piercing (Capillarys) with our routine HbA1c methods, which were an HPLC method (Tosoh G7) and an immunoassay (Tina-Quant on Roche Modular P) by analysing a large clinical material. Furthermore, we investigated sample stability. METHODS: HbA1c analysis was performed in parallel by all three methods for more than 600 patient samples including common and some rare haemoglobin variants, as well as for several controls, some with set target values. Sample stability at room temperature and refrigerated was assessed for up to seven days. RESULTS: Capillarys produced generally somewhat lower HbA1c values than both comparison methods, apparently due to positive bias for the comparison methods. Leaving out samples with haemoglobin variants, we found a mean bias (95% CI) for Capillarys compared to Tosoh G7 (without factorization) and Modular of -0.39 (-0.40 to -0.38) and -0.16 (-0.17 to -0.14) % HbA1c, respectively. HbA1c results were similar between instruments for samples from dialysis patients and for samples with heterozygous common haemoglobin variants, except that Tosoh G7 reported too low results in the presence of Hb E. For heterozygous Hb Raleigh, Capillarys and the immunoassay gave similar results. CONCLUSION: Capillarys is a convenient instrument for routine HbA1c analysis.

Association Between HLA Haplotypes and Increased Serum Levels of IgG4 in Patients With Primary Sclerosing Cholangitis.

Increased serum levels of IgG4 have been reported in 9%-15% of patients with primary sclerosing cholangitis (PSC); it is not clear whether this increase contributes to pathogenesis. We performed genetic analyses of the HLA complex in patients with PSC from Norway, Sweden, and from the United States. We found an association between levels of IgG4 above the upper reference limit and specific HLA haplotypes. These patients had a significantly lower frequency of the strongest PSC risk factor, HLA-B*08, than patients without increased IgG4, and significantly higher frequencies of HLA-B*07 and HLA-DRB1*15. HLA genotype therefore might affect the serum concentration of IgG4, and increased IgG4 might be a marker of a distinct phenotype of PSC.