Liver Disease and Sickle Cell Disease: Auto-Immune Hepatitis more than a Coincidence; A Systematic Review of the Literature.

In patients with SCD, chronic liver damage is a common manifestation. More than 50% of SCD patients have elevated liver enzymes. Common underlying aetiologies include sickle cell hepatic crisis, viral hepatitis, sickle cell intrahepatic cholestasis and hepatic sequestration in the acute setting, and cholelithiasis and iron overload in the chronic setting. Autoimmune hepatitis (AIH) is a rare disease that appears to occur more commonly in the sickle cell disease (SCD) population than in the general population. There are many schools of thought as to why this is the case, including the phosphatidylserine hypothesis, the heme inflammatory hypothesis, the complement generation hypothesis, and the transfusion alloimmunization hypothesis. Due to the natural history of the two illnesses, SCD is almost always diagnosed first in cases of dual pathology. Symptoms such as jaundice, fatigue, and abdominal pain are common in SCD, as are abnormal liver function tests (LFTs). These abnormalities, attributed to the other more frequent liver involvements in SCD, can lead to delays in AIH diagnosis in this population. Corticosteroids, sometimes with other immunosuppressive agents, such as azathioprine, are the cornerstone of acute AIH treatment. However, corticosteroid use in the SCD population has been shown to carry an increased risk of vaso-occlusive crises, providing a treatment dilemma. The following is a review of AIH in the SCD population, where we explore the pathophysiology behind the association between the two disorders, discuss an approach to investigating abnormal LFTs in SCD, and examine treatment options in this population with co-existing diseases.

The Importance of Being Aware of Intrinsic Methods' Limitation in Low-Density Lipoprotein Cholesterol Determination to Correctly Identify Cardiovascular Risk: Is Direct Determination Obtained with the Roche System Systematically Overestimating LDL in Very High-Risk Patients with Triglycerides Concentration of Less than 2.25 mmol/L?

BACKGROUND: low-density lipoprotein cholesterol (LDL-C) is a strong cardiovascular risk factor, but the methods used to correctly determine it are is still questioned. The aim of this study was to compare the direct determination of LDL-C levels, obtained with the Roche cobas c system, with LDL-C values calculated through Sampson's and Friedewald's equations in very high-risk patients with triglycerides concentrations of less than 2.25 mmol/L (<200 mg/dL). METHODS: in 127 consecutive patients with a recent diagnosis of acute coronary syndrome and triglycerides of less than 2.25 mmol/L, plasma LDL-C was measured directly and calculated with Sampson's and Friedewald's equations before hospital discharge, and the data were compared. RESULTS: median LDL values obtained with the Friedenwald and Sampson equations and with direct determination were 2.31 (IQR 1.59-3.21), 2.36 (IQR 1.66-3.26) and 2.64 (IQR 1.97-3.65) mmol/L, respectively. Direct measurements were higher by 0.35 and 0.40 mmol/L when compared to the levels calculated with Sampson's or Friedewald's equations, respectively (p < 0.01). The correlation between the two equations was almost perfect (rho 0.997) but decreased when the directly determined data were compared to those calculated with Sampson's equation (rho 0.954) or Friedewald's method (rho 0.939). CONCLUSION: direct determination generated higher values of LDL-C concentration through a probable systematic overestimation.

Utility of serum indices in a particular case of serum protein electrophoresis.

Screening and measurement of monoclonal (M) proteins are commonly performed using capillary zone electrophoresis (CZE). The identification of M-protein or monoclonal component (CM) is an essential requirement for diagnosis and monitoring of monoclonal gammopathies. The detection of CM has been largely improved by CZE. Capillary electrophoresis estimates CM more accurately, because absence of variation due to different dye binding affinities of proteins as instead seen with agarose gel electrophoresis. However, interferences can be present in CZE. This occurs because all substances absorbing at 200 nm can be identified. Recognition and handling of specimens exhibiting such interferences is essential to ensure accurate diagnostic and patient safety. We herein report on an unusual case of serum protein electrophoresis, to highlight that laboratory staff must be aware of and familiarise with the information provided by laboratory instruments. For example, in the case of serum indices, about specimen quality.

Autoimmune disease and sickle cell anaemia: 'Intersecting pathways and differential diagnosis'.

Sickle cell disease (SCD) is an inherited disorder, which occurs due to a single gene mutation. It has multisystemic manifestations, affecting millions of people worldwide. The effect of SCD on joints and musculature can overlap with clinical features of autoimmune disease (AD). It is therefore difficult for clinical haematologists and physicians treating SCD patients to discriminate between these two conditions clinically. A delay in diagnosis leads to untreated symptoms and treatment differs considerably. An accurate knowledge of clinical findings and laboratory results of AD and SCD can help physicians avoid this. In the review that follows, we examine the existing literature on SCD and AD, and describe the features that may distinguish SCD and autoimmune disease such as systemic lupus erythematosus and rheumatoid arthritis. We aim to guide clinical haematologists and physicians towards a more rapid diagnosis of AD in sickle cell anaemia patients, by correct interpretation of the clinical assessment and commonly available diagnostics.

Comparison of Freelite and N-Latex serum free light chain assays: a critical review.

INTRODUCTION: The measurement of serum free light chain (FLC) represents a fundamental aspect on the assessment of patients with monoclonal gammopathies (MG). Different analytical methods for FLC have become available with the possibility to obtain different value with a substantial impact on the assessment of patients with MG. This study aimed to evaluate FLC results obtained with two different assays and how the difference value obtained can impact in the patient's assessment. MATERIALS AND METHODS: Ninety-three patient serum samples that underwent analysis for FLC with two different methods, Serum Freelite (The Binding Site, Birmingham, UK) and N-Latex FLC (Siemens, Marburg, Germany), were included in this retrospective study. Statistical analysis was performed to evaluate correlation, difference, and the grade of concordance between the results obtained with the two methods. RESULTS: Significant statistical differences between the results obtained from the two methods were found (P < 0.05). A good correlation was found (0.99 for κ FLC, 0.95 for λ FLC, and 0.94 for the κ/λ ratio, respectively). We found a weighted kappa value of 0.65 for κ/λ ratio, 0.65 for λ FLC and 0.90 for κ FLC. A positive bias found with the Bland-Altman plot mirrors overestimation of κ FLC and κ/λ ratio with Freelite compared to N-Latex, whilst a negative bias underscores underestimation of λ FLC by Freelite compared to N-Latex. CONCLUSION: Although in general the concordance between Freelite and N-Latex appears satisfactory, several discrepancies could be evidenced and consequently the two assays are not interchangeable.

Preliminary assessment of the new Sysmex XN parameter Iron-Def for identifying iron deficiency.

BACKGROUND: Although the clinical assessment of iron status is usually based on iron stores, a rapid and accurate diagnosis of iron deficiency is challenging since ferritin is often unavailable as an urgent test and its value is frequently increased in acute phase conditions. This study was therefore aimed at evaluating the diagnostic performance of the new Sysmex XN "Iron Deficiency?" (Iron-Def) parameter for identifying patients with iron deficiency. MATERIALS AND METHODS: The study population consisted of 688 consecutive patients (median age: 71 years; 341 women and 347 men), referred for routine diagnostics to the Laboratory of Clinical Pathology of Lecco Hospital, Italy. A complete clinical chemistry profile and haematological testing were performed for identifying iron deficiency anaemia. RESULTS: A significant negative correlation was found between Sysmex XN Iron-Def and ferritin, serum iron, mean cell haemoglobin concentration, mean cell haemoglobin, mean corpuscular volume and age, while a positive correlation was noted with transferrin, percentage of microcytic red cell, red blood cell count and red blood cell distribution width. The diagnostic accuracy of Iron-Def for identifying patients with a percentage of saturation of transferrin <15% (n=104) was 84%, with a sensitivity of 0.952 and specificity of 0.538. A sub-analysis of 71 patients with ferritin <20 ng/dL yielded an even better diagnostic performance (86%, with a sensitivity of 0.935 and specificity of 0.620). DISCUSSION: Although additional confirmatory investigations would be needed, the preliminary findings of our study attest that Iron-Def may be an easy, inexpensive, rapid and reliable parameter for screening iron deficiency anaemia.

Intravenous ferric carboxymaltose is effective and safe in patients with inflammatory rheumatic diseases.

BACKGROUND: The aim of this study was to evaluate the efficacy and safety of ferric carboxymaltose in rheumatic patients with iron deficiency anaemia. MATERIALS AND METHODS: The study retrospectively evaluated a cohort of 34 patients with iron deficiency anaemia affected by inflammatory rheumatic diseases that are refractory or intolerant to oral iron therapy. They were treated with ferric carboxymaltose for a total of 56 cycles of treatment. The primary end point was to evaluate the increase of haemoglobin after ferric carboxymaltose treatment. The secondary end point was safety, including the occurrence of disease flare. RESULTS: Median age of the cohort was 60 years (range 31-91 years), with a male/female ratio of 4/30. Nine (26.5%) were affected by rheumatoid arthritis, 10 (29.4%) by spondyloarthritis, and 15 (44.1%) by other autoimmune connective tissue diseases. Median time from diagnosis was 7 years (IQR 2-12). At time of treatment (T0), median haemoglobin was 9.3 g/dL (IQR 8.2-10.3), transferrin saturation 6.2% (IQR 3.8-9.8), and ferritin 8.5 ng/mL (IQR 6.0-12.8). Median ferric carboxymaltose dose was 1,000 mg. At 6 weeks from T0, median haemoglobin was 12.3 g/dL (IQR 11.6-13.3), with a mean increase of 3.0 g/dL (p<0.01). Twelve (35.3%) patients needed re-treatment with ferric carboxymaltose for recurrence of iron deficiency anaemia. Four (4.3%) patients developed mild grade side effects. One suspected flare reaction has been observed. DISCUSSION: In patients affected by inflammatory rheumatic diseases, ferric carboxymaltose is safe and effective in correcting iron deficiency anaemia.

Effect of a liberal versus a restrictive pre-donation blood pressure policy on whole-blood donor adverse reactions.

BACKGROUND AND OBJECTIVES: The role of pre-donation blood pressure (BP) as independent contributor to post-donation vasovagal reactions (VVRs) is still debated. Differences between a liberal (i.e., inclusion of hypotensive donors) and a restrictive policy (i.e., not accepting hypotensive donors) should be investigated. This study aims to investigate the consequences of a liberal policy in development of VVRs after whole-blood donations. MATERIALS AND METHODS: We compared the incidence of VVRs between 2015 (restrictive policy) and 2016 (liberal policy) and the associated risk factors. We evaluated respectively 22 789 vs. 21 676 blood donations obtained from 18 001 blood donors (12 501 donated in both years). RESULTS: Comparing the results we obtained between 2015 and 2016, donations showed an overlap of the cohorts. Two hundred fifteen VVRs (incidence rate 0·48%) were observed, 104 (0·46%) of which in 2015, and 111 (0·51%) in 2016. A preliminary univariate analysis showed that donors with systolic BP <110 mm Hg had a two-fold risk of VVRs compared to normotensive donors (VVR/donation rate of 0·99% vs. 0·46%; P = 0·001). The subsequent multivariable logistic regression model showed that VVRs were highly associated with weight, site of collection, age and number of donations, excluding a role for systolic and diastolic BP. CONCLUSION: A liberal pre-donation BP policy seems to be safe for blood donors. Our analysis confirms that older donors with higher body-weight who already had donated blood are unlikely to experience VVRs.

Insight into the complex pathophysiology of sickle cell anaemia and possible treatment.

Sickle cell anaemia (SCA) is the consequence of abnormal haemoglobin production due to an inherited point mutation in the ß-globin gene. The resulting haemoglobin tetramer is poorly soluble when deoxygenated, and when this is prolonged, intracellular gelation of sickle haemoglobin occurs, followed by haemoglobin polymerisation. If many cycles of sickling and unsickling occur, the red cell membrane will be disrupted leading to haemolysis and vaso-occlusive events. Recent studies have also shown that leucocyte adhesion molecules and nitric oxide (NO) depletion are involved in endothelial damage. New insights in SCA pathophysiology and vascular biology have shown that cell-derived microparticle (MP) generation is also involved in the vaso-occlusion. Endothelial damage is perpetuated by impaired production or increased consumption of protective modulators such as protein C, protein S and NO. New therapeutic interventions should address these aspects of SCA pathogenesis. To date, the only US-FDA-approved therapy to prevent painful vaso-occulsive episodes is hydroxyurea that reduces haemoglobin polymerisation in sickle cells by increasing the production of foetal haemoglobin and L-glutamine. However, several new drugs have been tested in the last years in randomised clinical trials. We here report an update on the current status of knowledge on SCA.

Validity and reliability of serologic immunophenotyping of multiple blood group systems by ORTHO Sera with fully automated procedure.

CONCLUSIONS: The increase of immunization against blood group antigens has reinforced the need for automated extensive blood typing. The aim of this study was to assess both the validity and reliability of red blood cell (RBC) automated agglutination technology in testing for antigens of Kidd (Jk), Duffy (Fy), and MNS (Ss) blood systems. ORTHO Sera (Ortho Clinical Diagnostics, Raritan, NJ) anti-Jka, anti-Jkb, Anti-Fya, anti-Fyb, anti-S, and anti-s reagents were each tested on RBC samples previously typed. Replicates were performed on three separate testing sessions with three consecutive repetitions within each session, thus obtaining 486 test results. Accuracy was assessed by aggregate analysis of sensitivity, specificity, and area under the receiver operating characteristics curve (AUC). Reliability was estimated by a cross-classified mixed-effect logistic model. All reagents tested yielded optimal accuracy (100% for sensitivity and specificity, and 1.00 for AUC), except for anti-S, for which performance was slightly lower (98%, 100%, and 0.99, respectively), owing to misclassification of one sample in a single replicate. Anomalous automated measurements were recorded in 38 of 486 tests (7.8%), which then needed additional manual interpretation. Different sessions and samples were the major contributors to measurement failures (38% and 18%, separately). Order of repetitions and antigen specificity across replicates did not contribute to the risk of failures, although weak evidence of enhanced risk was observed with Jk testing. Automated RBC typing with ORTHO Sera reagents against antigens in the Kidd, Duffy, and MNS blood group systems displayed nearly 100 percent accuracy. However, a sizable number of replicates needed additional ad hoc interpretation, thus suggesting that the reliability could still be improved. Automated agglutination technology represents a viable option for phenotyping large volumes of samples.

The impact of different sample matrices in delayed measurement of glucose.

OBJECTIVES: This study was designed to compare glucose values in serum or lithium-heparin samples immediately centrifuged with those paired specimens collected in tubes containing Naf-KOx and centrifuged and analyzed 2.5h after collection. METHODS: Three blood samples were drawn from 20 volunteers. Blood samples collected in tubes with and clot activator and gel separator but without anticoagulant (SST) as well as those collected in tubes containing Lithium-Heparin and gel separator were centrifuged within 30min and analyzed 2h thereafter. Blood samples drawn in tubes containing the glycolysis inhibitor NaF-KOx were centrifuged after 2.5h and then analyzed. RESULTS: The glucose median value was 4.72mmol/L in SST tubes, 4.67mmol/L in lithium-heparin and 4.44mmol/L in NaF-KOx tubes. The difference between SST and lithium-heparin tubes was not statistically or clinically significant, whereas that between SST and Naf-KOx tubes was both analytically and clinically meaningful, exceeding the current quality specifications for glucose measurement. CONCLUSIONS: The rapid centrifugation of blood collected in serum or lithium-heparin tubes with gel separator is seemingly more reliable for delayed measurement of glucose compared to the use of blood tubes containing NaF-KOx.

Evaluation of capillary haemoglobin determination for anaemia screening in blood donation settings.

BACKGROUND: An accurate assessment of hemoglobin (Hb) values before donation is unavoidable for safeguarding donors' safety and fulfilling the current specifications of Hb content in blood bags. This study was hence aimed to compare a finger-prick method for Hb measurement in capillary blood with Hb assessment in venous blood using a hematological analyser. MATERIALS AND METHODS: The study populations consisted in 1,014 consecutive blood donors, who had paired measurement of Hb values with HemoCue on capillary blood and UniCel DxH800 in venous blood. RESULTS: A significant overestimation was found with HemoCue compared to UniCel DxH800, but the correlation between methods was significant (comprised between 0.600 and 0.759; all p<0.01) and the bias always lower than the quality specifications. The prevalence of Hb values below the gender-specific thresholds for blood donation was also not significantly different (p=0.186). DISCUSSION: It can hence be concluded that the finger-prick method evaluated is a safe and reliable means for screening blood donors.

Fully-automated, chemiluminescence IgA and IgG anti-tissue transglutaminase (tTG) antibodies serum assays for the screening of celiac disease.

AIM: Celiac disease (CD) is a systemic immune-mediated enteropathy sustained by gluten ingestion in genetically susceptible subjects. The European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) has recently revised the diagnostic criteria, emphasizing the crucial role of serological testing in the diagnosis of this condition. This study was hence aimed to evaluate a new chemiluminescence assay for measuring anti-transglutaminase (tTG) and anti-endomysium (EMA) antibodies in a general population of unselected outpatients. MATERIALS AND METHODS: The IgA and IgG anti-tissue transglutaminase (tTG) antibodies (Quanta Flash® IgA and Quanta Flash® IgG tTG, Inova Diagnostics, San Diego, CA, USA) were measured with the fully-automated BIO-FLASH® analyzer (Inova Diagnostics) in serum samples of 727 consecutive patients without a diagnosis of CD. Data were compared with those of anti-endomysium antibody (EmA) obtained in the same population. RESULTS: A total of 96.4% samples display a negative concordance (anti-tTG negative and EMA negative), O% were positive for EMA and negative for anti-tTG IgA and IgG, 3.6% were both positive for tTG IgA and EMA, whereas 0.6% displayed discordant results (positive for anti-tTG and negative for EMA). The concordance (99%) and inter-rater agreement (Kappa Statistics, 0.943; p<0.001) between anti-tTG IgA and EmA antibodies were excellent, with sensitivity and specificity of 99% and 100%, respectively. CONCLUSION: The results of this study show that Quanta Flash® IgA assay alone may be regarded as a reliable approach for screening of CD, with no need to perform EMA detection.

Autoantibody Profiling in a Cohort of Pediatric and Adult Patients With Autoimmune Hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a rare condition characterized by the presence of autoantibodies distinctive of type 1 AIH (AIH-1) and type 2 AIH (AIH-2). The aim of this study was to evaluate the autoantibody profile in a cohort of pediatric and adult AIH patients, using both indirect immunofluorescence (IIF) and a new multiplexed line-blot assay. METHODS: Sera from 63 pediatric and 53 adult AIH patients were tested for antinuclear (ANA), antismooth muscle (SMA), anti-liver kidney microsome 1 (anti-LKM1), anti-liver cytosol 1 (anti-LC1) autoantibodies using IIF methods; for anti-LKM1, anti-LC1, and soluble liver antigen/liver-pancreas (anti-SLA/LP) autoantibodies using the line-blot; for anti-F-actin autoantibodies using IIF both on VSM47 cell-line and on rat intestinal epithelial cells. RESULTS: AIH-1 was the most common type of AIH in the adult cohort (73.6%), while AIH-2 was the most common AIH in the pediatric cohort (61.9%). Both in adult and pediatric AIH-2 anti-LKM1 were the prevalent autoantibodies. In pediatric AIH-2 anti-LC1 autoantibodies were more frequent than in adult AIH-2 (59 vs. 28.6%), and in 35.9% of cases they were present alone. In 17 patients anti-LC1 autoantibodies were detected only with the line-blot assay. The levels of anti-LKM1 and of anti-LC1 were not different between adult and pediatric AIH, and the overall agreement between the results obtained with the two IIF methods for F-actin detection was 98.8% (CI 95%: 94.4-99.7%). CONCLUSIONS: The line-blot assay showed a higher sensitivity than IIF for anti-LC1 detection. Anti-LKM1 and anti-LC1 autoantibody levels are not different in adults and children. An almost perfect agreement between the two IIF methods for anti-F-actin detection has been observed.

Spurious elevation of serum potassium concentration measured in samples with thrombocytosis.

BACKGROUND: Several factors that can lead to falsely elevated values of serum. Thrombocytosis is one of these factors, since breakage or activation of platelets during blood coagulation in vitro may lead to spurious release of potassium. The purpose of the study was to evaluate to which extent the platelet count may impact on potassium in both serum and plasma. METHODS: The study population consisted of 42 subjects with platelets values comprised between 20 and 750×109/L. In each sample potassium was measured in both serum and plasma using potentiometric indirect method on the analyzer Modular P800 (Roche, Milan, Italy). Platelet count was performed with the hematological analyzer Advia 120 (Siemens, Milano, Italy). RESULTS: Significant differences were found between potassium values in serum and in plasma. A significant correlation was also observed between serum potassium values and the platelet count in whole blood, but not with the age, sex, erythrocyte and leukocyte counts in whole blood. No similar correlation was noticed between plasma potassium and platelet count in whole blood. The frequency of hyperkalemia was also found to be higher in serum (20%) than in plasma (7%) in samples with a platelet count in whole blood >450×109/L. CONCLUSIONS: The results of this study show that platelets in the biological samples may impact on potassium measurement when exceeding 450×109/L. We henceforth suggest that potassium measurement in plasma may be more accurate than in serum, especially in subjects with thrombocytosis.

Pre-analytic variability in cardiovascular biomarker testing.

The impact of laboratory medicine on clinical cardiology has dramatically increased over the years and a lot of cardiovascular biomarkers have been recently proposed. In order to avoid clinical mistakes, physicians should be well aware of all the aspects, which could affect the quality of laboratory results, remembering that pre-analytic variability is an often overlooked significant source of bias, determining the vast majority of laboratory errors. This review addresses the determinants of pre-analitycal variability in cardiovascular biomarker testing, focusing on the most widespread biomarkers, which are cardiac troponins and natriuretic peptides.