Hemoglobin A2 and Heterogeneous Diagnostic Relevance Observed in Eight New Variants of the Delta Globin Gene.

BACKGROUND: Hemoglobin A (Hb A) (α2ß2) in the normal adult subject constitutes 96-98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the ß-thalassemia carrier. METHODS: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. RESULTS: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1-2% during the screening tests in our laboratories. CONCLUSIONS: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

Spurious hemolysis does not influence the reliability of digoxin testing on Siemens RXL MAX and Roche Cobas e601.

Little information is available on the influence of spurious hemolysis on digoxin immunoassays. Seventeen consecutive, non-hemolyzed, sodium-heparin samples were divided in three aliquots. The first was immediately centrifuged and tested for hemolysis index (HI), as well as plasma digoxin on Siemens RXL MAX using the Siemens Dimension Flex and Roche Cobas e601 by electrochemiluminescent (ECLIA) technique. The second and third aliquots were subjected to mechanical hemolysis by aspirating the blood one and two times through a thin needle. The concentration of digoxin measured on Siemens RXL MAX was significantly decreased from aliquot #A, to aliquot #B (-4%), and aliquot #C (-6%), but in none of the hemolyzed specimens the 10% bias was exceeded. No significant variation was observed by measuring plasma digoxin on Roche Cobas.

Erythrocyte mechanical fragility is increased in patients with type 2 diabetes.

BACKGROUND: Anemia is common among patients with type 2 diabetes. We determined whether type 2 diabetic patients significantly differed in erythrocyte mechanical fragility as compared with nondiabetic subjects. METHODS: We recruited 25 Caucasian patients with type 2 diabetes (14 men and 11 women; mean age 58±8 years) and 25 age-, race- and sex-matched nondiabetic individuals. The fragility of erythrocytes was tested by inducing mechanical hemolysis by double aspiration of K(2)EDTA blood through a 0.5 mL insulin syringe equipped with a very thin needle. The plasma was then separated from the blood cells by centrifugation at 2000 xg for 15 min at room temperature. A Beckman Coulter DxC 800 was used to measure the hemolysis index by direct spectrophotometry. RESULTS: Compared with matched nondiabetic controls, type 2 diabetic patients had a significantly increased mechanical fragility of erythrocytes (hemolysis index ratio 21±13 vs. 14±10, p=0.02). Univariable linear regression analysis revealed that there was a strong positive association between percent hemolysis and fasting plasma glucose (r=0.669, p<0.001) and hemoglobin A1c (r=0.549, p<0.005) in type 2 diabetic subjects, but not in matched nondiabetic controls. CONCLUSIONS: Our data suggest that patients with type 2 diabetes have a significantly higher erythrocyte mechanical fragility than matched nondiabetic subjects, and that fasting plasma glucose is the strongest correlate of increased mechanical fragility of erythrocytes in this patients group.

The measurement of hemoglobin A2 and F in stored whole blood samples.

BACKGROUND: Little information is available on the influence of the storage conditions on the results of hemoglobin A2 (HbA2) and F (HbF) testing. METHODS: The stability of HbA2 and HbF was assessed on stored whole blood samples tested immediately after collection and after one week of storage at either + 4 degrees C or - 80 degrees C. RESULTS: An excellent correlation was observed between fresh specimens and those stored at either + 4 degrees C (HbA2: r = 0.994; p < 0.001 and HbF: r = 0.999; p < 0.001) or - 80 degrees C (HbA2: r = 0.995; p < 0.001 and HbF: r = 0.999; p < 0.001). The mean biases from the reference fresh specimens were very modest for samples stored at + 4 degrees C (HbA2: -0.06%; HbF: 0.02%;) and - 80 degrees C (HbA2: - 0.06%; HbF: 0.05%). CONCLUSIONS: The results of our investigation demonstrate that whole blood specimens for HbA2 and HbF testing can be stored at + 4 degrees C or - 80 degrees C for up to one week.

Influence of temperature and period of freezing on the generation of hemolysate and blood cell lysate.

BACKGROUND: There is little information on temperature and period of freezing for generating hemolysate and blood cell lysate. MATERIALS AND METHODS: Primary tubes containing whole blood were frozen at -20°C and -80°C and then serially thawed to assess cell lysis and clinical chemistry tests in centrifuged plasma. RESULTS AND CONCLUSIONS: Massive amount of blood cells lysis could only be obtained by storing samples 12h at -20°C, or 2h at -80°C.

Unusual false-positive case of urinary screening for buprenorphine.

Buprenorphine is a centrally acting analgesic drug that is administered for the management of opioid dependence and as an analgesic drug for the treatment of chronic pain. The growing use of this substance has determined an increased need for laboratory testing for either detection and confirmation of the illicit use or monitoring compliance as a substitution therapy for opioid dependence. We describe here the case of urinary sample adulteration with exogenous buprenorphine (6,952 ng/ml), which has led to afalse-positive immunoassay test result (14.9 ng/ml) on a subsequent sample due to a phenomenon of instrumental carry-over. This unusual case confirms the importance to take into account adulteration when screening urines for buprenorphine in patients undergoing substitution therapy for opioid dependence, routinely perform a confirmation assay on positive samples, and rule out instrumental carry-over.