1 Factores de confusión en el Análisis de HbA1cServicio de Análisis Clínicos Sesión de Actualización Otoño 2015 Eloísa Urrechaga, PhD Consultor en Análisis Clínicos

2 HbA1c una molécula únicaUna modificación post-traducción glicación de una proteína Hemoglobina en un tejido sangre se relaciona con una enfermedad Diabetes Mellitus No se produce por biosíntesis su síntesis procede como si el sistema fuera inerte

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4 HbA1c una molécula únicaEritrocitos mantienen un metabolismo residual Glucosa penetra en la célula por difusión facilitada La glicación de Hb es un proceso espontáneo no mediado por enzimas y acumulativo Es proporcional a la glucemia media y otros factores la vida media de los eritrocitos cinética de la reacción

5 Medicina basada en al Evidencia gold standard control metabólico DMDCCT Study Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulindependent diabetes mellitus: the Diabetes Control and Complications Trial N Eng J Med 1993; 329: 977–986 UK Prospective Diabetes Study Group. UKPDS 33: intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with Type 2 diabetes Lancet 1998; 352 : 837–853

6 1995 IFCC crea el grupo de trabajo para la standarización de HbA1CControvertida molécula…….. 1995 IFCC crea el grupo de trabajo para la standarización de HbA1C Definir el analito Preparar HbA0 y HbA1C puros Desarrollar un método de referencia Establecer a nivel mundial una Red de Laboratorios de Referencia Preparación de materiales de referencia secundarios

7 Hb glicada ≠ HbA1c La glicación se produce en los aminoácidos N terminales de las cadenas α y β como así también los grupos ε-amino de los residuos de lisina en la molécula de hemoglobina, resultan en una variedad de hemoglobinas glicadas, incluyendo HbA1c HbA1C se define por la Hb glicada en la Valina animo terminal de la cadena β β – N –(1-deoxi)-fructosil Hb (DOF Hb)

8 Método de referencia ifcc

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10 Clinical Chemistry & Laboratory Medicine 2007; 45(8): 942-4Diabetes Care 2007; 30 (9): Diabetologia 2007; 50 (8):2042-3

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14 Hemoglobinas Familia de moléculas Fisiológicas PatológicasModificadas químicamente

15 Durante la vida embrionaria, fetal y post-natalvarios son los genes que codifican las diferentes globinas z2 yz1 ya2 ya1 a2 a1 Cr.16 3’ Hb Grower-1 y -2 z2 e2 a2 e2 Hb Portland z2 g HbF 80-90% a2 g2 HbA2 2.5% a2 d2 HbA 97% a2 b2 0.5% Embrional Fetal Post-natal Cr.11 e e Gg Gg Ag Ag yb d d b b = Pseudo (y) genes sin expresión Fuente: presentación Prof P C Giordano

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17 Hb modificadas químicamenteInterferencia analítica es el error sistemático producido por una sustancia distinta de la que se pretende medir en un procedimiento analítico Hb modificadas químicamente son potencialmente interferentes debido a la separación incompleta de HbA1c HbA1c lábil esta formada por la unión inestable de glucosa al grupo amino N-terminal de las cadenas β (aldimina) La mayoría libera rápidamente la glucosa volviendo al estado no glicado, tras la reagrupación de Amadori se forma la cetoamina estable La carbamilación de forma muy acentuada en pacientes con función renal reducida y niveles elevados de urea en suero La acetilación in vivo del grupo N-terminal de las cadenas de globina tiene lugar en pacientes alcohólicos, mujeres embarazadas y en pacientes con altas dosis de aspirina La acetilación tiene lugar principalmente en las cadenas γ (HbF) Imagen tomada de presentación Sr M. Castellet. Sebia

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20 Diabetes y hemoglobinopatíasLas Hemoglobinopatías es la enfermedad autosómoca recesiva más prevalente en el mundo 7 % de la población mundial son portadores de genes de Hb mutados Cada año nacen más de medio millón de niños afectados World Health Organization 2008 IDF 2010

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22 Hb C β 6 Glu → Lys Hb D β 121 Glu → Gln Hb E β 26 Glu → Lys

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26 Sistemas analíticos basados en la separación de HbsNormal A1c/A A1c/A0 + A1c HbX Aglic + Xglic / Anoglic + Xnoglic Sacks D, Clin Chem 2003; 49(8):

27 Más que interferencia analítica: metodológica + biológicaFactores que interfieren con la medida de HbA1c La presencia de Hbs modificadas químicamente, variantes genéticas o Hb F elevada pueden afectar a la exactitud de la cuantificación de HbA1c El efecto final sobre el valor de HbA1c varía dependiendo del tipo de Hb variante y del método utilizado Factores que afectan a la interpretación de los resultados de HbA1c Cualquier condición que afecte a los eritrocitos y/o acorte su vida media va a rendir valores falsamente bajos Sangrados y hemólisis suelen ser tenidos en cuenta, pero otros factores pasan desapercibidos en nuestra práctica diaria Las Hbs variantes reducen la vida media de los eritrocitos No hay evidencia de que la cinética de la reacción de glicación sea equivalente a la de Hb A0

28 Hb Vida media RBC (días) HbA 120 HbS 93 HbC 87 HbD 115 HbE ?

29 Prevalencia de Diabetes Mellitus en regiones donde las Hb variantes son más habitualesRhea JM et al. Impact of Hemoglobin variants on HbA1c interpretation: Do we assume too much? Med Lab Obser 2012; 6: 8-14

30 Hb J y G interpretación es más complicada Sin conclusiones definitivasClin Chem & Lab Med 2015; 53 (9):e207-e210. Evidencia indirecta tasa de glicacion Hb S, C, D, y E es equivalente a la de Hb A0 Hb J y G interpretación es más complicada Sin conclusiones definitivas

31 Año Determinaciones HbA1c 2002 18461 2003 20505 2004 23960 2005 27036 2006 29984 2007 31306 2008 32826 2009 35806 2010 39667 2011 40447 2012 43944 2013 46627 2014 48035

32 2002 Una propuesta audaz

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34 Hb A1c y otras Hemoglobinas: amenaza / oportunidad / fortalezaAmenaza otras Hb químicamente modificadas Amenaza pero Oportunidad Hb variantes Oportunidad Fortaleza HbA2 Detección de portadores de β talasemia

35 Hb variante/TalasemiaNúmero de pacientes Beta talasemia 620 Alfa talasemia 177 Hb AS 130 Hb Lepore 44 Hb AC 30 Delta beta talasemia 13 Hb AD 9 Hb AE 4 Hb AJ 2 Hb Shelby 1 Hb SC Hb Setif 1032 COMARCA INTERIOR DE VIZCAYA Area que comprende el Este de Vizcaya y el Norte de Alava habitantes nivel socioeconómico medio, divididos entre medio rural y pequeñas villas de habitantes No hay datos oficiales de la prevalencia de hemoglobinopatías en el País Vasco Se considera un área “libre de Hbpatías” ………. Nuestra experiencia en el período 2002-noviembre 2015 ……….

36 Nuestra rutina diaria

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38 HA8160 vs HA8180V

39 Incidental detection of haemoglobin (Hb) variants during high performance liquid chromatography (HPLC) analysis of HbA1c: is it time for a standardised approach to reporting? J. Reeve et al Ir J Med Sci 2015; 184(2):353-5 BACK GROUND: Haemoglobin (Hb) variants are genetic variations in the globin genes that code for an abnormal globin protein structure. The prevalence of Hb variants has increased in Ireland due to the number of emigrants from Africa and Southeast Asia. The rate of incidentally detected Hb variants, in laboratories employing HPLC to measure HbA1c, has increased in parallel. The presence of a Hb variant can compromise HbA1cmeasurement and interpretation. In such cases, HbA1c cannot be used to diagnose diabetes or to assess concordance with glycaemic targets. AIM: To establish the number of incidentally identified Hb variants during 10 months of routine HbA1c analysis, and the percentage of HbA1c reports alerting the requesting clinician to the presence of a Hb variant. METHODS: The laboratory database was interrogated to extract all records of HbA1c requests and incidentally identified Hb variants from March to December 2012. RESULTS: A total of 32,636 HbA1c analyses were performed during the evaluation period. Seventy-three Hb variants were identified in a total of 46 patients. In 32.6 % (15 of 46) the haemoglobinopathy status was known prior to testing and 97 % of HbA1c reports communicated the presence of the Hb variant to the requesting clinician. Conclusion : Hb variants may invalidate the results of HbA1c analysis and could result in a missed diagnosis or a misdiagnosis of diabetes or mismanagement of a patient with diabetes mellitus. It is, therefore, imperative that a comment alerting the requesting clinician to the presence of the Hb variant is appended to the HbA1c result.

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41 Fleming RE, Ponka P. N Engl J Med 2012;366:348-359.Iron Cycle. Iron cycle Until several findings on basic research in the field of iron metabolism and erythropoiesis modifed the traditional concepts on anemia: the hormone Hepcidin which controls the iron homeostasis Now we even know how controls the controller . erythroferron, erythroferrone (ERFE), which is secreted by the erythroblasts to maintain adequate iron absorption and efficiency in erythropoiesis. Hepcidin levels are depressed in response Key sites are Enterocytes, , macrophages and liver We now know Liver produces hepcidin the hormone which regulates the iron metabolism Iron absorption from the diet takes place in the duodenum Intestinal iron acquisition is closely regulated, There are no substantial physiologic mechanisms that regulate iron loss bone marrow were erythropoiesis occurs Most of the body iron is associated to hemoglobin in circulating erythrocytes. After their 120 days lifespan RBCs are removed by tissue macrophages. Macrophages play a central role in the organism as they recycle iron after phagocytosis of senescent erythocytes The process is quantitative important only 1–2 mg /day are absorbed while the continuous recycling of iron by macrophages following catabolism of senescent RBC supplies 25–30 mg of iron to BM Hepcidin, which is secreted by the liver is now recognized as the central regulator of iron metabolism. There is now substantial evidence that hepcidin plays the main role in determining how much iron is absorbed from the gut and in influencing the release of iron from sites of storage macrophages inflammation induce hepcidin production; in fact is an acute phase response Hepcidin is secreted into the circulation, where it down-regulates the ferroportin-mediated release of iron from enterocytes, macrophages and hepatocytes  ACD is observed in many conditions associated with chronic inflammation. The main concept is functional iron deficiency : iron in the stores but reduced availability of iron so inefficent erythropiesis; Direct consequence of an imbalance between the erythroid marrow iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypromic mature red cells and reticulocytes Fleming RE, Ponka P. N Engl J Med 2012;366:

42 Camaschella C. N Engl J Med 2015;372:1832-1843.Iron cycle breaking news Figure 1. The Iron Cycle — Mechanisms of Adaptation to Iron Deficiency. The mechanisms of adaptation to iron deficiency are centered on the suppression of the hepatic hormone hepcidin and the tissue hypoxia that develops consequent to anemia. The production of erythropoietin (EPO) by the kidney increases in response to enhanced levels of hypoxia-inducible factor 2α (HIF-2α). As a consequence of the stimulation of erythropoietin, erythropoiesis is increased and hypochromic microcytic red cells are produced owing to the low availability of iron. Senescent red cells are destroyed by macrophages, and their iron is recycled. The increase in erythropoiesis suppresses the production of hepcidin. In mice, this function is mediated by erythroferrone (ERFE), which is secreted by the erythroblasts21 to maintain adequate iron absorption and efficiency in erythropoiesis. HIF-2α increases the expression of the duodenal divalent metal transporter 1 (DMT1) 22 on the apical surface of enterocytes to increase the transfer of dietary iron from the lumen to enterocytes. Hepcidin levels are depressed in response to a reduction in the physiologic signals that maintain its production (e.g., increases in levels of iron-bound transferrin and in the iron content of the liver), 2, 18 to the increased activity of the inhibitor transmembrane protease, serine 6 (TMPRSS6), 23 to the reduction in levels of the activator bone morphogenetic protein 6 (BMP6), and to increased inhibition from erythropoietin-stimulated erythropoiesis. Ferroportin (FPN), which is no longer being degraded because of the low levels of hepcidin, exports the available iron across the enterocyte basal membrane and from macrophage stores 17 to the circulation. Once stores are exhausted, levels of circulating iron decrease, even if absorption from the lumen is increased. Reduced levels of iron in the liver trigger increases in the synthesis of the iron carrier transferrin (referred to as apotransferrin when not bound to iron), further decreasing levels of iron-bound transferrin, the ligand of the transferrin receptor. Consequently, the uptake of iron from transferrin receptors by all cells and organs (e.g., skeletal muscles and the heart) is reduced. Camaschella C. N Engl J Med 2015;372:

43 Webster & Kumar . Models to enhace the value of information from current Laboratory plattforms Clinical Chemistry 2012; 58(3): Model for the typical volume and hemoglobin changes occurring in circulating red blood cells. Reticulocytes enter the circulation with a range of volumes and hemoglobin masses (blue contours). The volume and hemoglobin mass of each cell decrease over time such that the cell's hemoglobin concentration tends toward the mean concentration—the mean corpuscular hemoglobin concentration (MCHC). Most cells are removed from the circulation and recycled before reaching a clearance threshold, which is represented schematically as a dashed line perpendicular to the MCHC line. Reproduced with permission from Weatherall (2). A new promising this mathematical approach based on the model by Higgins Studying the evolution of volume and Hb concentration of red cells since they enter the blood and during their life until they are removed Life span can be modulated ie by iron content In the model red cells are considered a whole dynamic system, this is an extended information that's telling us a lot more about what's happening in vivo, Currently, both Siemens Healthcare Diagnostics and Abbott Laboratories, whose high-throughput analyzers Higgins used in creating the model, are looking into advancing the scientific

44 Conclusions/interpretation HbA1c is likely to be affected by iron deficiency and IDA with a spurious increase in HbA1c values; conversely, non-IDA may lead to a decreased HbA1c value This may lead to confusion when diagnosing diabetes using HbA1c. This review clearly identifies the need for more evidence, especially in identifying the types and degrees of anaemia likely to have significant impact on the reliability of HbA1c

45 Influence of iron deficiency on Hb A1c levels in diabetic patients EInfluence of iron deficiency on Hb A1c levels in diabetic patients E. Urrechaga, U. Unanue, O. Boveda, L. Romero, C. Izcara, L. Salinas Talk Walk Aims: Hemoglobin A1c (HbA1c) is gold-standard for the assessment of glycemic control in diabetic patients. Previous studies have reported that iron deficiency may elevate A1c concentrations, independent of glycemia. This study aimed to analyze the effect of iron status on HbA1c levels in diabetic patients. Methods: 661 patients 336 females (228 menopausal and 108 premenopausal) and 325 males (237 age> 50 years and 88 age < 50 years) were recruited. HbA1c, ferritin, fasting plasma glucose , hemogram and medical history were recorded. Analysis of variance ANOVA and Pearson’s regression were applied. Results: patients were divided according gender, age, glycemia and iron status (normal, latent iron deficiency LID, iron deficiency anemia IDA).All groups presented increasing HbA1c values in parallel with iron deficiency, subclinical and anemia, but the level of significance was not homogeneous in the different groups. Controlled premenopausal women HbA1c in normal iron status and IDA groups P= , between normal and LID, P=0.033. Not controlled premenopausal women Normal group and IDAP< 0.001, normal iron status and LID P=0.019. Controlled menopausal women normal group and IDAP< , LID and IDA P=0.01. Not controlled menopausal women normal group and IDA P= 0.04. Controlled men over 50 years normal and IDA groups P= 0.002, LID and IDA P=0.02. Controlled young men normal group and LID P= 0.03. Conclusion: This study found a positive correlation between iron deficiency and increased HA1c levels. In diabetic patients with IDA should be interpreted with caution, due to the possibility of spurious increment in HbA1c.

46 Female>50 years n (%) Females <50 years Males >50 years Males <50 Normal Iron status 124 (54.4) 45 (41.6) 138 (58.2 ) 61 (69.3) LID 50 (21.9) 41 (38.1) 39 (16.5 ) 27 (30.7) IDA 54 (23.7) 22 (20.3) 60 (25.3 ) 0 (0) TOTAL 228 (100) 108 (100) 237 (100) 88 (100)

47 FPG < 7.0 mmol/L Females>50 y HbA1c % mean (SD) Females< 50 y HbA1c Males> 50 y Males <50 y Normal Iron status 6.0 (0.8) 5.5 (0.9) 6.3 (0.8) 5.5 (1.1) LID 6.2 (0.8) 6.1 (1.4) 6.4 (1.1) (1.7) IDA 7.0 (1.2) 6.4 (1.4) ---- FPG > Males<50 y 7.8 (1.3) 6.6 (1.2) 7.4 (1.3) 7.7 (1.4) (1.2) (1.4) (1.4) (2.1) (1.5) (1.5) (1.8)

48 Females <50 y FPG < 7. 0 mmol/L Females <50 y FPG >7Females <50 y FPG < 7.0 mmol/L Females <50 y FPG >7.0 mmol/L Females >50 y FPG < 7.0 mmol/L Females >50 y FPG >7.0 mmol/L

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