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Laboratory Diagnosis of Anemia: Are the Old and New Red Cell Parameters Useful in Classification and Treatment, How?

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Laboratory Diagnosis of Anemia: Are the Old and New Red Cell Parameters Useful in Classification and Treatment, How? International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology REVIEW ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY Laboratory diagnosis of anemia: are the old and new red cell parameters useful in classification and treatment, how? M. BUTTARELLO Clinical Pathology Laboratory, SUMMARY Hospital of Adria, Adria (RO), Italy Introduction: Anemia is a global problem affecting the population in both developing and developed countries, and there is a debate on correspondence: Mauro Buttarello, Clinical which hemoglobin level limit should be used to define anemia in Pathology Laboratory, Hospital general population and particularly in the elderly. We present of Adria, ULSS 19 Piazza degli herein a laboratory approach to diagnosing the possible causes of Etruschi, 45011 Adria (RO), anemia based on traditional and new erythroid parameters. In this Italy. Tel.: 00393407719537; E-mail: [email protected] article, we provide practical diagnostic algorithms that address to differential diagnosis of anemia. Based on both morphological and kinetic classifications, three patterns were considered: microcytic, doi:10.1111/ijlh.12500 normocytic, and macrocytic. Methods: Main interest is on the clinical usefulness of old and new accepted for publication 4 April parameters such as mean cell volume (MCV), red blood cell distri- 2016 bution width (RDW), hypochromic and microcytic erythrocytes, Keywords immature reticulocyte fraction (IRF), and some reticulocyte indices Anemia, red cell indices, reticu- such as reticulocyte hemoglobin content and mean reticulocyte locytes, immature reticulocyte volume. The pathophysiologic basis is reviewed in terms of bone fraction, reticulocyte hemoglo- marrow erythropoiesis, evaluated by reticulocyte count (increased bin content, mean reticulocyte volume or normal/decreased) and IRF. The utility of reticulocyte indices in the diagnosis of iron-deficient erythropoiesis (absolute or func- tional) and in monitoring of response to treatment in nutritional anemia (iron and cobalamin) was also investigated. Results: For each parameter, the availability, the possible clinical applications, and the limitations were evaluated. A discussion on intraindividual biological variation and its implication on the use- fulness of conventional reference intervals and in longitudinal monitoring of the patients was also reported. Conclusion: Red cell parameters and reticulocyte indices play an essential role in differential diagnosis of anemia and in its treat- ment. More efforts are needed in harmonizing parameters whose results are still too different when produced by different analyzers. © 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 1 2 M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA underestimate Hct in hypochromic red cells. In this last INTRODUCTION condition, the use of Hct rather than the more accurate Anemia is when blood hemoglobin (Hb) concentra- measured Hb can overestimate the diagnosis of anemia tion is below the lower limit of the reference interval in subjects with iron deficiency (7). Also, the optical- stated for age, sex, race, and altitude. The commonly based instruments with isovolumetric sphering provide a accepted lower limits for adult population are the falsely elevated Hct when they analyze sickle red cells WHO criteria suggested by an expert committee that cannot be sphered. A typical artifactual dissociation nearly 50 years ago: 130 g/L in men and 120 g/L in with all the automated analyzers between Hb result women, without the distinction between age and race (usually correct) and Hct (underestimated) is the pres- (1). The definition of anemia has attracted interest in ence of red blood cell (RBC) agglutinates. Because the recent years because epidemiologic studies suggest upper volumetric threshold to consider cells as RBC is that anemia may be associated with poor prognosis in between 200 and 300 fL according to the analyzer, the many different diseases, particularly among aged peo- large RBC clumps are not counted as RBC. This causes a ple. A recent large population survey based on WHO spuriously low RBC count and low Hct. In contrast, Hb criteria (NHANES-III) (2) showed that nearly ten per- is measured after RBC lysis and is unaffected by agglu- cent of men and woman older than 65 years were tinins. As a consequence, MCHC is abnormally high, anemic. These percentages rose to 26% in males and usually greater than 360 g/L. Moreover, also Hb can be 20% in females older than 85. It is not clear whether erroneously overestimated, although more rarely, in the difference in lower limits, justified in androgen- subjects with severe hypertriglyceridemia or receiving dependent age, should be continued after 65 years of an intravenous administration of fat emulsions, or with age. Many of these subjects were apparently healthy, high WBC counts, due to the excessive turbidity. and in most cases, clinical investigations did not uncover a specific cause of anemia. These results sug- OLD ERYTHROCYTE INDICES gest that somewhat lower limits than ‘normal’ might be used in the elderly. Nevertheless, the too easy Maxwell Wintrobe 80 years ago proposed the anemia acceptance of mild anemia as physiologic in the classification based on the mean cell volume (MCV) elderly runs the risk of ignoring an underlying dis- obtained by Hct/RBC ratio from the measurement of ease. There is a debate on which hemoglobin lower spun Hct and manual hemocytometric RBC count. The limit should be used to define anemia in general pop- MCHC was calculated as Hb/Hct ratio, where Hb was ulation and particularly in the elderly (3). Two differ- also based on manual measure (8). Of these two ent, relatively recent, large databases (NHANES-III indices, which allowed to classify the anemia as micro- and Scripps-Kaiser) (4, 5) in which the hemoglobin cytic, normocytic, and macrocytic based on MCV value determination was carried out with standardized auto- and hypochromic, normochromic or hyperchromic mated methods obtained a good agreement and new based on MCHC, only MCV has survived as key lower limits are proposed (6). It would seem that parameter for the classification of anemia with auto- these limits (5% of normal distribution) are 137 g/L mated hematology analyzers (Figures 1–3). With the in white men (20–59 years) and 132 g/L for men after data collected on a cell-by-cell basis, modern instru- the age of 60; the corresponding value for women is ments generate a histogram of erythrocytes size distri- 122 g/L independently of age. In Afro-Americans, bution. From this histogram, an index of these limits are lower: 129 g/L in younger men and heterogeneity referred to as red cell distribution width 127 g/L in men older than 60, while the correspond- (RDW) can be determined. This is almost always ing value for women is 115 g/L at all ages. expressed as percentage coefficient of variation and, For many practical approaches, a decrease in hemat- less frequently, as standard deviation. The possibility ocrit (Hct) is considered equivalent to a decreased of a quantitative, nonsubjective measurement of an hemoglobin concentration, but this simplification is not anisocytosis index has rewakened interest. Bessman always correct. All hematology impedance-based analyz- et al. (9) in the early 1980 proposed a classification of ers falsely overestimate Hct in erythrocytes with a high anemia based on both MCV and RDW: homogeneous mean hemoglobin concentration (MCHC) and (with normal RDW) and heterogeneous (with © 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA 3 MICROCYTIC ANEMIA NORMOCYTIC ANEMIA (MCV < 80 fL) (MCV 80 - 100 fL) Reculocyte count Reculocyte count (20 - 100 X 109/L) (20 - 100 X 109/L) Normal or decreased Increased Normal or decreased Increased Reculocyte hemoglobin % Hypochromic RBC Reculocyte hemoglobin - Anemia of renal Markers suggesve of Markers not suggesve of content / reculocyte volume content / reculocyte volume insufficiency hemolysis: Haptoglobin, hemolysis (< 28 pg / < 100 fL) LDH, bilirubin (< 28 pg / < 100 fL) % Microcyc RBC - Anemia of chronic diseases - Acute infecons - Primary bone marrow disorders - Aplasc anemia Decreased Increased Decreased Peripheral blood smear Blood loss - βthalassemia trait - Early response to iron - Iron deficiency treatment Other findings (not Fragmented erythrocytes - Chronic diseases - βthalassemia diagnosc): consider - Membrane defect: PNH - Enzymopathy (G6PDH, PK) - Hemoglobinopathy Microangiopathy Spherocytosis % Micro / % Hypo rao Direct Coombs test posive Direct Coombs test negave Decreased Increased - Autoimmune hemolyc - Hereditary spherocytosis anemia - Cold agglunin disease Iron deficiency β-thalassemia trait Biochemical markers: HbA2 /hemoglobin analysis ferrin, %TSAT, … Figure 2. Diagnostic algorithm for normocytic anemia - Absolute iron deficiency - Funconal iron deficiency The mean hemoglobin content (MCH), which is - Chronic diseases strongly correlated with MCV, is calculated as Hb/RBC ratio. These last measurements with automated ana- Figure 1. Diagnostic algorithm for microcytic anemia. lyzers are more accurate and precise than MCV, which is derived by a direct measurement of a single cell size using different analytical methods (impedance increased RDW) erythrocyte population. The former with or without hydrodynamic focusing, or light scat- includes hypoproliferative anemia, marrow aplasia, tering). Moreover, MCV, different from MCH, is and thalassemia heterozygosity; the latter includes
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