Teste de screening hematologic pentru anemia latentă la populația vârstnică – studiu practic de medicină de laborator

Introduction

Epidemiologic studies have suggested that anemia may be associated with poorer outcomes in a variety of disorders. In many studies, the definition of anemia is the one suggested by a World Health Organization (WHO) expert committee over 40 years ago. Traditionally, anemia in general population has been defined by criteria developed by WHO in 1968.

The World Health Organization traditionally defines anemia in adults as a hemoglobin (HGB) concentration below 130 g/L (13 g/dL) for adult males or below 120 g/L (12 g/dL) for adult non-pregnant females.

These foundational thresholds were established by WHO, and they are still used as standard benchmarks for assessing anemia in individuals and populations. However, these thresholds are generalized baselines. The actual criteria for what are considered normal or low can vary depending on individual factors, including age (older adults may require adjusted interpretations), pregnancy status, altitude (people living at high altitudes often have higher HGB requirements) and specific underlying health conditions (like some chronic diseases, such as kidney disease)⁽¹⁾.

Defining anemia in the elderly

There is some evidence that HGB may decline slightly with age. For this reason, anemia can be defined as a decrease of HGB with 10% from initial normal value in people aged 66 and above. Results from the 1988-1994 United States National Health and Nutrition Examination Survey (NHANES III) indicate that fewer than 3% of adults aged 65 and older have hemoglobin levels below 110 g/L, and therefore most anemia cases among community-dwelling older adults are mild⁽²⁾. Nonetheless, recent evidence indicates that even mild anemia is independently associated with an increased risk of recurrent falls, poorer physical function, hospitalization and mortality in older adults⁽³⁾.

While some studies have reported differential distributions of anemia by age and sex, less attention has been devoted to disparities in anemia by race^(4-6). According to NHANES III estimates, older non-Hispanic Blacks were three times more likely to have anemia compared to older Hispanic Whites (27.8% versus 9%). Similar disparities in anemia prevalence have been observed in other population-based studies of older Blacks and Whites with hematologic parameters in red blood disorders from peripherical blood. The types of anemias and hematology/red blood cells disorders of the peripheral blood are presented in Figure 1.

Anemia in the elderly is an extremely common problem that is associated with increased mortality and poorer health-related quality of life, regardless of the underlying cause of the low HGB. A recent study in Iceland defined mild anemia as a HGB concentration between 10 and 11.9 g/dL in women and between 10 and 12.9 g/dL in men⁽⁷⁾. This cross-sectional analysis provides evidence of HGB in 36.7% of hospitalized patients, and it reveals an association between anemia, poor nutritional status and inflammation⁽⁸⁾. Future research on HGB in the elderly should focus on age-related physiologic changes underlying this condition and whether anemia correction can reduce anemia-associated risks and improve the quality of life⁽⁹⁾.

Materials and method

The purpose of this study was to determine the prevalence and causes of HGB among older adults hospitalized in an academic medical center. The prospective study consisted of 140 consecutive patients over 65 years old (80 males and 60 females), hospitalized for stroke (n=25), chronic hepatic diseases (n=45), complications of atherosclerosis (n=35), chronic cardiac disorders (n=20) and breast carcinoma (n=15), admitted in the hospital after a preliminary ambulatory consult of specialist physicians. A second cohort of 40 patients over 65 (25 males and 15 females) with chronic renal failure (prior to institution of hemodialysis) was also evaluated in the prospective study. Patients with evidence of acute or chronic blood loss were excluded from the study.

Laboratory tests were performed within the first 4-5 days of hospitalization. Clinical diagnoses were established by the physicians caring for the patients. For medical analyses, in the hematologic department, all patients had complete blood count analyzed on Coulter HMX Analyzer with 22 parameters and measurements of serum iron and iron binding capacity done on Vitros 700, Biochemic Analyzer. Reticulocyte count (RET %) was calculated after microscopic analysis with brilliant cresyl blue stained slides (normal ranges in adults: 0.5-1.5%).

To evaluate rate of erythropoiesis, the Reticulocyte Production Index (RPI) was calculated using the formula: [RPI = RET% x HCT patient/45/reticulocyte time maturation], where maturation time (reticulocytes survival days in peripheral blood) was considered 1 day for HCT 36-45%, 1.5 days for HCT 26-35%, 2 days for HCT 16-25% and 2.5 days for HCT<15%⁽¹⁰⁾. The reference interval for RPI in healthy individuals is 1-2; RPI(11).

The card with expected values to normal control group, in average mean, to 40 health patients, at HMX Coulter Hematologic analyzer, showed WBC=9700/dl; RBC=4,500,000/dl; HGB=13.9 g/dl; HCT=42.2%; MCV=93 fl; MCH=30.6 pg; MCHC=33 μg/dl; RDW=15.1 units; PLT=270,000/dl; MPV=8.2 fl; LIMPH=26-28%; MONO=6.3%; GRAN=62-68%.

Complete blood count (CBC), with blood cells, red blood cells and platelets, were measured by assaying one specimen from every patient for HGB, the number of red blood cells (RBC), hematocrit (HCT), mean cellular volume (MCV), Red Cell Distribution Width (RDW) and white blood cells (WBC), on automated hematology analyzer, Coulter HMX. Reticulocyte measurement included the percentage of reticulocytes (%, ret) and the absolute reticulocyte count (#RET = number of red blood cells x % RET).

Results

Abnormal CBC results were common in hospitalized elderly individuals. Only 33 patients (23.58%) had normal results for all hematological parameters in the CBC, while another 16 patients (11.4%) had normal HGB and HCT, but low MCV (average = 72 fL; SD=2.1) or MCH (average = 24.3 pg; SD=1.6). Of the 91 anemic individuals, 63 (69.2%) had mild anemia (HGB decreased, but higher than 10.6 g/dL), while the remaining 28 (30.8%) had severe anemia. All individuals in the group with severe anemia had low RET (mean 1.2%; range 0.5-1.5%) and RPI<1.4, indicating a hyporegenerative type of anemia.

It is very important to apply the criteria very carefully to individuals in order to ensure that the reference interval for hematological indices and biochemical markers of iron metabolism is correct. The controls for this study were selected by the following procedure (Table 1).

Because the total iron binding capacity (TIBC) is considered a sensitive analyte for the detection of iron deficiency, the reliability of this marker was tested using Vitros Fe slide and sample TIBC Calibrators Kit 4. The diagnostic testing was done on principle of the procedure with the Vitros TIBC reagent Fe slide

The most common cause of anemia was anemia of chronic disease (ACD), responsible for almost half of cases. Additional 12% of patients had anemia due to breast cancer, but with a laboratory picture similar to that seen in anemia of chronic disease. One-fifth of cases were due to chronic renal insufficiency and the lack of erythropoietin production. Only 15% of cases were due to iron deficiency, a common cause of anemia in younger individuals.

Anemia of chronic disease, referred to anemia of inflammation, was responsible in 66% of 140 cases of elderly patients. Anemia of chronic disease is more common in the elderly than in other groups. In ACD, the correlation of hematological testes was the following: 22% with normal hemoglobin and hematocrit, 31.5% with only MCV or MCH decreased, 22.5% of patients with hemoglobin (values: 10.8 g/dl for women and 11.08 g/dl for men), 15% of patients with Hb 9.8-10.6 g/dL and 9% of patients with Hb 7.8-9.6 g/dL (mean SD=2.2) and the average number of reticulocytes 1.2%. Production index reticulocytes (PIR) = 1.4 (below 2) in patients with anemia means hyporegenerative anemia, with globular value (GV) = 1.2.

Also, 72.50% of patients with anemia of renal chronic diseases (RKD) associated with inflammations had HTC of 29-25%, normal TIBC (average: 28 µ/dL; SD: 2.5), low RPI in 1.33%, and low IST in 7.62% of cases. In anemia of chronic renal diseases without inflammation, but with ineffective erythropoiesis, HCT was 18-24%, RPI was low (1.21), with normal TIBC (368 microgram/dL; SD: 2.4) and low IST (6.5%). In patients with chronic renal diseases (19.88%), there were registered ineffective erythropoiesis and severe IDA (Graphic 1).

In the cohort of older adult patients with CKD, only three patients (7.5%) did not have anemia. In contrast to the unselected group of elderly patients, in whom anemia was mild in the majority, more severe anemia was typical in the patients with renal failure. Of the total group of 40 patients, more than 75% had hemoglobin below 9 g/dL: 7-8.9 in 23 patients (57.5%) and below 7 g/dL in eight patients (20%). As a group, the dialysis patients had normal RPI (mean: 1.52), low TIBC (mean: 225 mcg/dL) and normal saturation of transferrin (mean: 29.1%).

Anemia of CKD is considered a distinct form of anemia, and it is typical due to decreased EPO production in 20%. As indicator of erythropoiesis, it was used RPI; when RPI

In anemia of CKD, hemoglobin concentration begins to decrease when the level of blood nitrogen (UREA) rises with 30 mg/dL above normal values. This anemia is typical normochromic and normocytic, and the HGB levels are frequently between 7 and 9 g/dl. Poikilocytosis is moderate to severe, with burr cells (echinocytes), abnormal red blood cells with 10 to 30 short, evenly spaced, blunt spicules on their surface, on hematological slide, acanthocytes (or spur cells), which are abnormal red blood cells with irregular, spiked, thorn-like projections on their surface, and schistocytes, which are fragmented, irregularly shaped pieces of red blood cells, are also present (Figure 2).

Megaloblastic anemia (MA) is a macrocytosis with numerous morphologic abnormalities evident in erythrocytes, including macroovalocytes, Howell-Jolly bodies and basophilic stippling. MCV is usually above 110 Fl, RDW is elevated (>15), and RET is normal or decreased. Hyper-segmentation of neutrophils is an early and common indicator of MA, and pancytopenia is typical (Figure 3).

Assays for serum cobalamin and serum folate are needed to document deficiencies of one or both as cause of megaloblastic anemia. Other relevant laboratory tests include assay for methylmalonic acid, antibodies for parietal and gastric cells, bilirubin, lactic dehydrogenase (LDH) and homocysteine. Other causes of macrocytosis include alcoholism, liver disease and hypothyroid disease. Generally, MCV is less elevated than in MA, and elevated RET is a key feature of hemolytic anemia and severe blood loss. An elevated MCV to a normal hemogram should not be ignored, because the patient is especially suspected of alcohol abuse.

In three patients with no obvious cause of anemia, bone marrow aspiration was performed by sternal puncture. Morphologic abnormalities seen on bone marrow examination included bilobed or unsegmented nuclei (pseudo-Pelger-Huet abnormality) and granulation abnormalities in varying forms, and, finally, these patients were diagnosed as having myeloproliferative disorders. These patients also had neutropenia and thrombocytopenia, with blast count below 5% in the peripheral blood and macrocytosis. The algorithm of thrombocytopenia follow is presented in Figure 4⁽¹⁰⁾.

In three patients with no obvious cause of anemia, bone marrow aspiration was performed by sternal puncture. These patients also had neutropenia and thrombocytopenia, with blast count below 5% in the peripheral blood and macrocytosis. Morphologic abnormalities seen on bone marrow examination included bilobed or unsegmented nuclei (pseudo-Pelger-Huet abnormality) and granulation abnormalities in varying form, and, finally, these patients were diagnosed as having myeloproliferative disorders. Hematologic Analyzer Coulter HMX with 22 parameters showed neutrophilia and lymphopenia (Figure 5).

The hematologic analyzer revealed neutrophilia and lymphopenia, while the microscopy exam on peripheral blood smears showed dimorphic pictures, anisocytosis, with poikilocytosis and ovalocytes also presented. Ancillary tests revealed an increased LDH value (710 U/L; n=313-618], glucose = 143 mg/dl (n=75-110), urea = 111 mg/dl (n=25-45 mg/dl), creatinine = 1.3 mg/dl (n=0.5-1.5 mg/dl), triglyceride = 251 mg/dl (n=50-150), iron = 35 ug/dl (n=49-181), serum ferritin = 15 ng/dl (30-150), total bilirubin below 0.2 mg/dl (n=0.5-1.2 mg/dl) and indirect bilirubin = 0.1 mg/dl (n=0.2-0.75 mg/dl); ALT and AST with normal values.

Other laboratory tests that can help discern the causes of hemolysis include the following: quantitative Hb electrophoresis, RBC enzyme assays, flow cytometry, cold agglutinins and osmotic fragility. At the bone morrow examination, on the blood film with May-Grünwald-Giemsa dye, increased erythropoiesis occurred which was megaloblastic in character, volume, with giant band forms and large metamyelocytes. In conclusion, bone morrow was with hyperplasic series erythrocytes approximately 45% and deficiency of erythropoiesis (predominant acidophil and polychromatophilic erythroblasts). Biopsy results from gastric mucosa showed lesions of chronic gastritis, non-atrophic.

Other paraclinical examinations (abdominal echography, barium Rx exam] were normal. B12 vitamin dosage on the Siemens IMMULITE 2000 xpi, Luminescence Measurements – Advanced Technology in peripheral blood showed 150 pg/mL (biological reference interval =193-982 pg/m L) and folic acid dosage into erythrocytes was normal (1632 nmol/L). In three patients with no obvious cause of anemia, bone marrow aspiration was performed by sternal puncture. These patients also had neutropenia and thrombocytopenia, with blast count below 5% in the peripheral blood and macrocytosis. Morphologic abnormalities seen on bone marrow examination included bilobed or unsegmented nuclei (pseudo-Pelger-Huet abnormality) and granulation abnormalities in varying forms, and, finally, these patients were diagnosed as having myeloproliferative disorders.

Discussion

The issues in diagnosing anemia occur when the iron reserves are depleted and no clinical symptoms are found. This is iron deficiency without any clinical symptoms, also known as iron deficiency without anemia. The second problem is represented by chronic inflammation, infections and neoplasms. Quantitative analysis of iron in serum without TIBC and transferrin in the serum is of little importance. Anemia due to iron deficiency (IDA) is microcytic and hypochromic, especially once the HGB level fall below 10 g/L, this being emphasized in 15% of studied cases. Typically, HGB and HCT are somewhat lower than that seen in anemia of chronic diseases (Table 2).

In severe cases, elliptocytes, which are abnormally shaped red blood cells that appear elongated, oval, cigar-shaped, or pencil-like under a microscope, may be accompanied by microcytes and hypochromic cells on blood film. Also, stomatocytes, which are abnormally shaped red blood cells that appear under a microscope, with a slit-like, mouth-shaped or “fish-mouth” center instead of the normal circular pale area, have a slit like appearance in the area of central pallor; many chemical agents may cause this abnormality.

In a longitudinal study in healthy elderly subjects, HGB slowly but predictably declined with aging. An inverse relationship between hemoglobin and all-cause mortality was observed; the lowest risk for mortality occurred at hemoglobin values between 13 and 15 g/dL for women and 14-17 g/dL for men. Anemia is associated with an increased risk for hospitalization and death in community-dwelling older adults⁽¹⁰⁾.

This study confirms that abnormal hematologic parameters are common in elderly individuals (54.2% in men versus 40.6% in women). The most common cause of anemia was chronic disease anemia, which can include a variety of underlying disorders (Table 3).

Myelodysplastic syndromes (MDS) are a group of rare blood cancers where the bone marrow produces abnormal, immature blood cells that fail to mature or function properly.

The peptide hormone hepcidin, secreted by the liver, controls plasma iron concentration by inhibiting iron export from macrophages and decreased absorption of iron from the intestine. Hepcidin is increased in anemia of chronic diseases^(11,12). Hypochromic cells, microcytic anemia due to iron deficiency (IDA) was an uncommon cause of anemia in the elderly patients in the current study (15%), while it is a common cause of anemia in younger individuals⁽¹³⁾.

In this study, the presence of hypochromic cells representing >10% of total RBC was considered functional iron deficiency (ID), when confirmed by low serum iron. All types of anemia can be classified using the reticulocytes values, then the Coombs hematologic test, which can be with positive or negative results, according to the type of anemia (Figure 6).

Various cutoff values for functional iron deficiency are reported in literature, ranging from 2% to 10% hypochromic cells⁽¹⁵⁾. Iron deficiency anemia (IDA) must be differentiated from AC and thalassemia⁽¹²⁾, which can also cause microcytosis. Measuring TIBC is an indirect method of assessing transferrin, which is typically low in ACD, normal or low in thalassemia and high in IDA. Low serum iron (SI), transferrin saturation index (TSI) and serum ferritin (SF) combined with elevated RDW and elevated total iron binding capacity (TIBC) are typically for IDA, and must be differentiated from ACD and thalassemia^(14,15).

Measuring of TIBC is an indirect method of assessing transferrin and provides comparable information. TIBC (or transferrin) are typically performed along with TSI. Taken together, these determinations are useful in the differential diagnosis of many disorders affecting iron metabolism, including hereditary hemochromatosis and iron deficiency anemia. Transferrin and TIBC are typically low-normal or decreased in hereditary hemochromatosis and are increased in iron deficiency⁽¹⁶⁾.

TSI is usually reported along with serum iron and TIBC. Transferrin saturation index indicates the percentage of iron binding sites on transferrin that are carrying iron. Transferrin saturation index is derived from a calculation using the formula: TSI = (SI/TIBC) x 100TSI. The results are reported as percentages. Typical reference intervals for transferrin saturation index are 20% to 30% for males and 15% to 40% for females. TSI is generally considered to be the most sensitive laboratory test for detecting altered iron metabolism in hereditary hemochromatosis. It may be elevated prior to significant deposition of tissue iron. Serum transferrin (ST) levels increase as additional iron is accumulated. A drawback to using TS is that it is dependent on performing both the serum iron and TIBC. Current guidelines from the American College of Physicians include a normal level of transferrin saturation index between 20% and 40%, a cutoff level of TSI >55%, identifying iron overload, and TSI<16%, meaning IDA⁽¹⁷⁾.

The serum ferritin level reflects the amount of storage iron in tissues. This hematologic parameter can’t be used in acute or chronic inflammations, infectious diseases and malignant diseases, being increased as a protein of acute phase (higher in inflammation). RDW is a mathematical expression of size variation used to quantify anisocytosis. The higher the RDW, the greater the anisocytosis. RDW is increased in iron deficiency and tends to be low in thalassemia minor and intermedia. An increased RDW may be an early indication of iron deficiency, where it may precede the onset of microcytosis. In this study, it was revealed that MCV of red cells decreased below normal before hemoglobin in IDA. Because iron deficiency anemia in elderly adults is most commonly a result of iron loss due to chronic gastrointestinal bleeding, the treatment includes stopping the loss and replacing iron with an oral supplement.

Anemia of chronic diseases and iron deficiency anemia may be found in combination, and in these situations, the interpretation of confirmatory tests is difficult. When patients are treated with hemodialysis, bleeding and loss or iron or folate results in anemia due to other causes. In this study, it was presented a case of atypical megaloblastic anemia with iron deficiency anemia in an elderly patient who was admitted in neurological and psychiatry department without a specific diagnostic of hematological disease⁽¹⁸⁾.

Iron deficiency anemia can be present in three forms:

1. Prevalent anemia with low serum ferritin, when ferritin decreases in the early stages of iron deficiency, before changing the hemoglobin concentration, the size of erythrocyte or the amount of iron or high TIBC.

2. Latent anemia with low serum ferritin and low circulating serum iron. TIBC is increased, urine iron is low, and erythrocytes are with low iron, such as in hypochromic anemia.

3. Installed anemia with deficiency of erythropoiesis, low ferritin (

Patients with a ratio erythrocytes target cells/erythrocytes normal cells >0.9, examined in 100 power field on microscopy blood film, were suspicious for beta-thalassemia, and this was discovered in 1% of studied cases. RBC count is higher in thalassemia minor groups compared with iron deficiency anemia. Microcytic, hypochromic and polyglobulia (an abnormally high number of red blood cells) are more evident in thalassemia minor compared with IDA and hemoglobin, and it can be normal, but only MCV and HMC are decreased in thalassemia silent carrier.

Unexplained hematocrit with anemia is generally a condition specific to elderly persons. It appears more commonly with advancing age, being rarely – if ever – encountered in younger adults⁽¹⁹⁾.

Even with the advent of better tests such as serum ferritin, methylmalonic acid and soluble transferrin receptor, a significant proportion of elderly persons with anemia will continue to have unexplained anemia. Bone marrow examination, including staining for hemosiderin, has been recommended in most elderly patients to determine if anemia is due to myelodysplastic syndrome⁽²⁰⁾.

Conclusions

In the elderly, the etiologies of anemia differ sufficiently from those in younger adults, to justify considering anemia in elderly persons as a distinct entity. In this study, anemia of aging was found in 65% of patients overall, and in more than 90% of older adults with chronic renal failure.

The results of these studies suggest that hemoglobin concentration may decline with age, and, therefore, reference intervals specific to geriatric patients are needed.

Most anemic adults are asymptomatic until hemoglobin fall below normal values. For this reason, routine anemia screenings are recommended (for hemoglobin, HCT, MCV, HCV and MCHC) in people older than 65. Anemia of the aging exists and must be managed by laboratory medicine in collaborative with the clinician. The diagnosis of causes must rely on laboratory medicine results along with the clinician’s interpretation.

Abbreviations: ACD=anemia of chronic disease; CFR=chronic renal failure; CBC=complete blood count; EPO=erythropoietin; HGB=hemoglobin; HCT=hematocrit; IDA=iron deficiency anemia; IST=index saturation transferrin; MA=megaloblastic anemia; MCV=mean cell volume; MCH=mean cellular hemoglobin; MCHC=mean cell hemoglobin concentration; RPI=reticulocyte production index; TS=transferrin saturation; RDW=red cell distribution width; RET=reticulocyte count; SI=serum iron; sTR=soluble transferrin receptor; TIBC=total iron binding capacity.

Funds Statement

Grant/funding support: Not applicable.

Financial disclosures: Not applicable.

Acknowledgement: The authors express their appreciation to Dr. Robert Dufour, MD, AACC/NACB Director, for editing the manuscript.

Corresponding author: Aurelian Udriștioiu E-mail: aurelianu2007@yahoo.com

Conflict of interests: none declared.

Financial support: none declared.

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