Red blood cells disorders diagnosis

Red blood cells disorders diagnosisAnemia is strictly defined as a decrease in red blood cell (RBC) mass. Methods for measuring RBC mass are time consuming, are expensive, and usually require transfusion of radiolabeled erythrocytes. Thus, in practice, anemia is usually discovered and quantified by measurement of the RBC count, hemoglobin (Hb) concentration, and hematocrit (Hct). These values should be interpreted cautiously because they are concentrations affected by changes in plasma volume.

Standard measurements for RBC Electronic, automated red blood cell counters provide a considerable amount of information that is useful in determining the severity, pathophysiology, and etiology of anemia.

The hemoglobin (Hgb), measured in grams per deciliter, represents the total amount of hemoglobin in all the erythrocytes in 100 mL of blood.

The hematocrit (Hct) is the percentage of the total blood volume that is composed of erythrocytes.

The mean corpuscular or cell volume (MCV) is measured directly on automated cell counters but can be calculated as MCV (m3 or fL) = Hct (%) 10/RBC count ( 106/L of whole blood).

The mean cell hemoglobin (MCH) is calculated by automated cell counters as MCH (pg) = Hgb (g/dL) 10/RBC ( 106/L).

The mean cell hemoglobin concentration (MCHC) is calculated by automated cell counters as MCHC = Hgb (g/dL) 100/Hct (%).

!!! MCH and MCHC are of limited value because of relatively poor sensitivity for any individual disorders, whereas MCV is extremely useful in classification and determination of the cause of anemia.

In addition to these standard measurements:

The RBC distribution width (RDW/CV) is a ratio of the width of the RBC size distribution curve at 1 Standard deviation from the mean size divided by the MCV. Because this value is a ratio with the MCV as the denominator, it tends to magnify any variation in cell size in patients with microcytosis but is relatively insensitive to mild or early macrocytosis.

A less frequently used value, RDW/SD, is the width of the RBC size distribution curve that encompasses 80% of the erythrocytes in the measured population. This latter measurement is particularly sensitive even to small populations of microcytic or macrocytic RBCs.

automated absolute reticulocyte counts per microliter of blood evaluations of new methylene bluestained peripheral blood smears for the percentage of positive-staining erythrocytes (reticulocytes) give a measure of the number of newly released (generally 1- to 2-day-old) erythrocytes. These newly formed erythrocytes still contain residual ribosomal RNA, which can easily be recognized on supravital staining with new methylene blue. Because ribosomal RNA is lost from the cell within the first 1 to 2 days in circulation and erythrocytes in blood survive an average of about 120 days, reticulocytes account for about 1 to 2% of all erythrocytes in the circulation; a normal, nonanemic adult has 40,000 to 100,000 reticulocytes per microliter of blood.

Automated blood cell counters also provide the total white blood cell (WBC) count, the WBC differential count, and the platelet count. All of this information is useful in assessing the mechanism of anemia.

NORMAL VALUES FOR RED BLOOD CELL MEASUREMENTS MeasurementUnitNormal Range (Approximate)[*]

Hemoglobing/dLMales: 13.517.5

Females: 1216

Hematocrit%Males: 4052

Females: 3648

Red blood cell (RBC) count 106/L of bloodMales: 4.56.0

Females: 4.05.4

Mean cell volume (MCV)fL8199

Mean cell hemoglobin (MCH)pg3034

Mean cell hemoglobin concentration (MCHC)g/dL3036

Red blood cell size distribution width

RDW-CV%1214

RDW-SDfL3747

Reticulocyte count (absolute number)No./L of blood40,000100,000

Reticulocyte percentage% of RBCs0.51.5

*Actual normal ranges for many of these values may vary slightly, depending on factors such as the location and type of laboratory instruments used, altitude above sea level, and patient age.

SELECTED LABORATORY STUDIES THAT ARE USEFUL IN THE DIAGNOSIS OF ANEMIAS If This Is Considered to Be a Possible Cause of a Patient's AnemiaThese Are Potentially Useful Diagnostic Laboratory Tests

HYPOPROLIFERATIVE ANEMIAS

Bone marrow aplasia/hypoplasia or myelophthisisPlatelet count, white blood cell count with differential, bone marrow aspirate and biopsy

MyelodysplasiaBone marrow aspirate and biopsy (including Prussian blue stain of iron), karyotype analysis

Acute leukemiaBone marrow aspirate and biopsy, flow cytometry, immunohistochemical staining, karyotype analysis

MyelofibrosisBone marrow biopsy with stains for collagen (trichrome stain) and reticulin (silver stain)

Iron deficiencySerum iron, TIBC, ferritin, soluble transferrin receptor ( bone marrow iron stain)

Anemia of inflammationSerum iron, TIBC, ferritin, soluble transferrin receptor ( bone marrow iron stain)

Folate deficiencyRed blood cell folate level, serum folate level, bone marrow aspirate

Vitamin B12 (cobalamin) deficiencySerum vitamin B12 level, urine ( serum) methylmalonic acid level, bone marrow aspirate, Schilling tests

HEMOLYTIC ANEMIAS

General measures of hemolysis (intravascular [I] and extravascular [E])Reduction in serum haptoglobin (I > E), presence of urine hemoglobin (I) and/or urine hemosiderin (I), increased serum LDH (I > E) and serum unconjugated bilirubin (I > E)

ThalassemiasHemoglobin A2 level, globin DNA analysis (Southern blotting, polymerase chain reaction, sequencing), globin chain synthesis ratios, hemoglobin electrophoresis (looking for mutated globins with altered electrophoretic mobility, which result in a thalassemia phenotype)

Sickle cell disordersHemoglobin electrophoresis

Autoimmune hemolysisDirect antiglobulin (Coombs) test, quantitation of red blood cell surface antibodies, cold agglutinin titer

Alloimmune hemolysisDirect and indirect antiglobulin (Coombs) test with specificity analysis of eluted antibodies

Traumatic (microangiopathic or macroangiopathic) hemolysisHistory and physical examination findings of hypertension, pregnancy, prosthetic heart valves or vascular grafts, systemic vasculitis, neurologic changes, fever; schistocytes, anemia, and destructive thrombocytopenia; BUN and creatinine; urinalysis; DIC panel; von Willebrand factor multimers, ADAMTS13

Hereditary spherocytosis, elliptocytosis, pyropoikilocytosis, and stomatocytosisPrimarily morphologic diagnoses; specific mutations detected by sequencing spectrin, ankyrin, band 3, or protein 4.1 DNA

Red blood cell enzymopathiesG6PD assay (1-2 months after acute hemolysis), Heinz body preparation, specific enzyme assays

Unstable hemoglobinsHeat/isopropanol denaturation tests, hemoglobin electrophoresis

Paroxysmal nocturnal hemoglobinuriaAcid hemolysis (Ham) or sucrose hemolysis test, flow cytometry analysis of GPI-anchored cell surface proteins (e.g., CD55, CD59)

BUN = blood urea nitrogen; DIC = disseminated intravascular coagulation; G6PD = glucose-6-phosphate dehydrogenase; GPI = glycosylphosphatidylinositol; TIBC = total iron-binding capacity.

RED BLOOD CELL MORPHOLOGIC ABNORMALITIES AS CLUES TO THE DIAGNOSIS OF ANEMIAS Red Blood Cell MorphologyRepresentative Causes of Anemia

MicrocytosisIron deficiency, anemia of inflammation, thalassemia, and rarely, lead poisoning, vitamin B6 deficiency, or hereditary sideroblastic anemias

MacrocytosisPolychromatophilia (reticulocytes), vitamin B12 (cobalamin) or folate deficiency, myelodysplasia, use of drugs that inhibit DNA synthesis

Basophilic stipplingHemolysis, lead poisoning, thalassemia

Target cellsThalassemia; hemoglobins C, D, E, and S; liver disease; abetalipoproteinemia

MicrospherocytesAutoimmune hemolytic anemia, alloimmune hemolysis, hereditary spherocytosis, some cases of Heinz body hemolytic anemias

Schistocytes and fragmented RBCsThrombotic thrombocytopenic purpura, disseminated intravascular coagulation, vasculitis, malignant hypertension, eclampsia, traumatic hemolysis secondary to a prosthetic heart valve or damaged vascular graft, thermal injury (burns), post-splenectomy status

Teardrop cellsMyelofibrosis, myelophthisis (bone marrow infiltration by neoplastic cells)

Sickle cellsHemoglobin SS, SC, or S-thalassemia

Acanthocytes (spur cells)Severe liver disease, malnutrition, McLeod blood group phenotype

Echinocytes (burr cells)Renal failure, hemolysis from malnutrition with hypomagnesemia and hypophosphatemia, pyruvate kinase deficiency, common in vitro artifact

StomatocytesAlcoholism, hereditary stomatocytosis

Bite cells or blister cellsGlucose-6-phosphate dehydrogenase deficiency, other oxidant-induced hemolysis, unstable hemoglobins

Howell-Jolly bodiesPost-splenectomy status, hyposplenism

Intraerythrocytic parasitic or bacterial inclusionsMalaria (parasites), babesiosis (parasites), bartonellosis (gram-negative coccobacilli)

Agglutinated RBCsCold agglutinin disease, in vitro artifact

Rouleaux formationMultiple myeloma, monoclonal gammopathy of undetermined significance

LABORATORY FINDINGS FOR IRON STUDIES IN MICROCYTIC HYPOCHROMIC ANEMIAS AnemiaSerum Iron

50150 g/dLTotal Iron Binding Capacity (TIBC) 250400 g/dLPercent Transferrin Saturation

2050%Serum Ferritin

20350 g/LSerum Transferrin Receptor (sTfR)

928 nMMarrow RE Iron

23+Marrow Ringed Sideroblasts

2080%

Iron deficiency anemiaLowHigh015Low (< 30 /L)HighAbsentAbsent

Anemia of chronic diseaseLowNormal or low515Normal or highNormalNormal or highAbsent

Sideroblastic anemiaHighNormal6090HighNormal or highHighPresent

RE = reticuloendothelial.

Diagnostic algorithm for polycythemia vera (PV) that incorporates mutation screening for JAK2V617F. CBC = complete blood count; JAK = Janus kinase; MPD = myeloproliferative disorder.

Algorithm for the diagnosis of anemias. DIC = disseminated intravascular coagulation; G6PD = glucose-6-phosphate dehydrogenase; HELLP = hemolysis, elevated liver (function tests), low platelets; HUS = hemolytic uremic syndrome; MCV = mean corpuscular volume; RBC = red blood cell; TTP = thrombotic thrombocytopenic purpura.

THE PERIPHERAL BLOOD SMEARWright-Giemsastained peripheral blood is examined for its formed elements: red blood cells (RBCs), white blood cells (WBCs), and platelets. A careful review of the peripheral smear is useful when a patient has an abnormal WBC count or platelet count with or without anemia, has a report of atypical WBCs, or is suspected of having a condition for which the smear is especially useful. Even in other circumstances, it is important for the physician to have an understanding of the morphologic characteristics that underlie the report of an automated complete blood count so that the likelihood of reaching the correct diagnosis is enhanced.

DISORDERS OF RED BLOOD CELLSSize and Color of Red Blood CellsRBCs are normally biconcave discs measuring about 7 m in diameter (Fig. 1), or about the same as the nucleus of a small lymphocyte (Figs. 2). The central pallor of the concavity in a normochromic RBC is about a third of the cell's diameter, and the remaining cytoplasm has a homogeneous pinkish color. The diameter of RBCs is proportional to mean corpuscular volume.

FIGURE 1Normal peripheral blood smear. These normal red cells are biconcave discs. Central pallor is less than a third the diameter of the cell. There are also scattered normal platelets (1000).

FIGURE 2Normal peripheral blood smear. The red cell diameter is about the same size as the nucleus of the small lymphocyte (400).

Excessive numbers of RBCs of various size on a peripheral smear is termed anisocytosis. If RBCs are smaller than the normal range, the term is microcytosis (Fig. 3). Microcytosis occurs in iron deficiency anemia, some cases of anemia of chronic disease, - and -thalassemia, the sideroblastic anemias, and vitamin B6 deficiency.

FIGURE 3Microcytic red cells from a case of thalassemia minor. The red cell diameter is smaller than that of the nucleus of the small lymphocyte (1000).

Hypochromia, in contrast to normochromia, denotes a central pallor greater than a third the diameter of the RBC. This finding appears in iron deficiency anemia, some cases of anemia of chronic disease, and the thalassemias. Hypochromic, microcytic red cells indicate more advanced iron deficiency anemia (Fig. 4).

FIGURE 4Microcytic, hypochromic anemia. The red cell diameter is smaller than that of the nucleus of the small lymphocyte, and the central pallor is greater than a third the diameter of the red cells (400).

Larger than normal RBCs, or macrocytosis, occur in folate and vitamin B12 deficiencies, liver disease, some myelodysplastic disorders, and conditions with reticulocytosis (Fig. 5). In more severe instances of folate and vitamin B12 deficiency, macrocytic RBCs may have an oval shape called macro-ovalocytosis. Reticulocytes, which are RBCs that have just been released from the bone marrow, retain RNA for about 48 hours and are also slightly larger than normal-sized RBCs. Polychromasia, which is evidenced by increased numbers of bluish tinged RBCs, occurs in any condition with an outpouring of reticulocytes from the marrow (Fig. 6).

FIGURE 5Macrocytic anemia. Here the red cell diameter is greater than that of the nucleus of the small lymphocyte (1000). A macro-ovalocyte is indicated by the arrow.

FIGURE 6Polychromasia. A polychromatic red cell is indicated by the arrow. Also present are two red cells with small, round, blue Howell-Jolly bodies (1000).

Shapes of Red Blood CellsPoikilocytosis denotes a peripheral smear with RBCs of various shape. Spherocytic RBCs, which lack central pallor (Fig. 7), appear when antibody coats the RBC surface and in congenital spherocytosis, a heterogeneous condition with cytoskeletal defects in RBCs. The target or Mexican hat cell, which has a central bull's-eye of hemoglobin surrounded by an area of pallor (Fig 8), is typical of liver disease, hemoglobinopathies, and sometimes iron deficiency anemia.

FIGURE 7Congenital spherocytosis. These red cells lack central pallor because of their more spherical shape (1000).

FIGURE 8Target cells. These cells have a central knob of hemoglobin surrounded by an area of pallor and then a peripheral area of hemoglobin (1000).

Sickle cells have a crescentic shape with pointed ends as a result of distortion by elongated curvilinear tactoids of hemoglobin S within their cytoplasm (Fig 9). This irreversible change is regularly observed in the homozygous SS state. Sickled red cells may be seen in patients heterozygous for hemoglobin S under conditions leading to hypoxia.

FIGURE 9Sickle cells. These sickle-shaped cells give sickle cell anemia its name (1000).

Fragmented RBCs with cytoplasmic projections, helmet-shaped forms, and burr cells are called schistocytes. These RBCs are associated with disseminated intravascular coagulation and thrombotic thrombocytopenia purpura, conditions in which RBCs undergo mechanical fragmentation related to microangiopathic hemolytic anemia (Fig. 10). RBCs with a single cytoplasmic projection, termed acanthocytes, are associated with disorders of abnormal lipid metabolism, including abetalipoproteinemia.

FIGURE 10Disseminated intravascular coagulation. Fragmented red cells with helmet shapes, cytoplasmic projections, and burrs are present (1000).

Elliptical RBCs (Fig. 11) occur in hereditary elliptocytosis (ovalocytosis), a heterogeneous condition sometimes associated with hemolytic anemia. Pencil poikilocytosis signifies elliptocytosis on a peripheral smear. A few elliptocytes may be seen nonspecifically in various conditions, including iron deficiency anemia. Burn patients may exhibit RBCs in markedly varying size, including microspherocytes and irregular RBC fragments related to thermal damage.

FIGURE 11Elliptocytosis. Elliptocytes (ovalocytes) are seen in a patient with hereditary elliptocytosis (1000).

Teardrop RBCs (Fig. 13) have a pointed end resembling a tear, hence the other term, dacryocyte. When an RBC must course through a particularly narrowed lumen of a vessel, it may assume this shape. These teardrop forms occur in myelofibrosis, myelophthisic states secondary to bone marrow replacement by tumor or granulomas, and megaloblastic anemia. An RBC with central pallor in the shape of a mouth, termed a stomatocyte, is associated with liver disease but can also appear as a genetic condition Fig. 14).

FIGURE 13Teardrop red blood cells. A teardrop-shaped red cell (dacryocyte) is present in the center of the photo (1000).

FIGURE 14Stomatocyte. Red cells with mouth-shaped central pallor (stomatocyte) are seen scattered throughout this smear (400).

Inclusions in Red Blood CellsA usually single, 1- to 2-m nuclear fragment retained within the red cell, termed a Howell-Jolly body (see Fig. 6), is seen most frequently in patients with an absent or nonfunctional spleen. Small, rounded iron inclusions, termed Pappenheimer bodies (Fig. 15), occur in patients with sideroblastic anemia. On an iron stain, these inclusions are termed siderotic granules. Hemoglobin C crystals are hexagonally shaped rods that occur in the red cell cytoplasm of patients with homozygous hemoglobin C Fig. 16). These crystals may also be extracellular.

FIGURE 15Pappenheimer body. Pappenheimer bodies are the small, rounded inclusions present in the red cell at the arrow (1000).

PlateletsPlatelets are 2 to 4 m in size and normally stain bluish with a granular appearance (Fig. 17). When there is increased turnover of platelets, giant platelets appear (Fig. 18). Increased platelet counts occur after acute hemorrhage, during hemolysis, in neoplastic states, and in the myeloproliferative disorders, including essential thrombocythemia. Thrombocytopenia is seen with the immune thrombocytopenias, hypersplenism, drugs and toxins, and hemopoietic malignancies.

FIGURE 17Normal platelet indicated by the arrow. Platelets are granular, although this is not apparent in this view (1000).

FIGURE 18Giant platelets. They are as large as or larger than the diameter of a normal red cell as shown by the arrow. Giant platelets indicate increased platelet turnover (1000).

Rarely, true thrombocytopenia can be masked by fragments of leukocytes in patients with acute leukemia. An incorrectly low automated platelet count can be recognized by discovery of platelet clumpinga phenomenon occasionally seen in blood collected in ethylenediaminetetraacetic acid (EDTA).

HepcidinHepcidin is a peptide hormone produced by the liver. It was discovered in 2000, and appears to be the master regulator of iron homeostasisThe 25-amino acid peptide of hepcidin is secreted by the liver, which seems to be the "master regulator" of iron metabolism. This binds the iron channel ferroportin, which is located on the basolateral surface of gut enterocytes and the plasma membrane of reticuloendothelial cells, and the degrading ferroportin shuts off the iron transport out of these cells that store it.[4] Ferroportin is also present on enterocytes and macrophages. By inhibiting ferroportin, hepcidin prevents enterocytes of the intestines from secreting iron into the hepatic portal system, thereby functionally reducing iron absorption. The iron release from macrophages is also prevented by ferroportin inhibition, therefore the hepcidin maintains iron homeostasis. Hepcidin activity is also partially responsible for iron sequestration seen in anemia of chronic disease and levels are elevated in people with renal failure.[5]Several mutations in hepcidin result in juvenile hemochromatosis. The majority of juvenile hemochromatosis cases are due to mutations in hemojuvelin, a regulator of hepcidin production.

Hepcidin has shown fairly consistent antifungal activity. Hepcidin's antibacterial activity currently seems to be inconsistent. The current scientific evidence suggests that hepcidin is a central regulatory hormone and its main action is to regulate systemic iron homeostasis.

-thalassemia is one of the most common congenital anemias arising from partial or complete lack of -globin synthesis. Excessive iron absorption is one of the main features of -thalassemia and can lead to severe morbidity and mortality. The serial analyses of -thalassemic mice indicate hemoglobin levels decreases over time, while the concentration of iron in the liver, spleen, and kidneys markedly increases. The overload of iron is associated with low levels of hepcidin. It was found that patients who have -thalassemia also have low hepcidin levels. The observations led researchers to the hypothesize that more iron is absorbed in -thalassemia than is required for erythropoiesis and if the concentration of hepcidin is increasing in the body of such patients might be therapeutic, limiting iron overload. It was demonstrated that a moderate increase in expression of hepcidin in -thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. The data led the researchers to suggest therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with -thalassemia and related disorders.[9]Gardenghi S, Ramos P, Marongiu MF, Melchiori L, Breda L, Guy E, Muirhead K, Rao N, Roy CN, Andrews NC, Nemeth E, Follenzi A, An X, Mohandas N, Ginzburg Y, Rachmilewitz EA, Giardina PJ, Grady RW, Rivella S (November 2010). "Hepcidin as a therapeutic tool to limit iron overload and improve anemia in -thalassemic mice". J Clin Invest. doi:10.1172/JCI41717. PMID21099112.