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.