Chapter 3 Laboratory Evaluation of Red

Blood Cell Disorders

Hu Yiqun

OVERVIEW

• It is helpful to consider the circulating red blood cell and its progenitors as a single functional unit, the erythron. Many of the diseases afflicting the erythron may then be seen to result from the same basic processes

• genetic disorders, disorders of immunity, neoplasia, infectious diseases, nutritional disease, and environmental disorders. The result of the majority of these disease processes is anemia.

• It is important, however, to understand both classification schemes and how they relate, as evaluation of the anemic patient requires a thorough and careful history, physical examination, laboratory investigation, and examination of the peripheral blood smear.

IRON-DEFICIENCY ANEMIA

• If one excludes the clinically silent thalassemia syndromes, iron deficiency is the primary cause of anemia worldwide. To understand the multiple causes of iron deficiency and the consequent biochemical changes that are used clinically as aids in establishing a diagnosis of iron-deficiency anemia

Iron Metabolism

• Iron is supplied exogenously through the diet in two forms: heme iron and nonheme iron. Heme iron is derived from hemoglobin, myoglobin, or other heme proteins in foods of animal origin.

• The absorption of iron is regulated at the level of the mucosal cell, and hence, the tight balance of iron is controlled (Fig.3-1).Under normal circumstances, 5% to 10% of ingested iron is absorbed; in stance of iron deficiency, absorption may increase several fold. Exactly how this process occurs and how it is controlled remain unclear.

Fig.3-1 Fe lost with sloughed cell

Laboratory Findings

• Serum Chemistry The appearance of a microcytic hypochromic

anemia is actually a finding late in the course of iron deficiency.

Decreased iron stores, reflected by decreased serum ferritin, are found early in the course of iron deficiency, as stored iron is liberated to transferrin to maintain adequate delivery of iron to the developing erythroblast.

• Blood

Early in the course of iron deficiency, the anemia appears normocytic and normochromic. As heme synthesis is impaired, hypochromic erythrocytes become evident. Microcytosis usually develops in tandem. Consequently, the MCV, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are typically proportionally reduced.

Poikilocytes that are often seen include ovalocytes, pencil cells, teardrop forms, fragments, and target cells.

The leukocyte count is typically normal, although slight granulocytopenia may be seen. The platelet count is often elevated, commonly as much as twice normal. Values reaching 1000×109/L may rarely be seen, mimicking essential thrombocythmia.

HEMOLYTIC ANEMIA

• The hemolytic anemias are anemias resulting primarily from increased red cell destruction.

• Disorders associated with defective erythropoiesis leading to marked intramedullary red cell destruction are, by convention, also excluded. The hemolytic anemias are therefore the disorders in which premature extramedullary red cell destruction is the primary cause of the anemia.

HEMOLYTIC ANEMIA

• Accellerated Erythrocyte Destruction

Normal Erythroid Senescence

Erythrocytes undergo a number of age-related changes, including diminished intracellular concentrations of glycolytic enzymes, water, and solutes; altered membrane composition; and reduced surface area and volume. These factors undoubtedly have detrimental effects on deformability and functions vital to cellular integrity

Hemoglobin Catabolism

• Senescent red cells liberate hemoglobin, the most of which are catabolized within the reticuloendotheliai system of the liver, spleen, and bone marrow.

• Within the reticuloendothelial system, hemoglobin is dissociated into its three main building blocks: globin, iron, and protoporphyrin.

Fig.3-2 Hemoglobin liberated from senescent erythrocytesis

catabolized in the reticulendothelial system

• The promporphyrin ring is initially cleaved by heme oxygenase, yielding biliverdin and carbon monoxide. This reaction provides the only source of endogenous carbon monoxide in the body.

• Laboratory Findings Elevated bilirubin levels, however, are nonspecific and

may be found as a sign of hepatocellular or biliary disease.

Decreased levels of serum haptoglobin are also found in hemolysis. Although often suggested as an indicator of intravascular red cell destruction, diminished levels of haptoglobin are also found with extravascularly or reticuloendothelial-mediated red cell destruction.

Fig.3-3 intravascular destruction of erythrocytes liberates

hemoglobin,which is bound in circulation by haptoglobin.

• haptoglobin is depleted through hepatic degradation of the haptoglobin-hemoglobin complex, free hemoglobin emerges in the circulation

Pathophysiologic Classification

• A pathophysiologic classification of disorders resulting in shortened red cell survival is presented in Table 3-1.Broad separation is based on the nature of the abnormality

TABLE 3-1 CLASSEHCAHON OF HEMOLYHC DESORDERS

Inherited hemolytic disordersDefects in the erythrocyte membrane

Hereditary elliptocytosisHereditary pyropoikilocytosis

StomatocytosisAbetalipoproteinemia(acanthocytosis)

Enzyme deficiencies of the pentose phosphate pathway

Giucose-6-phosphate dehydrogenaseDefects in globin structure and synthesis

HemoglobinopathiesThalassemias

Unstable hemoglobin diseaseEnzyme deficiencies of the glycolytic pathway

Pyruvate kinaseHexokinase

Glucose-phosphate isomerasephosphofructokinase

AldolaseOther

Defects in nucleotide metabolismPyrimidine 5`nucleotidase

Acquired hemolytic disordersImmune-mediated erythrocyte destruction

Transfusion of incompatible bloodHemolytic disease of the newborn

AutoimmuneInfectious agents

ProtozoansBacteria

Paroxysmal nocturnal hemoglobinuriaTraumatic erythrocyte destruction

MacrovascularMicrovakular

Chemicals/drugs/venomsPhysical agents

Intrinsic and Inherited Red Cell Abnormalities

• Intrinsic erythrocyte abnormalities are commonly grouped as membrane, metabolic, or hemoglobin defects.

• As inherited defects, these disorders may present a number of clinical features not typically seen with acquired red cell abnormalities.

• Chronic hemolytic disorders may be complicated by periods or crises of profound anemia. As previously described, individuals may experience an aplastic crisis.

Hereditary Spherocytosis

• General

Hereditary Spherocytosis is the descriptive name given to a group of inherited disorders with characteristic spheroidal red cell morphology. It is the most common hereditary hemolytic anemia among people of Northern European ancestry.

Approximately 75% of individuals exhibit an autosomal dominant mode of inheritance and cytoskeletal defects, which have been linked cither to defective interaction of spectrin with protein 4.l or to defects in ankyrin(Fig.3-4).

Fig.3-4 Schematic illustration of the erythrocyte cytoskeleton

Hematologic Findings

• The morphologic hallmark of hereditary spherocytosis is the spherocyte, a generally smaller, spheroidal red cell lacking central pallor and consequently appearing densely hemogiobinized. The spherocytes contrast sharply with the increased number of large polychromatophilic reticulocytes indicative of compensatory erythroid hyperplasia.

Fig spherocyte

spherocytes

Hereditary Elliptocytosis

• Hereditary elliptocytosis(HE) is a clinically, genetically, and morphologically heterogeneous group of disorders that are linked through the morphologic finding of more than 20% elliptocytes in the peripheral blood.

Fig elliptocyte

elliptocytes

Hereditary Pyropoikilocytosis

• HPP is a rare congenital hemolytic anemia occurring most commonly in blacks. Red cells in this disorder demonstrate increased susceptibility to thermal injury. In contrast to normal erythrocytes which can withstand temperature to 490C,red cells in HPP begin to fragment and disintegrate at 45 to 46 .℃

• The peripheral blood findings in HPP are striking and nearly pathognomonic. Poikilocytosis is unmatched by any other disorder. Erythrocytes appear as if they are disintegrating, with fragments, numerous microspherocytes, and wisps of red cell membrane.

Hereditary Stomatocytosis

• Clinically, hereditary stomatocytosis varies in its expression. Most individuals are asymptomatic or exhibit only mild anemia because of brisk compensatory erythroid hyperplasia.

• The morphologic hallmark of hereditary stomatocytosis is the stomatocyte a cell with a slitlike or “fish-mouth” area of central pallor. In suspension, these cells appear uniconcave or bowl shaped.

Fig. Stomatocyte

stomatocytes

Glucose-6-Phosphate Dehydrogenase Deficiency

• Glucose-6-phosphate dehydrogenase (G6PD), the initial and rate-limiting enzyme of the HMP pathway, accounts for more than 99% of all cases of hemolytic anemia attributable to enzyme deficiencies of this pathway.

• The structure and synthesis of G6PD are controlled by genes on the X chromosome. Numerous variants of the enzyme are found, based on a variety of physiochemical properties.

• the clinical expression of G6PD deficiency is largely limited to hemizygous males and homozygous females.

• Three distinct clinical syndromes are recognized: acute hemolytic anemia induced by oxidant stress, chromic hereditary nonspherocytic anemia, and favism. Although favism is an idiosyncratic reaction occurring on exposure to the fava bean in a subset of G6PD-defect individuals, the severity of the enzyme defect is the essential element in defining the two remaining clinical syndromes.

Pyruvate Kinase Deficiency

• Pyruvate kinase (PK) deficiency is the most common enzyme deficiency of the EM pathway. Numerous dysfunctional variants of the PK enzyme are known to exist, affecting, for example, enzyme activity, stability, or kinetic rate.

• Red cell morphology is often unremarkable. In more severe cases, echinocytes may be present, which become even more plentiful after splenectomy. Diagnosis of PK deficiency is based on enzymatic assays demonstrating quantitative or qualitative abnormalities.

Autoimmune Hemolytic Anemia

• Autoimmune hemolytic anemias are caused by autoantibodies self-induced antibodies directed at one's own red cells. The autoimmune hemolytic anemias are divided into two broad categories based on the class and the corresponding thermal activity of the responsible antibody: warm (IgG) and cold (IgM) autoimmune hemolytic anemias. Both warm and cold autoimmune hemolytic anemias may be further stratified into idiopathic and secondary forms.

Warm Autoimmune Hemolytic Anemia

• The warm autoimmune hemolytic anemias are predominantly mediated through the binding of IgG antibodies to the erythrocyte's surface, resulting in splenic sequestration and destruction.

• Red cells are typically coated with IgG occurring alone or in combination with varying amounts of intact C3.Rarely,the red cells exhibit only C3 on their surface. The responsible antibody may be serologically nonspecific

Cold Autoimmune Anemias: Cold Agglutinin Disease

• Primary Cold Agglutinin Disease.

Primary cold agglutinin disease is marked by episodic painful acrocyanosis induced by agglutination of red cells in the peripheral circulation on exposure to cold. It is typically a disease seen in the elderly and is managed largely by avoidance of cold environments.

• Secondary Cold Agglutinin Disease.

Secondary cold agglutinin disease is associated with a variety of infections, the prototype being mycoplasma pneumonia and infections mononucleosis. Cold agglutinin titer are often used as a supportive test in establishing apresumptive diagnosis of mycoplasma pneumonia liters rise within l to 3weeks after the onset of symptoms liters typically reach l:640, although on occasion they may reach values exceeding l:1,OOO and may be associated with symptomatic anemia.

• Paroxysmal Cold Hemoglobinuria Paroxysmal cold hemoglobinuria(PCH)is a

rare acquired hemolytic anemia that may be seen as a complication of syphilis or a recent viral illness, particularly measles, mumps, or infectious mononucleosis. It is the result of an anti-P autoantibody occurring in individuals expressing the nearly universal P1 and P2 red blood cell antigens.

• Hemolysis occurs only after the blood has been chilled and then warmed to 37 .Bound ℃IgG can be detected in the DAT only when performed at temperatures less than 24 ; at ℃body temperature, only C3 is detected. Clinical hemolysis is precipitated by exposure to cold and results in symptoms similar to those seen in acute intravascullar hemolysis associated with ABO incompatibility.

Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria(PNH)is an uncommon cause of hemolysis with a pathophysiology uniquely distinct from the hemolytic disorders previously discussed. PNH is an acquired clonal hematopoietic stem-cell disorder

Two of the most important proteins lacking on the membrane are decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59), which ordinarily protect cells from complement-mediated attack.

• Traditional diagnosis of PNH has relied on the demonstration of complement-mediated hemolysis. In the acidified serum lysis test (Ham test), PNH red cells will lyse in acidified serum, which activates complement.

• The median survival in PNH patients is between l0 and I5 years from the time of diagnosis. The natural course of the disease is often prolonged but marred by episodic bouts of hemolysis and a number of often facial complications

• CD55, CD59, CD16, and CD66b are shown to be much more practical and reliable for diagnosis of PNH. Flow cytometric analysis of granulocytes or red cells incubated with monoclonal antibodies appears to be sensitive and specific and can give information regarding the proportion of abnormal cells.

• A small number of patients may develop AML However, the incidence of AML in PNH appears to be similar to the risk of aplastic anemia patients developing AML Therefore it is possible that the PNH clone by itself may not be responsible for the increased risk of developing AML but rather that the aplastic anemia predisposes to clonal hematopoietic disorders such as AML

Thalassemia And Hemoglobinopathy Syndromes

• Structure and Function of Normal Hemoglobin Molecules

Normal human hemoglobin molecules consist of two Pairs of polypeptide (globin)chains ,each of which carries a heme portion.

The hemoglobins that are produced at various stages of embryonic, fetal, and adult life are listed in Table 3-3. Each of these hemoglobins consists of a pair ofα,or αlike(ξ)globin chains and a pair of β-or β-like globin chains (δ,γ,ε).

TABLE 3-3 THE NORMAL HUMAN HEMOGLOBENS

Hemoglobin Globin Chains Major Source Embryo Neonate Adult

% % %

Gower1 ζ2/ε2 Yolk sac 50 0 0

Gower2 α2/ε2 Yolk sac 25 0 0

Portland ζ2/γ2 Yolk sac 25 0 0

Hb F α2/Gγ2a Liver,spleen 0 75 ＜ 1

α2/Aγ2

Hba α2/β2 Bone marrow 0 25 97

HbA2 α2/δ2 Bone marrow 0 ＜ 1 3

• Classification Nomenclature

Most hemoglobinopathies are inherited abnormalities. A few result from de novo mutation, and there is a small but fascinating group of acquired defects mimicking α thaiassemia and persistence of fetal hemoglobin .

• Because many of these disorders are common, combinations occcur as a result of compound heterozygosity for abnormal genes. Furthermore, structural defects, such as Hb Lepore, or Hb E may cause an imbalance in globin-chain production and hence a thalassemia syndrome.

• Normal adult hemoglobin is designated Hb A, and fetal hemoglobin as Hb F. As hemoglobin variants were discovered, they were given letters of the alphabet, beginning with Hb C .

Laboratory Approach

• The diagnosis of all hemoglobinopathies eventually lies in the laboratory. The number and degree of sophistication of the tests needed depend on the clinical needs of the patient population and the interests of the investigator.

• Blood Count and Red Cell Morphology Three types of abnormal blood counts are

associated with clinically significant hemoglobinopathies:

●. Microcytic hypochromic red cells often without significant anemia (as in thalassemia minor);

●. Ahemolytic anemia (as in sickle ceil anemia and Hb C disease);

●.A combination of the two (as in thalassemia intermedia and major)

Fig. Red Cell Morphology

Microcytic hypochromic red cells

Target cells

• Red cell morphology may provide a general or specific indication of a hemoglobinopathy. The latter include sickle cells, Hb C crystals, and the bizarre-shaped red cells seen in Hb SC disease.

• A suspension of red cells containing Hb H when incubated at 37 for l hour with a few ℃drops of 1% citrate/saline solution of brilliant cresyl blue will contain many round, small-blue, stained inclusions of precipitated Hb H, giving the cell a golf-ball appearance.

• Solubility Test

The relative insolubility of deoxygenated Hb S compared with other hemoglobins is the basis of a simple test for its presence.

A positive solubility test indicates only the presence of Hb S or any hemoglobin containing the Hb S mutation (such as Hb C-Harlem, a doubly substituted hemoglobin variant)but does not provide a quantitative measure and does not therefore distinguish sickle cell trait, sickle cell anemia, or any combination of Hb S and another hemoglobinopathy.

Electrophoresis

• Electrophoresis on various media is usually the first step in demonstrating and specifying a hemoglobin variant. Four commonly used techniques are outlined here electrophoresis at alkaline pH, acid electrophoresis, isoelectric focusing(IEF), and electrophoresis of separated globin chains.

• Alkaline electrophoresis is a popular method used in the evaluation of hemoglobinopathies .It is also often called cell1dose acetate electrophoresis, as this is the support medium used by many laboratories.

• The amino acid substitution in many hemoglobin variants alters their net charge and thus their electrophoretic mobility (Fig.3-5).

Fig.3-5 Hemoglobin electrophoresis at pH8.6

• EF is another popular method used by laboratories that have a large-number of specimens or very small sample volumes, such as laboratories that perform newborn screening. This electrophoretic method utilizes carrier ampholytes, small proteins that are able to carry both current and pH(Zwitterions). These compounds have MWs of 300 to 1,000 and are used in mixtures of 50 to l00 individual compounds.

• Quantitation The quantitation of normal and variant

hemoglobins is often of diagnostic importance. With a well-prepared electrophoretic pattern,"eyeballing” the plate is often as useful as more specific quantitation. Estimating the relative proportions of Hb S and Hb A can often be done in this manner.

• For some hemoglobins, specific methods of quantitation are readily available and simple to perform. Hb F can be quantitated base on its resistance to alkali;toluene may be used in place of carbon tetrachloride called for in this method. Hb A2 can be measured by elution from an anion-exchange column , Hb Barts and Hb H by elution from a cation-exchange column.

High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC)is a method that has been available hr many years but not routinely used in the analysis of hemoglobinopathies

These instruments are approved by the U.S. Food and Drug Administration for the measurements of Hb S, A2, and F but also give useful information for other hemoglobin variants that may be present(Fig 3-6).These instruments generally utilize a weak cation exchange column.

Fig. 3-6 HPLC

A, Normal adult;

B, Neonatal specimen with high Hb F(a hemoglobin variant is also present));

C, -thalassemia triat with elevated Hb A2;

D, Hb S triat.