Hematologic Disorders

Hongbo Yu

INTRODUCTION

This chapter covers blood diseases encompassing pathology of the formed elements of blood (red cells, white cells, and platelets), plasma cell neoplasms, hemorrhagic and thrombotic diseases, and lastly metabolic disorders that have a major impact on hematologic parameters.

RED BLOOD CELL DISORDERS

ANEMIAS

□ Definition

Anemia is a reduction in hemoglobin (Hb) leading to decrease in oxygen supply to peripheral tissues. Normal Hb range is established by population studies, but range should be adjusted for different age groups, especially for children, and levels are lower in women and in African Americans. There is some debate whether people of older ages have physiologically lower Hb levels. Most likely lower values reflect underlying pathology. Hb values are more accurate than hematocrit (Hct) values, because Hb is measured directly by automated analyzers, whereas the Hct is a calculated value.

□ Diagnosis

There are many ways to classify anemias, but the differential diagnosis of anemia can be narrowed by using the RBC size, as reflected in the mean corpuscular volume (MCV) and the reticulocyte count. See Figure 11-1.
In addition, insight into mechanism and etiology complements the differential diagnosis.
Onset of anemia has a great impact on symptoms and diagnosis.

Figure 11-1. A-C: Workup of anemias based on the mean corpuscular volume (MCV).

Figure 11-1. (Continued)

Onset

Acute
- ▼ Bleeding
- ▼ Hemolysis
- ▼ Acute bone marrow disease (e.g., leukemias)
Chronic
- ▼ Deficiencies: iron (most common), folic acid, vitamin B₁₂, nutritional
- ▼ Congenital (hemoglobinopathies, hereditary spherocytosis)
- ▼ Neoplasia, especially metastatic or hematologic malignancies
- ▼ Renal disease
- ▼ Chronic inflammatory disorders
- ▼ Many others

When to Suspect Anemia

Children
- ▼ Young child who fails to thrive and is not as active as expected for age.
- ▼ Anemia detected at ages 3-6 months suggests a congenital disorder of Hb synthesis or structure.
Adults
- ▼ Nonspecific symptoms and signs such as weakness, dizziness, progressive lack of energy, pallor, and shortness of breath in the absence of serious heart or lung disease (overt congestive heart failure [CHF] may develop as a consequence of severe anemia)
- ▼ Protracted GI or vaginal bleeding
- ▼ A family history of anemia
- ▼ Jaundice or red urine

□ Laboratory Findings

Initial laboratory investigation should include a complete CBC with a reticulocyte count and examination of the peripheral blood smear (PBS). The reticulocyte count reflects bone marrow response to anemia.
Once the suspicion of anemia is confirmed by finding a reduction in Hb (the RBC count may be normal or even higher in certain conditions, such as thalassemia trait), the type of anemia must be determined by subsequent laboratory investigations, based mostly on the MCV, and subdivided by pathophysiology.
RDW provides a useful measurement of the variation in size of RBCs, indicating the presence of anisocytosis when elevated.
Once anemia is documented, subsequent investigations depend on the type of anemia suspected based on indices and the reticulocyte count (see Figure 11-1). More complex laboratory tests or bone marrow biopsy may be indicated to ascertain its precise etiology.
Various types of anemias are described subsequently.
- ▼ Microcytic
- ▼ Macrocytic
- ▼ Normocytic
- ▼ Aplastic
- ▼ Hemoglobinopathies
- ▼ Hemolytic anemias
- ▼ Sickle cell
- ▼ HbC, HbD, HbE diseases
- ▼ Thalassemias

MACROCYTIC ANEMIAS

□ Who Should Be Suspected?

A patient with macrocytic anemia, hypersegmented neutrophils on PBS, and symptoms of malabsorption, poor diet, chronic hemolysis without folate supplementation, chemotherapy, or hypothyroidism. Folate deficiency is seen with alcoholism; in third-world countries, it may be associated with sprue-like syndromes. Vitamin B₁₂ (cobalamin) deficiency increases in incidence with aging and should be searched for even in the absence of anemia in the elderly with neurologic deficits. Cobalamin and folic acid deficiencies often coexist. Other causes of macrocytic anemias are liver cirrhosis, myelodysplastic syndrome (MDS), azidothymidine (AZT) therapy for AIDS, Down syndrome, and normal newborns.

□ Laboratory Findings

Laboratory investigation of macrocytic anemias must differentiate between macrocytic anemias without megaloblastosis and true megaloblastic anemias resulting from vitamin B₁₂ and/or folate deficiency. Megaloblastic anemia is a morphologic definition based on bone marrow examination. B₁₂ deficiency may be the result of pernicious anemia (PA) (lack of intrinsic factor [IF]) or may have other etiologies.

CBC:
- ▼ Anemia with oval macrocytes, poikilocytosis and anisocytosis, and small teardrop cells
- ▼ High RDW
- ▼ Thrombocytopenia and leukopenia in severe cases
- ▼ Hypersegmented polymorphonuclear cells and giant metamyelocytes in megaloblastic anemias
- ▼ Reticulocyte count: inadequate for the degree of anemia
Serum or RBC folate and serum cobalamin are obtained if another etiology is not obvious. The specific metabolites methylmalonic acid and homocysteine accumulate in these deficiencies; they are additional assays and may help discriminate between cobalamin and folate deficiencies and other etiologies for macrocytic anemias. These assays, as well as RBC folate, are more expensive and should be reserved for patients with borderline folate or cobalamin values but strong suspicion of one or the other.
Serum cobalamin if <200 pg/mL is consistent with vitamin B₁₂ deficiency.
Serum folate if <2 ng/mL is consistent with folate deficiency.
Serum or urine methylmalonic acid if increased confirms vitamin B₁₂ deficiency. It may be normal in folate deficiency.
Homocysteine (total) if elevated is compatible with either cobalamin or folate deficiency. If normal, both can be excluded.
Documentation of cobalamin deficiency does not establish the diagnosis of PA, an autoimmune disease characterized by deficiency of IF and lack of HCl gastric secretion. PA was traditionally diagnosed by the absorption of orally administrated radiolabeled cobalamin, the Schilling test (no longer available in the United States). In its absence, the assays mentioned above are helpful, but not specific for PA. Fifty percent to 70% of PA patients will have positive serum anti-IF antibodies, thus documenting PA (100% specificity). The patients who are negative for IF antibodies cannot be distinguished from non-PA cases of cobalamin malabsorption but will respond to oral vitamin B₁₂ if not PA. Antiparietal antibodies are less sensitive or specific. Recently, chronic Helicobacter pylori infection has been implicated in the etiology of PA and the lack of IF.
- ▼ Bone marrow aspirate (indicated in very selected cases) may reveal marked red cell hyperplasia and megaloblastic maturation in both vitamin B₁₂ and folate deficiencies. Otherwise, it may uncover other reasons for macrocytosis, such as MDS.
- ▼ Serum LDH and indirect bilirubin are elevated in folate and vitamin B₁₂ deficiency.

□ Limitations

In the presence of coexisting iron deficiency, MCV may not be elevated, even in cases of overt folate or cobalamin deficiency.
Low cobalamin levels develop during pregnancy.
One hospital meal may normalize serum folate level (but not RBC).

Methylmalonic acid increases in renal insufficiency.

MICROCYTIC ANEMIAS

□ Who Should Be Suspected?

Suspect iron deficiency if the following are present:

History of GI, vaginal, or massive, repeated urinary bleeding
Microcytosis, hypochromic
Poor diet

□ Laboratory Findings

First line of investigation: serum ferritin. It has a specificity of 98% but a sensitivity of only 25% for a 12 µg/L threshold. Because ferritin is an acute-phase reactant, it may be normal or even increased despite iron deficiency when the patients have serious medical problems, such as chronic inflammatory conditions and active liver disease. As a consequence, a normal ferritin value does not exclude iron deficiency. Very low values are definitely diagnostic, iron deficiency is confirmed, and there is no need to obtain serum iron and total iron-binding capacity (TIBC). Investigation of etiology (history, stool examination for occult blood, GI investigation, pelvic and rectal examinations) is mandatory.
If serum ferritin is normal or borderline, serum iron and transferrin (usually reported as TIBC) are the next assays to be ordered.
If the serum iron is very low and TIBC is elevated (with the ratio of serum iron divided by TIBC <16%), diagnosis is confirmed.
Normal serum iron and TIBC: iron deficiency is excluded in most cases.
Low serum iron, low TIBC: most likely anemia of chronic disease; workup underlying etiology.
High serum iron, normal TIBC: the most likely diagnosis is thalassemia.
Two additional blood tests: the soluble transferrin receptor and the reticulocyte Hb content. They are optional. When used in conjunction with ferritin, these tests improve further our ability to accurately diagnose iron deficiency. However, they are not widely used.
As a last resort, if the diagnosis is still in doubt, bone marrow aspirate/biopsy is performed for Prussian blue stain. If it is negative, iron deficiency is definitely present.

NORMOCYTIC ANEMIAS

□ Who Should Be Suspected?

Patients with anemias secondary to an underlying nonhematologic disease (also known as “anemias of chronic disease” [ACD]). The term “anemia of chronic inflammation” may be used too, but it does not cover all situations (see the following paragraph). The most common conditions leading to ACD:

Anemia of chronic inflammation (infections, rheumatologic diseases) is the prototype of normocytic anemias. The red cells may occasionally be borderline microcytic.
The etiology of anemia of chronic renal failure is in part the reduced production of erythropoietin. Additional factors are shortened red cell survival and frequent bleeding.
Anemia in cancer patients is a common, multifactorial finding. Microangiopathic hemolytic anemia and myelophthisic anemia may be an additional feature resulting from disseminated carcinoma.
Aplastic anemias (AAs) can be congenital or acquired. In AA, hematopoiesis fails. All blood lineages are decreased (pancytopenia), with the possible exception of lymphocytes. Pure red cell anemia is a variant of AA in which only, or mostly, the red cell line is affected.

□ Laboratory Findings

CBC: Moderate anemia, normal to slightly reduced MCV in inflammatory conditions; normal red cell morphology, with only mild variation in RDW. In anemia of chronic renal failure, burr cells can be seen on the PBS.
Inadequate reticulocyte response.
Increased serum ferritin, reduced serum iron and TIBC.
Serum erythropoietin is inadequate for the level of anemia, especially in renal failure.

APLASTIC ANEMIA (AA)

□ Etiology

AA may be acquired or congenital (Fanconi anemia; see below). More than 50% of the acquired cases are idiopathic, most likely due to an autoimmune mechanism that destroys or suppresses the hematopoietic stem cell via cytotoxic T lymphocytes and the cytokines they produce.
Other cases may result from drugs, such as chemotherapy, anticonvulsants, and many more. It is essential to obtain a history of drug or toxin exposure.
- ▼ Immunologic disorders such as graft versus host disease.
- ▼ Thymomas.
- ▼ Exposure to ionizing radiation.
- ▼ Viral infections: EBV, human immunodeficiency virus-1 (HIV-1), the putative agent of seronegative hepatitis.
- ▼ Severe malnutrition: kwashiorkor, anorexia nervosa.
- ▼ Leukemia may be the underlying disease in 1-5% of patients who present with AA.
- ▼ Paroxysmal nocturnal hemoglobinuria (PNH) develops in 5-10% of patients with AA. Conversely, AA develops in 25% of patients with PNH.

□ Who Should Be Suspected?

An individual who presents with a clinical picture of increasing symptoms of anemia, mucosal bleeding, fever, mucosal ulcerations, and bacterial infections
due to neutropenia, in whom an initial CBC demonstrates pancytopenia. Pancytopenia from other causes, such as chemotherapy, should be ruled out (see below). The disease is frequent in East Asia.

□ Laboratory Findings

RBC: anemia is normocytic and normochromic. Hb may be <7 g/L. RDW is normal. MCV is frequently elevated at presentation.
Reticulocytes are invariably decreased to absent.
WBC: neutropenia (absolute neutrophil count <1,500/µL) is always present, often accompanied by monocytosis. Abnormal WBCs are not seen. Lymphocyte count is normal (false lymphocytosis if one observes the percentage of WBC rather than the absolute count).
Platelets are decreased, but severity of thrombocytopenia varies.
Bone marrow (BM) is markedly hypocellular (<25% of normal for age), with an “empty” marrow in severe cases. Hematopoiesis is not megaloblastic. The appearance of the BM in inherited or acquired AA is identical. Aspiration and biopsy are both necessary to rule out MDS, leukemias, granulomatous disease, or tumors. The BM examination must also exclude the viral hemophagocytic syndrome.
Cytogenetics: normal karyotype.
Flow cytometry phenotyping shows virtual absence of CD34 hematopoietic stem cells in blood and marrow. AA and PNH overlap in approximately 40-50% of cases.
Serum iron studies are normal.

Suggested Reading

Sheinberg P, Young N. How I treat acquired aplastic anemia. Blood. 2012;120:1185-1196.

PANCYTOPENIA

□ Etiology

Pancytopenia may be the result of congenital anomaly, neoplasia, or autoimmunity or may be iatrogenic (Figure 11-1). The following mechanisms may account for pancytopenia:

Decreased production of hematopoietic cells by the bone marrow resulting in a hypocellular marrow
Ineffective hematopoiesis with a cellular (or even hypercellular) marrow
Infiltration of the bone marrow by extraneous elements
Systemic conditions

Bone marrow aspirate and biopsy are mandatory in most cases without a clear etiology. Deciding the bone marrow cellularity may at times be difficult, because of imprecise quantitation of cellularity or sample error due to unequal distribution of bone marrow tissue. In some cases, biopsies from multiple sites may be necessary. Moreover, hypocellular marrow due to AA may evolve over time into a hypercellular marrow. This happens, for instance, when acute leukemia or PNH develops.

A thorough history and physical examination also play a prominent role in establishing the etiology of pancytopenia, with important clues, such as a history of any drug or toxin exposure or splenomegaly, directing the clinician to possible etiologic causes.

When to suspect pancytopenia:

Finding a persistent decrease in all three hematopoietic lines on a routine CBC
Clinical symptoms suggestive of anemia, bleeding, or prolonged fever
Repeated infections

Tests recommended:

CBC with differential.
Chemistry, immunology, or infectious investigations as suggested by systemic manifestations.
Flow cytometry analysis to rule out PNH or hematologic malignancies.
Bone marrow aspiration and biopsy (see above).
Cytogenetic and FISH analysis may establish the precise diagnosis in MDSs or other hematologic malignancies. Newer whole-genome scanning technologies such as chromosomal microarray analysis may be an additional diagnostic technology.
Histochemistry for infiltrative congenital disorders.

Suggested Readings

Nester CM, Thomas CP. Atypical hemolytic uremic syndrome: what it is, how is it diagnosed, and how is it treated. Hematology Am Soc Hematol Educ Program. 2012;2012:617-625.

Scheinberg P, Young NS. How I treat acquired aplastic anemia. Blood. 2012;120:1185-1196.

PURE RED CELL APLASIA (PRCA)

□ Definition

Chronic condition of profound anemia, characterized by severe reduction or absence of reticulocytes, and absent bone marrow erythroid precursors. (The congenital PRCA is described below under Diamond-Blackfan anemia.) All other cell lines are normal. Most cases are mediated by IgG autoantibodies. PRCA may be associated with certain drugs, thymomas, collagen vascular syndromes, or chronic lymphocytic leukemia (CLL) or parvovirus B19 infection. It may also be part of the 5q- MDS. PRCA may also develop following administration of recombinant erythropoietin due to the development of antierythropoietin antibodies.

□ Laboratory Findings

CBC: severe reduction but normal-appearing RBCs; normal WBC and platelet counts.
Reticulocytes are severely decreased or absent.
Bone marrow is normocellular, but erythroid precursors cells are absent (giant normoblasts may be seen if the etiology is a parvovirus infection). White cell precursors and megakaryocytes (except in the 5q- syndrome) are normal.
- ▼ Serum iron and transferrin saturation are increased.

FANCONI ANEMIA (FA)

□ Laboratory Findings

The hematologic findings evolve over months or years: macrocytic anemia, leukopenia due to neutropenia, and mild to moderate thrombocytopenia.

Cytogenetics: Normal chromosome numbers but structural instability causing breaks, gaps, constrictions, and rearrangements. The diagnosis is made by the presence of increased chromosomal breakage in lymphocytes cultured in the presence of DNA cross-linking agents.
Genetics: Multiple genes appear to be responsible for FA. The genes are dispersed through the genome and are involved in DNA damage detection and repair pathway.
Fetal Hb is increased (>28%).
i antigen may be observed.
- ▼ Serum alpha-protein levels are frequently elevated.

DIAMOND-BLACKFAN ANEMIA (DBA)

□ Laboratory Findings

RBC: severe macrocytic anemia that is refractory to conventional therapies.
Reticulocytes are <1%.
WBC, differential white cell count, and platelet count are normal.
Bone marrow is normocellular but presents with a marked decrease in erythroid precursors. All other cell lines are normal.
Fetal Hb is increased.
Adenosine deaminase is increased in RBCs.
Serum iron and all other hematologic parameters are normal.
Serum erythropoietin is elevated.

HEMOGLOBINOPATHIES

Hemoglobinopathies constitute the most common inherited disorders in humans as a result of selective pressure of endemic falciparum malaria. Human hemoglobins (Hb) are proteins containing a heme moiety and two pairs of globin genes. Normal adult Hb is composed of two alpha (α)- and two beta (β)-chains, which together add up to 97% of total Hb in red blood cells (RBCs). The balance globins are composed of HbA2 (approximately 2.5%) and fetal Hb (HbF) (usually 0.8-2%). More than 1,000 mutations involving the globin genes have been described; they result from amino acid substitution or from abnormalities of synthesis. The majority of these variants do not cause clinical or hematologic problems. Several variants, such as sickle cell disease (SCD) and β-thalassemias (described below), are protective and asymptomatic in the heterozygous; however, they result in severe morbidity in the homozygous. Initial screening and definitive diagnosis for Hb variants are described in Chapter 16. Table 11-1 describes the most common hemoglobinopathies encountered in North America: sickle cell syndromes, HbC disease, and β- and α-thalassemias. Genetic analysis may be necessary for uncommon or unknown variants. In North America, it is done in a few specialized laboratories.

SICKLE CELL ANEMIA

□ Definition

The term sickle cell disease describes all the conditions associated with sickling of RBC. SCD encompasses a group of conditions with autosomal inheritance of abnormal Hb β-chain resulting from a substitution of valine for glutamic acid in the β-globin chain. This substitution results in polymerization of poorly soluble deoxy-HbS, leading to a marked decrease in red cells’ deformability and irreversible distortion of red cells into the sickle cell shape, with their removal by the spleen (prior to the occurrence of autosplenectomy) and macrophages. SCD is encountered mostly in populations of African or Arab ancestry, as well as in some Indian groups:

Sickle cell anemia (SCA) is the homozygous state where the majority of Hb is S. This results in the precipitation and polymerization of Hb, causing rigid crystals that deform red cells (sickling), leading to microvascular occlusions and hemolysis.
Sickle cell trait (SCT) is the heterozygous form, in which the CBC is normal. Although generally asymptomatic, its diagnosis is important for genetic counseling.
Sickle cell syndromes (diseases) represent combinations of SCT with other hemoglobinopathies, most commonly with β-thalassemia or HbC.

TABLE 11-1. Hemoglobinopathies

Condition	HbA (%)	HbA₂ (%)	HbF (%)	HbS (%)	HbC (%)	Other (%)
Normal	≥94	2-3.5	0.5-1	0	0
Sickle cell trait	50-70	2-4.5	0.5-1	30-45	0
Sickle cell anemia	0	2-4	1-25	75-95	0
HbC trait	50-60	sl ↑	0.5-1	0	30-40
HbC (homozygous)	0	<3.5	sl ↑	0	95
HbS/HbC disease	Trace	0	<1	50-55	45-50
β-Thalassemia minor	90-95	3.5-7	1-5	0	0
β-Thalassemia major	β+: Trace β-: 0	2-sl ↑	60-95;95-98	0	0
α-Thalassemia with 1-2 abnormal genes	Varies with genetic type	2-3.5	0.5-1	0	0
α-Thalassemia: 3-gene defect (HbH disease)	<60	<2	<1-1	0	0	HbH: 5-40
α-Thalassemia: four-gene defect (hydrops fetalis)	0 or trace	<2	0 or trace	0	0	Hb Bart’s 70-80
HbS-β-thalassemia	10-30	4-6	<1-10	70-90	0
Hereditary persistence of fetal Hb (HPFH)	Heterozygous: 70-85 Homozygous: 0	1-2.1 0	15-30 100	0	0

□ Who Should Be Suspected?

SCA should be suspected in a child with a family history of SCD, failure to thrive, progressive hemolytic anemia, and vaso-occlusive crises (repeated painful episodes that lead to organ damage).
Clinical manifestations are not present at birth, but become apparent after 3-6 months of life, as the concentration of HbF declines and that of Hb S increases. By 2 years of age, 61% of children have already had painful vaso-occlusive episodes.
Aplastic crises are self-limited episodes of erythroid aplasia lasting 5-10 days. They are due to infections (most commonly parvovirus B19) and may require emergency transfusions.
Bilirubin gallstones are present in 30% of patients by age 18 and 70% by age 30.
Organ damage develops by the time SCA patients are in their teens, with involvement of the lungs, kidneys, heart, and liver. Cerebrovascular accidents are also common.

□ Laboratory Findings

A “sickle cell screen” can be obtained for a rapid preliminary diagnosis. It is positive in SCA, SCT, some non-S sickling hemoglobinopathies, and combined SCD with other hemoglobinopathies.
Hb variant analysis (HPLC or electrophoresis) is used to identify different hemoglobins. Newborns have predominantly HbF with a small amount of HbS and no HbA1. Because other sickle cell syndromes may have similar patterns, it is recommended to study the parents, or repeat the test after 1 year of age, when the adult pattern of SCA (i.e., very high HbS) is established. HbF may be slightly elevated (1-4%) especially in patients treated successfully with hydroxyurea where it may reach 15% or more, resulting in marked diminution in morbidity.
The newborn with SCT will have HbA, HbF, and HbS. Adults have >50% HbA1 and 35-45% HbS.
Prenatal testing: gene analysis of fetal DNA may be performed on chorionic villi (7-10 weeks of gestation) or amniocytes (15-20 weeks of gestation). DNA testing may be also useful in newborns or children in cases with high levels of HbF if hereditary persistence of fetal hemoglobin is suspected.
Patients with HbSC disease (see below) have equal amount of HbS and HbC.
Patients with SCT-β-thalassemia (+) have HbA1, elevated HbA2, and HbS.
CBC in patients with SCA:
- ▼ RBC: mild to moderate chronic hemolytic anemia (Hct 15-30%, Hb 5-10 g/dL), punctuated by aplastic crises (sudden, life-threatening episodes of very severe anemia) (see above).
- ▼ Reticulocytes 3-15% (they may account for an elevated MCV).
- ▼ MCV is in general normal (except as noted above); MCHC is elevated. Microcytosis and hypochromia may be present, however, if there is coexisting α- or β-thalassemia, or iron deficiency in nontransfused patients.
- ▼ Peripheral blood smear (PBS): visible sickle cells, polychromasia, and Howell-Jolly bodies in older children, reflecting hyposplenism due to autosplenectomy. Nucleated red cells, basophilic stippling, and Pappenheimer bodies are usually found.
- ▼ WBCs may be higher than normal. A persistent leukocytosis augurs a poor prognosis.
- ▼ Platelets may be elevated, in part the result of loss of splenic function.
- ▼ Bone marrow aspirate (not necessary for diagnosis) is hyperplastic.
- ▼ Serum erythropoietin may be inappropriately low in some patients, possibly as the result of progressive renal disease.
- ▼ Serum iron and ferritin may be low and transferrin elevated, due to iron loss in urine.
- ▼ Serum folate is low due to overutilization, if not replaced therapeutically.
- ▼ Serum LDH is elevated.
- ▼ Serum bilirubin is commonly elevated.
- ▼ Serum haptoglobin is decreased.
- ▼ Serum aminotransferase is often elevated.
- ▼ Ferritin becomes very elevated in multiply transfused patients.
- ▼ Urine hemosiderin and urobilinogen are present (not necessary for diagnosis).

HEMOGLOBIN S-HEMOGLOBIN C DISEASE

□ Laboratory Findings

Hb electrophoresis: HbA is absent; HbS and HbC are present in approximately equal amounts. HbF is ≤6%.
CBC:
- ▼ Anemia: mild to moderate; normochromic and normocytic.
- ▼ Peripheral blood smear (PBS): tetragonal crystals within the RBC in 70% of patients. Target cells and SC poikilocytes (blunt-angled sickle cells), rather than typical sickle cells (with pointed ends), are identified.
- ▼ MCV is low, or low normal; MCHC is high.

SICKLE CELL-α-THALASSEMIA DISEASE

α-Thalassemia modifies the severity of SCA. Otherwise, it is usually clinically insignificant.

SICKLE CELL-β-THALASSEMIA DISEASE

□ Laboratory Findings

Hb electrophoresis: HbS varies between 20% and 90% and HbF between 2% and 20%. If the HbS is very high and HbA1 is suppressed, the disease is severe. In milder cases, HbA1 is 25-50%. HbA2 is increased (due to the presence of β-thalassemia), but it has to be differentiated from HbC, which has a similar migration pattern.
CBC:
- ▼ RBC: hypochromic, microcytic anemia with decreased MCV (iron deficiency must be ruled out).
- ▼ Peripheral blood smear (PBS): target cells are prominent; other findings resemble those of SCA.

SICKLE CELL-PERSISTENT HIGH FETAL HEMOGLOBIN

□ Laboratory Findings

Hb electrophoresis: HbF is 20-40%; HbA1 and HbA2 are absent; HbS is approximately 65%.
RBC: HbF is unevenly distributed among RBC.

SICKLE CELL-HEMOGLOBIN D DISEASE

□ Laboratory Findings

Intermediate between those of SCA and SCT.
Hb electrophoresis cannot distinguish HbS from HbD at alkaline pH (8.2-8.6) but can be separated at acid pH (6.2).

Suggested Readings

Vichinsky EP, Mahoney DH Jr. Diagnosis of sickle cell syndromes. In: Basow DS, ed. UpToDate. Waltham, MA: UpToDate, Inc.; 2013.

Ware RE. How I use hydroxyurea to treat young patients with sickle cell anemia. Blood. 2010;115:5300-5311.

HEMOGLOBIN C DISEASE

□ Laboratory Findings

HbC trait: Hb variant analysis shows 50% HbA1 and 30-40% HbC.
Homozygous condition: There is no HbA1, and HbC forms the majority variant Hb; HgF is slightly increased. PBS shows a variable number of target cells (≤40%), a variable number of microspherocytes, occasionally nucleated RBCs, and a few tetragonal crystals within RBCs.

HEMOGLOBIN C-β-THALASSEMIA

HbC-β-thalassemia is a form of β-thalassemia (see below). Affected individuals are commonly asymptomatic, although moderate hemolysis may be present. These individuals have a moderate microcytic, hypochromic, hemolytic anemia, and splenomegaly. Their red cells may show HbC crystals.

HEMOGLOBIN D DISEASE

□ Laboratory Findings

Hb variant analysis demonstrates the abnormal Hb at acid pH (it has the same mobility as HbS at alkaline pH). There are no other laboratory abnormalities in the heterozygous individual.
RBC: mild hemolytic, microcytic anemia in homozygous individuals; the PBS shows target cells and spherocytes.

HEMOGLOBIN E DISEASE

□ Laboratory Findings

Hb variant analysis shows 95-97% HbE in the homozygous (the rest is HbF) and 30-35% in individuals carrying HbE trait. Electrophoretic mobility is the same as for HbA2, but it is present in much higher concentrations. It separates from HbC and HbO on citrate agar electrophoresis at acid pH.
CBC:
- ▼ Mild hemolytic, microcytic (MCV 55-70 fL) anemia or no anemia in the homozygous.
- ▼ Erythrocytosis may be present (RBC approximately 5,500/µL) in both the trait and the homozygous.
- ▼ Peripheral blood smear (PBS) shows 25-60% target cells and microcytes in the homozygous individuals.

HEMOGLOBIN E-β-THALASSEMIA

□ Laboratory Findings

Hemolytic anemia varies from moderate to severe, similar to β-thalassemias (see below).
Peripheral blood smear (PBS) shows severe hypochromia, macrocytosis, and marked anisopoikilocytosis with many teardrop and target red cells. Nucleated RBC and basophilic stippling may be present.

HEMOGLOBIN E-α-THALASSEMIA

A mild hemolytic anemia encountered in Southeast Asia. It causes microcytosis. The severity depends on the number of α-genes deleted (see α-thalassemia below).

THE THALASSEMIAS

Thalassemias are chronic, microcytic, hemolytic anemias. They result from defective synthesis of either β- or α-globin subunits of the Hb A molecule. Thalassemias are classified into β- or α-thalassemia according to which of the globin chain is affected. The thalassemias are among the most common genetic disorders worldwide. They have an autosomal recessive inheritance resulting in either homozygous (thalassemia major) or subtle (thalassemia minor) clinical abnormalities. The β-thalassemia syndromes are extremely heterogenous. In addition to β-thalassemia trait and β-thalassemia major described below, there are combinations with other hemoglobinopathies and variants described above.

β-THALASSEMIA MAJOR

□ Definition and Who Should Be Suspected

A severe condition resulting from impaired or absent production of the β-globin chains of Hb. The resulting excess α-chains precipitate inside the red cells with dire consequences: severe hemolysis, skeletal changes, liver abnormalities, gallbladder bilirubin stones, splenomegaly, aplastic crises, impaired growth, endocrine and cardiopulmonary complications, and hemosiderosis resulting from RBC transfusions. The clinical expression of the severe phenotype is extremely heterogenous. A milder form of β-thalassemia, β-thalassemia intermedia, is seen in patients with one β (-)-allele mutation that produce no β-globin chains and with a β (+)-mutation from the second allele. It produces a small amount of β-chains; thus, these patients are less severely affected.

β-Thalassemia is most common in individuals of Mediterranean ancestry (mutations result from protection against endemic malaria in the Mediterranean basin); it is also found in African Americans and in some groups in India.
Infants are well at birth, depending on high levels of HbF (no β-chains, just α- and fetal globins), for tissue oxygenation. The diagnosis is usually established at 6-12 months of age due to increasing symptoms, which include pallor, irritability, growth retardation, and abdominal swelling due to hepatosplenomegaly, followed by abnormal skeletal development, the result of an expanding extramedullary hematopoiesis.
Coinheritance of an α-thalassemia trait may ameliorate the morbidity of β-thalassemia major.

□ Laboratory Findings

CBC:
- ▼ RBC: profound anemia, microcytosis, reduced MCV and MCHC, and very elevated RDW. Hb levels may be as low as 3-4 g/dL. The anemia may become acutely life threatening during aplastic crises, mostly provoked by infection with parvovirus B19, which infects precursor erythroid cells.
  - Red cell morphology shows extreme hypochromia, poikilocytosis, teardrop cells, and many target cells. Heinz bodies are readily identified when the smears are stained with supravital stains.
- ▼ WBC is elevated (in part falsely so, due to enumeration of nucleated RBCs as WBCs by some automated counters), but true leukocytosis is usually present.
- ▼ Platelets may be reduced due to hypersplenism but become elevated in splenectomized patients.
- ▼ Peripheral blood smear (PBS): marked poikilocytosis with many target cells, teardrop cells, nucleated RBCs, and basophilic stippling of RBCs.
- ▼ Reticulocyte count is inappropriately low, in part the result of ineffective erythropoiesis. It may become 0 during aplastic crises.
Bone marrow aspirate shows red cell hyperplasia with marked shift to early red cell progenitors due to intramedullary hemolysis, in turn the result of accelerated apoptosis. Megaloblastic morphology may be observed in the absence of folate supplements. Extramedullary hematopoiesis develops in the skeletal bones, liver, and spleen.
Hb variant analysis shows absence of HbA1 in β(0) thalassemia, where only HbA2 and HbF are present. HbA2 may increase to 3-6% (unless iron deficiency is also present). HbA1 is present after RBC transfusions.
Serum iron and ferritin increase progressively throughout life due to RBC transfusions.
Serum bilirubin is elevated.
Liver function tests are abnormal, in part the result of transfusional viral hepatitis. This problem is becoming rare with current transfusion practice.
LDH and uric acid are elevated.
Haptoglobin is decreased.
Endocrine abnormalities are related to extensive iron deposits, with laboratory evidence of hypogonadism and diabetes.
Hypercoagulability: abnormalities in the level of clotting factors and their inhibitors have been reported in some cases.

β-THALASSEMIA MINOR (TRAIT)

□ Laboratory Findings

CBC shows microcytic anemia. The anemia is milder (Hb 10-13 g/dL), but the microcytosis is more profound (MCV 60-70 fL) than seen in iron deficiency. RBC count may be higher than normal (another contrast to iron deficiency anemia). RDW is normal, since the RBCs are uniformly microcytic and hypochromic. On PBS, basophilic stippling of RBCs and target cells may be observed. During pregnancy, carriers may develop a more profound anemia, than attributable to the physiologic anemia of pregnancy.
Hb variant analysis: HbA2 is elevated, sometimes as high as 7-8% with the ratio HbA2/HbA1 being 1:20 instead of the normal 1:40; HbF is slightly elevated in 50% of cases. Some forms of β-thalassemia trait may have a normal concentration of HbA2. Definitive diagnosis can only made by molecular genetic techniques.

α-THALASSEMIA SYNDROMES

□ Who Should Be Suspected?

α-Thalassemia should be suspected based on a family history of anemia and geographic and ethnic background. The condition is prevalent in populations of African, Middle East, or Southeast Asian ancestry. The diagnosis is further suspected in cases of microcytic, hypochromic anemia not due to iron deficiency, with normal levels of HbA2 on hemoglobin variant analysis.

□ Diagnosis

The severity of the syndrome depends on the number of α-genes affected.

Loss of all four α-globin loci results in hydrops fetalis with Hb Bart, condition incompatible with extrauterine life. This condition is not seen in populations of African ancestry, but it is encountered in Asian populations. Hb Bart is composed of four γ-globin chains, which fails to deliver oxygen to tissues. It is fast moving on Hb electrophoresis.
Loss of three α-loci results in hemoglobin H disease. These patients have a moderate microcytic, hypochromic anemia with inclusion bodies present on the PBS. Hb levels are usually 8-10 g/dL. Hb electrophoresis or chromatographic techniques show 5-30% HbH, which is the result of tetrameric β-chains. HbH disease can be acquired in hematologic malignancies, especially in MDSs.
Loss of two loci results in α-thalassemia-1 trait (a-thalassemia minor). There are two variants, depending if the two affected genes are on the same chromosome, or one per chromosome. Adult patients may have a mild microcytic, hypochromic anemia. In these cases, the red cells are microcytic and hypochromic, and target cells are present. Hb electrophoresis is normal. Definitive diagnosis can only made by molecular genetic techniques.
Loss of only one locus results in α-thalassemia-2 trait (α-thalassemia minima or silent carrier of α-thalassemia). There are no hematologic abnormalities, and Hb electrophoresis is normal. The diagnosis can only be made by DNA analysis.
Hemoglobin Constant Spring is a common structural variant associated with α-thalassemia in Asia. It is associated with a normal α-chain, but the Constant Spring allele functions as a severe α-thalassemia gene. Patients show a minor, very slowly migrating abnormal Hb component of Hb electrophoresis. Homozygosity results in a mild form of HbH disease.
Couples at risk for having offsprings with homozygous thalassemia may choose antenatal diagnosis by direct gene analysis of the fetus.

Suggested Readings

Benz EJ. Newborn screening for α-thalassemia-keeping up with globalization. N Engl J Med. 2011;364:770-771.

Forget BG. Thalassemia. Hematol Clin North Am. 2010;24:1-140.

Rachmilewitz EA, Giardina PJ. How I treat thalassemia. Blood. 2011;118:3479-3488.

HEMOLYTIC INTRINSIC RED BLOOD CELL DEFECTS

ENZYMOPATHIES

The most common enzymopathies are glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiencies. Other rare deficiencies of RBC enzymes do occur but will not be discussed here.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY

□ Definition

G6PD deficiency is an X-linked inherited RBC enzyme deficiency. Incidence is high in regions where malaria is or was prevalent. More than 300 variants have been described. G6PD deficiency can be divided into three classes, with the normal genotype being designated G6PD type B.

Class 1 (Mediterranean variant, also designated G6PD type B): <5% of normal RBC enzyme activity. It results in a chronic hemolytic anemia exacerbated by oxidant drugs or febrile illnesses. Very severe hemolytic attacks develop after the ingestion of fava beans (favism).
Class 2 (African variant, G6PD type A): <10% of normal RBC enzyme activity; patients have episodic hemolytic attacks produced by certain infections, oxidant drugs, or diabetic ketoacidosis. It is not triggered by ingestion of fava beans.
Class 3: 10-60% of the normal enzyme activity. There is no hemolysis except for limited episodes (2-3 days) after ingestion of oxidant drugs or following infections. Similar G6PD levels are found in female carriers.

□ Who Should Be Suspected?

G6PD deficiency should be considered in the differential diagnosis of a patient with nonimmune (Coombs negative) hemolytic anemia.

□ Laboratory Findings

Basis of diagnosis:

Generation of NADPH from NADP as detected by either quantitative spectrophotometric analysis or, more rapidly, by screening tests, such as a fluorescent spot test.
G6PD levels may be normal during and shortly following a hemolytic episode in type A cases, because very young RBCs contain sufficient enzyme. Assays for G6PD should be postponed for at least 6 weeks after an acute episode.

CBC: Hemolytic anemia—chronic in class 1 and intermittent in classes 2 and 3. It is seen 2-4 days after ingestion of an oxidant drug (primaquine and sulfa drugs are the most common offending agents) or after fava beans consumption. Female carriers: possible mild hemolytic episodes that are difficult to diagnose with conventional methodology; they can be diagnosed by genetic methods.

Peripheral blood smear (PBS): Heinz bodies in RBCs (require brilliant cresyl blue supravital special stain), nucleated RBCs, spherocytes, poikilocytes, fragmented RBCs, and bite cells
Reticulocytosis.
- ▼ MCV may be elevated in type A patients if not supplemented with folic acid.
- ▼ Bilirubin is elevated correlating to the degree of hemolysis. Neonatal jaundice develops in 5% of affected newborns of African or Mediterranean ancestry after the first 24 hours of life. Serum indirect bilirubin reaches a peak (often >20 mg/dL) at 3rd to 5th day with resulting kernicterus if not treated promptly.

PYRUVATE KINASE (PK) DEFICIENCY

□ Who Should Be Suspected?

A patient with chronic, sometimes severe, nonimmune (Coombs negative) hemolytic anemia.

There is a wide range of clinical and laboratory findings. The severity of anemia varies from severe neonatal anemia requiring transfusions to a fully compensated hemolytic process in adults who have 10-20% of the normal enzyme in their RBCs. The severely affected patients may require frequent red cell transfusions and as a consequence develop iron storage overload. The severe cases present with jaundice, pallor, and splenomegaly. Such patients may also develop gallstones. The anemia may worsen after certain infections (aplastic crises). PK deficiency is more common in persons of northern European extraction and possibly in Chinese. The disease is particularly severe among the Amish of Pennsylvania.

□ Laboratory Findings

Peripheral blood smear (PBS) shows no characteristic changes, particularly no spherocytes.
The laboratory diagnosis is based on demonstrating low erythrocytic PK enzymatic levels. A screening test using crude hemolysate detects heterozygous carriers in persons who are hematologically normal. This assay may miss some variants. Quantitation of the enzyme can be performed in specialized laboratories.
Genetic tests are the most definitive approach to diagnosis.
Elevated levels of LDH and decreased haptoglobin can be seen.

HEREDITARY SPHEROCYTOSIS (HS)

□ Definition

HS is a congenital red cell membrane abnormality resulting from defects in one of six genes encoding proteins involved in vertical linkages that tie the membrane skeletal network of the RBCs with the lipid bilayer. The ankyrin gene is the one most commonly involved. HS is inherited as autosomal dominant transmission in 75% of affected individuals. The condition is recessive or presents as a new mutation in the remaining 25%. HS is seen mostly in patients of northern European origin.

□ Who Should Be Suspected?

Patients with mild to severe anemia, jaundice, splenomegaly, and cholelithiasis early in life and a family history of a hereditary hemolytic anemia.
Exacerbations of anemia may occur in aplastic (infections with parvovirus B19 or other viruses), hemolytic crises (with some viral infections) or due to the development of megaloblastic anemia, usually the result of folate deficiency.

□ Laboratory Findings

CBC: anemia of varying severity, but with acute exacerbations (see above). Moderately severe anemia occurs in approximately 70% of cases. Approximately 20% have mild, compensated hemolysis. Approximately 10% of HS patients have severe, debilitating anemia and are transfusion dependent, unless splenectomized (splenectomy ameliorates the anemia, but spherocytosis persists).
Indices: normal or slightly low MCV (except elevated when the reticulocyte count is very high or if the patient is folate deficient), elevated MCHC (the most helpful red cell index in HS), and RDW.
Reticulocytosis (5-20%).
Peripheral blood smear (PBS): Spherocytosis of various degrees is invariably present. Howell-Jolly bodies indicate previous splenectomy. The presence of spherocytes on PBS is not pathognomonic: it may be due to acquired hemolytic anemias rather than HS.
Osmotic fragility reveals increased RBC fragility, but it may be abnormal (increased) also in patients with acquired hemolytic anemias.
Ektacytometry, acidified glycerol lysis test, cryohemolysis test, and especially the flow cytometric eosin-5-maleimide tests have surpassed the osmotic fragility test in sensitivity and specificity but may only be available in specialized laboratories.
Haptoglobin: decreased.
Coombs test: negative.
Hemoglobin: usually normal at birth but decreases sharply during the subsequent 20 days of life.
Bilirubin is slightly elevated, except in neonatal cases with severe hemolysis, when it may be elevated at birth, resulting in kernicterus if not treated promptly.
Genetic tests: offered in some research laboratories, usually unnecessary.

□ Other Considerations

Laboratory findings may reflect cholelithiasis or aplastic crises.
Falsely elevated potassium (hyperkalemia) is due to potassium leaking from RBCs.

HEREDITARY ELLIPTOCYTOSIS (HE)

□ Definition

HE is a congenital heterogeneous disorder of the membrane skeleton of RBCs, most commonly a defect in spectrin. It is transmitted in an autosomal dominant mode of inheritance. Occasionally, the transmission is recessive. Individuals who are heterozygous for HE are asymptomatic. Individuals who are homozygous or compound heterozygous (10% of patients) exhibit mild to severe anemia. The disorder is more frequent in African Americans and in patients of Mediterranean origin (previous areas of endemic malaria). Affected neonates may have transient overt hemolytic anemia until adult Hb supervenes.

□ Laboratory Findings

Peripheral blood smear (PBS): more than 50% of RBCs are ellipsoidal or rod shaped. Other markers of hemolysis are uncommon, except in the approximately 10% of severely affected patients. In severe cases of HE, severe poikilocytosis is common.
Indices: decreased MCV, MCH, MCHC; increased RDW.
Variant Hb studies and osmotic fragility (see above under HS) are normal.

□ Other Considerations

Some degree of elliptocytosis may be seen in PBS of other types of anemia.

HEREDITARY PYROPOIKILOCYTOSIS (HPP)

□ Laboratory Findings

Peripheral blood smear (PBS): RBCs are markedly misshapen (fragments, microspherocytes, elliptocytes, pyknotic forms). The RBCs fragment when heated at 45-46°C (normal RBCs show budding and fragmentation only when heated at 49°C). Severe microcytosis and micropoikilocytosis are present.

HEREDITARY OVALOCYTOSIS (HO)

□ Laboratory Findings

Peripheral blood smear (PBS): oval-shaped RBCs with one or two transverse ridges or a longitudinal slit.

□ Other Considerations

Hereditary ovalocytosis can be confused with HE.

HEREDITARY STOMATOCYTOSIS

□ Laboratory Findings

Peripheral blood smear (PBS):
- ▼ Homozygous individuals: >35% of RBCs show slit-like areas of central pallor, producing a mouth-like appearance.
- ▼ Heterozygous individuals: 1-25% stomatocytes.
Anemia: similar to that of HO.
- ▼ Homozygous individuals: varying degrees of hemolysis.
- ▼ Heterozygous individuals: no anemia.

□ Other Considerations

Stomatocytes may be seen on the PBS of many acquired disorders, such as alcoholism, liver disease, and drug-induced hemolytic anemias.

HEMOLYTIC EXTRINSIC RED BLOOD CELL DEFECTS

AUTOIMMUNE HEMOLYTIC ANEMIAS (AIHAS)

AIHAs may be classified on the basis of the type of antibody present: warm (binding optimally at 37°C), cold (binding optimally at 4°C), or occasionally combined warm and cold antibodies. Each of these AIHAs may be idiopathic or secondary to other diseases.

□ Definition

Warm-Reactive AIHAs

Warm-reactive AIHAs are due to the presence of IgG antibodies that react with RBC antigens at body temperature. About 60% are idiopathic, and the remaining 40% are the result of lymphomas, leukemias, other neoplasms, or autoimmune disorders such as SLE. They may also accompany HIV or other viral infections.

Cold-Reactive AIHAs and Cold Agglutinin Disease

These AIHAs result from the presence of IgM antibodies that react to polysaccharide antigens on the RBC surface at temperatures below that of the body’s. The antibodies fix complement.

Most of the chronic cases, for which the term cold agglutinin disease is commonly used, have an underlying B-cell neoplasm (chronic lymphocytic
leukemia/small lymphocytic lymphoma [CLL/SLL], lymphomas, macroglobulinemia) as their etiology. Some cases are idiopathic. Acute cases either are the result of viral infections such as mycoplasma pneumonia and infectious mononucleosis or belong to a group known as paroxysmal cold hemoglobinuria. There are variable degrees of hemolysis and the disease can be intravascular or extravascular. The symptoms are exacerbated in cold weather. Raynaud phenomena are common, with vascular obstruction due to RBC clumps, cyanosis of exposed parts, and pallor. Splenomegaly is uncommon since the liver is the site for sequestering the coated RBCs.

□ Laboratory Findings

Warm-Reactive AIHA

Hb: moderately to severely decreased, in the range of 7-10 g/dL.
Reticulocytes: elevated in most cases.
Indices: increased MCV due to reticulocytosis; increased MCHC reflects the presence of spherocytes.
Peripheral blood smear (PBS): microspherocytes, polychromasia, and occasionally nucleated RBCs.
Coombs test: direct IgG and C3d are positive. The warm antibodies are in most cases directed against IgG1 and less frequently against IgG3.
Unconjugated bilirubin, LDH, urine, and fecal urobilinogen: elevated.
Haptoglobin: decreased.

Cold-Reactive AIHA and Cold Agglutinin Disease

Anemia (severity depends on cold agglutinin titer) with anomalous high MCV and MCHC (artifacts due to RBC clumping at room temperature).
Peripheral blood smear (PBS): RBC clumping.
Reticulocyte count: high.
Anticomplement (C3) Coombs test (positive). Anti-I antibodies are best detected using cord blood red cells.
Cold agglutinin titers: elevated.

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)

□ Definition

PNH is an acquired disorder of hematopoietic stem cells, characterized by intravascular hemolysis and hemoglobinuria. Only 25% of cases present with classical nocturnal and paroxysmal hemolysis. The clinical triad of intravascular hemolysis, venous thrombosis (the leading cause of death), and bone marrow failure is typical. The chronic intravascular hemolysis results in iron deficiency. There is a high risk of evolution into AA, MDS, or AML. The International PNH Interest Group (I-PIG) has proposed a working diagnostic classification that includes the following three categories:

Classic PNH: PNH with clinical evidence of intravascular hemolysis, without another bone marrow abnormality.
PNH in the context of another bone marrow disorder: evidence of hemolysis as well as another primary bone marrow abnormality such as AA, a MDS, or primary myelofibrosis (PMF).
Subclinical PNH: a small population of PNH cells (i.e., blood cells lacking GPI-anchored proteins), without clinical or laboratory evidence of hemolysis. This is more commonly detected in patients with another bone marrow disorder, although this association may reflect more extensive evaluation of this population rather than a truly greater incidence of subclinical PNH in individuals with other bone marrow disorders.

□ When to Suspect Paroxysmal Nocturnal Hemoglobinuria

Patients with Coombs-negative intravascular hemolysis, especially if concurrently iron deficient.
Patients with hemoglobinuria.
Patients with venous thrombosis involving unusual sites (mesenteric, hepatic, portal, cerebral, or dermal veins) and especially patients with otherwise unexplained Budd-Chiari syndrome. Such patients should also be investigated for the JAK2 V617F mutation if the etiology remains unclear.
Patients with unexplained refractory anemia.

□ Laboratory Findings

Highly Recommended

Flow cytometry analysis (high sensitivity and specificity).
- ▼ FLuorescently labeled AERolysin (FLAER): This reagent is derived from the bacterial toxin aerolysin, which binds directly to the glycosylphosphatidylinositol (GPI) anchor. It is more sensitive and specific than the available monoclonal antibodies. This allows the simultaneous detection of PNH clones in monocyte and neutrophil lineages in a single-tube, multiparameter flow cytometric assay.
- ▼ At least two different monoclonal antibodies, directed against two different GPI-anchored proteins, either absent or greatly diminished in PNH, on at least two different cell lineages, should be used to diagnose a patient with PNH. In fact, the preferred approach includes the detailed evaluation of leukocytes, because some red blood cells may be lost secondary to hemolysis or diluted from their true frequency by repeated transfusions.
- ▼ CD59 and CD55 are the most commonly assessed. Other monoclonal antibodies directed against determinants on leukocytes such as CD14, CD16, and CD24 can be used.
Direct Coombs test: negative.

Recommended

CBC: RBC indices—macrocytic anemia evolving into a microcytic picture. Reticulocytes are increased, but not commensurate with the degree of anemia. Mild leukopenia and thrombocytopenia may be present; if severe, a combination with AA or another bone marrow failure syndrome should be considered.
Bone marrow: normoblastic hyperplasia; indicated if an additional underlying hematologic disease is suspected.
Haptoglobin: reduced.
Serum iron and ferritin: decreased.
Karyotype: normal.
LDH: increased.
Leukocyte alkaline phosphatase (LAP): absent or reduced.
Liver function studies: unconjugated bilirubin, increased; AST/ALT, normal; and ALP, normal.
Methemalbumin: reduced.
Hemoglobin, plasma: increased (hemoglobinemia).
Urinalysis: hemoglobinuria, hemosiderinuria, and no intact RBCs in urine sediment.

Suggested Readings

Brodsky RA. Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria. In: Post TW, ed. UpToDate. Waltham, MA: UpToDate, Inc.; 2017.

Hill A, Kelly RJ, Hillmen P. Thrombosis in paroxysmal nocturnal hemoglobinuria. Blood. 2013;121:4985-4996.

Parker CJ. Management of paroxysmal nocturnal hemoglobinuria in the era of complement inhibitory therapy. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29.

PAROXYSMAL COLD HEMOGLOBINURIA (PCH)

□ Laboratory Findings

Plasma appears scarlet and becomes maroon or brown after a few hours (free Hb is oxidized to metHb, as well as due to the formation of methemalbumin).
Peripheral blood smear (PBS): spherocytes, nucleated RBCs, anisocytosis, and poikilocytosis.
Donath-Landsteiner test: the patient’s serum is incubated with normal red cells and pooled normal human serum as a source of complement at 4°C for a period of time (during which antibody and the early components of complement were fixed) and then incubated at 37°C in order to allow the later components of complement to be activated. Hemolysis occurs if antibody is present but does not occur if the reaction mixture is continuously maintained at 37°C.
Complement-directed Coombs test: may be positive but the IgG Coombs is negative.

Drug-Induced Hemolytic Anemias

This hemolytic anemia is due to anti-RBC antibodies that develop as the result of drug effects. The drugs most commonly implicated and the mechanisms involved are described in Table 11-2.

TABLE 11-2. Drugs Most Commonly Implicated in Hemolytic Anemias

Mechanisms	Offending Drugs
Acute intravascular: positive direct Coombs test in the presence of the drug	Sulfonamides, quinidine, quinine, stibophen
Chronic extravascular: positive direct and indirect Coombs test without the drug present	α-Methyldopa, mefenamic acid, levodopa
Unknown mechanism	Ribavirin
Intravascular and extravascular: positive Coombs test in the presence of the drug	High-dose penicillin and analogs, cephalothin, streptomycin

HEMOLYTIC DISEASE OF THE NEWBORN

□ Definition

Hemolysis occurs when fetal RBCs cross the placenta and the mother is immunized with a fetal RBC antigen that is not present on her RBCs. Some women may be immunized with more than one type of RBC antigen. The resulting immune response triggers the production of IgG antibodies that are then transferred to the fetus and cause hemolysis of the fetal RBCs. The most frequent cases are due to immunization against the D antigen of the Rh blood group. The next most common are due to immunization against the Kell antigen.

□ Laboratory Findings

Laboratory findings are those of hemolysis in the newborn.
After birth, the by-products of RBC destruction occur, especially increased unconjugated bilirubin, with attended complications (bilirubin encephalopathy and kernicterus).

MECHANICAL HEMOLYSIS

□ Definition

Physical trauma to RBCs damages them, resulting in RBC fragmentation and intravascular hemolysis. Mechanical hemolytic anemias can be divided into two groups:

Microangiopathic: endothelial cell injury in small blood vessels due to fibrin strands in vessel lumens, as seen in DIC, TTP, HUS, and disseminated malignancy; malignant hypertension; vasculitis; HELLP syndrome; scleroderma insertion of foreign bodies into the circulation; Kasabach-Merritt syndrome (giant hemangioma); chemotherapy; and the “catastrophic” antiphospholipid antibody syndrome
Macroangiopathic: RBC injury from malfunctioning valvular prosthesis, severe cardiac valve deformities, or aortic atheromata (Waring blender syndrome)

Mechanical hemolysis may also occur in hypersplenism, march hemoglobinuria (runner’s hemoglobinuria), and freshwater drowning or inadvertent infusion of water.

□ Laboratory Findings

Laboratory diagnosis: directed to the causative disease.
Anemia: commensurate with the severity of the underlying process.
Peripheral blood smear (PBS): >5 of 500 RBCs are deformed (schistocytes) or helmet cells (a subtype of schistocytes) or are microspherocytes.
Platelets: varying degrees of thrombocytopenia, occasionally without anemia.
D-dimer and fibrinogen degradation products (FDPs): elevated if DIC is present.
Plasma Hb and urine hemosiderin: elevated.
Plasma haptoglobin: decreased.

EVANS SYNDROME

□ Laboratory Findings

As described for AIHAs (see p. 959 under Anemia, hemolytic) and ITPs (see p. 1029 under Platelets, thrombocytopenias). When neutropenia is present, studies for antileukocyte antibodies should be undertaken.

Suggested Reading

Michel M, Chanet V, Dechartres A, et al. The spectrum of Evans syndrome in adults: new insight into the disease based on the analysis of 68 cases. Blood. 2009;114:3167-3172.

ERYTHROCYTOSIS

Increased red cell mass (>25% above predicted hemoglobin or >18.4 g/dL in males or >16.4 in females)
Causes of erythrocytosis (see algorithm on Figure 11-2)
Clonal:
- ▼ Polycythemia vera
- ▼ Familiar erythrocytosis
Nonclonal:
- ▼ Relative erythrocytosis (hemoconcentration)
- ▼ Hypoxia, high altitude, pulmonary disease, right to left shift, sleep apnea, high-affinity hemoglobin, and carbon monoxide poisoning
- ▼ Renal disease
- ▼ Certain tumors (hypernephroma, hepatoma, cerebellar hemangioblastoma, adrenal tumors, pheochromocytoma, uterine fibromyoma)
- ▼ Androgen therapy and testosterone or erythropoietin abuse

Figure 11-2. Erythrocytosis algorithm.

WHITE BLOOD CELL DISORDERS

LEUKOCYTOSIS AND LEUKOPENIAS

Leukocytosis refers to a total white cell count >10,300/µL (in our laboratory). Counts up to 11,000 may be considered physiologic by allowing two standard deviations above the upper limit. Leukocytosis may reflect an absolute increase of neutrophils, lymphocytes, eosinophils, monocytes, basophils, or combinations. Leukopenia is defined as a total white cell count <4,300/µL.

□ Causes of Neutrophilia (Neutrophilic Leukocytosis)

In adults, neutrophilia is defined as an increase in the absolute neutrophil count >7,500/µL (or >72%). A relative neutrophilia is seen when the other cellular elements (mostly the lymphocytes) are decreased. The absolute neutrophil count, as reported by automated counters, is a more reliable parameter than the percent count. Spurious neutrophilia may be reported by automated counters in the presence of clumped platelets or cryoglobulins. The counters will flag such
results as not acceptable. Causes of neutrophilia can be divided into primary (clonal) and secondary.

Primary Neutrophilia

Myeloproliferative neoplasms
Neutrophilic leukemia
Hereditary, giant neutrophilia (occasional large neutrophils with multiple nuclear lobes)
Hereditary neutrophilia, a rare autosomal dominant condition without medical problems
Chronic idiopathic neutrophilia, condition not associated with medical problems

Secondary Neutrophilia

Acute infections
- ▼ Localized (e.g., pneumonia, meningitis, tonsillitis, abscess, acute otitis media in children).
- ▼ Systemic (e.g., septicemia). Certain bacteria, such as pneumococcal, staphylococcal, and clostridial species, may cause very elevated neutrophil and band counts.
Inflammation, especially during chronic diseases’ flare-ups.
Vasculitis.
Acute rheumatic fever.
Crohn disease and ulcerative colitis.
Rheumatoid arthritis.
Chronic hepatitis.
Metabolic (uremia, acidosis, eclampsia, acute gout).
Poisoning by chemicals (mercury) and venoms (e.g., black widow spider).
Parenteral (foreign proteins, vaccines).
Drugs: epinephrine, steroids, lithium, retinoic acid therapy for acute promyelocytic leukemia, therapeutic cytokines, especially granulocyte (or granulocyte-monocyte) colony-stimulating factors.
Acute hemorrhage.
Acute hemolysis.
Tissue or tumor necrosis.
Acute myocardial infarction.
Tumor necrosis.
Burns.
Gangrene.
Bacterial necrosis.
Physiologic conditions:
- ▼ Strenuous exercise
- ▼ Emotional stress
Labor.
Smoking.
Leukoerythroblastic reaction (myelophthisis): neutrophilia associated with immature granulocytes, nucleated red cells, and teardrop red cells;
it is associated with tumor invasion of the bone marrow, TB, and other granulomatous diseases.

NEUTROPENIA

□ Causes of Neutropenia

Decreased bone marrow production
- ▼ Myelodysplastic syndromes
- ▼ Aplastic anemia
- ▼ Chemotherapy
- ▼ Acute leukemia
- ▼ Radiation therapy or accident
- ▼ Folic acid or vitamin B₁₂ deficiency
Increased bone marrow production but decreased survival of neutrophils
- ▼ Autoimmune and isoimmune neutropenia
- ▼ SLE and RA
- ▼ Felty syndrome
- ▼ Hypersplenism
- ▼ Large granular lymphocytosis
Viral infections (various mechanisms)
- ▼ Infectious mononucleosis
- ▼ HIV infection
- ▼ Hepatitis
- ▼ Influenza
- ▼ Measles
- ▼ Rubella
- ▼ Psittacosis
Bacterial infections
- ▼ Overwhelming sepsis
- ▼ Miliary TB
- ▼ Typhoid and paratyphoid
- ▼ Brucellosis
- ▼ Tularemia
Rickettsial infections
- ▼ Scrub typhus (tsutsugamushi disease)
- ▼ Sandfly fever (caused by Sicilian or Naples virus)
Other infections
- ▼ Malaria
- ▼ Kala-azar
Drugs
- ▼ Sulfa drugs (TMP/SMX)
- ▼ Antibiotics (chloramphenicol, vancomycin, cephalosporin, macrolides)
- ▼ Antimalarials (chloroquine, quinine, amodiaquine)
- ▼ Antifungal agents (amphotericin B, flucytosine)
- ▼ Antidiabetics (chlorpropamide, tolbutamide)
- ▼ Anti-inflammatory (sulfasalazine, gold salts, phenacetin, phenylbutazone)
- ▼ Anticonvulsants (carbamazepine, phenytoin, valproate, ethosuximide)
- ▼ Psychotropic drugs (clozapine, phenothiazines, tricyclic and tetracyclic antidepressants, meprobamate)
- ▼ Cardiovascular (procainamide, ticlopidine, ACE inhibitors, propranolol, dipyridamole, digoxin)
- ▼ Diuretics (thiazides, furosemide, spironolactone, acetazolamide)
- ▼ Antithyroid drugs (thioamides)
- ▼ Dermatologic drugs (dapsone, isotretinoin)
Chronic idiopathic neutropenia
Neonatal and infantile neutropenia
- ▼ Maternal immune neutropenia
- ▼ Maternal isoimmunization to fetal leukocytes
Congenital neutropenia as seen with certain inborn errors of metabolism and other congenital syndromes

Suggested Reading

Boxer LA. How to approach neutropenia. Hematology Am Soc Hematol Educ Program. 2012; 2012:174-182.

AGRANULOCYTOSIS

□ Who Should Be Suspected?

Agranulocytosis should be suspected in anyone started recently or restarted on any drug, who suddenly develops fever, chills, and signs of infection. Sore throat is a common presenting symptom. Patients may develop overwhelming sepsis.

□ Laboratory Findings

CBC: normal Hb and platelets (except under special circumstances, such as postchemotherapy); absent or extremely decreased neutrophils and bands.
The granulocytes may show pyknosis or vacuolization. Normal lymphocytes and monocytes (but relative lymphocytosis and monocytosis).
Bone marrow shows an absence of cells in the granulocytic series but normal erythroid and megakaryocytic series.
ESR is increased.
Other laboratory findings reflect the infection.
Hb, RBC count and morphology, platelet count, and coagulation tests are normal.

LYMPHOCYTOSIS

□ Primary (Clonal) Lymphocytosis

Chronic lymphocytic leukemia (CLL)
Monoclonal B-cell lymphocytosis (<5,000 clonal lymphocytes)
Prolymphocytic leukemia
Hairy cell leukemia
Follicular, mantle cell and splenic marginal zone lymphomas (MZLs) in leukemic phase
Large granular lymphocytic leukemia

□ Secondary (Reactive) Lymphocytosis

Infections (e.g., pertussis, infectious mononucleosis [EBV], infectious lymphocytosis [especially in children], infectious hepatitis, CMV, mumps, German measles, chickenpox, toxoplasmosis, babesiosis, chronic TB, cat scratch disease)
Noninfectious causes (e.g., hypersensitivity reactions, stress)
Drugs: efalizumab (Raptiva)

LYMPHOCYTOPENIA

□ Causes

Corticosteroid therapy or Cushing syndrome; epinephrine injection
Certain infections (e.g., acute and chronic retroviral infections, TB)
Sarcoidosis
Congenital immunoglobulin disorders
Chemotherapy and radiation therapy
Neoplastic diseases, especially Hodgkin lymphoma
ARDS
Autoimmune disorders
Idiopathic CD4⁺ lymphocytopenia
CHF
Increase loss via the GI tract (e.g., intestinal lymphectasia, thoracic duct drainage, obstruction to intestinal lymphatic drainage)

MONOCYTOSIS

□ Causes

Acute monocytic or myelomonocytic leukemia and chronic myelomonocytic leukemia (CMML) (as part of MDSs or MPNs)
Hodgkin lymphoma, non-Hodgkin lymphomas, multiple myelomas
Carcinomas of the ovary, stomach, and breast
Lipid storage diseases (e.g., Gaucher disease)
Postsplenectomy
Recovery from agranulocytosis and chemotherapy or subsidence of acute infection
Protozoan infections (e.g., malaria, kala-azar, trypanosomiasis)
Some rickettsial infections (e.g., Rocky Mountain spotted fever, typhus)
Certain bacterial infections (e.g., bacterial endocarditis, TB, syphilis, brucellosis)
Ulcerative colitis, regional enteritis, sprue
Sarcoidosis and other connective tissue diseases (e.g., SLE, RA)
Tetrachloroethane poisoning
Chronic corticosteroid therapy
Acute minor viral infections (counts should be rechecked in 1 month)
Diurnal variations

EOSINOPHILIA

□ Associated Conditions

Primary
- ▼ Hematologic: hypereosinophilic (HES) syndrome
- ▼ Neoplastic disorders: chronic eosinophilic leukemia (CEL), myelomonocytic leukemia with inversion 16 mastocytosis, and T-cell lymphomas that secrete interleukin-5
Secondary
- ▼ Allergic diseases: atopic and related diseases, medication related
- ▼ Infectious diseases: parasitic infections, mostly helminths, some fungal infections, and infrequently other infections
- ▼ Collagen vascular disorders
- ▼ Autoimmune disorders such as the vasculitis of the Churg-Strauss syndrome
- ▼ Tumors with secondary eosinophilia: T-cell lymphomas (e.g., mycosis fungoides, Sézary syndrome) and Hodgkin lymphoma
- ▼ Pulmonary diseases: hypersensitivity pneumonia and Loeffler pneumonia
- ▼ Endocrine: adrenal insufficiency
- ▼ Immunologic reactions, transplant rejection
- ▼ Cholesterol embolism syndrome

PERSISTENT EOSINOPENIA

□ Associated Conditions

Drugs: corticosteroids or epinephrine administration
Cushing syndrome
Infections in conjunction with neutrophilia
Inflammation: acute

BASOPHILIA

□ Associated Conditions

Basophilia frequently accompanies MPNs, and its progression may herald a blast crisis in chronic myelogenous leukemia. The existence of basophilic leukemia is controversial. One case was recently described by our group.
Other causes of basophilia are
- ▼ Hypersensitivity states (drugs, foods, foreign protein injection)
- ▼ Myxedema
- ▼ Anemias, chronic hemolytic, iron deficiency (in some patients)
- ▼ Ulcerative colitis
- ▼ Postsplenectomy
- ▼ Hodgkin lymphoma
- ▼ Chronic sinusitis
- ▼ Chickenpox
- ▼ Smallpox
- ▼ Nephrotic syndrome (in some patients)

□ Basophilopenia (No Lower Limit Can Be Determined Because Some Normal Subjects Have 0% Basophils)

Hyperthyroidism
Irradiation or chemotherapy
Drugs: corticosteroids
Ovulation and pregnancy
Stress

LEUKEMOID REACTIONS

□ Definition

A count >50,000/µL white cells in nonleukemic conditions defines a leukemoid reaction. The PBS shows increase in and shift to the left of myeloid cells (bands, metamyelocytes, myelocytes, some promyelocytes, and rare myeloblasts), increased primary granules in the myeloid cells (toxic granulation) and Döhle bodies, and cytoplasmic vacuolization. If the left shift consists of an elevation of bands only (>700/µL), the term bandemia is applied. Frequently, it signals the onset of a septic episode such as acute appendicitis.

□ Causes of Leukemoid Reactions

Severe sepsis (osteomyelitis, empyema, disseminated TB)
Burns
Tissue necrosis (gangrene, mesenteric vein thrombosis)
Therapy with granulocyte colony-stimulating factor (G-CSF) or granulocyte-monocyte colony-stimulating factor (GM-CSF)
Metastatic infiltration of the marrow

ACUTE LEUKEMIAS

B LYMPHOBLASTIC LEUKEMIA/LYMPHOMA (B-ALL)

□ Who Should Be Suspected?

B-ALL is the most common form of cancer in childhood, constituting >85% of leukemias in children. The disease can however occur at any age. Children (peak incidence at age 2-3 years) or adults older than 65, presenting with acute onset of fever, infection, bleeding, fatigue, musculoskeletal pain (particularly in adolescents), and characteristic findings on the CBC. Lymphadenopathy and hepatosplenomegaly are present in the majority of patients but are not massive.
Predisposing factors: children with certain genetic disorders such as Down syndrome, neurofibromatosis type 1, Bloom syndrome, and ataxia-telangiectasia.
Poor prognostic signs at presentation: WBC count >100,000/µL, platelet count <50,000/µL, CD10 negativity, certain karyotypic abnormalities, occurrence of the disease before age 1 (probably having occurred before birth) or after age 10, and induction failure. Mature B leukemic phenotype rather than the precursor B cell is associated with poorer prognosis.

□ Laboratory Findings

Laboratory diagnosis is based on morphology, immunophenotype, and cytogenetic/genetic analysis.

Morphology

Blood: CBC
- ▼ Anemia, moderate to severe.
- ▼ Thrombocytopenia.
- ▼ WBC is usually elevated, with lymphocytosis and neutropenia, but approximately 50% of children have WBC counts <10,000 at presentation.
- ▼ Lymphoblasts are usually identified on the PBS.
- ▼ Bone marrow generally shows diffuse infiltration of lymphoblasts. It should be obtained before starting therapy to determine immunophenotype, cytogenetics, and overall cellularity. Peripheral blood may be sufficient for these studies in cases with high peripheral blood blast count. Once the diagnosis of leukemia is confirmed, definitive assignment to the subtype of B-ALL, as provided by immunophenotyping and cytogenetic studies, is mandatory before deciding on therapeutic protocol.

Immunophenotype

Seventy percent to 80% of childhood ALL are of the B-precursor lineage. The expression of markers on the leukemic lymphoblasts does not correlate strictly with normal lymphoid maturation. B-ALL lymphoblasts are positive for CD19, cytoplasmic CD79a, and cytoplasmic and surface CD22, CD24, PAX5, and TdT. The expression of CD34, CD10 (CALLA antigen), and CD20 is variable. Myeloid markers CD13 and CD33 may also be present. The aberrant immunophenotype serves to identify minimum residual disease in the bone marrow following therapy.
The degree of differentiation of precursor B-lineage lymphoblasts has clinical and genetic correlates. B-progenitor ALL present in 80-85% of childhood B-ALL. Eighty to 90% express CD10. The majority have an immunoglobulin gene rearrangement, predominantly involving the IGH gene. Different subsets are based on various cell markers: early precursor B-ALL or pro-B ALL (CD10-, no cytoplasmic Ig [cIg]), common B ALL (CD10+, but no cIg), and pre-B ALL (CD10+, cIg positive). The prognosis among these various forms of immature B-ALL depends mostly on their genetic etiology as reflected in karyotypes or by interphase FISH (see below).

Cytogenetic/Genetic Analysis

In addition to immunophenotype, cytogenetic and molecular genetic abnormalities are used in the prognostic evaluation and therapy of B-ALL.
Both numerical and structural abnormalities of the chromosomes are associated with prognosis and influence treatment.
- ▼ t(9;22)(q34;q11.2); BCR-ABL (the Ph chromosome) is present in about 25% of adults and 3% of children. Its presence denotes poor prognosis in B-ALL patients, but patients may respond to tyrosine kinase inhibitors.
- ▼ t(12;21)(p13;q22); ETV6-RUNX1: favorable prognosis.
- ▼ t(1;19)(q23;p13.3); TCF3-PBX1: intermediate to poor prognosis.
- ▼ MLL (also called KMT2A) (11q23) rearrangements, most commonly t(4;11)(q21;q23) with AFF1(AF4)/MLL and t(11;19)(q23;p13.3) with (MLL/MLLT1(ENL): poor prognosis.
- ▼ t(5;14)(q31.1;q32.1); IGH/IL3: associated with eosinophilia, similar prognosis
- ▼ iAMP21: amplification of a portion of chromosome 21, poor prognosis
- ▼ Hypodiploidy (the blasts contain <44 chromosomes, poor prognosis).
- ▼ Hyperdiploidy (54-58 chromosomes especially if associated with the combined trisomies of chromosomes 4 and 10 have the best prognosis). Note that hyperdiploidy may represent doubling of a hypodiploid clone. This doubled clone still confers poor prognosis. High hyperdiploidy may also be seen in combination with BCR-ABL1 and with t(1;9) and also carries a poor prognosis.
In addition to the genetic abnormalities demonstrated by chromosome and FISH studies, high-density chromosomal genomic array with copy number and single nucleotide polymorphism (SNP) probes and gene expression profiles are being increasingly used to stratify patients and determine prognosis and therapeutic protocols.
Once the initial profile of the leukemic cells had been established, the information is used to establish the effect of therapy as revealed by the presence of minimal residual disease (MRD), which correlates well with clinical outcome.

Additional Information

CSF may show increased protein and cells, some recognizable as lymphoblasts. Because of high incidence of meningeal involvement, examination of CSF is part of all protocols.
Serum LDH and sedimentation rate are elevated.
Hypercalcemia, hyperpotassemia, hyperphosphatemia, and hyperuricemia may be present at diagnosis or develop as the result of therapy.
Acute lysis syndrome may develop as the result of therapy.

Suggested Readings

Borowitz MJ, Chan JKC, Downing JR, et al. B lymphoblastic leukaemia/lymphoma not otherwise specified. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Revised 4th ed. Lyon, France: International Agency for Research on Cancer; 2017:200-202.

Mullighan CG. The molecular genetic makeup of acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program. 2012;2012:389-396.

T LYMPHOBLASTIC LEUKEMIA/LYMPHOMA (T-ALL)

□ Who Should Be Suspected?

Presentation is similar as for B-ALL, but there is more predominant extramedullary involvement, including frequent CNS and anterior mediastinal thymic masses.

□ Laboratory Findings

CBC: See B-ALL, but note higher leukocytosis at presentation.
Immunophenotype: CD3 is T-lineage specific. The lymphoblasts are TdT positive and express CD1a, CD2, CD4, CD5, CD7, and CD8 to variable degrees. CD10 and CD34 may also be positive.
Molecular genetics: Clonal rearrangement of the T-cell receptor gene (TCR) is almost always present.
Cytogenetics: Abnormal karyotypes are present in 50-70% of cases. The most common recurrent abnormality involves the alpha (α) and delta (Δ) TCR loci at 14q11.2.

Suggested Reading

Borowitz MJ, Chan JKC, Bene MC, et al. B lymphoblastic leukaemia/lymphoma not otherwise specified. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Revised 4th ed. Lyon, France: International Agency for Research on Cancer; 2017:209-212.

ACUTE MYELOID LEUKEMIA (AML)

□ Classification

The fourth and revised fourth WHO classifications (published in 2008 and revised in 2016) will guide the description of AML variants in this section. AML is divided into six major groups:
- AML with recurrent genetic abnormalities: these abnormalities impact prognosis. The most common ones are balanced abnormalities that create a fusion gene encoding a chimeric protein. The best examples are acute promyelocytic leukemia (APL) with PML-RARA, AML
  with inv(16)(p13.1q22), AML with t(8;21)(q22;q22.1), AML with t(6;9)(p23;q34.1), and AML with gene mutations such as NPM1, or RUNX1, or CEBPA.
- AML with myelodysplasia-related changes constitutes three subgroups: AML arising from previous MDS or MDS/MPN, AML with an MDS-related cytogenetic abnormality, and AML with multilineage dysplasia. This group has a poor prognosis.
- Therapy-related myeloid neoplasms: the leukemia occurs as a late complication of cytotoxic chemotherapy or radiation therapy.
- AML, not otherwise specified: cases that do not fulfill criteria for the other groups. These cases of AML are classified basically by morphology, and follow closely the FAB^* classification, except for having eliminated APL (formerly M3).
- Myeloid sarcoma: extramedullary myeloid tumor. It may precede or coincide with overt AML.
- Myeloid proliferations related to Down syndrome: Down syndrome individuals have a 50- to 150-fold increase in the incidence of AML in the first 5 years of life. Among these cases, acute megakaryoblastic leukemia accounts for approximately 70%. In addition, 10% of Down syndrome newborns have a transient episode of abnormal myelopoiesis expressed mainly as thrombocytopenia and marked leukocytosis.

□ Who Should Be Suspected?

AML is the most common acute leukemia in adults. It should be suspected during the first months of life (initiating events are in utero), in middle age, or in elderly, in a patient who is acutely ill, and with nonspecific presenting signs and symptoms that reflect profound disturbances in hematopoiesis, which include fatigue, malaise, infections, ulcerations of mucous membranes, bleeding, diffuse bone tenderness, joint pain, and swelling.
Other findings:
- ▼ Modest splenic enlargement is present in 50% of cases.
- ▼ Lymphadenopathy is not present. Isolated masses (myeloid sarcoma [chloroma]), which are collection of blasts in extramedullary sites, may precede systemic AML.

□ Laboratory Findings

Morphologic, cytochemical, immunophenotypic, cytogenetic, and molecular studies, if available, should be performed in every case to maximize the precise diagnosis and prognosis classification.

CBC:
- ▼ Anemia, normochromic and normocytic, is universally present. Nucleated red cells may be identified on the PBS.
- ▼ Thrombocytopenia is severe in most cases.
- ▼ WBC: Leukocytosis with neutropenia is present in more than half the cases; some patients may present with leukopenia, especially if AML follows MDS. Greater than 20% of white blood cells are blasts in some cases. There are few or no intermediate granulocytic cells (myelocytes, metamyelocytes, bands). Auer rods are present in certain subtypes with granulocytic differentiation and help to establish the diagnosis, especially by determining myeloid rather than lymphoid etiology at the first inspection of the patient’s PBS.
Bone marrow aspirate and biopsy are mandatory for cytochemical, immunophenotypic, cytogenetic, and genetic studies. The WHO classification defines AML as with either >20% blasts in bone marrow or PBS or with specific cytogenetic findings: t(8;21)(q22;q22.1) RUNX1-RUNX1T1, inv(16)(p13.1q22) or t(16;16)(p13.1;q22) CBFB-MYH11 and PML-RARA. The bone marrow is hypercellular in most cases, with a predominance of early progenitor cells (myeloblasts and promyelocytes or monoblasts and promonocytes) depending on the leukemic subtype. Initial assessment is based on counting 500 cells on the aspirate. Careful assessment of megakaryocytes and the degree of marrow fibrosis are also part of the initial studies for evaluation of acute megakaryoblastic leukemia and acute panmyelosis with myelofibrosis.
Coagulation studies. Bleeding, a severe complication of AML, is usually due to the severe thrombocytopenia, compounded by platelet functional defects. In addition, patients with the t(15;17) and hypergranular promyelocytes frequently develop a proteolytic state akin to DIC either spontaneously or following the initial chemotherapy. The mechanism is thought to be release of tissue factor from the promyelocytes’ granules. PT and PTT are elongated, FDP and latex D-dimers are elevated, and fibrinogen, initially elevated, decreases dramatically.
Metabolic and electrolyte abnormalities are common; the patients must be monitored carefully, especially during induction chemotherapy. Renal failure from multifactorial causes is common.
- ▼ Hyperuricemia is the most frequent biochemical abnormality. Hyperuricuria may also be present.
- ▼ Tumor lysis syndrome may develop during induction chemotherapy. It is characterized by rapid development of hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia.
- ▼ Acute promyelocytic leukemia differentiation syndrome (previously the retinoic acid syndrome) develops in 2-27% of patients in the 1st to 3rd week after initiating all-trans retinoic acid (ATRA) therapy for this type of AML. The most susceptible patients are those with hyperleukocytosis and abnormal serum creatinine. Lactic acidosis has been described in patients with AML.
- ▼ Hypokalemia is common and may be profound.
- ▼ Lysozyme is released from the leukemic cells and may induce renal tubular damage.
- ▼ Hypercalcemia and hypocalcemia have been reported.
- ▼ Spuriously high potassium and decreased serum glucose may be the result of circulating metabolically active white cells.
CNS involvement is infrequent in AML (5-7% of patients). It is more common in patients with a monocytic predominant clone and with hyperleukocytosis and patients under 2 years of age. KMT2A (previously called MLL) rearrangements, inv(16), and complex karyotypes may also predispose to CNS involvement.
Cytochemistry, although extremely useful in the past, is taking a secondary role in the era of cytogenetic/genetic and immunophenotyping diagnosis and classification. It plays a role when a rapid result is beneficial, such as rapidly differentiating AML from ALL. The most commonly used stains are as follows:
- ▼ Myeloperoxidase or Sudan Black B: positive in AML without maturation, AML with maturation, acute myelomonocytic leukemia, and APL (strongly positive); negative in ALL, AML with minimal differentiation, acute monoblastic and monocytic leukemia, pure erythroid leukemia, and acute megakaryoblastic leukemia.
- ▼ Chloroacetate esterase: positive in AML without maturation, AML with maturation, and acute myelomonocytic leukemia; negative in ALL, AML with minimal differentiation, acute monoblastic and monocytic leukemia, pure erythroid leukemia, and acute megakaryoblastic leukemia.
- ▼ Nonspecific esterase: positive (and inhibited by sodium fluoride) in acute myelomonocytic leukemia and acute monoblastic and monocytic leukemia; negative in ALL, AML with minimal differentiation, AML without maturation, AML with maturation, pure erythroid leukemia, and acute megakaryoblastic leukemia.
- ▼ Periodic acid-Schiff (PAS): positive in pure erythroid leukemia, acute megakaryoblastic leukemia, and ALL; negative in other types of acute leukemia.
- ▼ Lysozyme is positive in AML with monocytic differentiation.
Immunophenotype: Most cases of AML are characterized by their complex immunophenotypes. There is great variation in immunophenotype depending on the leukemic subtype. Blasts are positive for CD34 (except for APL and some cases with monocytic differentiation, where CD34 may be weakly expressed or absent) and in some cases HLA-DR (except for APL) and CD117. The AML variants with differentiation toward the granulocytic phenotype express CD13, CD33, CD15, and CD65. Those with monocytic characteristics are positive for CD14, CD4, CD11b, CD11c, CD64, and CD36. The megakaryoblastic leukemias express platelet antigens, such as CD41 and/or CD61. CD2 is expressed in subsets of APL, more often in microgranular variant. CD19 is expressed in AML with RUNX1-RUNX1T1. CD56 expression can be seen in a fraction of AMLs, especially APL and AML with RUNX1-RUNX1T, and it is associated with worse prognosis. Other T- or B-cell antigens are expressed in acute leukemia of ambiguous lineage (mixed phenotype acute leukemia).
Because of this possibility and its impact on prognosis (poor), their panel of antigens used at the time of diagnosis must contain multiple myeloid, B- and T-cell markers.
Cytogenetic/molecular genetic investigations determine to a great extent prognosis and therapeutic protocols and have become the major criteria the WHO uses for subclassification of AML. Cytogenetics is also critical in distinguishing AML from chronic myeloid leukemia in blast crisis. There are specific cytogenetic abnormalities seen only in AML, for example, t(1;22)(p13;q13). Complex karyotype has consistently been associated with poor outcome. Although cytogenetic studies are essential for diagnosis and classification, many of the variant translocations can be detected by real-time polymerase chain reaction (RT-PCR) that has higher sensitivity and as such is useful for residual disease monitoring. Gene expression profiling leads to further subclassification of AML, with prognostic and therapeutic implications.
- ▼ AML with t(8;21)(q22;q22.1) with RUNX1-RUNX1T1 fusion is found in approximately 5% of AML cases. Generally, it shows maturation in the neutrophilic lineage, occurs in a younger population, and may present as myeloid sarcomas. Good response to chemotherapy.
  
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Tags: Wallach's Interpretation of Diagnostic Tests Pathways to Arriving at a Clinical Diagnosis

Mar 20, 2021 | Posted by drzezo in PATHOLOGY & LABORATORY MEDICINE | Comments Off

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