blood cell identification: 2010-c mailing: hemoglobin sc disease · acid electrophoresis, run at a...

Blood Cell Identification: 2010-C Mailing: Hemoglobin SC Disease

All material is © 2010 College of American Pathologists, all rights reserved

1 - Education

Please Note: To view the Figures and Images contained within this education activity in color, access the

electronic version of the reading.

CASE HISTORY

The patient is a 37-year old female with a history of multiple sickle cell crises. She now presents with

avascular necrosis of the left hip. Laboratory data include: WBC = 12.8 x 109/L; RBC = 3.05 x 1012/L;

HGB = 8.7 g/dL; HCT = 26.4%; MCV = 86.4 fL; MCHC = 33.0 g/dL; RDW = 16.9; and

PLT = 331 x 109/L.

DISCUSSION

This case illustrates some of the morphologic findings seen in the blood smear of a woman experiencing

multiple sickle cell crises. The features illustrated here include several sickle cells (drepanocytes; see Figure

6. on page 8 and Figure 10. on page 12), target cells, and moderate anemia. These findings suggest a

sickling disorder, although the morphology does not specifically point to the exact etiology. In this particular

instance, the sickling disorder is secondary to Hemoglobin SC (Hb SC) disease, and the blood smear also

shows some misshapen cells with crystalline material, often referred to as SC poikilocytes (see Figure 10.

on page 12). Hemoglobin SC disease is a hemoglobinopathy, a term that refers to production of abnormal

hemoglobin molecules. In contrast, the term thalassemia refers to decreased production of the normal

globin chains that comprise hemoglobin.

Hb SC disease is a compound heterozygote phenomenon, signifying that it is a genetic combination of two

beta globin abnormalities, Hb S and Hb C. Both of these hemoglobin abnormalities are seen most frequently

in the United States in African Americans where the hemoglobin S gene is present in ~8% of individuals

(heterozygous Hb S; Hb S trait) and the hemoglobin C gene in ~2-3% (heterozygous Hb C; Hb C trait). The

frequency of Hb SC disease in African Americans is ~0.05%, which is less common than Hb SS (0.16%

of African Americans) and more common than Hb CC (0.02% of African Americans) disease.

In order to better understand Hemoglobin SC disease, it is important first to review the structure of normal

adult hemoglobin and methods used to identify abnormal hemoglobins, then discuss homozygous

Hemoglobin SS and homozygous Hemoglobin CC in comparison to Hb SC disease.

NORMAL ADULT HEMOGLOBIN

Hemoglobins are each composed of heme groups and protein groups. The heme group contains an iron

molecule bound within a porphyrin ring and is identical in all hemoglobins. The protein part of the molecule

is a tetramer, comprised of 2 dimers. Each dimer contains an α–like globin and a β-like globin, and each

globin chain binds a heme group.



2 - Education

Hemoglobin A is the major normal adult hemoglobin (~97% of total normal hemoglobin). It consists of two

identical α-chains, each comprised of 141 amino acids; and two identical β-chains, each comprised of 146

amino acids, and thus, can be referred to as α2β2 . Each chain is linked to one heme group, with the heme

groups at the surface of each molecule, thereby having the ability to combine reversibly with oxygen. In

addition to Hb A, adult red cells also contain small quantities of Hb A2 (α2δ2; 1.5-3.5%) and Hb F (α2γ2;

< 2%); see Table 1. for a comparison of hemoglobins present in a normal adult with those seen in Hb SS

disease, Hb CC disease, and Hb SC disease.

Table 1. Comparison of Degree of Anemia, Hb values, MCV, and HPLC Fractions

Genotype Degree of Anemia

Hb (mg/dL) MCV (fL)

HbS (% of total Hb)

HbC (%)

HbA (%)

Normal Normal 12-14 80-100 - - >95 SS Severe 6-10 85-95 >80 - - CC Asymptomatic to

mild 12-14 70-72 - >90 -

SC Mild to moderate 11-12 85-95 ~50 45-50 -

DIAGNOSIS OF HEMOGLOBINOPATHIES

Several laboratory methods are available to evaluate hemoglobin composition, including electrophoresis

(acid and alkaline), high performance liquid chromatography (HPLC), capillary electrophoresis, isoelectric

focusing, and amino acid/DNA sequencing. With the exception of DNA sequencing, these methods

generally rely on differential charge of various hemoglobin types to identify abnormalities. In addition to

these other methods, if a sickling disorder is a consideration, a solubility test for sickling hemoglobins may

also be useful.

Solubility Test for Sickling Hemoglobins

This test is relatively simple to perform, but has significant limitations in overall interpretation. In this test,

lysates of red blood cells are placed in a high phosphate buffer. Sodium hydrosulfite is added to the

solution lowering the oxygen tension. If Hb S is present, a cloudy solution will form. Unfortunately, this

test will be positive in both heterozygous and homozygous Hb S, as well as in other mixed disorders such

as Hb SC disease. In addition, some hemoglobinopathies contain two amino acid substitutions, and if one

of these is similar to that of Hb S, such as in Hb C-Harlem, the test may also be positive. So, while this is

an easy to perform and rapid test, overall interpretive conclusions are limited. In addition, if Hb S represents

less than 15-20%, a false negative test may occur.

Hemoglobin Electrophoresis Hemoglobin electrophoresis is generally performed at both alkaline and acid pH. Using cellulose acetate or

agar at an alkaline pH of 8.6, hemoglobins are negatively charged and will move on the gel towards the

positive electrode (anode). Hemoglobins containing an amino acid substitution that changes the overall

charge of the molecule will have differing mobility from Hb A (Figure 1. on the following page). Alkaline



3 - Education

electrophoresis is quite useful; however, many hemoglobin variants will migrate in a similar position. For

example, hemoglobins S, D, and G migrate in the same location, as do hemoglobins C, E, O, and A2.

Acid electrophoresis, run at a pH of 6.2, complements information obtained from alkaline electrophoresis.

By this method, the most common abnormal hemoglobins, Hb S and Hb C, are effectively separated from

Hb A, as well as most others that migrate in similar locations by alkaline electrophoresis (Figure 2.).

Unfortunately, many of the other abnormal hemoglobins migrate in a pattern similar to Hb A, and thus, acid

electrophoresis is often not useful for characterization of other abnormal hemoglobins.

Figure 1.

Figure 2.

Hemoglobin electrophoresis performed at alkaline pH. Hemoglobins are negatively charged and move towards the positively charged electrode (anode). Mobility within the gel differs based on the overall charge of the hemoglobin. This method allows separation of many hemoglobins from normal Hb A; although multiple abnormal hemoglobins may migrate in the same position. For example, hemoglobins S, D, and G, all migrate in the S postion, while hemoglobins C, E, O, and A2 migrate in the same position.

Hemoglobin electrophoresis performed at acid pH. By this method, the most common abnormal hemoglobins, Hb S and Hb C, are effectively separately from Hb A, as well as most others that migrate in similar locations by alkaline electrophoresis.

Isoelectric Focusing Isoelectric focusing is an additional electrophoretic method in which a pH gradient from approximately 6-8

is established within the gel. This differential pH gradient allows hemoglobins to migrate within the gel to

their isoelectric point, the point at which they contain zero charge. Isoelectric focusing allows better

discrimination of hemoglobins that migrate in similar locations on alkaline electrophoresis. Isoelectric

focusing has largely been replaced by high performance liquid chromatography (HPLC) as a method of

hemoglobin analysis.

High Performance Liquid Chromatography (HPLC) Although historically used in the research setting, more recently compact HPLC instruments designed for

hemoglobin analysis have become available for routine use in the clinical laboratory. Generally, these

instruments utilize a weak cation exchange column to which hemoglobins bind. As the ionic strength of the

eluting liquid phase increases, hemoglobin variants will come off the column at a specific retention time,



4 - Education

thus allowing identification of the hemoglobin variant based on the overall charge characteristics of the

protein. The pattern seen by alkaline electrophoresis demonstrates some correlation with retention time by

HPLC since both methods are dependent on the charge of the hemoglobin molecule; although the specific

retention time by HPLC is dependent on the column and eluting solution used in the instrument. In general

terms, amino acid substitutions leading to more overall negative charge will result in faster migration by

alkaline electrophoresis and a shorter retention time on the column by HPLC. One advantage of this method

is that Hb C does not migrate with Hb A2 as it does on alkaline electrophoresis, thus allowing measurement

of Hb A2 in a patient with heterozygous or homozygous Hb C. Unfortunately, Hb E does elute with Hb A2

by this method, precluding accurate measurement of Hb A2 when Hb E is present. Examples of a normal

adult hemoglobin pattern by HPLC, as well as some common hemoglobinopathies, are shown in Figure 3.

The proportions of the various hemoglobins in normal adults, sickle cell disease, Hb CC disease, and Hb SC

disease are compared in Table 1. on page 2.

Figure 3.

HPLC in a normal patient, Hb SS disease, Hb SC disease, and Hb C trait. Elution times on these plots shows Hb F at 1.08-1.13

minutes, Hb A at 2.37-2.49 minutes, Hb A2 at 3.61-3.62 minutes, Hb S at 4.41-4.46 minutes, and Hb C at 5.17-5.19 minutes. Note

that in Hb SS disease a dominant peak in the S window is present, without detectable Hb A, while in Hb SC disease, similar peaks are

noted in the S and C windows. In Hb C trait (heterozygous), normal Hb A is present, as is Hb C.



5 - Education

Capillary Electrophoresis Most recently, automated capillary electrophoresis (CE) instruments have been making their way in to the

clinical laboratory for hemoglobin analysis. By this method, electrophoresis is performed by adding patient

sample to a thin capillary tube containing a buffer, most often an alkaline buffer. Voltage is applied to allow

separation of hemoglobins based on their charge, similar to the traditional gel electrophoresis methods

mentioned above. This method has the advantage over HPLC of allowing accurate quantitation of Hb A2 in

the presence of Hb E; although on some CE instruments adequate separation of Hb A2 from Hb C to allow

accurate quantitation may not be possible in all cases.

DNA Sequencing While most common hemoglobin variants can be identified by other methods, some uncommon variants

require DNA sequencing for further identification. In particular, sequencing may be required for some

clinically important variants, including unstable hemoglobins, low or high oxygen affinity hemoglobins, and

M-hemoglobins, that fail to separate from Hb A by other methods, often due to their neutral charges.

Generally, polymerase chain reactions (PCR) are performed to amplify the exons of the ß and/or α-globin

genes. Then these PCR products are put through a sequencing reaction that determines the nucleotide

sequence of these genes (Figure 4.). The nucleotide substition that results in the abnormal amino acid

sequence is usually easily identified, and can be compared to known sequences for identification. In

addition to its use in hemoglobinopathies, DNA sequencing can also be useful to identify nucleotide

substitutions associated with ß-thalassemia, a group of disorders resulting in decreased production of a

normal ß-globin chain.

Figure 4.

DNA sequencing of the ß-globin chain of hemoglobin in the region of codon 6. The reference gives the normal DNA sequence in this

region, which is the same as that from a normal individual. In Hb SS disease, there is a single DNA nucleic acid substitution of a T

instead of A (see box). This changes codon 6 from GAG that codes for the amino acid glutamic acid to GTG, encoding valine. Hb S

differs from Hb A by only this single nucleic acid.



6 - Education

Now let’s move on to discuss Hb SS disease, Hb CC disease, and Hb SC disease. The hemoglobin

mutations in these disorders are compared in Table 2. and the laboratory findings and clinical features of

these disorders are compared in Table 3.

Table 2. Prevalence of Hemoglobin Disorders and Comparison of Mutations

Prevalence in African Americans

Genotype Mutation

Hemoglobin SS 0.16 Homozygous; β6 glu val

Glutamic acid in 6th position on β-globin chain replaced by valine

Hemoglobin CC 0.02 Homozygous; β6 glu lys

Glutamic acid in 6th position on β-globin chain replaced by lysine

Hemoglobin SC 0.05 Heterozygous; one allele with β6 glu val, other allele with β6 glu lys

Table 3. Laboratory Findings and Clinical Features

Clinical Manifestations

Sickling Test

Blood Smear Findings Clinical Expression Life Expectancy

Hemoglobin SS

• Severe anemia

• Normocytic

Positive • Sickle cells • Polychromasia • Nucleated RBCs • Target cells,

numerous • Howell-Jolly

bodies • Pappenheimer

bodies • + Neutrophilia • + Thrombocytosis

• Severe sickling crises

• Markedly decreased

• Fatal • In the US,

median survival ~45 years of age

Hemoglobin CC

• Mild anemia • Microcytic

Negative • Hemoglobin C crystals (hexagonal or rod-shaped)

• Target cells • Microspherocytes • Polychromasia,

minimal

• Often asymptomatic

• + Occasional jaundice

• + Occasional abdominal discomfort

• Normal life expectancy

Hemoglobin SC

• Mild to moderate anemia

• Normocytic

Positive • Sickle cells / “S/C poikilocytes” – misshapen crystals

• Target cells, numerous

• Anisocytosis, mild to severe

• Poikilocytosis, mild to severe

• Usually mild hemolytic anemia

• Splenomegaly • Less frequent

sickling crises • Less painful

crises • Normal to stocky

body habitus • More retinopathy

than Hb SS

• Slightly to moderately shortened



7 - Education

HEMOGLOBIN SS

Hemoglobin S is comprised of two normal α-globin chains and two abnormal β-globin chains that contain a

single amino acid substitution, from glutamic acid to valine, on the 6th amino acid of the beta globin chain,

hence the nomenclature of β6 glu val.

Patients who are homozygous for Hb S (sickle cell disease) generally have debilitating illness with only

partial compensation of their hemolytic anemia and baseline Hgb level of 6-10 g/dL. The mutation in the β-

globin chain results in decreased solubility of de-oxygenated hemoglobin S that then forms rigid polymers

that distort the red cells in to the characteristic sickled shape. Classically, these red cells appear in the

shape of a thin crescent with two pointed ends and will lack central pallor. The polymerization of

deoxygenated hemoglobin S may cause red cells to appear in one or more of the following forms: crescent-

shaped, boat-shaped, filament-shaped, holly-leaf form, or envelope cells (Figure 5.).

Figure 5.

Sickle cells can be seen in a variety of sickling disorders, including sickle cell anemia (homozygous Hb S),

hemoglobin SC disease, SD disease, and S-beta-thalassemia. In addition to sickled forms, the blood smear

findings include target cells, increased polychromasia, nucleated red cells, and findings associated with

hyposplenism, such as Howell-Jolly and Pappenheimer bodies (Figure 6. on the following page and Table 3.

on page 6).



8 - Education

Figure 6.

Blood smears from Hb SS patients demonstrating sickled cells, as well as occasional target cells.

As with other hemolytic anemias, these patients are prone to gallstones, and suffer consequences of

microvascular occlusion due to the decreased deformability of the sickled red cells. Microvascular occlusion

leads to pain crises, cerebrovascular accidents (strokes), aseptic necrosis of the hip, renal injury, acute

chest syndrome with pulmonary compromise, and skin ulcers. Repeated splenic infarction results in

autosplenectomy in adult patients and increased suseptibility to infection. See Figure 7. on the following

page and Table 4. on page 10 for a summary of sickle cell anemia and complications due to sickling crises.

Vascular occlusive crises can be precipitated by infection, acidosis, cold exposure, and hypoxia.

Unfortunately, life expectancy is significantly decreased in patients with sickle cell disease with median age

of death of approximately 45 years.



9 - Education

Figure 7. Sickle Cell Formation

Summary of sickle cell disease, including the complications arising from red cell sickling.



10 - Education

Table 4. Complications of Sickling Crises Due to Ischemia

Affected Site/Organ

Manifestation Consequence

Hand-foot syndrome, aka sickle cell dactylitis

Distal extremities Bilateral swelling of hands and feet

Pain

Splenic sequestration

Spleen Sudden pooling of blood and rapid enlargement of spleen

Hypovolemic shock

Functional asplenia Spleen Inadequate antibody production, impaired ability of reticuloendothelial system to clear bacteria from blood

Increased risk of certain infections, like Salmonella or pneumococcal infection

Renal papillary necrosis

Kidney Blood in urine Chronic kidney disease

Vaso-occlusive crises

Abdomen Abdominal pain

Aseptic bone necrosis

Bone Bone pain Possible osteomyelitis; bone fracture

Acute chest syndrome

Lungs

Shortness of breath

Hypoxia

HEMOGLOBIN CC

Similar to hemoglobin S, Hemoglobin C results from a single amino acid substitution on the 6th amino acid

of the beta globin chain. Only, in hemoglobin C, the substitution is from glutamic acid to lysine

(β6 glu lys).

Patients who are homozygous for Hb C generally have mild red cell microcytosis (MCV of approximately

70-72 fL) and a mild to moderate hemolytic anemia that is less severe than that seen in homozygous Hb S.

In patients with homozygous Hb C the hemolysis is well compensated, and anemia may not even be

present. These patients are largely asymptomatic; although some degree of splenomegaly may be present.

Hb C is also less soluble than Hb A, and forms crystals in the oxygenated state. These crystals may

contribute to hemolysis; although some authors attribute the hemolysis in these patients to potassium

efflux from the red cells. Characteristic hemoglobin C crystals within red cells of patients with this

homozygous mutant are composed of dense structures with rhomboidal, tetragonal, or rod shapes (Figure

8. on the following page). They often distort the cell and project beyond its rim. The classic crystal shape

resembles the Washington monument. The crystals are often surrounded partly by a clear area or blister

devoid of hemoglobin. Hemoglobin C crystals are readily seen after splenectomy in patients with

hemoglobin C disease or SC disease. In addition to Hb C crystals, the blood smear frequently shows

numerous target cells and occasional microspherocytes. Hb C appears identical to Hb E on alkaline

electrophoresis but can be easily identified on acid electrophoresis since Hb E migrates with Hb A by this

method.



11 - Education

Figure 8.

Blood smears from Hb CC patients demonstrating Hb C crystals.

HEMOGLOBIN SC

Hb SC disease can be viewed as a hybrid of hemoglobin S and C; an “intermediate” type of disease, if you

will. The symptoms are not as severe as Hb SS, and yet its manifestations are not quite as benign as Hb

CC.

Diagnosis of Hemoglobin SC

Hemoglobin SC disease is easily diagnosed, based primarily on the essentially equal amounts of HbS and

HbC detected on hemoglobin electrophoresis (Figure 9.) and HPLC (Figure 3. on page 4). On alkaline

electrophoresis, however, recall that hemoglobin C can migrate with hemoglobins A2, E, O-Harlem, and O-

Arab. Hb C is easily distinguished on acid electrophoresis, thankfully, as it is the only hemoglobin that

migrates to the C position.

Figure 9.

Alkaline and acid electrophoresis in a patient with Hb SC disease (see starred gel lanes).



12 - Education

Blood Smear Findings Anemia is frequently seen, albeit often a mild hemolytic anemia. On occasion, moderate, normochromic and

normocytic anemia is present; although the Hb level usually remains above 10 g/dL. While true sickled cells

are rare, as in the present case (Figure 10A.), irregularly shaped cells, appearing to contain crystalline

material, are frequently present and often referred to as SC poikilocytes (Figure 10B. and C.). Other cells

may have a boat shaped (resembling plump sickled cells) or clam shell appearance, and target cells are

frequently seen. The typical polyhedral crystals characteristic of homozygous C disease are unusual in SC

disease. Our current case is an excellent example where target cells predominate, affecting up to 85% of

all red blood cells, and true sickled cells are fairly uncommon.

Figure 10A.

Figure 10B.

Figure 10C.

Photomicrographs from the blood smear in our patient with Hb SC disease. Panel A shows a sickled cell, an uncommon finding in SC disease. Panels B and C show crystalline material in SC poikilocytes.

Pathophysiology of Hemoglobin SC The patholophysiology of sickling manifestations and hemolytic anemia in SC disease remain controversial.

It is not clear that Hb S and Hb C co-polymerize to result in sickling in these patients. Two possible factors

contribute to the sickling manifestations in SC disease, namely the increased proportion of Hb S in

comparison to S trait (50% vs. 40%) and the cellular dehydration associated with the presence of Hb C.



13 - Education

Clinical Implications Interestingly, Hb SC presents most often as a mild hemolytic anemia; although there is a wide spectrum of

disease severity in these patients. Some individuals are largely asymptomatic while others suffer frequent

sickling crises and resulting in severe consequences. Sometimes, splenomegaly may be the only sign of Hb

SC. While this disorder typically presents in childhood, it may not be detected until later in life.

In patients who suffer from sickling crises, their symptoms correlate with the sites affected by hypoxia: hip

and low back pain may signal avascular necrosis of the femoral head. Blood in the urine may indicate

infarcts in the kidney parenchyma. Abdominal pain may be a manifestation of splenic infarction. Acute

chest syndrome, similar to that seen in Hb SS homozygotes, can present with fatigue, shortness of breath

while resting and/or upon exertion. In contrast to Hb SS, life expectancy is only slightly to moderately

shortened.

CASE HISTORY, CONCLUSION

As mentioned previously, the Case History and photographs (images) are from a patient with Hb SC

disease. Although SC disease is generally less severe than homozygous Hb S, our patient demonstrates

relatively severe clinical findings, including significant anemia, and a history of repeated sickling crises, the

most recent of which was associated with avascular necrosis of the hip.



14 - Education

References

1. McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Management By Laboratory Methods.

21st ed. Philadelphia, PA: Saunders Elsevier; 2007.

2. Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing.

Northfield, IL: College of American Pathologists; 1998.

3. Hoyer JD, Kroft SH, eds. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency

Testing. Northfield, IL: College of American Pathologists: 2003.

4. Nagel RL, Fabry ME, Steinberg MH. The paradox of hemoglobin SC disease. Blood Reviews.

2003:17;167-178.



15 - Education

Education Activity Authors

Joan Etzell, MD, FCAP: Joan Etzell, MD, is a Professor of Clinical Laboratory Medicine and the Director

of the Clinical Hematology Laboratory at the University of California, San Francisco (UCSF). She is AP/CP

and Hematology Board certified by the American Board of Pathology. Dr. Etzell is actively involved in the

education of medical technologists, medical students, residents, and fellows in hematology /

hematopathology. She serves as the Hematopathology Fellowship Director and Associate Residency

Program Director in Laboratory Medicine in UCSF. Dr. Etzell has authored over 50 papers, book chapters,

educational activities and abstracts in the areas of hematology and hematopathology. Dr. Etzell currently

serves as the Vice-Chair of the Hematology and Clinical Microscopy Resource Committee for the College of

American Pathologists (CAP).

Maria E. Vergara-Lluri, MD: Ria Vergara-Lluri, MD, is a Fellow in Surgical Pathology at the University of

California, San Francisco (UCSF) Medical Center in San Francisco, California. She is in her fourth year of

postgraduate training in anatomic pathology and clinical pathology. Dr. Vergara-Lluri is co-chief resident for

the anatomic pathology department at UCSF (2010-2011), serving as leader, liaison and advocate for

resident education and training. She is the junior member of the Hematology and Clinical Microscopy

Resource Committee for the College of American Pathologists (CAP).

Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML)


16 - Education

Please Note: To view the Figures and Images contained within this education activity in color, access the

electronic version of the reading.

DISCUSSION

The most common of the myeloproliferative neoplasms, chronic myelogenous leukemia, BCR-ABL1 positive

(CML), accounts for about 20 – 35% of all adult leukemias. It typically occurs at ages 40 – 60, with

approximately 20 – 40% of patients being asymptomatic at diagnosis. The diagnosis of CML may be

suggested by hepatosplenomegaly on physical examination or abnormal results—leukocytosis, anemia, or

thrombocytosis—on routine hematologic testing. When symptoms occur, they usually relate to

splenomegaly (left upper quadrant discomfort or early satiety), problems from increased white cell

production (bone pain, mild fever, night sweats, weight loss), or anemia (dyspnea, fatigue, pallor); see

Table 1 below. The median survival is now about 5 – 7 years, and data thus far from clinical trials with

imatinib mesylate (Gleevec®) and similar targeted therapies suggest that this figure will continue to

improve.

Table 1. Signs and Symptoms of CML

Symptoms Signs Fatigue Splenomegaly Weight loss Sternal tenderness Abdominal fullness and early satiety Lymphadenopathy Easy bruising or bleeding Hepatomegaly Abdominal pain Purpura Fever Retinal hemorrhage

Adapted from Wintrobe’s Atlas of Clinical Hematology. Chronic myeloproliferative syndromes. George TI. 2007:115.

If untreated, the disease often progresses through three stages (Figure 1. on the following page):

Chronic phase (CML-CP)

Accelerated phase (CML-AP)

Blast phase (CML-BP)



17 - Education

Figure 1. Natural History of CML

By definition, blasts represent less than 10% of marrow nucleated cells in the chronic phaseof the disease. In blast phase or blast crisis, 20% or more blasts are present in the bone marrow. Accelerated phase of the disease typically shows 10 – 19% marrow blasts. Most patients present in the chronic phase of the disease and if untreated progress to either accelerated phase or blast crisis.

In the chronic phase, the blood studies typically show mild anemia and leukocytosis that usually exceeds

25 x 109/L (median white count of about 170 x 109/L), primarily composed of neutrophils in various stages

of maturation, particularly due to increases in myelocytes and mature neutrophils (Figure 2. on the

following page). Basophils are universally increased, and eosinophilia is common, which may be helpful in

differentiating CML from a reactive neutrophilia. The platelet count is normal or elevated and may exceed

1000 x 109/L, but resulting thrombosis is unusual. The serum lactate dehydrogenase (LDH) and uric acid

are commonly increased, reflecting the underlying excessive marrow cell proliferation. The bone marrow

shows hypercellularity in the neutrophil line, but myeloblasts usually constitute < 5% of the cells. Unlike

other myeloproliferative diseases, megakaryocytes are often small and hypolobated (Figure 3. on the

following page). Reticulin fibrosis may be increased, particularly in the 50% of patients where

megakaryocytes are increased in number. Because of the increased and uncontrolled hematopoiesis, the

number of cells undergoing proliferation and eventual cell death is increased, and macrophages containing

the lipids from the dead cells may be visible as sea-blue histiocytes or pseudo-Gaucher cells.



18 - Education

Figure 2. Peripheral Blood Smear

Figure 3. Bone Marrow Biopsy

Peripheral blood smear, chronic phase of CML. The blood smear shows a marked leukocytosis with a predominance of segmented neutrophils and myelocytes. (Wright Giemsa, original magnification x600). George TI, Arber DA. Pathology of the myeloproliferative diseases.Hematol Oncol Clin North Am. 2003;17:1104, Fig 1.

Bone marrow biopsy, chronic phase of CML. The biopsy is markedly hypercellular with very little fat. A myeloid hyperplasia is present with many segmented neutrophils. Increased numbers of dwarf megakaryocytes are present, some in a cluster. (Hematoxylin & eosin, original magnification x 400). George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am.2003;17:1104, Fig 1.

The accelerated phase is defined by at least one of the following (also, see Table 2. below):

1. Persistent or increasing white count (> 10 x 109/L) and/or spleen size despite therapy

2. Persistent thrombocytopenia (< 100 x 109/L) or thrombocytosis (> 1000 x 109/L) despite

treatment

3. Cytogenetic evidence of clonal evolution

4. Peripheral blood basophilia ≥ 20%

5. 10 – 19% blasts in the bone marrow or peripheral blood

The first 3 criteria have been associated with the transformation from chronic phase to accelerated phase,

with the last 2 criteria associated with the transition from accelerated phase to blast phase. Other bone

marrow findings that are suggestive of, but not definitive for, CML-AP are marked dysplasia of neutrophil

precursors or the appearance of abundant aggregates of small, dysplastic megakaryocytes associated with

marked reticulin or collagen fibrosis.

Table 2. Accelerated Phase of Chronic Myelogenous Leukemia

Criteria Occurrence Blood/marrow blasts 10-19% Blood basophils ≥ 20% Cytogenetics Clonal evolution Persistent thrombocytopenia < 100 x 109/L Persistent thrombocytosis* > 1,000 x 109/L Splenomegaly, rising WBC* Morphology/histopathology Megakaryocyte proliferation, ↑fibrosis

and/or granulocytic dysplasia *unresponsive to therapy



19 - Education

The blast phase is defined by one or more of the following features:

1. Blasts account for ≥ 20% of peripheral blood white cells or nucleated bone marrow cells

2. Blasts proliferate in extramedullary sites, such as the skin, lymph nodes, and spleen

3. Large aggregates of blasts occur in the bone marrow

The blasts are usually myeloid, but in about 20 – 30% of cases they are lymphoid, usually precursor B

lymphoblasts. The ability of CML to transform to either a myeloid or lymphoid leukemia confirms that this

disorder arises from a pluripotent bone marrow stem cell that is capable of multilineage differentiation.

Occasionally, both types of leukemic blasts are present simultaneously.

Genetic abnormalities now define CML. All cases of CML have a BCR-ABL1 fusion gene resulting from a

translocation involving chromosomes 9 and 22. In 90 – 95% of cases the characteristic translocation—

t(9;22)(q34;q11)—results in the Philadelphia chromosome and is detected on routine cytogenetic studies.

The Philadelphia chromosome refers to the abnormal chromosome 22 created from fusion of part of the

ABL1 gene on chromosome 9 to part of the BCR gene on chromosome 22 (Figure 4. below).

Figure 4. Cytogenetics Karyotype

Arrows indicate the characteristic translocation involving chromosomes 9 and 22 of chronic myelogenous leukemia. In addition, this karyotype also has a translocation between chromosomes 4 and 5, as well as an extra Y chromosome. Clonal evolution, that is additional chromosomal abnormalities other than the t(9;22) translocation often signify acceleration of disease. Courtesy of Stanford Cytogenetics Laboratory, Stanford, CA.



20 - Education

Most of the remaining cases of CML have variant genetic abnormalities that result in the BCR-ABL1 fusion

gene, but involve other chromosomes in addition to chromosomes 9 and 22. A small number of cases of

CML have cryptic BCR-ABL1 translocations that cannot be identified by routine cytogenetic analysis. When

this is suspected, molecular genetic studies, such as reverse transcriptase polymerase chain reaction

(RT-PCR) analysis and FISH analysis, are indicated (Table 3. and Figure 5. below). In all cases the

BCR-ABL1 fusion gene results in production of an abnormal BCR-ABL1 protein with enhanced tyrosine

kinase activity that leads to the marked cellular proliferation characteristic of CML.

Table 3. Genetic Evidence of Chronic Myelogenous Leukemia

Technique Sensitivity (%) Cytogenetic karyotype 90-95 RT-PCR 99 FISH > 99

RT-PCR = reverse transcriptase polymerase chain reaction FISH = fluorescent in situ hybridization

Figure 5. FISH

Interphase dual fusion fluorescent in situ hybridization (FISH) for detection of the BCR-ABL1 fusion gene resulting from the t(9;22) translocation. In interphase FISH, a probe directed at 9q34 (ABL1 gene) is labeled with a green fluorophore and a probe directed at 22q11.2 (BCR gene) is labeled with a red fluorophore. When a fusion gene is present, the red and green combine to form a yellow signal. The presence of two yellow signals indicates that both BCR-ABL1 and ABL1-BCR fusion genes are present. Courtesy of Stanford Cytogenetics Laboratory, Stanford, CA.



21 - Education

The differential diagnosis of chronic myelogenous leukemia includes both reactive and neoplastic disorders.

The reactive disorders are called leukemoid reactions and mimic the features of CML. In leukemoid

reactions the white blood cell count is typically below 50 x 109/L, while CML often presents with much

higher white blood cell counts. Leukemoid reactions can be due to any strong stimulation of the bone

marrow, including bacterial infection, growth factors, carcinoma or other metastatic disease, tuberculosis

and even certain viral infections. While a leukemoid reaction will show a neutrophilia and may show a left-

shift in granulocytes, they do not show the typical predominance of segmented neutrophils and myelocytes

seen in CML. Leukemoid reactions also lack the eosinophilia and basophilia found in CML. Leukemoid

reactions often show features of neutrophil infection/activation (i.e., toxic granulation, vacuolated

neutrophils, Dohle bodies). Neoplastic disorders that can mimic CML include chronic myelomonocytic

leukemia (CMML) and atypical chronic myelogenous leukemia, BCR-ABL1 negative (aCML), (Table 4.

below).

Table 4. Differential Diagnosis of Chronic Myelogenous Leukemia

Feature CML aCML CMML BCR/ABL1 + - - WBC +++ ++ + Basophils* ≥ 2% < 2% < 2% Monocytes* < 3% 3 – 10% > 10% Immature granulocytes* > 20% 10 – 20% ≤ 10% Blasts* < 2% > 2% < 2% Granulocyte dysplasia - ++ + Marrow erythroid hyperplasia - - +

CML = chronic myelogenous leukemia BCR-ABL1 positive aCML = atypical chronic myelogenous leukemia BCR-ABL1 negative CMML = chronic myelomonocytic leukemia *In peripheral blood Adapted from George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am. 2003;17:1101-1127.

All can show elevated white blood cell counts, but the highest counts are typically seen in CML. CMML and

aCML can show basophilia, but typically only CML has peripheral blood basophilia more than 2%. All three

disorders may show a monocytosis, but a monocytosis of more than 10% of white blood cells is usually

only seen with CMML. Large numbers of immature granulocytes in the peripheral blood (i.e., more than

20%) are typical of CML. Whereas immature myeloid forms may be seen in CMML and aCML, immature

granulocytes are less than 20% of the blood leukocytes. Blasts may be observed in the peripheral blood of

any of these disorders, but the highest blast counts are seen in aCML (> 2%), while blasts counts less

than 2% are more typical of chronic phase CML and CMML. Granulocytic dysplasia is most marked in

aCML and is seen to a lesser extent in CMML. Although granulocytic dysplasia can be seen in CML, it is

less marked than in the other 2 neoplastic disorders. If dysplasia is seen in CML, it is usually manifested by

hypogranular leukocytes, giant metamyelocytes, hypersegmented neutrophils, leukocytes with mixed

basophil-eosinophil granules, and Pelgeroid-like neutrophils. Other morphologic abnormalities include

nucleated red blood cells, target cells, giant platelets, mitotic cells and megakaryocyte nuclei. However, as

noted above, the defining feature of CML is the presence of the BCR-ABL1 fusion gene, allowing for



22 - Education

definitive diagnosis with molecular or cytogenetic testing in most cases. Review of cases that were

previously called BCR-ABL1 negative CML has resulted in reclassification of many of these cases as either

CMML or aCML.

In the past, CML was considered a progressive disease with allogeneic bone marrow transplant as the only

potentially curative treatment. Median survival was approximately 5 years. The therapy of CML was

markedly altered by discovery of imatinib mesylate, a tyrosine kinase inhibitor that directly targets the BCR-

ABL1 tyrosine kinase fusion protein. This has allowed for targeted therapy specific for CML. Newly

diagnosed patients have a 95% chance of reaching a hematologic response within 18 months (resolution of

blood and marrow abnormalities with normalization of blood counts) or a 75% chance of complete

cytogenetic response (loss of detectable t(9;22)). Responses are also seen in accelerated and blast phases

of CML, although the response rate is not as high. Treatment of CML is not curative in most cases, and

small numbers of abnormal cells remain that can be detected by molecular (RT-PCR) monitoring to detect

the BCR-ABL1 transcript. All patients treated with imatinib mesylate have blood or marrow monitored on a

routine basis by quantitative molecular testing using RT-PCR to identify changes in BCR-ABL1 transcript

levels. Significant increases in the levels of the BCR-ABL1 transcript correlate with developing resistance to

imatinib therapy, usually due to mutations in the drug-binding site in the neoplastic cells. Mutations in the

drug-binding site may also be identified by gene sequencing in patients who do not respond initially to

therapy. When drug resistance is suspected, increasing doses of imatinib or use of second-generation

tyrosine kinase inhibitors often allows control of the disease. With this approach, patients with CML can be

effectively treated and maintained for long periods of time without disease progression or transformation.



23 - Education

References

1. George TI. Chronic myeloproliferative syndromes. In: Tkachuk D, Hirschmann JV, eds. Wintrobe’s Atlas

of Clinical Hematology. Philadelphia, PA: Lippincott Williams and Wilkins, Inc; 2007: 105-136.

2. George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am.

2003;17(5):1101-1127.

3. George TI. Pathology of the myeloproliferative diseases. In: Greer JP, Foerster J, Rodgers GM,

Paraskevas F, Glader B, Arber DA, Means RT, eds. Wintrobe’s Clinical Hematology. 12th ed.

Philadelphia, PA: Lippincott Williams and Wilkins, Inc; 2008.



24 - Education

Education Activity Authors

Tracy I. George, MD, FCAP: Tracy George, MD, is Director of Hematology for Stanford University

Medical Center Clinical Laboratories, which serves Stanford Hospital and Clinics and Lucile Salter Packard

Children’s Hospital. She is an assistant professor of Pathology at the Stanford University School of

Medicine in Stanford, CA. Dr. George has written over 70 papers, book chapters, books, educational

activities and abstracts in the areas of hematology, hematopathology and surgical pathology. She teaches

medical students, residents, and fellows, participates in clinical service work in hematopathology, and

performs translational research in the areas of myeloproliferative neoplasms and laboratory hematology. Dr.

George is currently Chair of the Hematology and Clinical Microscopy Resource Committee and a member of

the Council on Scientific Affairs for the College of American Pathologists (CAP).

Kyle T. Bradley, MD, MS, FCAP: Kyle T. Bradley, MD, is an Assistant Professor in the Department of

Pathology & Laboratory Medicine at Emory University Hospital in Atlanta, GA. He is board certified in

anatomic pathology, clinical pathology, and hematology by the American Board of Pathology. His primary

responsibilities are in clinical service work and resident/fellow teaching in the areas of surgical pathology

and hematopathology. Dr. Bradley has authored a number of original articles, abstracts, and educational

activities in the fields of hematopathology and anatomic pathology and is a member of the Hematology and

Clinical Microscopy Resource Committee for the College of American Pathologists (CAP).

Sherrie L. Perkins, MD, PhD, FCAP: Sherrie L. Perkins, MD, PhD, is a professor of Pathology at the

University of Utah Health Sciences Center and the Chief Medical Officer for ARUP Laboratories in Salt Lake

City, UT. She is the Director of Hematopathology for ARUP Laboratories and has responsibilities in

teaching, resident training, clinical service and research. Dr. Perkins has written over 140 peer-reviewed

papers and 70 book chapters in the areas of hematology and hematopathology. Dr. Perkins is currently a

member of the College of American Pathologists (CAP) Hematology and Clinical Microscopy Resource

Committee.

blood cell identification: 2010-c mailing: hemoglobin sc disease · acid electrophoresis, run at a...

Documents