Blood Cell Identification – Graded€¦ · lymphocytes as a reference for the size of normal red blood cells (RBC), the marked degree of red cell size variation (anisocytosis) can

2

Blood Cell Identification – Graded Case History

This peripheral blood smear is from a 15-year-old boy from Vietnam. Laboratory data include: WBC = 14.8 × 109/L; RBC = 3.68 × 1012/L; HGB = 9.1 g/dL; HCT = 28%; MCV = 77 fL; MCHC = 32 g/dL; RDW = 28.5 and PLT = 766 × 109/L. Identify the arrowed object(s) on each image. (BLOOD, WRIGHT-GIEMSA)

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P-11

Referees Participants Identification No. % No. % Evaluation Neutrophil, segmented or band 95 96.0 4988 94.5 Good Neutrophil with hypersegmented nucleus 2 2.0 200 3.8 Unacceptable Neutrophil, toxic 1 1.0 70 1.3 Unacceptable Neutrophil, polyploid 1 1.0 4 0.1 Unacceptable The arrowed cell is a segmented neutrophil as correctly identified by 96.0% of the referees and 94.5%

of the participants. It has a characteristic nucleus divided into three lobes by a thin filament of nuclear material. The cytoplasm is pale pink with fine granules. There is a marked degree of red cell size variation (anisocytosis) and increased number of polychromatophilic red cells. Multiple nucleated red cells are present.

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Blood Cell Identification – Graded

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P-12

Referees Participants Identification No. % No. % Evaluation Nucleated red cell, normal or abnormal

morphology 99 100.0 5131 97.3 Good

The arrowed cells are nucleated red cells present in the peripheral blood as correctly identified by

100.0% of the referees and 97.3% of the participants. These cells have small dark nuclei, with clumped chromatin and abundant cytoplasm. In this peripheral blood smear, macrocytes, microcytes and a few normocytes are represented. The majority of the red cells are hypochromic. Increased numbers of polychromatophilic red cells and target cells are present. Many of the red blood cells contain small inclusions on the periphery of the cell, known as Pappenheimer bodies. Basophilic stippling is also present.

4


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P-13

Referees Participants Identification No. % No. % Evaluation Monocyte 99 100.0 5216 98.9 Good The arrowed cell is a monocyte as correctly identified by 100.0% of the referees and 98.9% of the

participants. It is round, slightly larger than a neutrophil, with an indented nucleus. The edges of this cell are smooth; however some monocytes have pseudopod-like cytoplasmic extensions. There is abundant gray-blue cytoplasm with fine pink azurophilic granules. There are several empty vacuoles present. Similar to the other blood films associated with this case, there is marked anisocytosis of the surrounding red cells with the presence of nucleated red cells and polychromasia.

5


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P-14

Referees Participants Identification No. % No. % Evaluation Eosinphil, any stage 98 100.0 5271 99.9 Good The arrowed cell is an eosinophil as correctly identified by 100.0% of the referees and 99.9% of the

participants. The cytoplasm is evenly filled with coarse, uniform sized, orange- red granules which rarely overlie the cell nucleus. This cell has a bilobed nucleus separated by a barely visible thin filament. Two nucleated red cells are also present.

6


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P-15

Referees Participants Identification No. % No. % Evaluation Target cell (codocyte) 99 100.0 5261 99.7 Good The arrowed cells are target cells as correctly identified by 100.0% of referees and 99.7% of the

participants. These cells are red cells that have the appearance of a bull’s-eye, with a dark central hemoglobinized area within an area of relative pallor, surrounded by a peripheral hemoglobinized zone. Target cells have a disproportionate increase in the ratio of surface membrane to volume. In this peripheral smear, several microspherocytes are located in the lower center area of the slide. Spherocytes stain more densely and lack the central pallor of a normal red blood cell. One nucleated red blood cell can be seen at the left edge. Marked anisocytosis with hypochromia, increased polychromatophilic red cells, and Pappenheimer bodies are also noted.

Janet Piscitelli, MD Hematology and Clinical Microscopy Resource Committee

Blood Cell Identification: 2012-B Mailing: Hemoglobin E-Beta ( ) Thalassemia

7- Education

All material is © 2012 College of American Pathologists, all rights reserved.

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 15-year-old male from Vietnam. Laboratory data include: WBC=14.8 x109/L; RBC=3.68 x

1012/L; HGB=9.1 g/dL; HCT=28 %; MCV=77 fL; MCHC=32 g/dL; RDW=28.5; and PLT=766 x 109/L

Figure 1: Peripheral Blood Smear

INTRODUCTION

This case involves a patient from South East (SE) Asia with anemia, microcytosis, and a wide red cell

distribution width (RDW). In the peripheral smear provided (Figure 1), using the nuclei of the small

lymphocytes as a reference for the size of normal red blood cells (RBC), the marked degree of red cell size

variation (anisocytosis) can be appreciated. Macrocytes, microcytes and a few normocytes are represented.

The majority of the red cells are hypochromic. Increased polychromasia and target cells are present. Many

of the red blood cells contain small inclusions on the periphery of the cell, known as Pappenheimer bodies.

Nucleated red blood cells are also noted. These morphological findings, while frequently associated with

hemoglobinopathies and thalassemia, are non-specific. Additional studies, including hemoglobin separation

techniques were consistent with a Hemoglobin (Hb) E/ beta ( ) thalassemia.


8- Education


Thalassemia; alpha ( ) and beta ( ):

Thalassemias are globin gene mutations that result in impaired synthesis of the globin chains. The

thalassemias have a high prevalence in South East Asia and in the Mediterranean basin. Alpha ( )

thalassemia is common in Southeast Asia, Malaysia, and Southern China whereas beta ( ) thalassemia is

common in the Indian subcontinent and in people of African ancestry. The frequency of the mildest type of

alpha ( ) thalassemia can reach greater than 90% in some populations. The high prevalence areas of

thalassemias overlap with hemoglobin variants common in the “malaria belt” and co-inheritance of Hb S or

Hb E with thalassemia is not unusual.

Hemoglobin A, the major hemoglobin in adult life, is a tetramer composed of two alpha ( ) and two beta ( )

globin chains. Each globin chain is linked to a heme group via histidine residues. The heme within the

tetramers contains iron in the ferrous form (2+), allowing it to reversibly bind and transport O2. Decreased

production or any alteration in the structure of the hemoglobin chain can reduce the oxygen carrying

capacity and the solubility of hemoglobin in the aqueous environment of the blood.

In adults, Hb A comprises approximately 97% of the total normal hemoglobin. Additionally, red cells

contain small quantities of Hb A2 and Hb F. Hb A2 consists of two alpha ( ) globin chains and two delta ( )

globin chains, whereas Hb F is formed from two alpha ( ) globin chains and two gamma ( ) globin chains.

Any variation in the relative percentages of these hemoglobin fractions may be representative of a

hemoglobinopathy. Various methods to determine the proportion of the hemoglobin fractions include:

electrophoresis (alkaline, acid or capillary), isoelectric focusing, high performance liquid chromatography

(HPLC) and amino acid/DNA sequencing. Frequently definitive diagnosis will require integration of data from

several different methodologies.

The alpha ( ) thalassemias are a group of conditions resulting from a reduced rate of synthesis of the alpha

( ) globin chains, and usually occur secondary to large deletions in one or more of the four alpha ( ) globin

genes. The alpha ( ) globin gene cluster on chromosome 16 contains two functioning alpha ( ) globin

genes (designated as 1 and 2). Alpha 2 ( 2) genes contribute 1.5-3.0 times more to the production of

globin chains than the 1genes.

The clinical spectrum of patients with alpha ( ) thalassemia ranges from asymptomatic to a fatally severe

anemia (Hb Barts, hydrops fetalis), depending on the number of alpha ( ) genes deleted. A summary of the

alpha ( ) thalassemias and associated disorders are listed in Table 1. The pathophysiology of the anemia in

these conditions is secondary to the imbalance between the and chains. In some of the more severe

forms of alpha ( ) thalassemia, the excess beta ( ) chains form an abnormal tetramer ( 4) known as

Hemoglobin H (Hb H). Hemoglobin H has a high affinity for oxygen: it is unstable and precipitates in both

red cell precursors and mature cells. This leads to hemolytic anemia and ineffective erythropoiesis with

bone marrow expansion and splenomegaly.


9- Education


Alpha ( ) thalassemia is suspected when there is a microcytosis and compensatory red blood cell increase

in patients in which beta thalassemia or iron deficiency has been excluded. Some individuals with multiple

alpha ( ) gene deletions may have Hb Barts or Hb H by electrophoresis or HPLC, however, the hemoglobin

separation pattern in a number of cases can be normal and definitive diagnosis requires DNA analysis.

Table 1 – Summary of -Thalassemia (adapted from Hoyer & Kroft, 2003)

Subtype Genes

Deleted

Associated Disorder Clinical Effect

Normal 0 None None

Heterozygous -thal-2 1 Silent Carrier Asymptomatic

Homozygous -thal-2 2 Thalassemia minor Microcytosis +/- mild anemia

Heterozygous -thal-1 2 Thalassemia minor Microcytosis +/- mild anemia

-thal-1/ -thal-2 3 Hemoglobin H

disease

Chronic hemolytic anemia

Homozygous -thal-1 4 Hb Barts, hydrops

fetalis

Fatal

Beta ( ) thalassemias are a group of conditions in which there is absent or decreased production of beta ( )

globin chains. More than 200 mutations on Chromosome 11, which tend to affect transcription, translation

or RNA stability, have been recognized in association with beta ( ) thalassemia. Unlike alpha ( )

thalassemia, the associated mutations are usually due to point mutations, rather than large deletional

mutations. Beta ( ) thalassemia may exist in the heterozygous (one normal, one abnormal allele) or

homozygous (two abnormal alleles) states. Beta ( ) thalassemia mutations are divided into two broad

categories, beta zero ( 0) thalassemia in which there is a complete absence of beta ( ) chain production by

the abnormal allele and beta plus ( +) thalassemia with reduced formation, not absence, of the beta ( )

globin chain.

The severity of beta ( ) thalassemia varies greatly depending on the mutation and the zygosity. Inheritance

of one beta ( ) thalassemia mutation results in a rather benign condition characterized by microcytosis, a

compensatory increase in the red cell number and in some cases a mild anemia (Figure 2). This has been

termed beta ( ) thalassemia trait (minor).


10- Education


Figure 2 – Beta ( ) thalassemia trait – The blood smear demonstrates

microcytosis, hypochromia and rare target cells.

Figure 3- Beta ( ) thalassemia intermedia - There is marked anisocytosis,

poikilocytosis, and hypochromia.

Both beta ( ) thalassemia intermedia and beta ( ) thalassemia major are disease states in which there is

marked decreased or absent production of the beta ( ) globin genes. These patients are either homozygous

for beta ( ) thalassemia ( 0 0 or + +) or have a compound heterozygosity, 0 +. Patients with beta ( )

thalassemia intermedia do not require transfusion for survival, which differentiates this entity from beta ( )

thalassemia major. In both beta ( ) thalassemia major and intermedia, the blood smear will include

microcytosis, anisocytosis, poikilocytosis, basophilic stippling, increased polychromasia, target cells and

circulating erythroblasts (Figure 3). These patients not only have ineffective erythropoiesis but also

shortened red cell survival. This results in extramedullary hematopoiesis with hepatomegaly, splenomegaly,

and a hemolytic anemia leading to mild jaundice and gallstones. In most patients with beta ( ) thalassemia

the hemoglobin fractionation pattern has a characteristic increase in Hb A2.


11- Education


Hemoglobin E (Hb E):

In addition to mutations that give rise to thalassemias, there are over 500 structural hemoglobin variants in

the literature resulting from the alteration in the amino acid sequence of the globin chain. Most hemoglobin

variants are secondary to point mutations in the globin genes with single amino acid replacements,

however, deletions, insertions and fusion gene mutations have been described. The clinical significance of

the individual abnormal hemoglobin variants is varied, ranging from those variants that are totally

asymptomatic to those that are incompatible with life when present on both alleles.

Hb E is the second most common structural hemoglobin variant in the world, surpassed only by Hb S

(sickle cell). It is found commonly in South East Asia; with the highest prevalence at the border between

Thailand, Laos, and Cambodia, otherwise known as the Hb E triangle. This region also has a high

prevalence for alpha ( ) and beta ( ) thalassemia, therefore, coinheritance of these mutations is not

uncommon.

Hb E results from the substitution of lysine for glutamic acid at position 26 of the beta ( ) globin chain. It

can be identified through the various hemoglobin separation techniques; at an alkaline pH on cellulose

acetate, Hb E migrates to the Hb C position (Figure 4). The migration of Hb E to the Hb A position by acid

electrophoresis distinguishes it from Hb C (Figure 5). Hb E elutes slightly prior to the Hb A2 position by

HPLC (Figure 6) and separates to Zone 4 by capillary electrophoresis (Figure 7).

Figure 4: Hb E trait, alkaline electrophoresis Hb E migrates to the Hb C position.


12- Education


Figure 5: Hb E trait, acid electrophoresis. Hb E migrates to the Hb A position, and therefore can

be differentiated from Hb C.

Figure 6: Hb E trait, HPLC chromatogram.


13- Education


Figure 7: Hb E trait, Capillary electrophoresis.

The hemoglobin that results from the lysine substitution leads to a less functional beta ( ) globin mRNA,

with a decreased production of the affected beta ( ) chain. For this reason, although it is a structural

variant, Hb E presents with thalassemic manifestations. An individual who is heterozygous for the mutation

(Hb E trait) has approximately 25-35% of the total hemoglobin comprised of Hb E. The peripheral smear

usually displays hypochromia, microcytosis and the presence of target cells. Homozygous Hb E (inheritance

of two Hb E alleles), is also clinically insignificant, with mild microcytosis, however, the peripheral smear

reveals marked target cells. In these patients Hb A is absent, with Hb E being the predominant fraction (95-

97%). It should be noted that a concomitant alpha ( ) chain mutation may be masked in this disorder, as

the clinical presentation and laboratory findings are similar.

There are several other Hb E disorders that result from co-inheritance of thalassemia mutations. Hb E

trait/alpha ( ) thalassemia combination is asymptomatic when one or two alpha ( ) genes are mutated.

Similar to Hb E trait, there is microcytosis and hypochromia on the peripheral smear; however the

percentage of Hb E is less (20-25%). Hb E trait/Hb H disease occurs when there is co-inheritance of Hb E

mutation and at least three alpha ( ) gene deletions. This is a moderately severe condition with a

hematologic picture similar to Hb H disease, however the electrophoretic pattern consists of Hb A, Hb E

(approximately 10-15%) and Hb Barts (not Hb H). The formation of beta ( ) chain tetramers (Hb H) does

not occur since alpha ( ) chains have a greater affinity for Hb A beta ( ) chains than Hb E beta ( ) chains.

As a result, in Hb E trait/Hb H disease, there are not enough surplus normal beta ( ) chains to form beta ( )

tetramers (Hb H) and Hb Barts is present instead. Homozygous Hb E/Hb H disease is characterized by the

presence of Hb F and Hb Barts, with a higher percentage of Hb E (>40%). Hb A is absent. In this disorder,

both alleles for the beta ( ) globin chain carry mutations for Hb E and in addition multiple loci of the alpha

( ) genes are deleted resulting in clinical features of Hb H disease.

The most common form of severe thalassemia in South East Asians is Hb E trait/Beta ( +) thalassemia,

with varied clinical expression. Although some patients may present with a milder form, most patients have


14- Education


a syndrome similar to thalassemia intermedia and major. Patients experience severe anemia, complications

of expanded erythropoiesis including hepatomegaly and splenomegaly, fragility and distortion of the bones

and microcytosis. The increased erythropoiesis in the bone marrow leads to neurological manifestations due

to brain or spinal cord compression due to extramedullary hematopoietic tumors. Most require repeated

blood transfusions with resultant iron overload.

Diagnosis:

Although many clinically significant hemoglobin disorders display characteristic findings on peripheral blood

smear, the diagnosis of hemoglobinopathies includes assimilation of multiple laboratory tests, as well as

clinical features and family history, including ethnic origin. The red cell indices, including hemoglobin,

hematocrit, mean corpuscular volume (MCV) and RDW usually are the first indicators of an abnormality.

Quantification of the percentages of the various hemoglobins: Hb A, Hb F, Hb A2, and in the structural

hemoglobin variants (in this case Hb E), usually aid in rendering a diagnosis. In some cases, further studies,

including amino acids/DNA sequencing are required for confirmation. Table 2 summarizes the percentages

of the various syndromes and the associated red cell indices for the disorders discussed above.

Table 2 Summary of common beta ( ) thalassemia and Hb E syndromes

Subtype Genotype Clinical Picture Adult Hemoglobin

Normal / A A Normal Hb A, Hb F

Hb A2

Beta ( ) trait / + A

/ 0 A

Mild to no anemia

Microcytosis

Erythrocytosis

Hb A,

Hb F (may be )

Hb A2:

Beta ( ) thalassemia

intermedia & Beta ( )

thalassemia major

/ 0 +

/ + +

/ 0 0

Syndromes vary from mild anemia

to a severe hemolytic anemia

requiring transfusions

Hb A, Hb F:

Hb A2:

Hb E trait / A E Mild to no anemia

Slightly decreased MCV

Erythrocytosis

Hb A, Hb F

Hb E: 25-35%

Homozygous Hb E / E E Microcytosis without associated

hemolysis

Marked target cells

Hb A: Absent

Hb F: ≤ 5%

Hb E: 95-97%


15- Education


Hb E trait/alpha( )

thalassemia

/ - A E

-/ - A E

Mild to no anemia

Microcytosis

Erythrocytosis

Hb A, Hb F

Hb E: 20-25%

Hb E trait/Hb H disease -/-- A E


Hb A,

Hb E: 10-15%

Hb Barts

Homozygous Hb E /Hb H

disease

-/-- E E


Hb A: Absent

Hb F:

Hb E: > 40%

Hb Barts

Hb E trait/Beta ( +)

thalassemia

/ + E Syndromes vary from mild anemia

to a severe hemolytic anemia

Hb A

Hb F

Hb E: > 40%

Hb E trait/Beta ( 0)

thalassemia

/ 0 E Severe anemia, icterus,

microcytosis, hepatomegaly

Hb A: Absent

Hb F: 30-60%

Hb E: > 40%

Summary:

This case illustrates the coinheritance of both a structural variant and thalassemia, with a resulting clinical

syndrome of moderate severity. The differential diagnosis of a patient with anemia, microcytosis and a

wide RDW from South East Asia includes: thalassemia, structural hemoglobin variants, and iron deficiency.

The peripheral smear findings, although typical for a thalassemic disorder, are non-specific. Definitive

diagnosis requires additional tests including hemoglobin separation techniques and/or DNA sequencing.

Janet Piscitelli, MD


16- Education


(REFERENCES)

1. Bain BJ. Haemoglobinopathy Diagnosis. Cambridge, MA: Blackwell Publishing; 2006.

2. Clarke GM, Higgins TN. Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and

Update. Clin Chem. 2000;46(8):1284-1290.

3. Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology Basic Principles and Practice. 3rd ed. New York,

NY: Churchill Livingstone; 2000:1106-1129.

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

Testing. Northfield, IL: CAP Press; 2003.

5. Huisman THJ, Carver MFH, Efremov GD. A Syllabus of Human Hemoglobin Variants. Augusta, GA: The

Sickle Cell Anemia Foundation; 1996.

6. Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of Hemoglobin. Genetics,

Pathophysiology, and Clinical Management. Cambridge, UK: Cambridge University Press; 2001.

Janet Piscitelli, MD, FCAP: is the Medical Director for the Teterboro Laboratory of Quest Diagnostics. She

is a Diplomate of the American Board of Pathology, Anatomic and Clinical Pathology, and a Clinical

Assistant Professor of Pathology at the Albert Einstein College of Medicine in the Bronx, NY. Her primary

responsibilities are in clinical service work, with a special interest in hemoglobinopathies. Dr. Piscitelli is has

authored several clinical pathology articles and is currently a member of the Hematology and Clinical

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

7

Blood Cell Identification – Ungraded Case History

This peripheral blood smear is from a 16-year-old boy with a history of episodic hemolytic anemia since birth. Laboratory data included: WBC = 12.5 × 109/L; RBC = 1.95 × 1012/L; HGB = 8.5 g/dL; HCT = 25.3%; RDW = 19 and PLT = 698 × 109/L. Identify the arrowed object(s) on each image. (BLOOD, WRIGHT-GIEMSA)

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P-16

Referees Participants Identification No. % No. % Evaluation Pappenheimer bodies, presumptive

(Wright stain) 87 89.7 4618 88.8 Educational

Howell Jolly body (Wright stain) 7 7.2 381 7.3 Educational Pappenheimer bodies, (iron stain) 1 1.0 87 1.7 Educational Basphilic stippling (coarse) 1 1.0 50 1.0 Educational Plasmodium sp. (malaria) 1 1.0 16 0.3 Educational The arrowed objects within the cells are Pappenheimer bodies, as correctly identified by 89.7% of the

referees and 88.8% of the participants. Pappenheimer bodies are red cell inclusions represented by blue-purple inclusions located at the periphery of the RBC. They are less than 1 um in diameter and may be less than 0.5 um and may form doublets, as in these cells. They are evident on Wright Giemsa and supravital stains. As these inclusions are iron containing autophagosomes, they also stain with iron stains. Pappenheimer bodies are seen in the peripheral blood of patients with hyposplenic conditions, absent spleen, megaloblastic anemia and severe hemolytic anemia, as in this case. (Note the presence of anisocytosis and poikilocytosis, including echinocytes (burr cells, crenated cells).

8

Blood Cell Identification – Ungraded

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P-17

Referees Participants Identification No. % No. % Evaluation Platelet, normal 98 100.0 5177 99.4 Educational The arrowed cells are platelets, as correctly identified by 100.0% of the referees and 99.4% of the

participants. Mature platelets or thrombocytes range in size from 1 to 4 µm and are round or oval. They are not actually cells, but cytoplasmic fragments of their precursor cell, the megakaryocyte. Their cytoplasm is pale blue or grey and contains purple-red granules. Tight clustering of these granules may impart the appearance of a nucleus. This patient did have thrombocytosis of 698 x 109/L, which may occur in hemolytic anemias, including pyruvate kinase deficiency.

9


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P-18

Referees Participants Identification No. % No. % Evaluation Polychromatophilic (non-nucleated) red

cell 98 100.0 5157 99.1 Educational

The arrowed cells are polychromatophilic red blood cells, as correctly identified by 100.0% of the

referees and 99.1% of the participants. These immature red cells that is slightly larger than normal red cells. They are round or very slightly oval and contain ribonucleic acid (RNA) in addition to hemoglobin, which imparts a more grey-blue staining appearance. In adults and children over one week of age, an increase in these cells above the normal 1% is termed polychromasia. It is associated with increased bone marrow erythropoietic activity with release of reticulocytes into the circulation, in this case in response to red cell destruction, or hemolysis. Acute blood loss, bone marrow recovery following chemotherapy or other factors such as infections or drugs which can suppress the marrow will result in polychromasia.

10


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P-19

Referees Participants Identification No. % No. % Evaluation Echinocyte (burr cell, crenated cell) 69 70.4 3807 73.1 Educational Acanthocyte (spur cell) 28 28.6 1370 26.3 Educational Erythrocyte with overlying platelet 1 1.0 5 0.1 Educational The arrowed cell is an acanthocyte, as correctly identified by 28.6% of the referees and 26.3% of the

participants. Acanthocytes, or spur cells, are spheroidal red cells lacking central pallor with his spine-like projections that are irregularly distributed over the cell surface. They stain densely like spherocytes. A large number of acanthocytes may elevate the MCHC. Acanthocytes differ from echinocytes in that the latter cells exhibit central pallor and tend to have evenly distributed, short blunt spicules. Echinocytes are often due to an artifact in smear preparation and found at the edge of a blood smear, but may also be seen in uremia, pyruvate kinase deficiency, severe burns and other disorders. The lack of definitive central pallor in the arrowed cell favors acanthocyte as the identification for this cell, although it should be recognized that acanthocytes and echinocytes may form part of a morphologic spectrum and transitional forms between acanthocytes and echinocytes may be seen. Both acanthocytes and echinocytes may be observed in pyruvate kinase deficiency.

11


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P-20

Referees Participants Identification No. % No. % Evaluation Nucleated red cell, normal or abnormal

morphology 97 99.0 5191 99.7 Educational

Howell Jolly body (Wright stain) 1 1.0 1 0.0 Educational The arrowed cell is a nucleated red blood cell, as correctly identified by 99.0% of the referees and

99.7% of the participants. This particular nucleated red cell is an orthochromatophilic normoblast. It represents the last stage in erythroid maturation prior to extrusion of the nucleus and release into the peripheral blood from the bone marrow. It follows the sequence of pronormoblast, or erythroblast, basophilic normoblast and polychromatic normoblast. The orthochromatic normoblast is round or slightly oval and contains dense nucleus that may be centrally or eccentrically located, as in this cell. Nucleated red blood cells, including more immature forms, such as polychromatic normoblasts may be seen in the peripheral blood of normal newborns, in hemolytic disease of the newborn and other hemolytic anemias, as in this patient. Nucleated red blood cells also occur in infiltrative conditions of the marrow such as metastatic carcinoma and in myelodysplastic and myeloproliferative conditions and acute leukemia.

References: 1. Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing.

Northfield, IL: College of American Pathologists; 1998. 2. Gulati G, Care J. Blood cells: An Atlas of Morphology with Clinical Relevance. Chicago, IL: ASCP

Press, 2007.

Martha R. Clarke, MD

Hematology and Clinical Microscopy Resource Committee

Blood Cell Identification: 2012-B Mailing: Pyruvate Kinase Deficiency

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 16-year-old male with a history of episodic hemolytic anemia since birth. Laboratory data

included: WBC=12.5 x 109/L; RBC=1.95 x 1012/L; HGB=8.5 g/dL; HCT=25.3 %; MCV=99 fL;

MCHC=33.5 g/dL; RDW=19; and PLT=698 x 109/L.

INTRODUCTION

This case represents pyruvate kinase deficiency (PKD), an autosomal recessive red cell disorder most

common in Caucasian Europeans, but seen in all ethnic groups. This patient demonstrates characteristic

anemia and macrocytosis due to the large number of polychromatophilic cells or reticulocytes. PK

deficiency may be suggested by the presence of echinocytes and/or acanthocytes on the peripheral blood

smear but they may be absent if the patient has a functioning spleen. The deficiency is caused by a defect

in the pyruvate kinase (PK) enzyme in the Embden–Meyerhoff (glycolysis) pathway, a metabolic pathway

used to convert glucose into pyruvate, while generating ATP and other metabolites that is the main source

of metabolic energy in the red cell Approximately one third of patients with pyruvate kinase deficiency

present in the neonatal period with jaundice and hemolysis. Rarely the disorder causes hydrops fetalis and

neonatal or intrauterine death. However, in some children the symptoms are quite mild and hemolysis may

go unnoticed for many years. Jaundice, splenomegaly, decreased energy (due to anemia), and gallstones

may occur in affected patients. In patients who are transfusion dependent, hemochromatosis commonly

occurs.


2- Education


Figure 1: Embden Meyerhoff (glycolysis) pathway


3- Education


RED CELL ENZYME DISORDERS

Red cells are strongly dependent on glucose metabolism to main cellular function and integrity. Red blood

cells anaerobically convert glucose to lactic acid via the Embden Meyerhoff (EM) pathway, also called the

anaerobic glycolysis pathway (see Figure 1). This pathway performs 3 essential functions in the RBC: ATP

production, 2,3 diphosphoglycerate (2,3 DPG) production, and NADH production. Enzyme deficiencies in

the EM pathway have been described, but are quite rare, except for glucose-6 phosphate dehydrogenase

deficiency (G6PD deficiency), which is the most common of all red blood cell (RBC) enzymatic

abnormalities, affecting more than 400 million patients world-wide. G6PD deficiency leads to decreased

NADPH production and increased sensitivity to oxidative stress and resultant hemolysis.

PYRUVATE KINASE DEFICIENCY

Although much more rarely seen, the next most common red cell enzymatic deficiency is pyruvate kinase

deficiency, which is inherited as an autosomal recessive disorder. Pyruvate kinase catalyzes the conversion

of phosphoenolpyuvate (PEP) to pyruvate and will lead to decreased production of red cell ATP and lactate.

A discrepancy between RBC energy requirements and ATP-generating capacity produces irreversible

membrane damage and an inability of the cell to maintain a gradient of ions across the cellular membrane,

resulting in loss of potassium and water from the cell. This results in cellular distortion, rigidity, and

dehydration of red blood cells, leading to premature red blood cell destruction or hemolysis by the spleen

and liver. In most patients this causes a congenital nonspherocytic hemolytic anemia that manifests during

infancy or early childhood, although some patients may not manifest symptoms until later in life.

Differences in clinical symptoms seen between patients are not always completely explained by levels of

the pyruvate kinase enzyme activity. Decreased ATP levels are also associated with loss of protection of

the RBC from damage by oxidation and free radicals that may exacerbate red cell destruction and hemolysis

during times of oxidative stress or infection

The location of pyruvate kinase near the end of the EM pathway means that a deficiency will lead to

accumulation of products that are created earlier in the pathway. In patients with pyruvate kinase

deficiency there is a 2-3X increase in 2,3 DPG levels, resulting in a significant rightward shift in the

hemoglobin-oxygen dissociation curve. As a result, affected individuals have an increased capacity to

release oxygen into the tissues, thus enhancing oxygen delivery. This results in increased exercise capacity

and fewer symptoms occur than may be expected for the decreased level of hemoglobin and hematocrit

caused by red cell hemolysis. This feature is especially advantageous during pregnancy because of

enhanced transfer of oxygen to the fetus. Women with PKD do not usually require transfusions during

pregnancy to maintain adequate levels of oxygen for the developing fetus.

Pyruvate kinase exists as 4 isoenzymes. Two isoenzymes (PKM1 and PKM2) are encoded on chromosome

15 (15q22) and are found in striated muscle, brain, fetal tissue, marrow cells, leukocytes, platelets, lungs,

spleen, kidneys and adipose tissue. The other two isoenzymes (PKL and PKR) are encoded by a locus on

chromosome 1 (1q21) and are found in liver, marrow red cell precursors, reticulocytes, and erythrocytes.

Red cell precursors initially use the PK-M2 isoenzyme, but as the cell matures, the PK-R isoenzyme replaces

the PK-M2 enzyme. Since mature red blood cells cannot produce any new proteins, they cannot

compensate for a deficiency in pyruvate kinase levels by increasing the synthesis of the PK-R isoenzyme.


4- Education


More than 156 mutations in 8 polymorphic sites have been reported on the PK- LR gene on chromosome 1.

Single nucleotide substitutions have been most frequently described. The 1529A mutation is especially

common, even among unrelated families. Based on the gene frequency and its relative abundance in

patients with nonspherocytic hemolytic anemia, the prevalence of PKD is estimated at 51 cases per million

within Caucasian populations. PKD is observed most frequently in Pennsylvania Amish populations, where

consanguinity is common but has been described in other ethnic groups

Pyruvate kinase is one of the more difficult red cell enzyme deficiencies to diagnose because the enzyme

has a complex structure. The residual enzyme activity is not always greatly decreased despite markedly

decreased levels of the enzyme being present. In such cases, establishing the diagnosis may depend on

showing that the level of 2,3-DPG or 3-phosphoglyeric acid is greatly elevated or that NADH levels are

decreased. It is also useful to measure the thermal stability of the residual enzyme, as mutant enzymes are

often very unstable on heating. The most generally useful means for differentiating red cell enzymes from

one another is semi-quantitative measurement of the enzymatic activity. Fluorescent screening tests for red

cell enzymatic deficiencies based on detection of NADH have been developed that have a high degree of

reliability. The accumulation of pyrimidine nucleotides (NADH) can be detected by measuring the ultraviolet

spectrum of perchloric acid extract of red cells. Specific quantitation of red blood cell enzyme activities is

more specialized and difficult to perform, due to the isoenzymes being encoded by genes two different

chromosomes. Since activity of PK in leukocytes is quite high, the presence of any leukocytes in a red cell

suspension may falsely elevate the PK enzymatic activity level. In addition, the blood of patients with

significant hemolysis may have many reticulocytes and young red blood cells with normal or near normal

levels of PK, which may affect the accuracy of quantitative enzyme measurements. It is therefore essential

to consider the age of the circulating red cells and the transfusion status of the patient when performing

and interpreting test results. It may be helpful to obtain blood samples from parents or children of the

patient to determine whether abnormal (usually half normal) PK activity can be documented in relatives, as

expected in an autosomal recessive condition.

DIFFERENTIAL DIAGNOSIS OF PYRUVATE KINASE DEFICIENCY

There are many etiologies of hemolytic anemia to consider in the differential diagnosis. Hemolytic anemias

are broadly described as cellular or extracellular. Physical examination, family history, and examination of

the peripheral blood smear in conjunction with appropriate laboratory testing will help in formulation of an

accurate diagnosis. Examples of causes of hemolytic anemia to consider are given below:

Hemolytic Anemia Etiologies

CELLULAR: Intrinsic abnormality of membrane, enzyme or hemoglobin structure

Membrane defects

Hereditary spherocytosis

Elliptocytosis

Pyropoikilocytosis

Stomatocytosis

Paroxysmal nocturnal hematuria (usually acquired)


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Enzyme deficiencies

Glucose-6-phosphate dehydrogenase deficiency (G6PD)

Pyruvate kinase

Other rare enzyme deficiencies: hexokinase, glucose 6 isomerase, aldolase

Hemoglobin abnormalities

Sickle cell anemia, other structurally abnormal hemoglobins

Thalassemias

EXTRACELLULAR: Antibodies, mechanical factors and plasma factors

Examples of extracellular processes that may lead to hemolysis include:

Autoimmune

Cold antibodies

Warm antibodies

Fragmentation hemolysis

Disseminated intravascular coagulation (DIC)

Thrombotic thrombocytopenic purpura (TTP)

Hemolytic uremic syndrome (HUS)

Extra corporeal membrane oxygenation (ECMO)

Prosthetic heart valves or mechanical destruction

Burns, thermal destruction

Plasma factors

Liver disease

Infections

Wilson's disease

The diagnosis of pyruvate kinase deficiency should be considered in a patient who may be jaundiced with

evidence of nonimmune hemolysis (anemia and hyperbilirubinemia), as documented by a negative direct

antiglobulin (DAT) test. Most patients will present in early life with a congenital nonspherocytic hemolytic

anemia. Clinical history and appropriate serologic tests must demonstrate absence of infection or exposure

to hemolytic agents, and may identify other family members with anemia. Hemoglobinopathies and red cell

membrane disorders should be ruled out by examination of the blood smear to document the absence of

target cells or other signs of abnormal hemoglobin production, and there should not be significant numbers

of spherocytes or bite cells present. Hemoglobin analysis by electrophoresis or other approaches may be

needed to completely exclude a hemoglobinopathy. Red blood cell morphology is usually normal in patients


6- Education


with PKD, although increased numbers of polychromatophilic cells are seen reflecting compensation for

hemolysis. A few dense cells with irregular or speculated appearing margins (echinocytes and/or

acanthocytes) are occasionally seen. Echinocytes and acanthocytes are not specific for PKD, and may also

be seen in some cases of glucose-6-phosphate dehydrogenase deficiency, vitamin E deficiency, and

hemoglobin H disease (alpha thalassemia). If hemolysis due to other causes is clinically excluded, screening

for red cell enzymatic abnormalities and additional testing for specific enzymes may be undertaken.

CLINICAL ASPECTS OF PYRUVATE KINASE DEFICIENCY

Pyruvate kinase heterozygotes are often clinically and hematologically normal, although they have roughly

half the normal amount of red blood cell pyruvate kinase activity. Most of the symptomology in

homozygous patients is limited to early life and times of physiologic stress. Patients with homozygous

pyruvate kinase deficiency may need blood transfusions periodically, and those cases with severe

hemolysis may require splenectomy. Most patients will have life-long compensated hemolytic anemia with

mild to minimal anemia after initial presentation with anemia in infancy. Because of increased demands for

red cell production in the bone marrow, many patients will require supplementation with folate.

Alice Werner, MD

REFERENCES:

1. Beutler E. Erythrocytes enzymopathies. In: Warrell DA, Cox TM, Firth JD, eds. Oxford Textbook of

Medicine. 5th ed. New York, NY: Oxford University Press; 2010.

2. Embden Meyerhoff Pathway. http://www.biologyguide.net. Accessed March, 2012.

3. Glader B, Allen G. Neonatal Hemolysis. In: DeAlarcon PA, Werner EJ, eds. Neonatal Hematology.

Cambridge, UK: Cambridge University Press; 2005.

4. Prchal JT. Genetics and Pathogenesis of methemoglobinemia. UpToDate. October 19, 2011.

http://www.uptodate.com. Accessed March 2012.

5. Segal GB. Enzymatic defects. In: Kligman RM, Behrman RE, Jensen HB, Stanton BF. Nelson Textbook

of Pediatrics. 18th ed. Philadelphia, PA: Saunders, Elsievier; 2007.

Alice Werner, MD: is Associate Professor of Pathology and Pediatrics at Eastern Virginia Medical School

and practices all aspects of Pediatric Pathology at Children's Hospital of The King's Daughters in Norfolk,

VA. She has published a number of articles and abstracts and participates in numerous collaborative

research activities with pediatric subspecialists at the institution. Dr. Werner is a member of the

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

http://www.biologyguide.net/

http://www.uptodate.com/

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