Please circulate to your laboratory staff.

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1 Please circulate to your laboratory staff. ALL staff can now earn free CME/CE credit with the Blood Cell Identification Survey (KP-B)! Complete the specified reading in the 2007 KP-B Participant Summary and the related online activity on the CAP Web site to earn free CME/CE credit. All staff from laboratories participating in the Blood Cell Identification Survey (2007 KP-B) can now individually earn CME/CE credit by following the instructions below. Please note that you will need the Kit ID number and CAP number found in the header of your KP-B evaluation or on your result form in order to access the online activity. You may enroll in and complete this activity until December 28, After this date the activity will no longer be available. Directions: 1. Complete the specified reading in the Participant Summary that was mailed to your institution. You may also obtain an electronic version of this reading by completing the following steps to access the online portion of this education activity. 2. Go to and log in with your individual User ID and Password. a. If you are unsure whether you have an individual Web account with the CAP, or do not remember your User ID and Password, click on the Forgot your user ID or password? link. You may either enter the requested security information and the system will recognize you and log you in, or you may enter your last name and address so that the system can send you a temporary ID and password. b. If you do not have an individual Web account, click on the Create an Account link. Complete and submit the account request form. You will be notified within one business day that your individual account has been activated. 3. Click on the Education Programs tab. Under Course Catalog, click on Surveys. 4. Click on the KP-B 2007 link. 5. Click on the Enroll link. This will bring up the validation screen. 6. Enter and submit the Kit ID and CAP numbers associated with the KP-B Survey. These numbers can be found in the header of your KP-B proficiency testing evaluation or on your result form and are required to access the online activity. 7. Click on the Launch link. The first page of the activity will open. 8. Read and follow the instructions provided online to complete the activity. For more information, call a CAP Customer Contact Center representative at ( ) option 1#. Kit ID #: CAP #: 6 - Education

2 Continuing Education Information Accreditation The College of American Pathologists (CAP) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. CME Category 1 The CAP designates this educational activity for a maximum of 1 Category 1 credit toward the AMA Physician s Recognition Award. Each physician should only claim those credits he/she actually spent in the educational activity. CME (International physicians) The American Medical Association has determined that physicians not licensed in the US who participate in this CME activity are eligible for AMA PRA category 1 credit. CE (Continuing Education for non-physicians) The CAP designates this educational activity for a maximum of 1 credit/hour of continuing education. Each participant should only claim those credits/hours he/she spent in the educational activity. This activity is acceptable to meet the continuing education requirements for the ASCP Board of Registry Certification Maintenance Program. This activity is approved for continuing education credit in the states of California and Florida. Disclosure Statement All authors/planners of a CAP educational activity must disclose to the program audience any financial interest or relationship with the manufacturer(s) of any commercial product(s) that may be discussed in the educational activity or with the manufacturer of a product that is directly competitive with a product discussed in the presentation. Relevant financial relationships are considered to be any financial relationships in any amount occurring within the past 12 months that create a conflict of interest. The College of American Pathologists does not view the existence of these interests or uses as implying bias or decreasing the value to participants. The CAP, along with the Accreditation Council for Continuing Medical Education (ACCME), feels that this disclosure is important for the participants to form their own judgment about each presentation Below you will find the financial disclosure relationships for anyone who was able to affect content of this educational activity. The CAP does identify and manage all potential conflicts of interest to ensure that all educational activities promote improvements or quality in healthcare and are free of commercial bias for or against a product/service. The following authors/planners have no financial relationships to disclose: Ramon Blanco, MD, FCAP; Lydia C. Contis, MD, FCAP; Patricia A. Devine, MD, FCAP; Tracy I. George, MD, FCAP; Alexandra Marie Harrington, MD; William Koss, MD, FCAP; Beverly Patricia Nelson, MD, FCAP; Robert William Novak, MD, FCAP; Deborah Ann Perry, MD, FCAP; Carla S. Wilson, MD, PhD, FCAP; Roberta L. Zimmerman, MD, FCAP; Jennifer Lynn Shinnick, MT(ASCP); Barb Flamich, MT(ASCP) The following authors/planners have financial relationships to disclose: Donald Warkentin, PhD Dade Behring, Inc., Honorarium, Occasional Lecture Dade Behring, Inc., Consulting Fee, Supply Specimens (clinical) The following In-Kind Support or Commercial Support has been received for this activity: None Learning Objectives Upon completing the reading and answering the learning assessment questions, you should be able to: 1. Identify the common causes for hypochromic microcytic anemia. 2. Realize the effect circulating nucleated red blood cells have on altering the automated white blood cell count. 3. Compare differences in hemoglobin production in thalassemias versus hemoglobinopathies. 4. Discuss why patients with thalassemia develop anemia. 5. Identify abnormal red blood cell morphology on a peripheral blood smear. 7 - Education

3 Discussion Educational Case Study, Presentation This 23-year-old male of Greek descent was recently seen for chronic anemia that required splenectomy at the age of 10 years. Laboratory data includes: WBC (corrected) = 9.9 x 10 9 /L; RBC = 3.42 x /L; Hgb = 8.9 g/dl; Hct = 29.7%; MCV = 78.1 fl; MCH = 24.1 pg; MCHC = 30.7 g/dl; RDW = 27.1%; PLT = 762 x 10 9 /L; NRBCs = 894/100 WBCs. COMPLETE BLOOD COUNT (CBC) The CBC in this educational case study is remarkable for a slightly decreased number of red blood cells. The CBC indices provide important information about the potential underlying cause for this young man s anemia. The red blood cells are small in size (low MCV) and have a decreased mean corpuscular hemoglobin concentration (low MCHC). Based on these parameters, the differential diagnosis for a hypochromic microcytic anemia must be considered. The most common causes of hypochromic microcytic anemia are the following: Iron deficiency anemia Thalassemia Sideroblastic anemias (hereditary or acquired secondary to a toxic effect such as lead poisoning, or drugs) Severe anemia of chronic disease Peripheral Blood Smear Review Examination of the peripheral blood smear aids in distinguishing between the potential etiologies for a hypochromic microcytic anemia. Particularly important is evaluating red blood cell shape and detecting red cell inclusions, such as Howell-Jolly bodies, Pappenheimer bodies (hemosiderin-containing granules), and basophilic stippling (altered ribosomes). The initial evaluation is usually aimed at distinguishing between iron deficiency anemia and thalassemia, as these are by far the most common causes for this type of anemia, as discussed below. However, if Pappenheimer bodies and a dimorphic red blood cell population are identified, this raises the possibility of a sideroblastic anemia. Coarse basophilic stippling suggests potential lead poisoning, although coarse basophilic stippling may also be seen in some types of thalassemia and refractory anemia. A virtual microscopy slide (online portion of this education activity) has been provided to enable all participants to review a digitized glass slide image of this patient s peripheral blood smear. Preparation of high quality peripheral blood smears is crucial for the accurate assessment of cell morphology and confirmation of cell types and number. In particular, the increased nucleated red blood cell count reported by the automated analyzer in this case would have been flagged for microscopic confirmation. 8 - Education

4 Initial review of the peripheral blood smear (online virtual slide) at low power (best viewed at magnification in green squares) shows no significant red blood cell clumping or rouleaux formation, but confirms increased gaps between red blood cells consistent with the reduced red blood cell number. Higher magnification allows for better visualization of red blood cell morphology and presence of inclusions. The two most prominent features observed are the numerous nucleated red blood cells present and the prominent anisopoikilocytosis with many target cells. Howell-Jolly bodies and basophilic stippling are also present. Red Blood Cell Morphology Target cells are thin red blood cells that have an overabundance of cell membrane, which causes the cells to assume a bell shape while in circulation. When the cells are flattened out on a smear, the top of the bell is pushed to the center creating a central target or bulls-eye. Target cells are most commonly seen in the following clinical conditions: Liver disease Hemoglobinopathies Thalassemia Post-splenectomy Iron deficiency Drying artifact with uneven distribution on smear Laboratories should have a mechanism in place to semi-quantitate the red blood cell abnormalities present, such as the quantity of target cells, so that this is reproducible among technologists reviewing smears from the same patient over time. In addition, semi-quantitation allows for interpretation of the significance of an abnormality. For example, target cells are infrequent in iron deficiency as compared to thalassemia. An increase in the number of target cells may signify worsening disease. When red blood cells are starting to undergo karyorrhexis, residual DNA occasionally remains in the cytoplasm as the cells are released into circulation, after extrusion of the nucleus. This DNA forms small round Howell-Jolly bodies that are typically removed by the spleen. However, when the spleen is hypofunctional or has been taken out, as in this patient, Howell-Jolly bodies are seen in the circulating red blood cells. Howell-Jolly bodies may also be formed in cells when aberrant chromosomes are separated from the nuclear DNA at the time the nucleus is removed. 9 - Education

5 Nucleated Red Blood Cells (NRBCs) The presence of circulating NRBCs, outside of the neonatal period or occasionally during pregnancy, generally indicates either increased red blood cell production or bone marrow infiltration by malignant cells, fibrosis, granulomas, etc. When bone marrow infiltration occurs, granulocytic precursors are usually concurrently seen in circulation. The most common circulating NRBC is at the orthochromic stage of differentiation, although the term NRBC is used for all normoblasts regardless of the stage of maturation. It is critical to correctly identify these cells as they may not be recognized by automated hematology counters and will artificially raise the WBC count. This impacts patients receiving chemotherapy whose treatments are based on the absolute neutrophil count; they may be treated inappropriately if the circulating NRBCs erroneously elevate the WBC count. The newer hematology instruments automatically correct the WBC count when NRBCs are detected. However, even the most sophisticated instruments will, at times, misidentify these cells necessitating a manual correction of the WBC count. Therefore, review of the peripheral blood smear for confirmation of the number of circulating NRBCs is important when NRBCs are flagged as being detected. The formula for correcting WBC counts when NRBCs are present is as follows: uncorrected WBC* x 100 = corrected WBC count # NRBCs * If the instrument has already attempted to correct the WBC, it is necessary to retrieve the uncorrected WBC count from the instrument for the calculation. Summary The composite finding of a hypochromic microcytic anemia, markedly increased target cells, and numerous circulating NRBCs, is most suggestive of thalassemia, a disorder of hemoglobin synthesis. HEMOGLOBIN Structure and Function of Hemoglobin The main function of hemoglobin in red blood cells is to carry oxygen from the lungs to tissue. Hemoglobin is composed of the following: 4 globin chains (2α chains and 2* chains) 4 heme groups (iron and protoporphyrin) 4 oxygen molecules *Chain changes during growth/development 10 - Education

6 The structure of hemoglobin can be seen in Figure 1. below. Figure 1. Heme and Globin Formation (Source: Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists: 1998:183.) 11 - Education

7 The heme and globin chains are made in the RBC cytoplasm. The two α chains remain constant throughout life, while the other chain type switches during development. At birth, gamma chains predominate to form fetal hemoglobin, Hb F (α2,γ2). After approximately one year of age, the main type of hemoglobin (>95%) becomes adult Hb A, composed of two α and two β chains (α2,β2). Hemoglobin Disorders Due to Altered Globin Chain Synthesis Two main types of disorders result from abnormalities in globin chain synthesis. These include the following: 1. Hemoglobinopathies production of structurally different hemoglobin than normally seen. Hemoglobins that cause the most clinical problems are usually the result of a genetic mutation in the β-globin gene that leads to a structurally different globin chain. Examples include: Hb S (sickle cell), Hb C, Hb E. 2. Thalassemias gene mutation or deletion that leads to decreased (or absent) production of structurally normal α-globin or β-globin chains. In summary, these genetic disorders are caused by either production of a different type of hemoglobin (hemoglobinopathies) or decreased production of normal adult Hb A (thalassemia). An important clue to the diagnosis of these disorders is the appearance of the red blood cells on the peripheral blood smear. Distinctive shapes characterize some of these entities, such as sickle cells in Hb S (sickle cell disease), and intracellular tetrahedral crystals (resembling Washington monument) in Hb C disease. THALASSEMIA The thalassemia syndromes are divided into two main groups. β-thalassemia is caused by deficient synthesis of structurally normal β-chain, whereas α-thalassemia is caused by deficient synthesis of structurally normal α-chain. The gene abnormalities in both of these syndromes are heterogeneous leading to significant variation in clinical manifestations. β-thalassemia is most prevalent in people of Mediterranean descent, while α-thalassemia is common in Southeast Asia and Africa, and occasionally in Mediterranean and Middle Eastern populations. A significant difference between these two disorders is the number of genes on the chromosome that encode for each of the globin chains Education

8 Globin Chain Genes β -globin genes Chromosome 11 2 genes total α-globin genes Chromosome 16 4 genes total Because every red blood cell has twice as many α-globin genes as β-globin genes, an abnormality involving 1 or 2 of these genes has much more significant impact when β-globin genes are affected. The clinical consequences of α-thalassemia depend on how many α-globin genes are deleted. Red blood cell morphology is altered in patients with all forms of thalassemia. Hypochromic microcytes and target cells are the main features in asymptomatic individuals. Patients with more severe forms of thalassemia have the following red blood cell findings: Hypochromic microcytic red blood cells Anisocytosis and poikilocytosis Target cells, ovalocytes, occasional fragmented red blood cells, basophilic stippling, increased polychromatophilic cells (but insufficient for the degree of anemia), Howell-Jolly bodies, circulating NRBCs, prominent basophilic stippling β-thalassemia β-thalassemia is considered the most common genetic disorder that occurs worldwide. More than 170 different mutations of the β-globin gene have been discovered and thus the clinical manifestations are extremely diverse. Different patients with β-thalassemia can produce varying quantities of β-globin depending on their specific genetic defect and whether one or both of the β-chain genes are affected. Inheritance of only one β-thalassemic gene (heterozygote) frequently results in either no or only mild hypochromic microcytic anemia and an elevated or normal red blood cell count. A more serious disorder is seen when two beta-thalassemic genes are inherited (homozygote); each gene may produce either decreased or no β-globin chains. The β-globin chains that are produced are structurally normal. Because of the tremendous diversity in the molecular abnormalities in β-thalassemia, patients are commonly separated into three categories based on the severity of their clinical condition rather than the underlying genetic abnormality as demonstrated on the following page Education

9 β-thalassemia minor β-thalassemia minor is clinically asymptomatic, usually hypochromic microcytic red blood cells, increased red blood cell number, and possible mild anemia. These patients generally have thalassemia trait with one normal β-globin gene and one β-thalassemia gene. The main reason to confirm this diagnosis is to prevent unnecessary lab testing (iron studies) or treatment (chronic iron supplementation). β-thalassemia major β-thalassemia major is RBC transfusion dependent. Individuals usually present within first six months of life with profound anemia and die within first two years of life if not treated with regular blood transfusions. These individuals have inherited two β-thalassemia genes. β-thalassemia intermedia β-thalassemia intermedia represents a wide clinical spectrum encompassing all patients who are not minor or major. Some patients are asymptomatic until adult life requiring no or only occasional blood transfusions. Other patients present between ages 2-6 years and require regular transfusion therapy for adequate growth and development. Thalassemia intermedia can result from inheritance of either one or two β-thalassemia genes with the later more common. Anemia of β-thalassemia The primary cause of anemia in β-thalassemia is an imbalance in the production of α-globin and β-globin chains. When normal α-globin chain is produced with insufficient β-globin chain, excess α-chains precipitate in red blood cell precursors and cause cell death in the bone marrow. The bone marrow attempts to compensate by increasing red blood cell production but insufficient numbers of cells, including immature cells, are released into circulation. The red cells that make it into circulation often have a shortened life span, in part, due to hemolysis of the cells with precipitated globin chains in the spleen. In patients with significant hemolysis (similar to the patient presented in the Blood Cell Identification challenges BCK/BCP- 11 through BCK/BCP-15), the MCV may be elevated to normal range as the reticulocytes are larger than the more mature circulating red blood cells. The high RDW reflects this range in the red blood cell size. The anemia in β-thalassemia is characterized by: Increased red blood cell production with death of red blood cell precursors in bone marrow leading to insufficient red cells released into circulation (ineffective erythropoiesis) Circulating red blood cells have a shortened life span with peripheral destruction (hemolytic anemia) Overall decrease in hemoglobin synthesis Thalassemic patients with chronic anemia have increased gastro-intestinal iron absorption and develop iron overload. This causes a number of complications of which the most important is cardiac failure leading to death. Progressive therapy with blood transfusions and iron chelation therapy have resulted in longer survivals and improved quality of life. Additional complications for patients such as this include splenomegaly and gallstones Education

10 Hemoglobin Electrophoresis Definitive diagnosis of β-thalassemia is made by hemoglobin electrophoresis or high performance liquid chromatography (HPLC). Increased amounts of Hb A2 and Hb F are associated with excess unpaired α- chains combining with delta chains (α2δ2) or gamma chains (α2γ2), respectively. Patients with β-thalassemia minor have elevated levels of Hb A2 (usually >3.5%) and approximately 30% will have slightly elevated levels of Hb F. Significantly elevated hemoglobin F establishes the diagnosis in β-thalassemia intermedia (20-40%) and β-thalassemia major (60-98%) when no additional abnormal hemoglobin species is identified and hereditary persistence of fetal hemoglobin has been excluded. Figure 2. below shows the electrophoretic work-up of a patient with β-thalassemia intermedia (row marked with *); the band to the left of A represents Hb F. Hemoglobin A2 may or may not be elevated in patients with severe forms of β-thalassemia. Figure 2. Alkaline Electrophoresis (ph 8.6) Pattern (Source: Hoyer JD, Kroft SH, eds. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency Testing. Northfield, IL: College of American Pathologists: 2003: 38.) Mild forms of α-thalassemia produce no electrophoretic abnormalities and only a few types are detected by Southern blot analysis, so this diagnosis is usually one of exclusion. However, the family history often supports this diagnosis. Summary The patients in the participant challenges (BCK/BCP-11 through 15) and in this educational case study were classified as having thalassemia intermedia, which in general means that the patients were not transfusiondependent from infancy, yet developed anemia related complications during their lifetimes that have required treatment Education

11 References 1. Bain BJ. Diagnosis from the blood smear. N Engl J Med. 2005;353: Lee S-H. Virtual microscopy: applications to hematology. Laboratory Hematology. 2005;11: Perkins S. Disorders of hemoglobin synthesis. In: Kjeldsberg CR, ed. Practical Diagnosis of Hematologic Disorders. Volume 1 Benign Disorders. Chicago, IL: ASCP: 2006; Thein SL. Genetic insights into the clinical diversity of beta-thalassemia. Br J Haematol. 2004;124: Thein SL. Genetic modifiers of beta-thalassemia. Haematologica. 2005;90: Taher A, Isma eel H, Cappellini MD. Thalassemia intermedia: revisited. Blood cells, molecules, and diseases. 2006;37: Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists: Hoyer JD, Kroft SH, eds. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency Testing. Northfield, IL: College of American Pathologists: Education Activity Author Carla S. Wilson, MD, PhD, FCAP: Carla S. Wilson, MD, is an Associate Professor of Pathology at the University of New Mexico Health Sciences Center and is Director of Flow Cytometry at TriCore Reference Laboratory in Albuquerque, New Mexico. Dr. Wilson has over 60 chapters, papers, and abstracts on hematopathology topics and is currently a member of the College of American Pathologists (CAP) Hematology and Clinical Microscopy Resource Committee Education