Peripheral blood and bone marrow: The beginning (and end) of many hematopathologic diagnoses

Transcription

1 Peripheral blood and bone marrow: The beginning (and end) of many hematopathologic diagnoses USCAP 2010 Jo-Anne Vergilio Children's Hospital Boston Boston, MA Adam Bagg University of Pennsylvania Medical Center Philadelphia, PA

2 Peripheral blood and bone marrow: The beginning (and end) of many hematopathologic diagnoses A complete blood count (CBC) is a simple, and often underappreciated, test that is typically performed as part of a routine medical evaluation. Detection of certain abnormalities in the CBC should prompt review of a peripheral blood smear. To the experienced eye, much information can be gleaned from these tandem laboratory studies that can offer invaluable clues, and direction, to the establishment of an underlying hematopathologic (or other) diagnosis while avoiding unnecessary and often invasive procedures. Major objective of this course: The primary aim of this course is to offer a practical and algorithmic approach to the hematologic review of CBCs and peripheral blood smears such that participants can offer a thoughtful differential, or when appropriate, definitive diagnosis, as well as recommendations for essential ancillary diagnostic studies. Specific objectives of this course: Review the assessment of a CBC and concurrent peripheral blood smear with elucidation of the predominant quantitative and qualitative abnormality(ies) Emphasize how the morphologic assessment of a peripheral blood smear may be used to hone the differential diagnosis Discuss appropriate utilization of ancillary diagnostic studies (e.g. immunophenotypic, cytogenetic, molecular diagnostic and other laboratory studies) Outline the indications that dictate performance of a bone marrow biopsy/aspirate smear Highlight, where appropriate, new insights into some long-standing hematologic diseases and how they may be applied diagnostically

3 Course agenda 1. Introduction 2. Case studies The purpose of these cases is to (a) explore some of the major categories of quantitative CBC abnormalities that should prompt review of a peripheral smear and (b) highlight how the morphologic/qualitative smear features are critical to refining the differential diagnosis 2.1 Macrocytic anemia - to discuss the differential diagnosis of macrocytic anemia with emphasis on vitamin B12 and folate deficiencies, including their underlying pathophysiology and laboratory evaluation Case 1: Megaloblastic anemia secondary to vitamin B12 deficiency 2.2 Microcytic anemia - to review the common, and not so common, causes of microcytic anemia, including iron deficiency, anemia of inflammation, thalassemia and the sideroblastic anemias; the mechanisms of (normal and abnormal) iron transport will be discussed as well as the laboratory studies that are critical to the evaluation of iron status Case 2: X-linked sideroblastic anemia 2.3 Hemolytic anemia to provide an approach to the determination of the cause of hemolytic anemia, with particular emphasis on the role of a detailed assessment of red cell morphology Case 3: Autoimmune hemolytic anemia secondary to underlying chronic lymphocytic leukemia 2.4 Absolute lymphocytosis to appreciate the many differential diagnoses of an elevated lymphocyte count and how the age of the patient and the morphology of the lymphocytes can direct the appropriate additional studies Case 4: Infectious mononucleosis

4 2.5 Thrombocytosis to consider the causes of both elevations in platelet number and their size, and to provide a contemporary approach to the non-cml myeloproliferative disorders Case 5: Essential thrombocythemia 2.6 Monocytosis to explore both reactive and neoplastic causes of monocytosis in adults and children; the myelodysplastic/myeloproliferative diseases of chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia will be reviewed; the acute myelogenous leukemias, particularly those associated with monoblastic/monocytic differentiation, will also be discussed. Case 6: Chronic myelomonocytic leukemia 2.7 Pancytopenia this particular CBC finding necessitates a bone marrow examination; although clues to etiology may be found in the peripheral smear, the differential diagnosis is approached from the perspective of an underlying cellular or hypocellular marrow; although a variety of entities will be discussed, emphasis is on the myelodysplastic syndromes as well as the inherited and acquired aplastic anemias Case 7: Aplastic anemia 3. Conclusion 4. Question and Answer Session

5 1. INTRODUCTION The complete blood count (CBC) The CBC offers a quantitative assessment of each of the blood s cellular elements. By now, we often refer to key components of the CBC reflexively; however, below is an abbreviated refresher of terms, definitions and calculations relevant to the red blood cell parameters. These will not be discussed during the course, but are simply provided for your benefit should you wish to revisit this information. Please refer to the reference list at the end of this syllabus for resources that offer a more comprehensive review of these parameters, of the current technologies essential to automated hematologic analysis and of the limitations inherent in these methodologies. Red blood cell count (RBC) Hemoglobin (HGB) Hematocrit (HCT) the number of red cells in a specified volume of blood [10 6 /µl = M/µL] the amount of hemoglobin in a specified volume of blood [g/dl] the ratio of the volume of red cells to the volume of whole blood [%] HCT = MCV (fl) x RBC (10 6 /µl) Mean corpuscular volume (MCV) the average volume of a red cell, typically directly measured using impedance technology [fl] MCV = HCT (%) RBC (10 6 /µl) Mean corpuscular hemoglobin (MCH) the average amount of hemoglobin in a red cell [pg] HGB (g/l) MCH = RBC (10 6 /µl)

6 Mean corpuscular hemoglobin concentration (MCHC) the average concentration of hemoglobin per red cell [g/dl] HGB (g/l) MCHC = HCT (%) Red cell distribution width (RDW) a measurement of the variability (anisocytosis) of red cell size [%]; = CV of the MCV The peripheral blood smear To examine a peripheral blood smear, first scan the entire slide under low power in order to assess the overall quality of preparation. Relevant questions to ask yourself include: (1) is the blood smeared adequately? (eg. not too thick or too thin), (2) is the stain quality sufficient?, (3) are artifacts present? (eg. preservative/anticoagulant artifacts, cell/platelet clumping and/or satellitism). Once you have established that the smear is adequate, find the optimal site for close examination. This is typically just inside of the feathered edge, where the red cells are just slightly separated from one another. In a normal/healthy smear, most of the red cells should exhibit central pallor, comprising approximately one-third of the cell diameter. In comparison, areas distant from the feathered edge contain cells that show an apparent loss of central pallor because they are too thinly spread while regions near the origin of the smear contain cells that exhibit apparent, though artifactual, rouleaux formation because they are too thickly spread. To visually assess red cell size, locate a small mature lymphocyte. An erythrocyte should be approximately equal in size to the lymphocyte nucleus. The main caveat here is to choose your lymphocyte with care. Activated/ reactive lymphocytes are inadequate for comparative purposes as they have enlarged nuclei. The platelet count may be approximated by examining a single field under 100x oil immersion; count the number of platelets and multiply by 10,000. This result should yield a rough estimation of platelet numbers.

7 2. CASE STUDIES Brief clinical histories and peripheral blood smear images were provided for seven cases [1-7]. The following exercises were to be performed for each case: 1. Offer a basic/broad descriptor of the most prominent abnormality(ies) noted on the CBC 2. Describe the various morphologic findings on the smear 3. Formulate a differential diagnosis 4. Suggest additional testing, if appropriate, that my help refine the differential 5. Indicate why (or why not) a marrow study should be performed 6. Finally, match each peripheral blood smear with its corresponding bone marrow aspirate &/or biopsy [designated A-G]

8 Section 2.1 Case 1: Clinical: 43-year-old female with several year history of fatigue, diagnosed with mild hypothyroidism and started on synthroid with some symptomatic improvement. Subsequently had this routine blood work performed at the time of a gynecologic examination. CBC: HGB 11.0 g/dl, MCV fl, RBC 2.53 M/µL, HCT 33.0%, MCH 43.6 pg, MCHC 33.5 g/dl, RDW 18.4% WBC 5.4 K/µL [50% neutrophils, 43% lymphocytes, 4% monocytes, 2% eosinophils, 1% basophils] PLT 333 K/µL Smear: macro-ovalocytes, moderate anisopoikilocytosis, rare basophilic stippling and hypersegmented neutrophils The first case is that of a patient who is noted to be mildly anemic on routine CBC while red cell indices demonstrate a profound macrocytosis and mildly elevated RDW. One approach to anemia using red cell size (MCV) When evaluating an anemic patient, a classical algorithmic approach begins with an assessment of the MCV, enabling us to separate cases into microcytic (MCV <80 fl), normocytic (MCV fl) and macrocytic (>100 fl) categories. The most prominent abnormality in case 1, therefore, is a marked macrocytic anemia. The differential diagnosis of macrocytic anemia includes the following entities: Alcoholic and nonalcoholic liver disease Hypothyroidism Nutritional deficiencies (eg. vitamin B12 and folate) Drugs [see Table 2.1] Marrow failure syndromes Reticulocytosis/accelerated erythropoiesis (eg. hemorrhage or hemolysis) Physiologic causes (eg. enlarged fetal cells of newborn) Artifactual (eg. severe hyperglycemia, red cell clumping/agglutination)

9 Table 2.1: Drugs that can induce a macrocytosis Chemotherapeutic Azathioprine Cyclophosphamide Cytosine arabinoside 5-Fluorouracil Hydroxyurea Mercaptopurine Methotrexate Anticonvulsants Phenytoin Primidone Valproic acid Hypoglycemics Metformin Antimicrobial Pyrimethamine Sulfamethoxazone Trimethoprim Antiretroviral Zidovudine Stavudine Anti-inflammatory Sulfasalazine Other Nitrous oxide A first step in dissecting these various etiologies is to examine a reticulocyte count. Increased numbers of reticulocytes indicate accelerated erythropoiesis and imply the presence of underlying hemorrhage or hemolysis whereas a normal or decreased reticulocyte count, in the setting of on-going anemia, indicates a hypoproliferative marrow response and implies a problem with erythrocyte production [to be discussed in detail in Section 2.3]. The reticulocyte count in this patient was not adequately elevated at 1%, thereby allowing us to eliminate etiologies from the differential that are associated with accelerated (and effective) erythropoiesis. The second step in this algorithm is to assess smear morphology for megaloblastic features; these are best characterized by the presence of macro-ovalocytes and/or hypersegmented neutrophils. Macro-ovalocytes, as the name implies, are large (MCV > 115 fl) and oval in shape as compared to normal erythrocytes that have a round discoid shape. Neutrophils normally exhibit three (to occasionally four) nuclear lobes, so the presence of a few 5-lobed or one 6-lobed form constitutes neutrophilic hypersegmentation. Other associated findings typically include non-specific anisocytosis and poikilocytosis with red cell fragments (generated secondary to shearing of the macro-ovalocytes). All of these features contribute to an elevated red cell distribution width (RDW). Basophilic stippling, Howell-Jolly bodies, Cabot rings (red ringed shaped or figure-of-eight structures within erythrocytes) and circulating megaloblasts may also be seen. The presence of megaloblastic morphology narrows the differential diagnosis to include vitamin B12 and folate deficiencies as well as specific drugs that

10 function as folate inhibitors. Other inherited defects in both vitamin B12 and folate metabolism, as well as defects in other pathways, must also be considered within the differential; however, these conditions are exceedingly rare and will not be discussed in detail herein. In comparison, the underlying etiology of a non-megaloblastic macrocytic anemia may be elicited from morphologic clues within a peripheral blood smear. Individuals with: either alcoholic or non-alcoholic (eg. hepatitis, obstructive jaundice) liver disease exhibit predominantly round macrocytes that typically include some targeted forms, an underlying myelodysplastic syndrome generally demonstrate dysmyelopoiesis with hypogranular and/or hyposegmented ( Pelger-Huet - like) neutrophils; circulating blasts may also be seen, aplastic anemia, another form of marrow failure, present with pancytopenia, often with only few evaluable, and largely unremarkable cells, seen on a peripheral smear. Drug exposures are best elicited from a detailed clinical history while clues to hypothyroidism should be evident on careful physical examination by the treating clinician. MEGALOBLASTIC ANEMIA The two main differential diagnoses to consider in megaloblastic anemia are deficiencies of either (or both) vitamin B12 and folate. Though distinct in many ways, they are intricately related in their contributions to DNA synthesis and it is the disturbance in this pathway that dictates many of the manifest morphologic features. Vitamin B12 and folate the fundamentals Vitamin B12 is part of the cobalamin family of compounds. This family is characterized by a corrin ring structure that is similar to porphyrin, though with a unique cobalt center. Vitamin B12 is produced by microorganisms, is ingested by a variety of animals, and thus, is derived from various foods of animal-origin, including meat, fish, seafood and dairy products such as eggs, cheese and milk. Body stores maintain approximately 2-3 mg of vitamin B12 and depletion of these stores takes approximately 3-6 years after cessation of its intake. The active forms include methyl-cobalamin, found predominantly in the plasma, and adenosyl-cobalamin, found in the liver, kidney and erythrocytes. Vitamin B12 functions as a cofactor in two reactions, both of which serve to detoxify potentially dangerous compounds. These reactions involve [1] the cytoplasmic synthesis of the amino acid, methionine, from homocysteine and 5-methyl-tetrahydrofolate (the major pathway for the regeneration of methionine in humans), and [2] the

11 mitochondrial conversion of methylmalonyl-coa to succinyl-coa, a component of the citric acid cycle. The folates represent a family of compounds derived from folic acid, a conjugate of pteroic acid and glutamic acid. The addition of multiple glutamic acid residues to folate yields polyglutamates. These are common in food folates and comprise the predominant intracellular form in humans. Folates are found in grains, cereals and particularly green leafy vegetables. Body stores maintain approximately mg of folate, half of which is retained within the liver; however, daily utilization is much higher than vitamin B12 such that stores are more rapidly depleted, typically within 3-6 months after cessation of intake. Folate is active only in its reduced form as a tetrahydrofolate (THF). Folates play an essential role in one-carbon transfers and are required for the de novo production of thymidylate that is needed in DNA synthesis. The metabolic pathways Though seemingly disparate, the metabolic pathways of vitamin B12 and folate are interconnected as diagrammed below. Figure methyl-THF methylated product homocysteine methyl transferase B12 methionine lipids, myelin DNA THF serine du glycine dump dttp dihydrofolate reductase 5,10-methylene THF DHF 5-methyl-THF, in the presence of methyl transferase, donates its methyl group to cobalamin forming methylcobalamin. Methylcobalamin transfers this methyl group to homocysteine forming the amino acid, methionine, as well as THF, the primary intracellular form of folate. THF, in conjunction with the conversion of serine to glycine, is converted to 5,10-methylene THF, a component in the rate-limiting step of DNA synthesis. In this step (requiring

12 the enzyme thymidylate synthetase), 5,10-methylene THF is converted to dihydrofolate (DHF) while deoxyuridylate (dump) is converted to thymidylate (dttp), a critical component in pyrimidine, and hence, DNA synthesis. Dihydrofolate reductase then reduces DHF back to THF, thereby replenishing intracellular stores. Note that various drugs and chemotherapeutic agents (such as methotrexate) inhibit dihydrofolate reductase such that THF cannot be regenerated intracellularly via this mechanism they secondarily induce folate deficiency. Effects of impaired DNA synthesis In the setting of vitamin B12 or folate deficiency, the resultant defect in DNA synthesis impacts both the morphologic and clinical manifestations of disease. With diminished DNA production per unit time, more cells remain in the DNA synthesis phase of the cell cycle. In contrast, cytoplasmic maturation is controlled by RNA synthesis - independent of the above factors. Thus, the outcome is asynchrony of nuclear and cytoplasmic maturation such that proliferating cells appear markedly enlarged (megalo-) and exhibit immature nuclei (-blastic) with comparatively mature cytoplasm. All hematopoietic lineages are affected. Though macro-ovalocytes are the eventual outcome of this impaired erythropoiesis, the most characteristic cell within erythroid precursors is the orthochromatophilic megaloblast as its mature-appearing pink-orange cytoplasm contrasts markedly with its enlarged and heterogeneous immature nucleus. Erythroid precursors can also exhibit mild dysplasia with irregular nuclear contours and mild multinucleation. Characteristic features within the myeloid lineage include giant metamyelocytes and/or band forms. These cells appear markedly enlarged with fine open chromatin. Megakaryocytes, though typically the least affected cell line, may exhibit abnormal nuclear lobulation, in particular hyperlobulation, and immature washed out chromatin in severely deficient states. One outcome of this impaired DNA synthesis is ineffective erythropoiesis. The abnormal erythroid precursors undergo destruction in the marrow with resultant mild indirect hyperbilirubinemia and jaundice. Consequently, the marrow mounts an attempted compensatory response yielding marrow hypercellularity and erythroid hyperplasia. With worsening deficiency and progressive involvement of all cell lineages, pancytopenia may eventually develop. The morphologic features detailed above can be misleading - marrows from individuals with vitamin B12 and/or folate deficiency have been misinterpreted as myelodysplastic syndromes (MDS) or acute myelogenous leukemias. Do not make this mistake! Although megaloblastoid erythroid maturation can be seen in MDS, in these syndromes, the erythroid precursors show more marked nuclear irregularities with extensive lobulation, detached fragments of nucleus and extreme multinucleation. Additionally, myeloid elements typically exhibit aberrant cytoplasmic

13 maturation evidenced by neutrophilic hypogranularity; however, they are not generally enlarged with open sieve-like chromatin as in megaloblastic anemia. Furthermore, giant metamyelocyte and band forms are not present in MDS while megakaryocytic nuclei are typically hypolobulated or multinucleate with hyperchromatic condensed nuclear chromatin. The cytomorphologic features of MDS will be reviewed in section 2.7, allowing for a more detailed comparison. In acute myeloid leukemia, unlike in megaloblastic anemia, myeloid elements typically predominate and myeloid maturation arrest is evident with an associated expanded population of blasts. Auer rods may also be identified. The extreme nuclear-cytoplasmic dyssynchrony of megaloblastic anemia is not typically seen in the erythroid or megakaryocytic elements of acute leukemia. In order to discriminate megaloblastic anemia from these other entities, all lineages must be carefully evaluated, with focused attention on nuclear and cytoplasmic features as well as on differentiation and maturation. Comparative Marrow Morphology Lineage Megaloblastic Anemia Myelodysplasia Erythroid Myeloid Megakaryocytic Mild multinucleation Mild nuclear contour irregularities Orthochromatophilic megaloblast Fine open (sieve-like) chromatin in all stages of myeloid maturation No expanded blast population Giant metamyelocytes and band forms Fine open (sieve-like) chromatin Nuclear hyperlobulation Extreme multinucleation Marked nuclear contour irregularities, nuclear lobulation and detached fragments of nuclear material Irregularly condensed chromatin in more mature myeloid forms Variably increased blasts Neutrophilic hypolobulation (Pelger-Huet-like neutrophilic forms) Cytoplasmic hypogranularity Hyperchromatic condensed nuclear chromatin Nuclear hypolobulation and/or multinucleation

14 A few more features are worthy of note, specifically with respect to red cell abnormalities, and may serve as clues to the appropriate evaluation and diagnosis of these nutritional deficiencies. Macrocytosis is one of the earliest manifestations of a deficiency state, developing months to years before overt anemia. Thus, vitamin B12 and folate status should be formally assessed in any individual with a persistent and progressive macrocytosis. Additionally, the magnitude of macrocytosis also correlates with the presence of a nutritional deficiency, such that an MCV >130 fl is much more predictive of vitamin B12 or folate deficiency than an MCV <130 fl. Because these cells are large, their hemoglobin content (eg. mean cell hemoglobin, MCH) is higher when compared to their normal counterparts; however, in relationship to their size, their hemoglobin concentration (eg. mean cell hemoglobin concentration, MCHC) is within normal limits. Other clinical manifestations Though patients typically present with macrocytosis and/or unexplained anemia, neurologic manifestations may also be evident in individuals with vitamin B12 deficiency. These manifestations most commonly include parasthesias, numbness and ataxia, although dementia and psychosis may occasionally develop. Such neurologic sequelae are more typically associated with vitamin B12, and not folate, deficiency and are secondary to a variety of mechanisms, including degeneration of the posterior and lateral columns of the spinal cord, peripheral neuropathy and central effects; however, the cause of this degeneration is not well understood. Referring back to Figure 2.1 above, impaired methylation of lipids and myelin (via the methionine, homocysteine, cobalamin reaction) may be one operative factor. Although hematologic manifestations typically precede neurologic manifestations, neurologic signs may present in isolation and be the sole clue to diagnosis. Parallel with this, neuropsychiatric symptoms in folate-deficient patients may suggest coexisting vitamin B12 deficiency. Once either the presence of a megaloblastic anemia has been established or the suspicion of a nutritional deficiency state has been invoked, how do we determine whether the underlying etiology is secondary to vitamin B12 deficiency, folate deficiency or both??? Laboratory studies are a logical starting point for this determination, so we need an appreciation of the pathways of vitamin B12 and folate absorption, transport and cellular uptake. Absorption, transport and cellular uptake These pathways are important in order to [1] understand the laboratory evaluation of vitamin B12 and folate status, and [2] ascertain the underlying etiology once a deficiency state has been established.

15 The absorption of vitamin B12 begins in the acidic environment of the stomach, where it is made accessible for transport. Acid (produced by parietal cells) and pepsin (generated by cleavage of pepsinogen that is produced by chief cells) digest food, thereby releasing B12. Vitamin B12 binds transport factors called R-binders (secreted by the salivary glands), which convey it into the small intestine. In the duodenum, pancreatic proteases degrade the R-binders, making B12 accessible for binding to intrinsic factor (IF) that is produced and released by parietal cells in the stomach. IF carries B12 through the jejunum and into the ileum, where IF receptors located within the ileal mucosa bind the IF-B12 complex. This complex is endocytosed, IF and B12 are released, and B12 is then made accessible for binding to transcobalamin II (TCII) which shuttles it into the portal circulation. The TCII-B12 complex is rapidly cleared by cellular uptake via TCII receptors. From there, B12 is released intracellularly for use in the cytoplasmic and mitochondrial metabolic pathways detailed above. In the systemic circulation, B12 is largely bound to yet another transporter, transcobalamin I (TCI); however, the mechanisms affecting regulation and action of TCI are not as well understood. In comparison, the absorption of folate takes place in the jejunum of the small intestine. The polyglutamic forms of food folate are hydrolyzed to monoglutamic forms via intestinal conjugases within the gut lumen and brush border as well as within the lysosomes of enterocytes. Monoglutamic and polyglutamic forms can be absorbed across the intestinal mucosa by both passive diffusion as well as active protein transport. Reduced folates are rapidly converted to 5-methyl-THF in the enterocyte and then enter the portal circulation they are typically detected here within 15 minutes of entering the stomach. Folate levels peak within 1 hour of ingestion and then decline slowly over several hours. Folate enters cells via a transport system that involves its exchange with intracellular divalent anions. Of note, red blood cell uptake of folate is vitamin B12 dependent a point to which we shall return momentarily. Laboratory testing Before we discuss the laboratory studies pertinent to vitamin B12 and folate status, what do we expect to find in each deficiency state? Please refer back to Figure 2.1 above. Expected results: With B12 deficiency, one would expect serum B12 levels to be low. Serum folate may be normal or elevated given that 5-methyl-THF does not accumulate intracellularly, but rather diffuses out of cells and into the circulation. RBC folate should be diminished because B12 is necessary for folate uptake in erythroid precursors while methylmalonic acid and homocysteine should both be elevated.

16 With folate deficiency, one would expect serum and RBC folate levels to be low and serum B12 to be normal. Homocysteine should be elevated while methylmalonic acid should be normal given that its metabolism is folate independent. With a combined deficiency (of both B12 and folate), one would expect low serum B12 as well as low serum and RBC folate with elevated methylmalonic acid and homocysteine. However, as will become apparent below, the interpretation of these tests is not necessarily as straightforward as one might anticipate. Serum B12: Serum B12 has been the mainstay for assessing vitamin B12 status with 97% sensitivity in detecting clinical B12 deficiency. By this is meant a deficiency state in which individuals manifest clinical signs and symptoms of disease. These manifestations may be hematologic or neurologic as outlined above. Definitionally, a serum B12 level below 200 ng/l (148 pmol/l) is considered abnormal. Only 2.9% of patients who are clinically affected and unequivocally B12 deficient demonstrate levels above this cut-off. Unfortunately, however, serum B12 is not that specific as various other factors may cause deceptively low or high levels. In fact, less than 10% of low B12 levels are associated with clinical signs of deficiency. Causes of misleading serum B12 levels Low levels (potentially in the absence of B12 deficiency) Pregnancy (last trimester) Folate deficiency (mechanism poorly understood) Drugs (eg. anticonvulsants, oral contraceptives) Other diseases (eg. HIV infection, plasma cell myeloma) Normal/high levels (possibly in the presence of B12 deficiency) Myeloproliferative neoplasms or leukemoid reactions - elevated transcobalamin I levels Intestinal bacterial overgrowth - production of biologically inactive cobalamin analogues Congenital transcobalamin II deficiency Liver disease - especially with hepatic necrosis (eg. hepatitis)

17 Additionally, a state of subclinical deficiency has been described in which clinical signs and symptoms are absent; however, patients may demonstrate abnormal electrophysiologic responses on neurologic testing. In this group, serum B12 levels range between ng/l, above the designated cut-off level; yet, these individuals typically demonstrate other mild metabolic abnormalities suggestive of B12 deficiency (eg. elevated methylmalonic acid and homocysteine levels). Most importantly, these metabolic abnormalities resolve after institution of B12 therapy, supporting the notion of a subclinical deficiency state. This subclinical condition is found in 10-20% of the elderly population as well as within a much lower proportion of younger individuals. Approximately 70% of low B12 levels and 30% of low-normal levels are believed to represent subclinical deficiency. Serum folate: Traditionally, folate deficiency has been defined as serum folate levels below 2.5 µg/l (5.7 nmol/l); however, this test boasts neither great specificity nor sensitivity for this determination. Although tissue stores may be normal, serum folate levels decrease within a few days to weeks of dietary restriction. In fact, one third of hospitalized patients may demonstrate low serum folate that is best considered a transient phenomenon rather than true deficiency. Alcohol, in particular, as well as other drugs and medications may cause a short-term decline in serum levels despite adequate tissue stores. Folate also has limited stability with long-term storage (even in samples maintained at -20 C); hence, determinations should be made from freshly procured specimens. Serum folate levels may also be artifactually elevated. Folate levels increase with oral intake; hence, fasting levels are a prerequisite when testing. B12 deficiency is a secondary cause of increased serum folate. As diagramed above in Figure 2.1, impairment of the conversion of homocysteine to methionine results in the intracellular accumulation of 5- methyl-thf, which then diffuses out of cells and into the circulation. Lastly, folate is stored in erythrocytes, so hemolysis of a blood sample will result in artificially elevated levels.

18 Causes of misleading serum folate levels Low levels (in the absence of folate deficiency) Transient poor dietary intake Drugs (oral contraceptives, acetylsalicylic acid, alcohol) Artifact -- improper specimen handling (eg. prolonged storage) -- renal failure (eg. abnormal folate binding protein) High levels (potentially in the presence of folate deficiency) Non-fasting determination B12 deficiency Artifact -- hemolysis of blood sample Red blood cell folate [normal range: µg/l]: Given the above caveats associated with serum folate determinations, erythrocyte folate is thought to better correlate with folate tissue stores. Red blood cells incorporate folate early during their maturation and these levels remain constant throughout the life of the cell. Mature erythrocytes neither acquire nor lose folate. Hence, red cell folate levels are less sensitive to dietary fluctuations in intake and are believed to better reflect true folate status. Despite the above attributes, RBC folate has its own limitations. Sensitivity and specificity remain problematic particularly in pregnant women and alcoholics. Spuriously high and low levels have been observed in these populations. One important cause of low red cell folate levels is vitamin B12 deficiency. As mentioned, vitamin B12 serves as a cofactor for the uptake of folate within erythroid precursors. In the absence of vitamin B12, folate cannot be incorporated. Serum methylmalonic acid (MMA) and homocysteine levels: These two tests can help to distinguish one deficiency state from the other. Although both are sensitive indicators of clinical deficiency when elevated, they too lack specificity, which compromises their utility when either is used in isolation. [Normal ranges: serum MMA ( nmol/l), homocysteine ( µmol/l)].

19 Causes of elevated MMA levels Renal insufficiency Hypovolemia Inherited metabolic defects (methylmalonyl-coenzyme A mutase deficiency) Causes of elevated homocysteine levels Renal insufficiency Hypovolemia Hypothyroidism Psoriasis Pyridoxine deficiency Inherited errors of homocysteine metabolism Given the various limitations and caveats associated with each of the above laboratory tests, one strategy to employ in the diagnostic evaluation of megaloblastic anemia is to require abnormal results in a minimum of two of these, particularly two tests that do not share common causes of falsely elevated or depressed levels. Establishing the diagnosis of vitamin B12 or folate deficiency with laboratory studies is only the first step in the evaluation process. The second, and equally important step, is to establish the underlying cause of deficiency as this may modify prognosis, management and the therapy itself. Underlying etiologies of nutritional deficiencies Vitamin B12 Dietary insufficiency can develop in vegans and/or breast-fed infants of vegan mothers. Gastric disorders Food cobalamin malabsorption inability to extract B12 from its binding proteins in food. This is a common condition, affecting 30-40% of all patients with low B12 levels, but its symptoms are typically mild. Common underlying causes include atrophic gastritis with achlorhydria, gastric acid suppression (often iatrogenic secondary to H 2 -blockers) and gastric surgical procedures. This can progress to pernicious anemia, but only when IF secretion ceases. Pernicious anemia (PA) malabsorption of B12 secondary to absence of gastric IF production. Arises in the setting of severe atrophic gastritis with destruction of parietal cells and resultant loss of both IF and acid production. Two forms have been described: (1) type A with severe fundic atrophy, antral sparing, secondary hypergastrinemia and associated autoimmune

20 phenomena, and (2) type B with a pangastritis (often secondary to Helicobacter pylori infection); hypergastrinemia and autoantibodies are not demonstrated in this variant. Within the type A form, 50-75% of patients possess circulating autoantibodies against intrinsic factor. Other autoimmune phenomena may also be seen, such as hypothyroidism or hypofunctional endocrinopathies (eg. hypoadrenalism, hypoparathyroidism, type I diabetes mellitus). Inherited IF deficiency rare condition, most commonly manifest in children. Gastric surgery partial or complete resection can impact various factors relevant to B12 transport and uptake as detailed above. Intraluminal intestinal disorders Pancreatic insufficiency with inadequate production of pancreatic proteases, R-binders are not digested and, therefore, do not release B12, making it inaccessible for binding to IF. Bacterial contamination bacterial overgrowth occurs secondary to poor bowel motility, blind loop syndromes, large diverticulae and/or strictures. Bacteria compete for B12 within the gut lumen and they secrete inactive B12 analogues. Parasitic infestation Diphyllobothrium latum is a parasite that infects fresh water fish and when ingested by humans can colonize the bowel and serve as a competitor for B12 uptake. Ileal disorders all impair the absorption of B12 across the intestinal mucosa Inflammatory conditions tropical sprue, celiac disease Ileal bypass surgery Radiotherapy regional to the abdomen Hereditary B12 malabsorption apparent absence of ileal B12 receptors, also called Imerslund-Gräsbeck syndrome; typically presents in infancy and childhood. Drugs/toxins long-term use of colchicines, neomycin, slow-release potassium chloride and alcohol, to name just a few culprits. Inborn errors of metabolism rare disorders affecting transport and metabolism; these include, but are not limited to, deficiencies of transcobalamin II, methylmalonyl-coa mutase deficiency and R-binders (haptocorrins). Folate Dietary insufficiency much less common in the United States, now that many foods are fortified with folate, offering involuntary dietary supplementation. When it does occur, it is typically secondary to malabsorption, so deficiency is not usually limited to folate; rather, multiple other nutrients/factors are also affected.

21 Malabsorption any disease affecting the upper half of the small bowel can affect folate absorption. Offenders include celiac disease, tropical sprue, Whipple s disease and any inflammatory condition of the small bowel. Hereditary folate malabsorption characterized by severe megaloblastic anemia and low folate levels in the first few months of life. Additional clinical manifestations include diarrhea, oral ulcers, failure to thrive and progressive neurologic deterioration. This entity is not that well understood, but is believed to alter the folate transporter that is present within intestinal mucosa. Drugs/toxins alcohol (mechanism not that well understood), methotrexate (binds to dihydrofolate reductase preventing its binding to DHF); trimethoprim and pyrimethamine (both folate inhibitors). Inborn errors of metabolism rare conditions that diminish various enzymes involved in folate metabolism. Increased folate requirements Growth-related needs relevant to infants and children as well as women who are pregnant and lactating Pathologic increased cell turnover hemolysis, exfoliative dermatitis, various hematologic malignancies (eg. acute leukemias and chronic myeloproliferative neoplasms) Acute folate deficiency an acute onset of megaloblastic changes that occur in catastrophically ill patients. Findings are limited to the bone marrow and are not seen in peripheral blood. This condition is believed to represent an acute folate deficiency state that is superimposed upon multiple other underlying medical problems. Additional diagnostic testing The Schilling Test: The Schilling test, though now less frequently employed, may still be utilized for complex cases of suspected vitamin B12 deficiency. This test permits the discrimination of a deficiency of (or defect in) IF from a malabsorption syndrome. Patients receive a physiologic oral dose of radiolabeled B12. A flushing dose of unlabeled B12 is subsequently administered intramuscularly in order to saturate liver and tissue binding sites, so that absorption of B12 (if it does indeed occur) is not restricted within B12-depleted tissues, but rather will result in its eventual excretion in the urine. If patients are able to secrete functional IF, absorption of B12 occurs, and this absorption is reflected by a specified minimum level of urinary excretion of radiolabeled B12. If the test results are abnormal, the test is repeated several days later with one modification. IF is administered in conjunction with the B12 in order to

22 determine if this corrects the outcome. If it does, a deficiency/defect of IF is invoked and if it does not, a malabsorptive problem is implicated. As one might expect, individuals with food cobalamin malabsorption have normal Schilling test results. However, a food Schilling test utilizing radiolabeled B12 bound to egg yolk can be employed in this scenario to confirm the diagnosis. The radiolabeled mixture is scrambled and fed to patients. Laboratory studies for case 1 The patient in this case demonstrated laboratory studies that were consistent with vitamin B12 deficiency. Autoantibodies against IF were subsequently detected, consistent with a diagnosis of pernicious anemia. Thyroid stimulating hormone was normal, indicating that ongoing hypothyroidism was not contributing to her macrocytosis. Serum cobalamin <150 ng/l [normal range, ng/l] Folate > 20 µg/l [normal range, µg/l] Methylmalonic acid 8386 nmol/l [normal range, nmol/l] Thyroid stimulating hormone 3.66 miu/l [normal range, ]

23 Case 1 - summary/answers to questions: 1. Basic/broad descriptor of the most prominent laboratory abnormality(ies). Macrocytic anemia 2. Describe the various morphologic findings Macro-ovalocytes Moderate anisopoikilocytosis Basophilic stippling Neutrophilic hypersegmentation = These features indicate megaloblastic anemia 3. Formulate a differential diagnosis Vitamin B12/folate deficiencies or defects Drugs/toxins 4. Additional testing to help refine the differential To establish a deficiency state o Serum cobalamin o Serum folate o Red blood cell folate o Methylmalonic acid o Homocysteine To help ascertain underlying etiology o Autoantibody testing (eg. parietal cells, IF) To exclude coincident causes of macrocytosis o Thyroid stimulating hormone (TSH) level 5. Is a bone marrow study required? No 6. Match with corresponding bone marrow aspirate/biopsy Bone marrow D o Hypercellular marrow with erythroid hyperplasia o Megaloblastic morphology with nuclear and cytoplasmic maturational dyssynchrony FINAL DIAGNOSIS: Vitamin B12 deficiency, secondary to pernicious anemia

24 Section 2.2 Case 2: Clinical: CBC: 8-year-old healthy male who had a routine CBC performed as part of a well child care visit. HGB 8.8 g/dl, MCV 52.6 fl, RBC 5.55 M/µL, HCT 29.2%, MCH 15.8 pg, MCHC 30.1 g/dl, RDW 25.4% WBC 5.95 K/µL [39% neutrophils, 46% lymphocytes, 3% atypical lymphocytes, 4% monocytes, 7% eosinophils, 1% basophils] PLT 295 K/µL Smear: dimorphic red blood cell population, marked anisopoikilocytosis (including targets, elliptocytes, teardrops, fragments, bizarre forms) and Pappenheimer bodies The second case is that of a healthy 8-year-old male, who on a routine CBC performed as part of a well child care visit, is noted to be modestly anemic with a profound microcytosis and modestly elevated RDW. The category of microcytic anemias, into which this patient is classified, generally implies an underlying problem with iron metabolism and/or processing, so let s begin with an overview of the major pathways relevant to this process. Iron trafficking in erythrocytes Iron is first taken up by erythroid precursors where it enters the mitochondrion and is incorporated into protoporphyrin, forming heme. Heme is released back into the cytosol where it combines with four globin chains to form hemoglobin, creating a functional erythrocyte that is capable of oxygen exchange. Iron is largely recycled in the body, so at the end of the roughly 120 day lifespan of an erythrocyte, macrophages phagocytose effete red cells and recirculate the iron, enabling it to be re-incorporated into newly formed erythroid precursors as part of on-going erythropoiesis. Differential diagnosis of microcytic anemia General blockades to this pathway include [1] iron deficiency, in which insufficient iron enters erythroid precursors, [2] sideroblastic anemia, in which iron cannot be incorporated into protoporphyrin such that heme is not formed, [3] thalassemia, in which globin chains are produced in insufficient

25 quantities for the formation of hemoglobin, and [4] anemia of inflammation, in which macrophages retain their iron, rendering it inaccessible for recycling and reutilization. To expand on these four major blockades, we can consider both [1] and [4] above to be part of generally disordered iron metabolism. Iron deficiency can result from excess loss such as with physiologic or non-physiologic hemorrhage. It may be the consequence of inadequate intake such as with an iron-deficient diet or malabsorption. Likewise, increased physiologic demands may exceed resources such as with growth during infancy and childhood or during pregnancy and lactation. Additionally, although the body may appear replete with iron, it may be functionally inaccessible to erythroid precursors as with inflammation, infection and/or malignancy. The sideroblastic anemias, item [2] above, are consequences of disordered heme synthesis. These conditions can be either hereditary or acquired, and in the latter instance, causation may be idiopathic or toxin-mediated such as with lead and alcohol poisoning. The thalassemic syndromes [3] result from disordered globin synthesis. These may be secondary to insufficient production of either the alpha, beta, gamma or delta globin chains; however, only alpha- and beta- thalassemia will be discussed herein. Of note, although our algorithmic approach to anemias necessitates an inquiry regarding the reticulocyte count, this result is not so helpful in this category of diseases, as all of the primary entities on the differential (when present in isolation) have ineffective erythrocyte production and are, therefore, expected to have an inadequate reticulocyte response. The most common causes of microcytic anemia are iron deficiency, thalassemia minor and anemia of inflammation; however, causation is age, gender and population dependent. Furthermore, many patients with thalassemia minor are microcytic without overt anemia. Young menstruating women are more likely to have iron deficiency given on-going physiologic iron loss. An otherwise healthy elderly male who presents with microcytic anemia is suspected of having an underlying (particularly gastrointestinal) malignancy with occult bleeding. Hospitalized patients commonly develop anemia of inflammation while a microcytosis with borderline anemia in an individual of Mediterranean descent might favor beta-thalassemia trait. Regardless of the presentation, iron studies are an inevitable part of the diagnostic work-up and evaluation of microcytic anemias. Iron homeostasis As mentioned above, most of the body s iron is recycled; consequently, only a minute amount (1-2 mg) of the total body iron stores (~4 gms) must be absorbed from the diet each day. Dietary iron comes in two basic forms, [a] heme iron, Fe 2+, derived from animal blood and muscle, and [b] non-heme iron derived from plants and salts. The former is more easily absorbed than the latter, which exists in the poorly soluble Fe 3+ state. Absorption of heme

26 iron occurs in the duodenum and this process is facilitated by the contributions of gastric acidity. Non-heme iron must first be reduced to Fe 2+ within the brush border prior to its uptake by enterocytes. Although iron can be stored within the enterocyte, most iron exits the cell and is converted back to Fe 3+, thereby enabling it to bind transferrin, its primary plasma transport protein. Transferrin receptors are located predominantly on erythroid precursors and hepatocytes. Erythroid elements contain approximately 65% of total body iron stores. Erythroid precursors mature, circulate as functioning erythrocytes and then undergo phagocytosis by macrophages within the reticuloendothelial system (RES). Iron can be stored within both hepatocytes and macrophages as ferritin, a nonreactive tissue form that can be easily mobilized upon increased iron demands. These various intracellular tissue storage compartments maintain approximately 30% of total body iron. The average daily loss of iron, 1-2 mg (ie. that needed to be replenished by dietary intake), is secondary to normal cell turnover and physiologic hemorrhage (sloughing of mucosal cells in the intestinal and urinary tracts, skin desquamation and menstruation). With an understanding of iron homeostasis, we are now prepared to explore the various laboratory studies that are so fundamental to the assessment of iron status... and there are many. Laboratory iron studies Serum iron - this is a measure of iron that is bound to transferrin. Methodology - under acidic conditions, iron is dissociated from transferrin, reduced to Fe 2+, and then quantitated via a colorimetric change. The main flaw of this test is that it has low sensitivity for the iron-deficient state. Serum iron does not decline substantially until iron stores have been completely depleted. Total iron binding capacity (TIBC) this is an indirect measure of transferrin concentration. Transferrin accounts for nearly all of the iron-binding capacity of serum. Methodology - Fe 3+ is added to serum, the excess iron (that is not bound to transferrin) is removed and iron is quantitated as outlined above for serum iron. Transferrin is synthesized by hepatocytes and its production is upregulated in iron deficiency; hence, TIBC will be increased in this scenario. The above two tests should always be performed simultaneously in order to differentiate iron deficiency from other disease states. Transferrin saturation a parameter that is calculated from serum iron and TIBC. Transferrin is approximately one-third saturated under normal conditions.

27 Methodology % transferrin saturation = serum iron/tibc x 100 Guidelines for interpretation: <10% saturation [considered by some to be diagnostic of iron deficiency] <16% saturation [considered by some to be highly suggestive of iron deficiency] This test suffers from poor specificity (eg. individuals with anemia of inflammation may also exhibit low/suggestive saturation levels). Serum ferritin reflects tissue iron stores and is, therefore, a good indicator of iron status; however, in comparison to tissue ferritin, its concentration is miniscule. Interestingly, unlike tissue ferritin, serum ferritin contains little if any iron. Special importance: Serum ferritin is decreased only in iron deficiency. Generally, it is the first laboratory test to become abnormal, even before morphologic changes are evident in erythrocytes. Guidelines for interpretation: <10 µg/l [considered diagnostic of iron deficiency, even when used in isolation] <20 µg/l [considered highly suggestive of iron deficiency] The main caveat to an interpretation of serum ferritin is that it is an acute phase reactant. So, increased levels do not exclude the diagnosis of iron deficiency. Soluble (serum) transferrin receptor (stfr) represents a proteolytic cleavage product of the transferrin receptor that is derived from erythroid precursors. Increased levels are seen in iron deficiency as well as ineffective erythropoiesis such as with the thalassemic syndromes. Importance: Levels are not elevated in anemia of inflammation; therefore, this parameter may help to distinguish that condition from iron deficiency. For this discrimination, utilize the stfr-ferritin index. STFR-F index = stfr/log ferritin Guidelines for interpretation: >2.0 [suggests iron deficiency alone or in conjunction with an inflammatory state] <2.0 [suggests anemia of inflammation] Unfortunately, this test also suffers from low specificity. Free erythrocyte protoporphyrin (FEP) in conjunction with ferrochelatase, heme is formed by the incorporation of Fe 2+ into protoporphyrin IX within the mitochondrion. FEP represents protoporphyrin that is lacking in iron (to be