Blood Cell Identification Graded

Transcription

1 Blood Cell Identification Graded Case History A 51-year-old female presented with dyspnea on exertion. Laboratory results were as follows: WBC=5.5 X 10 9 /L; Hgb=4.2 g/dl; Hct=13.9%; MCV=78.7fL; RDW=30; PLT=505 X 10 9 /L; Ferritin=1 ng/ml (normal = ); Serum Vitamin B12=101 pg/ml (normal= ); Serum Folate=17.7 ng/ml (normal=>2); TSH=24.2 mu/ml (normal= ). Microcyte with central pallor Good /-21 The arrowed red cell is the smallest one in the field and it clearly shows abundant central pallor. Ideally, a lymphocyte nucleus should be used as a size benchmark while evaluating blood smears and as a point of reference. An MCV is provided in this case history and abundant numbers of platelets are present to clearly identify this cell as a microcyte. Microcytes are small erythrocytes measuring less than 6μm in diameter and or less than 80 fl by electronic counters volume measurements. They should have clear central pallor as opposed to spherocytes or microspherocytes that do not. Microcytes are commonly seen in iron deficiency anemia, thalassemias and lead poisoning. 2

2 Blood Cell Identification Graded /-22 Platelet, normal Good The arrowed element is a normal platelet. Platelets represent fragments of megakaryocytic cytoplasm and vary in size from 1.5 to 3 μm. They can vary in shape but are usually round and in a well stained slide will display fine purple-red granules. Platelet counts are an important information element of the routine Complete Blood Cell counts (CBC), as physicians usually work up any unexpected thrombocytopenia (low platelet count) or thrombocytosis (high platelet count). Good laboratory practice requires a review of the blood smear to confirm thrombocytopenia before it is reported. Pseudothrombocytopenia, usually related to platelet clumping, is a relatively common phenomenon. 3

3 Blood Cell Identification Graded /-23 Neutrophil, hypersegmented nucleus Good The arrowed neutrophil clearly displays six lobes and is characteristic for a hypersegmented neutrophil. The neutrophils shown in /-24 have even a higher number of lobes. Hypersegmented neutrophils are seen in association with megaloblastic hematopoiesis related to impaired DNA synthesis. Vitamin B12 and Folate are cofactors in the DNA synthesis process and consequently, if the availability of one or both is decreased or absent, by whatever mechanism, it will result in megaloblastic hematopoiesis. The presence of hypersegmented neutrophils in the peripheral blood is recognized as a very specific feature of impaired DNA synthesis. Neutrophil hypersegmentation may precede megaloblastic erythropoiesis and therefore, it should always be reported when encountered as part of a manual differential review. Rarely hypersegmented neutrophils may be seen as a congenital anomaly, or in association with sepsis, renal disease and myeloproliferative states. 4

4 Blood Cell Identification Graded /-24 Tear-drop cell Good The arrowed red cell is pear shaped with a single blunted end and is typical of a tear-drop red cell (Dacryocyte). Tear-drop cells can represent an artifact of preparation. However, in these situations their tails are all pointed in the same direction. Tear-drop cells vary in size and may have a normal or increased amount of central pallor. Tear-drop cells are usually associated with an abnormal spleen or bone marrow function and or structure. Conditions that usually have the presence of tear drop red cells in the peripheral blood include myelofibrosis, pernicious anemia, anemia of renal disease, thalassemia, hemolytic anemia and a variety of disorders that result in bone marrow infiltration (hematologic and non hematologic malignancies). 5

5 Blood Cell Identification Graded /-25 Neutrophil necrobiosis (degenerated neutrophil) Good The arrowed cell shows multiple unconnected nuclear lobes with dark, dense and homogeneous pyknotic nuclear material consistent with degenerated nuclear material. The cytoplasm resembles the other shown cells with neutrophil granules present. This is an example of neutrophil necrobiosis also referred to as a degenerated neutrophil. This is a relatively common finding that can be seen in normal individuals as well as in a spectrum of medical conditions like infections, chronic inflammatory disorders and malignancies. This is a nonspecific finding but important to recognize, especially when there is only one single dark pyknotic nucleus, as the cell may be mislabeled as a nucleated red cell. 6

6 Discussion The work up and follow up of patients presenting with hypochromic microcytic anemia is one of the most common clinical situations. The patient in this case has a very severe anemia based on the recorded hemoglobin. The presenting dyspnea on exertion likely relates to the decreased oxygen carrying capacity by the fewer red blood cells in circulation. The most common entities in the differential diagnosis of hypochromic microcytic anemia include disorders of iron metabolism (most frequently iron deficiency and anemia of chronic inflammatory states), thalassemia (a disorder of abnormal globin synthesis) and sideroblastic anemias. The physician s initial evaluation of clinical and baseline laboratory findings is the determining factor in the selection of the laboratory tests used for identification and appropriate diagnosis of the underlying cause(s) for anemia. The provided laboratory information for this patient is confirmatory of an iron deficient state by a very low ferritin value. However, the mean corpuscular volume (MCV) is mildly decreased, while the anemia is very severe. Thus, in the absence of acute bleeding, the possibility of an additional cause for the anemia is likely. The red cell distribution width (RDW) is markedly elevated confirming the marked anisocytosis and presence of a dimorphic red cell population observed on smear review. As illustrated by the images, the presence of microcytic hypochromic red cells and hypersegmented neutrophils should have been recorded and reported as part of a manual smear review. The latter is a very important feature as it is characteristic of megaloblastic anemias. Therefore, in this case study, proper identification of the peripheral smear morphologic findings is critical in alerting physicians to the possibility of a combined deficiency. This exercise challenges participants to identify morphologic findings, although the chemistry results are also provided for additional information. The reader should remember that in the usual clinical scenario, the morphologic findings of the blood smear will ideally precede the ordering of any specific tests and are the ones that the physician will use as baseline information to further investigate the cause of the anemia. In this particular situation, noting and reporting the presence of hypersegmented neutrophils is the clue to suspect vitamin B12 deficiency in the absence of the usual high MCV. This exercise illustrates anemia caused by combined iron and vitamin B12 deficiencies. Iron deficiency is the most common cause of hypochromic microcytic anemia and one of the most common human diseases. The most common causes of iron deficiency include inadequate intake for metabolic demands (pregnancy and young age), poor absorption (stomach and small bowel related problems) and increased loss through bleeding (usually chronic situations like heavy menstrual periods, stomach ulcer, hemorrhoids). Laboratory testing is an essential arm in the support of an iron deficient state. Iron deficiency usually has a high RDW, while a microcytic anemia related to thalassemia trait will have a normal RDW. Serum iron, total iron binding capacity (TIBC) a measure of transferrin, percent iron saturation, soluble serum transferrin receptor and free protoporphyrin are among the most common tests available to distinguish iron deficiency anemia from the anemia of chronic inflammation and others. A bone marrow aspirate and biopsy to evaluate iron stores is presently a rare indication for this procedure, as in most clinical situations the question can be answered without the need of this invasive procedure. The tests that are used for the diagnosis of an iron deficient state vary with the physician preference and the clinical scenario. The low ferritin value in this patient was sufficient for establishing the diagnosis. However, a normal or elevated 7

7 ferritin level does not exclude the diagnosis of iron deficiency. Individuals with active inflammation may have elevated serum ferritin, as ferritin behaves as an acute phase reactant. The measurement of soluble serum transferrin receptor is advocated by some as a more specific test to document the presence of iron deficiency in patients with chronic inflammatory states. Megaloblastic anemias are a heterogeneous group of disorders that share common morphologic features. The most common causes for these anemias are cobalamin (vitamin B12) and folate deficiencies. This patient had a low vitamin B12 but a normal serum folate. The coenzyme forms of folate and vitamin B12 have an intrinsic role in DNA synthesis as part of a complicated biochemical pathway. Moreover, distinction between folate and cobalamin deficiency is very difficult by clinical grounds. Folate replacement may correct the megaloblastic change of vitamin B12 deficiency. Therefore, empiric treatments have been traditionally discouraged as permanent neurologic sequela may result when patients are given folate alone. A common cause of cobalamin deficiency is pernicious anemia (see below). Folate deficiency is most frequently due to folate poor diets but may also occur in patients with non tropical and tropical sprue, in patients who are pregnant and in patients taking antifolate or other medications. Folate deficiency was a very common occurrence before folate supplementation of foods was instituted in the recent past. However, dietary related deficiencies are still common in chronic alcoholics and individuals, usually in low socioeconomic conditions, that consume inadequate diets. Megaloblastic anemia is characterized by enlargement of all rapidly proliferating cells of the body, including the bone marrow cells. As these vitamin deficiencies affect DNA synthesis only, with RNA synthesis usually spared, cytoplasmic growth and maturation continue. This explains the cell enlargement (mega) and the characteristic nuclear to cytoplasmic asynchrony in maturation (blastic). Changes are observed in the erythroid, myeloid and megakaryocyte lineages with typical large normoblasts and giant metamyelocytes seen. Cells may display abnormal nuclear contours and the nuclear chromatin is open and less condensed than expected for the stage of maturation. The described nuclear changes together with the increased cell size are referred to as megaloblastic maturation. The bone marrow is characteristically hyperplastic but with ineffective medullary erythropoiesis resulting in anemia and typically high lactic dehydrogenase (LDH) levels in the serum. In some situations of severe megaloblastic anemias, patients may present with pancytopenia. One of the most commonly encountered clinical problems is decreased intrinsic factor production by stomach parietal cells which is required for adequate vitamin B12 absorption. This autoimmune disorder, called pernicious anemia, is associated with chronic atrophic gastritis and autoantibody production. Autoantibodies specifically directed against intrinsic factor and parietal cells are commonly detected in patients with pernicious anemia. As parietal cells are also responsible for the acid secretion of the stomach, anti-parietal cell antibodies and atrophic gastritis also create achlorhydria (deficient acid output by the stomach). This interferes with the absorption of iron. Therefore, the association of vitamin B12 deficiency and iron deficiency is not uncommon. Pernicious anemia has a genetic predisposition and is more commonly associated with human leukocyte antigen (HLA) types A2, A3 and B7 and type A blood group. The disease is called pernicious anemia because it was fatal prior to the discovery that liver therapy caused improvement and the vitamin deficiency was identified. Today, patients on appropriate vitamin B12 treatment replacement have a normal lifespan. The patient in this case study had a normal serum folate value. However, the reader should remember that the measurement of RBC folate is much more sensitive for the diagnosis of folate related megaloblastic 8

8 anemia and is the test of choice. Serum folate may be falsely elevated in hemolysis and also affected by a recent ingestion. The measurement of methylmalonic acid was popular for the early diagnosis of vitamin B12 deficiency many years ago and is now re-emerging as a useful test. Methylmalonic acid is normal in folate deficiency but may be abnormal before macrocytosis or anemia develop in cobalamin deficiency. The high thyroid stimulating hormone (TSH) in this case suggests a concurrent hypothyroid state. Patients with one autoimmune disorder are more likely to develop another one. Patients with autoimmune thyroiditis have a high prevalence of vitamin B12 deficiency and particularly pernicious anemia. In summary, this is a case of combined iron deficiency and vitamin B12 deficiency anemia. This association has been recognized for many decades. Laboratorians play a key role as they provide the alerting clues for its recognition based on the peripheral blood findings. References: 1. Perkins S. Hypochromic, Microcytic Anemias. Carl R. Kjeldsberg, ed. Practical Diagnosis of Hematologic Disorders, 4 th ed, Foucar K. Megaloblastic Anemias. Carl R. Kjeldsberg, ed. Practical Diagnosis of Hematologic Disorders, 4 th ed, Glassy E ed. Color Atlas of Hematology. An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists: Cook J. Diagnosis and management of iron deficiency anaemia. Best Practice and Research, Clin Haematology. Vol 18: , Bain B. Diagnosis from the Blood Smear NEJM. Vol 353: , Carmel R, Weiner J, Johnson C. Iron Deficiency Occurs Frequently in Patients with Pernicious Anemia. JAMA , Ness-Abramof R, Nabrinski D, Braverman L, Shilo L, et al. Prevalence and evaluation of B12 deficiency in patients with autoimmune thyroid disease. Am J Med Sci. 332: , Hershko C, Ronson A, Souroujon M, et al. Variable hematologic presentation of autoimmune gastritis: age related progression from iron deficiency to cobalamin depletion. Blood. Vol 107: , McPherson R and Pincus M eds. Henry s Clinical Diagnosis and Management by Laboratory Methods, 21 st ed. Saunders Elsevier, William Koss, MD Hematology and Clinical Microscopy Resource Committee 9

9 Blood Cell Identification Ungraded Case History The patient is a 51-year-old female with a 3-month history of fatigue. Laboratory data include: WBC=5.5 X 10 9 /L; RBC=3.19 X /L; Hgb=10.3 g/dl; Hct=29.6%; MCV=92.7 fl; MCH=32.3 pg; MCHC=34.8 g/dl; RDW=19.8; PLT=505 X 10 9 /L; /-26 Lymphoblast Educational Blast cell Educational Lymphocyte Educational Myelocyte Lymphoma cell (malignant) Educational Monocyte, immature Lymphocyte reactive (to include plasmacytoid and immunoblastic forms) Educational This cell is a lymphoblast. Lymphoblasts can be variable in size, even in the same patient. They range from μm, have an increased nuclear:cytoplasmic (N:C) ratio and fine nuclear chromatin. The amount of nuclear membrane irregularity is also variable. The lymphoblast depicted in this slide has a high N:C ratio, fine nuclear chromatin, slight nuclear membrane irregularity and absent nucleoli. These are all features of lymphoblast as typically seen in acute lymphoblastic leukemia. Distinguishing lymphoblasts from myeloblasts is often difficult and may be impossible on a morphologic basis alone. Features favoring lymphoblasts are a high N:C ratio, slightly small cell size, either no or fine cytoplasmic azurophilic granules, and no Auer Rods. Pending cytochemical stains, immunohistochemical stains or cell surface marker studies, classifying these cells simply as blasts is acceptable practice. 10

10 Blood Cell Identification Ungraded /-27 Blast cell Educational Lymphoma cell (malignant) Educational Monocyte, immature Educational (promonocyte, monoblast) Lymphocyte reactive (to Educational include plasmacytoid and immunoblastic forms) Lymphoblast Educational Lymphocyte Monocyte Educational Prolymphocyte Educational This cell is also a lymphoblast. This lymphoblast is larger than the one depicted in the previous slide. In addition, there is a nuclear cleave and a small nucleolus. The two blasts (/-26 and 27) demonstrate the variable cell size and morphologic features that can be seen in a patient with acute lymphoblastic leukemia. This larger blast can be especially difficult to distinguish from a myeloblast. Thus, pending additional studies, classification as a blast is acceptable. 11

11 Blood Cell Identification Ungraded /-28 Lymphocyte Educational Lymphocyte reactive (to Educational include plasmacytoid and immunoblastic forms) Neutrophil with dysplastic Educational nucleus and/or agranular cytoplasm Lymphocyte, large granular Educational Neutrophil, metamyelocyte Educational Neutrophil, myelocyte Educational Monocyte Educational The arrowed cell was a lymphocyte. Lymphocytes are small, 7-15 μm, with round to oval nuclei and a variable amount of cytoplasm. Most small lymphocytes have scant cytoplasm, while medium to large lymphocytes have a more moderate amount of pale blue, agranular cytoplasm. Nucleoli, if present, are small and inconspicuous. The dense nuclear chromatin pattern, the low N:C ratio and lack of nucleoli help to distinguish lymphocytes from lymphoblasts. 12

12 Blood Cell Identification Ungraded /-29 Eosinophil Educational Basophil Educational Myelocyte Educational Promyelocyte Educational The arrowed cell is an eosinophil. Eosinophils are round leukocytes with characteristic coarse, orangered uniform cytoplasmic granules. About 80.0% of eosinophils have a classic bilobed nucleus. Threelobed eosinophils constitute most of the remaining eosinophils in the peripheral blood. The eosinophil in this photo is at the myelocyte stage, with single nuclear lobe and numerous cytoplasmic granules. 13

13 Blood Cell Identification Ungraded /-30 Plasma cell, mature Educational Lymphocyte reactive (to Educational include plasmacytoid and immunoblastic forms) nrbc, megaloblastic Educational Plasma cell, abnormal (malignant, myeloma cell) Educational The arrowed cell is a plasma cell. While plasma cells are normally seen in the bone marrow, lymph nodes and spleen, plasma cells in the peripheral blood are an uncommon finding. The morphologic features of peripheral blood and bone marrow plasma cells are the same, with a round to ovoid, eccentrically placed nucleus, coarse, clumped nuclear chromatin and no nucleoli. The cytoplasm is gray-blue to deeply basophilic with a prominent paranuclear pale zone (hof area). Plasma cells in the blood may be seen in patients with bacterial infections, viral infections, protozoal infections, chronic liver disease, autoimmune and collagen vascular disorders, various malignancies, drug and toxin exposure, hypersensitivity reactions and serum sickness. 14

14 Discussion This 51-year-old woman has acute lymphoblastic leukemia (ALL), which was diagnosed using a combination of morphologic, cell surface marker, cytogenetics and clinical findings. Acute leukemia is the most common type of cancer seen in the pediatric population with approximately 2000 new cases diagnosed each year. Leukemia and lymphoma combine to account for about 40% of new malignant diagnoses in children each year. Acute lymphoblastic leukemia is a rare malignancy in adults and is seen primarily in those greater than 60 years of age. Adult ALL patients are less likely to have fever, infection, bleeding, peripheral lymphadenopathy, a mediastinal mass or splenomegaly, when compared to children with ALL. Hepatomegaly and central nervous system involvement is similar in both populations, as are the presenting WBC, hemoglobin and platelet counts. Examination of a peripheral blood smear reveals lymphoblasts, which have fine nuclear chromatin, a high N-C ratio, scant to moderate cytoplasm and smooth or irregular nuclear membranes. Bone marrow examination is the next step in confirming the diagnosis of ALL. Bone marrow aspirate material is submitted for cell surface marker studies to determine phenotype, for cytogenetics analysis and possibly for special studies. Utilizing the WHO Classification system, ALL is classified either as Precursor B- ALL or Precursor T-ALL. The majority of both pediatric and adult ALL is of precursor B-type. In adult ALL patients with successful cytogenetics analysis, clonal abnormalities are identified in many of the cases. Philadelphia (Ph) chromosome positive ALL is seen much more frequently in adult ALL, whereas it is quite rare in the pediatric population. Significant prognostic factors in adult ALL include patient age, white blood count, immunophenotype, cytogenetics abnormalities, treatment response and minimal residual disease. Patients over 30 years of age, those with a WBC >30,000/uL, pro-b phenotype, t(4:11) or t(9;22) cytogenetic abnormalities, poor response to chemotherapy and positive minimal residual disease studies are all poor prognostic factors. At the time of diagnosis of ALL, the leukemic prognostic biologic factors along with the clinical characteristics of the patient are used to stratify the patient and determine a treatment program. Following induction chemotherapy additional modifications in the treatment program may be made based on responsiveness to therapy, toxicity and minimal residual disease. Many new agents are being explored as overall adult ALL has a poor prognosis with a survival in the range of 35% at 5 years. References: 1. Schultz KR, Pullen DJ, Sather HN, et al. Risk- and response-based classification of childhood B- precursor acute lymphoblastic leukemia: a combined analysis of prognostic markers from the Pediatric Oncology Group (POG) and Children s Cancer Group (CCG). Blood. 1007;109: Larson RA. Acute Lymphoblastic Leukemia: Older Patients and Newer Drugs. Am Society of Hem Education Book. 2005: Gokbuget N and Hoelzer D. Treatment of Adult Lymphoblastic Leukemia. Am Society of Hem Education Book. 2006: Deborah A. Perry, MD Hematology and Clinical Microscopy Resource Committee 15

15 Actions Laboratories Should Take when a PT Result is Not Graded The College uses Exception Reason Codes for the proficiency testing (PT) analyte that has not been graded. The Exception Reason Code is located on the evaluation report in brackets to the right of the result. Identify all of the analytes with an Exception Reason Code and investigate the acceptability of performance with the same rigor as if it were an unacceptable performance. The actions accredited laboratories should take include but are not limited to: Codes Exception Reason Code Description Action Required 11 Unable to analyze. Document why the specimens were not analyzed (e.g., instrument not functioning or reagents not available). Perform and document alternative assessment (i.e., split samples) for the period that commercial PT was not tested to the same level and extent that would have been tested. 20 No appropriate target/response; cannot be graded. Document that the laboratory performed a self-evaluation using the allinstrument data presented in the Participant Summary. Perform and document this review. 21 Specimen problem. Document that the laboratory has reviewed the proper peer group statistics supplied by the PT Provider. Perform and document alternative assessment for the period that commercial PT was not tested to the same level and extent that would have been tested. 22 Result is outside the method/ instrument reportable range. Document the comparison of results to the proper peer group statistics and peer group information supplied by the PT Provider. Verify detection limits. 24 Incorrect response due to failure to provide a valid response code. Document the laboratory s self-evaluation against the proper peer group statistics supplied by the PT Provider. Perform and document the corrective action of any unacceptable results. Document corrective action to prevent future failures. 25 Inappropriate use of antimicrobial. Document the investigation of the result as if they were unacceptable and review the proper reference documents to gain knowledge of the reason your response is not appropriate. 26 Educational challenge. Response to the CAP is not required. Laboratory should document its review. 27,31 Lack of participant or referee consensus. Document that the laboratory compared its results to the modal (most common) result. Perform and document this review. 28 Response qualified with a greater than or less than sign; unable to quantitate. Document the laboratory s self-evaluation against the proper peer group statistics supplied by the PT Provider. Verify detection limits. 30 Scientific Committee decision. Document that the laboratory has reviewed the proper peer group statistics supplied by the PT Provider. 33 Specimen determined to be unsatisfactory after contacting the CAP. 40 Results for this kit were not received. 41 Results for this kit were received past the evaluation cut-off date. 42 No credit assigned due to absence of response. 44 This drug is not included in our test menu. Use of this code counts as a correct response. 77 Improper use of the exception code for this mailing. 91 There was an insufficient number of contributing challenges to establish a composite grade. *35, Various codes. 43, 88, 92 Document that the laboratory has contacted the CAP and no replacements specimens were available. Perform and document alternative assessment (i.e., split samples) for the period that commercial PT was not tested to the same level and extent that would have been tested. Document why results were not received, corrective action to prevent recurrence and the laboratory s self-evaluation of the results by comparing results to the proper peer group statistics supplied by the PT Provider. The Participant Summary booklet mailed with the proficiency testing evaluation indicates which tests are graded (see evaluation criteria) and which tests are Not Evaluated/Educational. Updates to grading will also be noted. If a test is educational, the laboratory is not penalized for leaving a result(s) blank. The code 42 that appears on the evaluation is not a penalty. However, if a test is graded (regulated and non-regulated analytes) and your laboratory performs that test, results cannot be left blank. The laboratory is required to submit results for all challenges within that test or use an appropriate exception code or indicate test not performed/not applicable/not indicated. Exceptions may be noted in the Kit Instructions and/or the Result Form. Document corrective actions to prevent future failures. Verify that the drug is not tested on patient samples and document to ensure proper future reporting. Document the identification of the correct code to use for future mailings. Document the investigation of the result as if it were an unacceptable result. Perform and document the corrective action if required. No action required by the LAP. 16 Rev 7/2007