Hematology Case 4: Reactive Leukocytosis The patient is a 55-year-old white male who presents to the emergency room complaining of chest pain and labored breathing. His temperature is 98.6° F. He is overweight and smokes two packs of cigarettes a day. His EKG and cardiac enzyme profile reveal changes compatible with an acute myocardial infarction. The complete blood count (CBC) shows: Hemoglobin (Hb) 13 gm/dl (reference range 12 - 15.2 gm/dL) Total white blood cell 15,500/mm3 Platelets (Plt) 250,000/mm3 A differential white cell count was included in the CBC report, showing 80% segmented neutrophils (polymorphonuclear neutrophils), 10% "band" neutrophils, 7% lymphocytes, and 3% atypical lymphocytes. Based on the reported neutrophilic leukocytosis and "bandemia," an extensive infectious disease work-up was initiated. This decision was confusing so you went to the laboratory and performed a 100-cell differential (white cell) count of the peripheral blood smear. The numbers you obtained basically agree with the original report except that you counted only 6% "bands" and you identified a myelocyte and a basophil. 1.Peripheral Blood Smear, 4x 2.Peripheral Blood Smear, 10x 3.Peripheral Blood Smear, 100x 3a.Segmented Neutrophil 3b."Band" Neutrophil 1. Leukocytosis - Peripheral Blood A low magnification view (4x) of the peripheral blood smear permits you to estimate the total white blood cell count (TWBC), which in this case was reported to be 15 x10^3/ml. [Note: a rule of thumb for assessing the TWBC: 15 to 20 WBC/4x field correlates with a TWBC of approximately 15 - 20x10^3/ml]. At a slightly higher magnification (10x), review of the smear now reveals that most of the leukocytes are granulocytes, specifically neutrophils. A superficial examination of these neutrophils reveals that some are fully segmented while others are "band" forms. A higher magnification (100x) reveals a segmented neutrophil demonstrating the typical thin chromatin strand connecting the lobes (segmented neutrophil -3a). In addition, the smear shows a typical "band" form with a horseshoe nucleus and no thin chromatin strands ("band" neutrophil -3b). Although the distinction between these two cells is clear in this picture, such is not always the case. 1. What findings in the CBC report prompted an infectious disease work-up? Neutrophilic leukocytosis and "bandemia" (i.e., a "left shift"). 2. Compare and contrast neutrophilic leukocytosis, leukemoid reaction, chronic myelogenous leukemia, and acute myeloblastic leukemia. Neutrophilic leukocytosis refers to an increase in the number of neutrophils in the peripheral blood. This may be caused by pyogenic infections or by non-microbial stimuli, such as tissue destruction. The neutrophil increase results primarily from release of mostly mature neutrophils from the marrow storage pool, and hence neutrophil precursors are rare in the peripheral blood. Leukemoid reaction refers to an extreme form of reactive neutrophilic leukocytosis in which the peripheral blood contains not only mature neutrophils but also immature leukocytes, resembling leukemia. However, in leukemoid reactions, there are fewer immature myeloid cells (myeloblasts are rare), leukocyte alkaline phosphatase is normal or increased, and cytogenetic abnormalities are absent. Acute and chronic myelogenous leukemias are clonal neoplastic disorders that are discussed later. Basophil (a) Neutrophil (b) Eosinophil (c) 2. Circulating Granulocytes The three frames in this image (100x) illustrate the different granulocytes that are seen commonly in the peripheral blood. Granulocytes are leukocytes, or white blood cells, that contain cytoplasmic granules. Frame (a) demonstrates a basophil with the obvious large basophilic granules typical of these granulocytes. The nuclear segmentation of these cells is often difficult to appreciate due to the heavy cytoplasmic granulation. Basophils are the least common of the granulocytes in the peripheral blood. Basophilia -- or increased number of basophils -- is frequently noted in myeloproliferative disorders. The most abundant of the circulating granulocytes is the neutrophil (b). The specific granules of these cells are not readily identified with the light microscope. An increase in their number (neutrophilia) may be seen in a variety of conditions, including infections or tissue necrosis (as in myocardial injury). The large red granules of eosinophils (c) are readily apparent in this picture. Eosinophilia, an increase in eosinophils, will be presented in the next image. Examples of Eosinophils Lymphocyte 3. Eosinophilia - Peripheral Blood This slide (100x) demonstrates a leukocytosis composed predominantly of eosinophils, i.e., eosinophilia. What are some examples of non-neoplastic eosinophilia? Eosinophilia may be seen in allergic reactions (including asthma, drug reaction), parasitic infestation, and skin diseases. Nucleus Cytoplasm 4. Lymphocytes - Peripheral Blood In the previous image, the lymphocyte manifests the cytologic features of a normal circulating lymphocyte: small size (about the size of a red cell), high nuclear-to-cytoplasmic ratio (N/C) and condensed nuclear chromatin. These small lymphocytes constitute about 90% to 95% of circulating lymphocytes. However, as the cells above demonstrate, larger lymphocytes may also be seen in the peripheral blood of normal individuals. These larger lymphocytes are given the designation "atypical lymphocytes" -- to distinguish them from "typical" small circulating lymphocytes -- and make up less than 5% of lymphocytes in circulation. One "atypical" characteristic is the frequent presence of nucleoli. Another "atypical" feature is the presence of abundant blue cytoplasm. This blue hue sometimes is noted only at the periphery of the cytoplasm. Lymphocytosis is found in viral syndromes, such as infectious mononucleosis, although it is not diagnostic of such infections. Frequently in these viral syndromes there are an increased number of atypical lymphocytes. The circulating atypical lymphocytes in these viral syndromes are T cells that have been transformed (or activated) in response to EBV-infected B cells in infectious mononucleosis. Are these activated lymphocytes mature or immature (i.e., blasts)? These activated T cells may resemble immature (primitive) cells, but they are in reality progeny of mature, resting lymphocytes. Atypical Lymphocytes Monocytes 5. Atypical Lymphocytes vs. Monocytes These pictures (100x) depict the cytologic differences between atypical lymphocytes (a & c) and monocytes (b). As can be appreciated from these pictures, the differentiation is at times difficult. However, the cytoplasm of monocytes usually is gray and frequently contains vacuoles (not demonstrated in this example), while the cytoplasm of atypical lymphocytes reveals the bluish tinge previously described. Lymphocytes, typical or atypical, can be differentiated by the expression of surface antigens. Monocytes frequently express CD14, while T cells lack this antigen but express CD3, CD4, CD5, CD7, CD8. B cells do not express any of these CD antigens; instead, they often have CD19, CD20, and CD22. What trends can you see regarding CD numbers and lymphocytes (T and B cells)? CD digits less than CD 10 frequently are associated with T cells. CD digits around CD 20 frequently are associated with B cells. Leukemic Blasts (Lymphoblasts) Atypical Lymphocyte 6. Lymphoblasts vs. Atypical Lymphocytes The differentiation between atypical lymphocytes and leukemic blasts may, on occasion, be quite difficult; however, as the two pictures (100x) in this image demonstrate, there are significant morphologic differences. Blasts often have a very high N/C (nuclear to cytoplasmic) ratio and stippled chromatin pattern. Atypical lymphocytes, as a rule, have abundant cytoplasm and coarser chromatin. The clinical and hematologic changes associated with these cases are helpful in this differentiation as well. Leukemic blasts, as seen in acute leukemia, tend to infiltrate the marrow and suppress the production of normal hematopoietic elements. Myeloblast Promyelocyte Myelocyte Metamyelocyte Band Neutrophil Segmented Neutrophil 7. Maturation of Neutrophils - Bone Marrow The different cells seen in the maturation sequence of the neutrophil are identified (100x), from a myeloblast to a segmented neutrophil. The overall maturation scheme of the neutrophilic line consists of nuclear shrinkage and segmentation with concurrent condensation of the chromatin. This process begins with the formation of a functionally immature myeloblast and culminates with the development of a phagocytic segmented neutrophil. This maturation takes about 15 days and occurs in the bone marrow. About half of this time is spent in the so-called "mitotic" compartment. The cells of this compartment -- myeloblast, promyelocyte, myelocyte -- multiply (i.e., are mitotically active) and undergo maturation. In the last stage of the mitotic compartment, the myelocyte begins to replace its primary (nonspecific) granules with secondary (specific) granules. The final three cellular stages of maturation comprise the so-called "storage" compartment. Cells in this pool consist of metamyelocytes, band neutrophils, and segmented neutrophils or "polys" (polymorphonuclear neutrophils). When are these cells released into the blood? These cells remain in the marrow until the segmented forms are preferentially released into the peripheral blood. Kidney Case 1: Nephrotic Syndrome: A previously healthy 35-year-old man, complained of recent fatigue, and swelling of his feet and ankles. He was also noted to have puffy eyes as well. Physical exam revealed an afebrile man, with lower extremity edema, extending to the knee, periorbital edema, and a small amount of ascites. Laboratory features were as follows: Creatinine 0.8 mg/dL Urinalysis: protein - 4+ BUN 18 mg/dL glucose - neg Albumin 2.8 g/dL blood - neg Hematocrit 40% bilirubin - neg Micro: rare RBCs, no WBCs, many oval fat Liver function tests normal bodies Triglycerides 400 mg/dL Cholesterol 375 mg/dL 24 hr urine 11.2 gm/24 protein hrs Drug history was negative. ANA, HIV, and hepatitis serology were negative. The patient developed right flank pain the following day. Ultrasound revealed renal vein thrombosis. Renal biopsy was performed. Capillary Loops Mesangial Areas 1. Nephrotic Syndrome, Renal Biopsy - Silver Stain This is a representative glomerulus from the patient's biopsy. The capillary loops show subtle, uniform thickening. The capillary loops are open (no inflammation) and the mesangial areas and cellularity are normal. (See image 2, high power.) Capillary Lumen Urinary Space GBM "Spikes" Epithelial Cell Endothelial Cell Mesangial Cells and Matrix 2. Nephrotic Syndrome, Renal Biopsy - Silver Stain Glomerular basement membranes (GBM's) and spikes stain black, while immune complex deposits do not stain. Notice that the "spikes" are a part of GBM. The immune complexes are situated in- between the spikes. Is there any inflammation seen? No inflammation is seen. What would the IF pattern look like? IF pattern would be granular and peripheral, which correlates with the immune complex deposits between spikes. What is the clinical syndrome that this patient presented with? This patient presented with nephrotic syndrome. He had marked albuminuria, low serum albumin, and consequent generalized edema. Hyperlipidemia is also often seen in patients with nephrotic syndrome. This gives rise to lipiduria, seen as oval fat bodies in the urine. Note the absence of red cells. Capillary lumens GBM 3. Nephrotic Syndrome, Renal Biopsy - Immunofluorescence With Anti-IgG This is a direct immunofluorescence preparation of the patient's biopsy using antisera against IgG. Note two features: A. Where is the staining? On the GBM. B. What kind of pattern is seen? Granular. This pattern corresponds to the immune complex deposition. It is the immune complexes that are stained. Capillary Lumen Urinary Space Dense Deposits GBM "Spikes" GBM Podocytes Schematic of EM 4. Nephrotic Syndrome - Electron Micrograph This is an electron micrograph of a glomerulus from the patient. Correlate this EM pattern with the previous IF Pattern. In what area are the deposits located? The deposits are located in the outer aspect of the basement membrane under the visceral epithelium of the Bowman's capsule. Thus, they are subepithelial. Are the spikes part of the deposits? No. The spikes are part of the GBM that lies between the deposits. Based on the results of the light microscopy, immunofluorescence, and electron microscopy of the renal biopsy, what is the patient's diagnosis? The biopsy reveals a membranous glomerulopathy, with subepithelial deposits of electron dense, immune complex type material. By light microscopy, this is illustrated by lack of increased glomerular cellularity, and uniformly thickened GBMs. A special stain (silver) shows "spikes" of GBM material projecting between the deposits, a feature better understood on the EM photo. Immunofluorescence shows the typical granular, peripheral capillary loop staining with IgG and C3, typical of membranous GN. What is the purpose of the additional tests the clinician ordered after the diagnosis was made? After the diagnosis of nephrotic syndrome was made, the clinician did serological tests to rule out some secondary causes of membranous glomerulonephritis that account for about 40% of cases. This is important, because they can in many cases be treated. These include SLE, hepatitis B, malaria, gold and penicillamine for rheumatoid arthritis, Captopril (an angiotensin-converting enzyme inhibitor), and occult carcinoma. Capillary Lumen Podocyte 5. Nephrotic Syndrome, Minimal Change Disease (MCD) - Silver Stain The glomerulus shows normal mesangial cellularity, with normal open tufts. The basement membrane is of uniform thickness. No inflammation is seen. The tubules are of normal shape and size. The interstitium is also unremarkable. MCD is the most common cause of nephrotic syndrome in children. Could MCD involve adults? Although the peak incidence is between two and six years of age, adults may also be affected. Compare this image with membranous GN (Images 1and 2). What does the IF look like in MCD? It is normal. Tubular "Lipoid" Cells 6. Nephrotic Syndrome, Minimal Change Disease (MCD)- Trichrome Stain Interstitial and tubular foam "lipoid" cells are seen in MCD and other conditions associated with nephrotic syndrome, hyperlipidemia, and hyperlipiduria. What are the general differences in the etiology of nephrotic syndrome in children vs. adults? In children primary renal disease accounts for 95% of the cases, the most common cause being minimal change disease. By comparison primary renal diseases are responsible for 60% of cases in adults; membranous glomerulonephritis is the most common primary renal cause. Podocytes Minimal Change Disease, Higher Power Basement Membrane Fused Foot Processes Schematic of EM-Minimal change Normal EM Podocytes Basement Membrane Endothelial Cell Nucleus Podocyte 7. Minimal Change Disease - Electron Micrograph In MCD, the EM shows effacement of the foot processes. Is there anything abnormal about the basement membranes of the capillary loops? No. The only abnormality is foot process effacement. Are there any immune deposits? No deposits are seen in MCD. What is the pathogenesis of MCD? This is not fully understood. There is no evidence of anti GBM antibodies or immune complexes. There seems to be some form of primary injury to glomerular epithelial cells that effaces foot processes and causes secondary alterations in GBM. Early Segmental Sclerosis High Power View Intracapillary Foam Cells Epithelial Cell Hyperplasia 8. Focal Segmental Glomerulosclerosis (FSGS), Early Lesion -Trichrome Stain FSGS is one of the common causes of nephrotic syndrome. Notice the collapse of capillary loops in a portion of a single glomerulus (segmental capillary collapse), accompanied by intracapillary foam cells and extracapillary epithelial cell proliferation. In a case of FSGS, what would the IF show in this segmentally sclerotic focus? IF will be positive in the sclerotic focus, and will stain with IgM and C3. in a non-sclerotic part? IF is negative in the nonsclerotic part. Is FSGS the result of immune complex injury? No. The basic lesion in primary FSGS is disruption of visceral epithelial cells by unknown mechanisms. The sclerosis and entrapment of plasma protein, including IgM and C3, are non specific. They result from hyperpermeability at sites of epithelial cell damage. Normal Looking Glomeruli Focal Sclerosis Interstitial Inflammation High Power View Segmental Sclerosis Atrophic Tubules 9. Focal Segmental Glomerulosclerosis (FSGS), Late Lesion -Trichrome Stain In a later stage of FSGS dense glomerular sclerosis is seen, with more interstitial fibrosis, tubular atrophy, and chronic inflammation. Is FSGS a descriptive term, a clinicopathologic entity, or both? FSGS is a descriptive term that implies sclerosis of some, but not all, glomeruli (i.e., focal), and in the affected glomerulus only a portion of the capillary tuft is involved (i.e., segmental). Such changes may occur in glomerular diseases secondary to HIV infection or heroin abuse; these may also occur in IgA nephropathy. In addition, idiopathic FSGS is a distinct clinicopathologic entity. What is the difference in the clinical presentation and prognosis of minimal change disease vs FSGS in children? Both FSGS and minimal change disease present with nephrotic syndrome, (the latter is more common). It is essential to distinguish them because their course and prognosis are different. FSGS is less steroid responsive and, unlike the benign course in minimal change disease, 50% of cases with FSGS end up with renal failure in 10 years. Lobular Pattern 10. Membranoproliferative Glomerulonephritis (MPGN) - H&E MPGN is another relatively common primary renal cause of nephrotic syndrome in both children and adults. Notice the lobular pattern, the hypercellularity, and the collapse of the capillaries. What is proliferating in MPGN? Mesangial cells, monocytes, and lymphocytes. What is another name for MPGN? Mesangiocapillary GN. "Tram-Track" Capillary Loops 11. Membranoproliferative Glomerulonephritis (MPGN), Later Stage - Silver Stain At a later stage of MPGN, many of the capillary loops show a double contour, or "tram-track," appearance. What causes the double contour appearance? The double contour appearance is caused by an apparent "splitting" of the basement membrane. This split appearance results from the intercalation of mesangial cell processes into the peripheral capillary loops. What do you expect to see in the IF? IF would show granular and peripheral patterns with/without mesangial pattern. Are there any spikes in MPGN? (Compare with images 1 and 2.) No spikes are seen in MPGN. Mesangial Staining Peripheral Staining 12. Membranoproliferative Glomerulonephritis - Immunofluorescence for C3 The pattern here is mesangial and peripheral. Compare with the IF of membranous GN. Could this pattern be seen in SLE? Yes, because in SLE the immune complexes can deposit in the mesangium or along the basement membrane. What are the types of MPGN? There are two types of primary MPGN. Type I, the more common form is an immune complex disease and shows granular pattern of IgG and C3 deposition by IF. The immune complexes are subendothelial by EM. Type II, the less common form has C3 but not IgG in a granular-linear pattern on the basement membrane and as aggregates in the mesangium. The two are distinguished best by electron microscopy Electron Dense Deposits Endothelial Cell Podocyte Schematic of EM 13. Membranoproliferative Glomerulonephritis - Electron Micrograph Notice where the deposits are. What cell makes the double track membrane? Extension of the mesangial cells. Name a systemic disease in which you see MPGN type disease. It can occur in association with SLE, hepatitis B or C antigenemia, and -1-antitrypsin deficiency. Hematology Case 2: Hemolytic Anemias The patient is a 6-year-old male who was seen by his pediatrician because of a low grade fever and sore throat. Mild yellow coloring of his sclera was noted by his mother two days previously. On physical examination, the child had mild jaundice. His spleen was moderately enlarged. Selected laboratory findings are shown below: Complete Blood Count (CBC) Hemoglobin 9.8 g/dL Reticulocyte count 14% Hematocrit 28% Leukocyte count 9,200/mm3 Erythrocyte count 3.4 x 106/mm3 Platelet count 375,000/mm3 MCV 84 µm3 Serum bilirubin Total 4.5 mg/dL Direct 0.4 mg/dL MCHC 35.0 g/dL RDW 20% Your microscopic examination of the peripheral blood smear reveals red cell changes as seen in Image 1. The child had not been known to have anemia previously. There was no history of bleeding. A paternal uncle had a splenectomy as a child for "low blood." A direct antiglobulin test (DAT) was negative. Blood was drawn for an osmotic fragility test. The following day both of the parents and the child returned to the pediatrician's office. A CBC and blood smear examination were performed on the parents. The father had a normal hemoglobin but an increased reticulocyte count; spherocytes were observed on his blood smear. His spleen was palpably enlarged. The mother's studies were normal. The osmotic fragility test on the child showed increased osmotic sensitivity, which was markedly accentuated after incubating the red cells in normal saline for 24 hrs. Peripheral Blood Smear, Patient Peripheral Blood Smear, Normal 1. Peripheral Blood Smear - Unknown Compare the peripheral blood smear obtained from the case's patient with the normal peripheral blood smear (Feature 2). Note the size and coloration of red cells (explanation on next image). Is this patient's anemia due to a production defect or due to hemolysis? Due to hemolysis. What laboratory tests are used to demonstrate hemolysis? Serum bilirubin, fecal urobilinogen, serum haptoglobin, chromium-labeling of RBCs (survival), and serum LDH. What is the cause of jaundice in such patients? Hyperbilirubinemia resulting from increased destruction of red cells. How are hemolytic anemias classified? Intrinsic (intracorpuscular) versus extrinsic (extracorpuscular). What is the diagnosis in this case? Hereditary spherocytosis. What is the inheritance pattern, red cell defect, mechanism of hemolysis, and treatment in this disorder? This disease is caused by a genetic defect (most commonly autosomal dominant) in the red cell membrane cytoskeleton. Abnormal red cells get trapped within the cords of the splenic red pulp and are phagocytosed by macrophages. Transfusions and/or splenectomy are sometimes necessary. Why was a DAT ordered? To exclude immune-mediated hemolysis. Spherocytes can also be seen in immune-hemolytic anemias. Polychromatophilic Red Cell Spherocytes Normal Red Cells Platelets 2. Hereditary Spherocytosis This image illustrates reticulocytes (largest cells with slightly greyish color), normal mature red cells (intermediate-sized cells with an area of central pallor), and spherocytes (smallest cells, which stain darkly and lack central pallor). Spherocytes result from membrane damage due to either a problem extrinsic to the red cells or an abnormality intrinsic to the red cells. What are the major categories of intrinsic red blood cell abnormalities? Intrinsic red blood cell abnormalities include membrane abnormalities, enzyme deficiencies, or hemoglobin abnormalities. Hereditary spherocytosis, which is the diagnosis in this case, is caused by a genetic defect in the red cell membrane. An abnormality of membrane skeletal proteins causes progressive loss of membrane, making the red cells less deformable. The red cells then get trapped within the cords of the splenic red pulp and are phagocytized by the macrophages. The process is an example of extravascular hemolysis. Spherocytes are not unique to hereditary spherocytosis and are seen in other hemolytic states, particularly in immune hemolysis. Polychromatophilic Erythrocytes Reticulocytes 3. Polychromasia/Reticulocytes The Wright stained slide, at left, demonstrates polychromatophilic red cells in the peripheral blood. (The term "polychromasia" refers to the bluish tinge of these cells.) These red cells have been recently released from the bone marrow and still contain significant amounts of RNA, which is the reason for the bluish tinge (remember, RNA stains blue with the Wright's stain). The right panel is a reticulocyte stain. In this procedure, RNA is precipitated inside the erythrocytes. Because precipitated RNA gives the reticulated appearance shown, the cell is called a reticulocyte. For all practical purposes, reticulocytes and polychromatophilic red cells represent red cells of similar age. These cells remain as reticulocytes in circulation for about 24 hours. Because these cells have been recently released from the marrow, it stands to reason that the number of reticulocytes would reflect the ability of the marrow to respond to the challenges of anemia. In other words, if a person is anemic and has an elevated reticulocyte count, the likely explanation for his anemia is hemolysis. That is to say, the red cells are being destroyed outside the bone marrow. What does an anemia with a low reticulocyte count signify? Anemia with a low reticulocyte response signifies a production problem within the marrow. Spleen (10x) White Pulp Red Pulp Spleen, Red Pulp (45x) Sinuses Cord 4. Spleen - Hereditary Spherocytosis The two slides in this image are from a section of spleen removed from a patient with hereditary spherocytosis. As stated in slide 2, the spleen is the site of destruction of red cells in this disorder. Why are red blood cells destroyed in this manner? The loss of membrane causes the red cells to lose their deformability and become unable to negotiate the splenic microvasculature. This low magnification (10x) picture of the spleen (1) permits review of the general microanatomy of the spleen. The red and white pulp are noted. It is in the red pulp that red cells are entrapped. A high magnification of the spleen (45x) (2) shows empty sinuses and trapping of red cells in the splenic cord. This is the typical finding in hereditary spherocytosis, reflecting the inability of the red cells in this disease to navigate the unique microvasculature of the spleen. Elliptocytes 5. Hereditary Elliptocytosis Hereditary elliptocytosis -- like hereditary spherocytosis -- is an example of a hemolytic state associated with an intrinsic red cell membrane defect. The red cells in this blood smear (100x) have an elliptical shape, which results from an abnormality in the red cell membrane protein skeleton. What are clinicopathologic aspects of this disorder? Most patients with this defect have normal or only a slightly shortened red cell survival time and no significant anemia. 1.Blood Smear 100x "Bite" Cell 2.Blood Smear, Heinz Body Preparation Heinz Bodies 6. G6PD Deficiency This hemolytic state is related to a different intrinsic red cell defect: specifically, an enzyme deficiency. This image (1) shows an abnormal red cell on a blood smear (100x) from a patient with G6PD deficiency who has been challenged by an oxidant stress. The configuration of this red cell has the appearance of an apple core and is sometimes referred to as a "bite" cell. Describe the pathophysiology of G6PD deficiency. Denatured hemoglobin precipitates in the form of Heinz bodies (Image 2). These precipitates are removed by splenic macrophages causing the configuration observed on this blood smear. These "pitted" cells are then prone to leak hemoglobin in circulation, which explains the hemoglobinuria -- a hallmark of intravascular hemolysis -- noted in these patients. Eventual removal of these red cells from the circulation by macrophages in the spleen -- indicating an extravascular hemolytic component -- contributes to the red cell destruction in these cases. (2) demonstrates a special Heinz body stain in a patient with a severe form of G6PD deficiency after an oxidant challenge. The Heinz bodies are the dark inclusions at the periphery of the red cell. (A special stain is required because the routine Wright stain used on blood smears does not stain Heinz bodies.) Spherocytes Polychromatophilic Red Cells 7. Immune Hemolysis - Warm Antibody Type An example of an extrinsic insult causing a hemolytic state is immune-mediated hemolysis. In this case, the blood smear reveals mainly spherocytes. Spherocytes in this condition are formed due to partial loss of the red cell membrane caused by attempted phagocytosis of Ig-coated red cells. The spherocytes are then removed in the spleen (i.e., extravascular hemolysis). So-called warm antibodies, which may be auto- or alloantibodies, are responsible. (The terms "cold" and "warm" antibodies relate to the temperature at which the antibody binds most avidly to the red cell antigen.) What are causes of warm antibody hemolytic anemias? Primary (idiopathic) or secondary (lymphomas, leukemias, cancers, autoimmune disorders, drugs). What is the isotype of the antibody in such cases? IgG. Red Cell Agglutination 8. Cold Agglutinin - Hemolytic Anemia The presence of a cold agglutinin antibody is another example of an extrinsic red cell insult resulting in an immune-mediated hemolysis. The blood smear in these cases often shows characteristic clumping of the red cells (as shown here). In contrast to warm antibodies described in the previous slide, cold agglutinin antibodies are always autoantibodies. The antibody involved is most often IgM. What is the cause of hemolysis in this process? The in vivo agglutination property of these antibodies is not the cause of the hemolysis; rather, their ability to fix complement on the red cell surface at low temperatures results in intravascular hemolysis when red cells move to warmer areas. In addition, at 37oC, IgM antibody is released from the cell surface, leaving a coating of C3b. This is an opsonin and such opsonized red cells are phagocytosed by monocyte-macrophages. Thus, there is extravascular hemolysis. Plasmodium Ring Forms Platelets Yet another example of an extrinsic insult which can result in a hemolytic anemia is the infestation of red cells by parasites. This blood smear, taken from a patient with Plasmodium falciparum malaria, shows two ring-form parasites in the red cell in the center of the field. This red cell will be destroyed as the parasites mature and are eventually released from the cell. What is the most common cause of infection-induced hemolytic anemia? Malaria. Hematology Case 3: Hemolytic Anemias The patient is a 15-year-old black male who presents to the emergency room (ER) complaining of severe abdominal pain. His mother informs you that these painful episodes began when he was about two years old. She also tells you that the patient, his brother, her husband, and she have been told they have the "sickle gene." However, only her husband has suffered similar painful episodes ("pain crises"). Physical examination demonstrates a soft abdomen and intact bowel sounds. The patient's pulse is rapid with a blood pressure of 125/90. His temperature is 98.8 F. Complete blood count (CBC) demonstrates: Hemoglobin 8 gm/dl (reference range 12 - 15.2 gm/dl WBC count 13,500/mm3 Platelets 350,000/mm3 Your microscopic examination of the peripheral blood smear reveals red cell changes as seen in Image 1; in addition, you note that there is significant polychromasia. Most of the white cells are neutrophils (segmented forms and bands). 1.Peripheral Blood Smear, Patient 2.Peripheral Blood Smear, Normal 1. Peripheral Blood Smear, Unknown Compare the peripheral blood smear obtained from the patient (1) with a normal peripheral blood smear (2). How are the morphologic abnormalities in the red cells seen here related to polychromasia and this patient's abdominal pain? Red cells in this smear are sickled and are therefore prone to undergo hemolysis in the spleen; in addition, patients with sickle cell anemia have episodes of vascular occlusion. The hemolysis causes reticulocytosis and polychromasia. The vascular occlusions cause painful episodes (crises). What is the pathophysiology of vaso- occlusive crises in these patients? The pathophysiology of vascular occlusions and resulting organ damage is not fully understood. It is thought that sickle Hb causes red cell membrane damage. Such cells, even if not sickled, have increased stickiness to endothelium and tend to retard blood flow in the microcirculation. In the sluggish blood flow, there is greater deoxygenation of HbS, and, hence, sickling is facilitated. The abnormal cells thus accumulate and obstruct blood flow. Why did this patient have no noticeable clinical problems before the age of two? HbF present in red cells during infancy inhibits polymerization of HbS. Why don't his mother and brother have abdominal crises but his father does? His mother and brother have HbAS, i.e., they have sickle cell trait. What is the correlation between the patient's symptoms and the number of irreversibly sickled cells (ISC)? Why? ISC number correlates with hemolysis, because irreversibly sickled cells undergo hemolysis in the spleen. How is the severity of clinical manifestations affected by the red cell concentration of hemoglobin S and by the presence of other types of hemoglobins? Many factors affect the severity of sickle cell disease, including intracorpuscular concentration of HbS. Homozygotes with HbSS have 100% HbS and full blown sickle cell disease; heterozygotes with HbSA have a milder disease called sickle cell trait. Other Hb's (A, F, C, etc.) inhibit polymerization of HbS. Hgb "C" Crystallized Hgb "C" Target Cells Hgb "S" Irreversibly Sickled Cells 2. Hemoglobinopathies - "C" and "S" The cases illustrated in these frames are examples of hemolytic states caused by a type of intrinsic red cell defect: specifically, hemoglobin abnormalities. The peripheral smear (100x) on the left is from a patient with Hemoglobin C disease. It shows many "target cells" characterized by a peripheral ring and central deposition of hemoglobin giving them the appearance of a "target" with a bull's-eye. Another abnormal cell, which stains deep red and has a rectangular shape, is the result of crystallization of hemoglobin C. Homozygous hemoglobin C or Hemoglobin C disease is usually associated with mild to moderate chronic anemia. The peripheral smear (100x) on the right is from a patient with sickle cell anemia. It shows several irreversibly sickled cells (ISC). How are ISC formed? ISC are the result of permanent membrane damage related to multiple bouts of polymerization-depolymerization of intracellular hemoglobin S. All the cells shown in this picture contain hemoglobin S and are prone to sickling; however, only those that have developed irreversible membrane damage (ISC) appear as sickled cells in the smear. Hb A Hb F Hb S Hb C Sickle Cell Trait Sickle Cell Disease Hemoglobin C Disease 3. Hemoglobin Electrophoresis on Agarose Gel Hemoglobin electrophoresis is a commonly used laboratory test for qualitative evaluation of hemoglobinopathies. Hemoglobin molecules in an alkaline solution have a net negative charge and move toward the anode in an electrophoretic system. Differences in the charge of various types of hemoglobin results in different mobilities, causing their separation. Lanes 1 and 2 are control lanes. Lane 1 has adult (HbA) and fetal (HbF) hemoglobins, whereas Lane 2 has HbA, sickle hemoglobin (HbS) and hemoglobin C (HbC). Lanes 5 and 7 are normal individuals containing only HbA. Lanes 3 and 8 are from patients with sickle cell trait showing both HbA and HbS. Lane 6 is a case of sickle cell disease with total absence of HbA; HbS is the predominant hemoglobin, with HbF making up the rest. Lane 4 is an individual with Hemoglobin C disease who has only HbC. What do you think is the diagnosis of the patient in lane 9? HbSC. Examples of Red Cell Precursors 4. Bone Marrow Changes in Hemolysis The marrow biopsy (left) demonstrates a hypercellular marrow; the marrow aspirate (right) shows predominantly a normoblastic proliferation, i.e., erythroid hyperplasia. This erythroid hyperplasia is the expected finding in a hemolytic state. (Note: Although in this example the marrow biopsy permits identification of the proliferating cells as normoblasts, this is not always the case). Gastrointestinal Case 3: Inflammatory Bowel Disease R.N. is a 56-year-old white male with a history of inflammatory bowel disease of 20 years duration and migratory polyarthritis. Three months prior to admission he experienced an exacerbation with a 40 pound weight loss and bloody diarrhea. He was admitted to the hospital with complaints of 20 bloody diarrheal bowel movements per day, diffuse abdominal pain, and nausea and vomiting. He stated that his flare-ups in the past were resolved with a short course of Prednisone. He did not report any family history of inflammatory bowel disease. On physical exam the patient was a cachectic white male in no acute distress. The abdomen demonstrated positive bowel sounds and was non-distended, soft, and non-tender. There was no hepatosplenomegaly or masses. The rectal examination demonstrated black, hemoccult-positive stool but no masses. The remainder of the physical exam was unremarkable. On admission the following lab results were noted: TEST RESULT NORMAL VALUE Hematocrit 23.3% 40-54% Albumin 2g/dl 3.9-4.8 g/dl CEA (Carcinoembryonic) Antigen 6.4 ng/dl 0-3 ng/ml The other laboratory values were unremarkable. The patient was admitted to the hospital and treated with steroid therapy. There was no symptomatic improvement after eight days of therapy, so the patient was placed NPO (nothing by mouth) and on TPN (total parenteral nutrition). He was treated with two units of packed red blood cells. Colonoscopic examination revealed disease in the descending and sigmoid colon and rectum. Biopsies were taken and the interpretation by the pathologist was more consistent with ulcerative colitis than with Crohn's disease. There was no evidence of dysplasia. Options were discussed with the patient, and he decided to have a partial colectomy. Diffusely Inflamed Rectal Mucosa Anus 1. Chronic Ulcerative Colitis, Rectum - Gross Rectum demonstrates a diffusely inflamed mucosal surface. Ulcerative colitis causes chronic diarrhea with grossly bloody, mucus-filled stools. Since the disease begins in the rectum and proceeds proximally without interruption (no skip areas), it is usually evident on proctoscopic exam, where pseudopolyps and ulcers can be seen. Compare the gross and microscopic features of ulcerative colitis with Crohn's disease. What factors are believed to underlie the pathogenesis of inflammatory bowel disease (IBD)? The etiology of inflammatory bowel disease (ulcerative colitis and Crohn's disease) is unknown. Familial aggregation suggests a genetic predisposition but no definite genetic markers have been identified except for the uniform presence of HLA-B27 in patients with IBD and ankylosing spondylitis. Current opinion favors abnormal host immunoreactivity to luminal or mucosal antigens. The occurrence of IBD in mice lacking selected cytokines (IL-2, IL-10) or disruption of T-cell receptors, favors this concept. The beneficial effects of immunosuppressive therapy also point to the role of an aberrant immune response in the pathogenesis of IBD. Diffusely Inflamed Mucosa Appendix 2. Chronic Ulcerative Colitis, Colectomy Specimen - Gross Large bowel demonstrates a diffusely inflamed mucosal surface extending from the rectum to the cecum. Note the appendix. The right colon appears to be less involved; however microscopically the mucosa would demonstrate an active colitis. Is the picture consistent with Crohns' disease? No, this is not consistent with Crohn's disease because of the uninterrupted involvement of the mucosa. Although Crohn's disease may involve the colon, the affected areas are sharply delimited with intervening unaffected areas. Areas of Ulceration Pseudopolyp Submucosa Muscularis Propria 3. Chronic Ulcerative Colitis, Pseudopolyp -Low Power This image demonstrates a pseudopolyp (bulging mass of inflamed residual mucosa) surrounded by an extensive area of ulceration. Inflammatory infiltrate is seen in the submucosa. What is the definition of a polyp? The term polyp is used to describe any nodule or mass that projects above the level of surrounding mucosa; it may be hyperplastic or neoplastic. Why are the lesions in ulcerative colitis called pseudopolyps? These lesions are not really the result of mucosal proliferation, but rather the results of mucosal ulceration. Focal areas that are unaffected by ulceration appear to project above the denuded mucosa surrounding it. Normal Crypts Distorted Regenerating Crypts Ulcer 4. Chronic Ulcerative Colitis - Low Power Image demonstrates ulcer base covered by fibrin, capillaries, and inflammatory cells. Note the distorted, regenerating crypts at the edge of the ulcer. Compare the shape of the regenerating crypts with the normal crypts. How is the extent of these lesions different from that seen in Crohn's disease? Crohn's disease causes transmural inflammation that ultimately results in fibrosis and thickening of the wall. Lamina Propria Crypt Abscess 5. Chronic Ulcerative Colitis - High Power The lamina propria demonstrates an increase in acute and chronic inflammatory cells. A mucosal crypt is distended by a collection of neutrophils, known as a crypt abscess. Because ulcerative colitis is a diffuse mucosal disease process, crypt abscesses are common. Crypt abscesses are also seen in Crohn's disease but are not as common. What histologic feature is seen in Crohn's disease that is not seen in ulcerative colitis? Granulomas. What are the complications of ulcerative colitis? The most serious complication is development of carcinoma; with disease affecting the entire colon, the risk is 10% at 20 years. Other life-threatening complications can include severe diarrhea and electrolyte disturbances, severe colonic dilatation (toxic megacolon) with potential for perforation and peritonitis, and massive hemorrhage. Normal Colon Diseased Segments Normal Small Bowel 6. Crohn's Disease, Regional Enteritis - Gross The specimen is a section of normal ileum, thickened ileum, and right colon. The intestinal wall is thick, the result of edema, inflammation, fibrosis, and hypertrophy of the muscularis propria. Linear ulcers are typically present in the diseased segment of bowel. Unlike ulcerative colitis, the diseased areas are sharply demarcated from adjacent, uninvolved bowel. Note the normal area of colonic mucosa on the left and normal small bowel on the right. Are any other organs affected in Crohn's disease? Ulcerative colitis? Both Crohn's disease and ulcerative colitis are systemic diseases, associated with varied extraintestinal manifestations of immunologic origin. These include polyarthritis, sacroiliitis, uveitis, sclerosing cholangitis, and erythema nodosum. Gut Lumen Ulceration Submucosa Outer Muscle Layer Lymphoid Aggregate 7. Crohn's Disease - Low Power The low power view shows focal areas of ulceration, submucosal edema, and transmural inflammation with lymphoid nodules. Fissures are tracks of necrosis and inflammation that start in the mucosa and penetrate deeply into the wall. When the entire bowel wall is traversed, a sinus is formed that may open to the outside skin or adhere to adjacent viscus. What is the risk of colon carcinoma in Crohn's disease? While the risk is increased, it is less so than with ulcerative colitis. Distorted Crypts Lymphoid Aggregate 8. Crohn's Disease - Medium Power Under this magnification, crypt distortion, marked submucosal edema, fibrosis, and lymphoid aggregates can be seen. In addition, these patients may have non-caseating granulomas in the bowel wall. They are not seen in every case. What are the complications of Crohn's disease? The fissures in the mucosa can extend through the wall and form sinus tracts resulting in fistula formation to other loops of bowel, urinary bladder or vagina; there may be localized peritonitis and abdominal abscesses; fibrosis of the gut wall may lead to strictures and obstruction. Mucosa Fissures 9. Crohn's Disease - Low Power In this view, the marked thickening of the bowel wall can be appreciated. The thickening results from edema, inflammation, and fibrosis. In addition, two fissures penetrating the wall can also be seen. These tracks of necrosis and acute inflammation start in the mucosa. Here on cross section, the mucosal connection of the fissures cannot be seen. Non-caseating Granulomas Mucosa 10. Non-caseating Granulomas - High Power A non-caseating granuloma is present in the lamina propria of an uninvolved region of colonic mucosa. Non-caseating granulomas are present in about half of the cases in all tissue layers, both within areas of active disease and in uninvolved regions of the bowel. Granulomas have been documented throughout the alimentary tract, from mouth to rectum.