Hematopathology

Hematopathology I
Hematopoiesis & Anemia

Introduction
Before discussing anemia(s), I am reformatting the normal or reference ranges for the red blood cells found in Robbins Table 12-2 and have added explanatory notes. It is not necessary for you to memorize the respective reference range, but it is necessary to know the meaning and significance of each parameter since you will be using this information the rest of your medical career. You will become fluent in this terminology as you study the anemias.


Anemia is the reduction of the body’s red cell mass and is manifested clinically by hypoxia. Anemia initially produces a decrease of the RBC count followed by a decrease in Hb and Hct.
The Red Blood Cell (RBC) Count is the number of red cells per mm3. The RBC count is decreased in anemia and increased in polycythemia. In anemia, the RBC count is the first parameter to decrease in the complete blood count or what is termed the CBC.
Hemoglobin (Hb) is the main constituent of the peripheral red blood cell (RBC). Hemoglobin is a conjugated protein that transports oxygen and carbon dioxide. Hb concentration is reduced in anemia.
The hematocrit (Hct) or packed cell volume is the ratio of the volume of red cells to that of whole blood. Hct is expressed as a percentage (%).
With hypoxia, erythropoietin (EPO) production is increased which stimulates proliferation, growth, and differentiation of red cell precursors. Increased red cell production (erythropoiesis) is manifested by an increase in the reticulocyte count (>2%). Reticulocytes are immature non-nucleated red cells in the peripheral blood that contain ribonucleic acid (RNA) which continue to synthesis hemoglobin after nuclear extrusion.


The Red Cell Indices are calculations used to help classify anemias based on morphology.
MCV is the average volume of a RBC. A decreased MCV implies microcytosis and increased MCV implies macrocytosis. The MCV is the key red cell index for classifying anemia on the basis of morphology.
MCHC is the average concentration of hemoglobin in a given volume of red cells. Cells with a decreased MCHC have a decreased hemoglobin concentration and are hypochromic.

MCH is the content (weight) of Hb of the average red cell. Red cells with a decreased MCH have decreased hemoglobin content and are hypochromic.

1. Disorders of the hematopoietic system includes red cells (erythrocytes), white cells (leukocytes), and the hemostatic mechanism. In postnatal life, erythrocytes, leukocytes, and platelets are normally produced only in the bone marrow (intramedullary). Lymphocytes are produced in the bone marrow and secondary lymphoid organs. These introductory lectures will cover diseases of red cells (erythrocytes). The most frequent manifestation of red cells disorders is anemia, and anemia is the most common of all hematopoietic disorders. Anemia can be defined as a reduction in the oxygen transporting capacity of blood because of a low hemoglobin (Hb) or low hematocrit (HCT).
Anemia can be classified according to the mechanism of its production (Robbins Table 12-1, page 342) or according to its morphology . Both the pathophysiologic mechanisms and morphologic manifestations of anemia need to be understood in order to make the correct diagnosis. Anemias classified according to morphology tend to correlate with the underlying etiology, but exceptions abound.
Some anemias have more than one pathogenic mechanism and go through more than one morphologic stage (e.g. low grade blood loss anemia starts with a normochromic, normocytic anemia and progresses to microcytic, hypochromic anemia).
AGAIN, ANEMIA IS A MANIFESTATION OF AN UNDERLYING DISEASE. Understanding the pathogenic mechanism of the anemia allows for one to properly treat the anemia.
2. Normal hematopoiesis is the production and development in the bone marrow (BM) of blood cells (primarily red cells, granulocytes (polymorphonuclear leukocytes, eosinophils, basophils), monocytes, and platelets and lymphocytes). Within the bone marrow are pluripotential stem cells (PSC) which are capable of self-renewal. Pluripotential stem cells ultimately differentiate into these aforementioned blood cells under the influence of various interleukins. Soluble or membrane bound biochemical factors that regulate the proliferation and differentiation of hematopoietic precursor cells and facilitate the function of mature blood cells are known as growth factors or interleukins. Erythropoietin (EPO) is the major growth factor that stimulates red cell production. EPO is secreted by the kidney in response to hypoxia and stimulates PSC to differentiate and mature into erythroid cells.
3. In the adult, blood cells are produced in the bone marrow in selected locations, primarily in the pelvis, sternum, and proximal humerus as illustrated in this diagram. In children, there is hematopoiesis in the long bones, but this gradually diminishes with age. In the fetus, hematopoiesis begins in the liver and spleen, and then shifts to the bone marrow. Splenic and hepatic hematopoieses can recur in the adult when the bone marrow becomes fibrotic or is overrun with malignancy. Abnormal production of myeloid cells outside of the normal bone marrow is known as extramedullary hematopoiesis (liver, spleen). With stimulation of the erythron in various severe pathologic states (e.g. beta-thalassemia major), hematopoiesis in children can be maintained in the long bones and can even occur in the skull.

4. Venipuncture. The drop of blood at the tip of the hypodermic needle shows poor technique. Blood consists of a liquid portion known as plasma [water (93%) and proteins (primarily albumin), coagulation factors primarily fibrinogen), lipids, electrolytes, etc.]. Plasma accounts for approximately 55% of the blood volume. The cellular portion of blood consists of mostly red cells, white cells (granulocytes, lymphocytes, monocytes, etc.), and platelets. The volume of “packed red cells” in blood is approximately 45% and measured clinically as the hematocrit (Hct). Blood is routinely analyzed by an automated cell counter and a peripheral blood smear is made, stained, and examined under the microscope.
5. The normal mature red blood cell (RBC) is an anucleated biconcave disk which measures approximately 7 microns (u) in diameter. This biconcavity gives the red cell the greatest surface area for a given volume thus allowing for the optimal transfer of gases (oxygen and carbon dioxide). This biconcave shape also allows the red cell to easily deform and squeeze through capillaries much smaller than its size. The red cell membrane is made up of a lipoprotein bilayer (Robbins fig. 12-2) supported by a protein cytoskeleton. Notice that normal red cells have a uniform size and shape.
6. Red blood cells in routine peripheral blood smears appear as homogeneous anucleated pink round cells with a uniform central area of pallor. The pink color by Wright-Giemsa (WG) staining reflects hemoglobin content of the individual red cell. The red cell with the normal uniform area of central pallor (~1/3 of the cells diameter), with a uniform rim of pink hemoglobin (~2/3 of the cell diameter) is termed normochromic. With microscopic examination, if the area of central pallor is increased (> 1/3 of the cell’s diameter) then the amount of hemoglobin in the cell is decreased and the red cell is termed hypochromic. Hypochromic cells are also detected by a decrease in the MCH & MCHC by automated cell counters. Red blood cells are normally of uniform size and shape and are termed normocytic. When red cells are decreased in size, they are termed microcytic. Microcytic cells will have a decreased MCV. Red cells that are increased in size are macrocytes and will have an increased MCV. The term used for variation in red cell size is anisocytosis. Variability in red cell shape is termed poikilocytosis. These descriptive and quantitative terms are used to classify anemia clinically and give some clue as the underlying etiology. Once classified then the cause of the anemia has to be identified.
The following list puts the morphologic findings and red cell indices “together” along with the various underlying pathogenic mechanism.

Hypochromic Microcytic Anemia- MCV & MCH/MCHC-Decreased
Iron Deficiency Anemia Thalassemia
Chronic Blood Loss Aplastic anemia

Normochromic, Normocytic Anemia-MCV & MCH/MCHC-Normal
Sudden Severe Blood Loss-Trauma
Renal Disease
Malignancy
Chronic Disease (SLE, RA, TB, etc.)

Macrocytic Anemia-MCV Increased
Folate Deficiency
Vitamin B12 Deficiency
Chemotherapy-interferes with DNA synthesis


7. Bone Marrow Biopsy Paraphernalia. The items labeled A, B, & C are used for a posterior iliac crest biopsy. The items D & E are for sternal aspirations.
8. The most common anatomic site for a bone marrow aspirate and biopsy is the posterior iliac crest. A skilled operator can perform a sternal aspiration.
9. The normal bone, microscopically, has areas of hematopoietic tissue (the dots) interspersed with fat (holes) and stromal cells. The pink homogeneous acellular bone is at the bottom and top of the image.
10. The erythrocyte goes through several steps of maturation. The proerythroblast, at the earliest stage of erythroid maturation, has a large nucleus with lacy chromatin and readily apparent nucleoli. The cytoplasm in the proerythroblast is basophilic reflecting the abundance of cytoplasmic RNA, which will synthesize hemoglobin. As the erythroid cell matures, the nucleus condenses, becomes pyknotic, and is ultimately extruded. Concomitantly, the cytoplasm becomes pink reflecting the synthesis of hemoglobin with diminution of RNA. The normal circulating erythrocyte is pink and anucleated and essentially a “bag of hemoglobin”. About 1-2% of red cells circulating in the peripheral blood cells are slightly larger and immature containing some residual RNA which gives the cell a polychromatophilic appearance on routine Wright-Giemsa stains. These polychromatophilic erythroid cells are also known as reticulocytes because of their particular reticulated appearance imparted by RNA with special stains. Polychromatophilia or reticulocytosis in the peripheral blood indicates the bone marrow is responding appropriately to an anemia by generating new red cells (accelerated erythropoiesis). The presence of more immature nucleated red cells however is evidence of pathologically accelerated erythropoiesis (e.g. sickle cell disease, hemolytic anemia, and bone marrow fibrosis).
11. Anemia is a reduction of the oxygen-carrying capacity (hypoxia) of blood because of a reduction in the RBC mass. Patients may present with the symptoms of anemia such as being fatigued, having pale mucous membranes, or even chest pain. There are compensatory mechanisms to anemia, which include adjustment of the cardiac output, respiratory rate, and oxygen affinity for hemoglobin. A patient may thus be asymptomatic and found to be anemic by the CBC with a decrease in RBC count, Hb, and Hct.

13. Using automated instruments, the Hb is directly measured and the Hct is calculated from the RBC number and RBC size (MCV). Of course, the Hct can be measured using a specialized microhematocrit tubes and a microcentrifuge. The diagnosis of anemia is first established by measuring the Hb/Hct.
14. Once anemia is detected, the red blood indices are used then to classify the anemia. (See Introduction above and # 6). The MCV determines the size of the red cell and is the first and essential step in this classification (microcytic, normochromic, or macrocytic). The MCH/MCHC assess the amount of hemoglobin in the cell and are sometimes helpful in further determining the etiology of the anemia.


The Red Cell indices can indicate the underlying etiology or mechanism for the anemia. The following table lists the types of anemias that we will be discussing.


Red Cell Distribution Width (RDW)
Hypochromic, Microcytic Anemia with a Normal RDW (No variation in RBC size) implies Thalassemia .
Hypochromic, Microcytic Anemia with an Increased RDW (Variation in RBC size-Poikilocytosis) implies Iron Deficiency Anemia.
15. The mechanisms causing anemia can be classified broadly as a production disorder or survival disorder. Production disorders can be further subclassified as either as a result of hematopoietic cell damage or a nutritional factor deficiency.
16. Common causes of red cell survival disorders are those in which the life of the red cell is diminished such as in hemolytic anemias wherein there is increased destruction of RBC’s.
17. The clinical symptoms of anemia are due to the diminished oxygen carrying capacity of blood causing tissue hypoxia.
18. Iron deficiency is the most common form of nutritional anemia and chronic blood loss is being the most common cause of iron deficiency in the Western world.. Iron is needed for the production of the protoporphyrin heme. Heme is then coupled with the protein globulin in the red cell cytoplasm to form hemoglobin. The red cells late in iron deficiency anemia are microcytic (decreased MCV) and hypochromic (decreased MCH & MCHC) with marked aniso- and poikilo-cytosis (increased RDW).
19. Iron balance is tightly regulated by the absorption of dietary iron primarily in the duodenum (in) and by the normal shedding of duodenal epithelial cells containing iron (out). The storage or “blocking” of iron in the duodenal epithelium maintains the balance between absorbed iron and iron transfer from the epithelial cell to the blood carrier protein transferrin. Normal serum iron concentrations range from 100-120 ug/dl.
20. Regulation of iron absorption (Robbins fig. 12-9). Iron is readily absorbed from food into the duodenal mucosa. The transfer of iron into plasma is rate limited from mucosal cells when tissue iron stores (e.g. bone marrow) are adequate. When iron is needed, iron is then transferred from duodenal mucosal cells to transferrin and carried to the erythroid marrow and liver for storage. Iron is normally “lost” through the shedding of duodenal mucosal cells. Loss of iron through bleeding is always abnormal.
21. Transferrin is the major serum iron transport protein and is approximately 30% saturated with iron. The total capacity of transferrin to bind iron is known as the total iron binding capacity (TIBC).

22. The primary storage forms of iron are ferritin and hemosiderin. Ferritin is found in all tissues but primarily in the liver, bone marrow, and spleen. The breakdown product of ferritin is hemosiderin. Hemosiderin is readily found in bone marrow macrophages and is detected visually with special bone marrow iron stains. In iron deficiency anemia, tissue stores of hemosiderin and ferritin are depleted first.
23. There is a subtle progression in the development of iron deficiency anemia, regardless of its cause. First there is the depletion of stored iron (ferritin and bone marrow hemosiderin), followed by an absolute decrease in serum iron then followed by an increase transferrin (or TIBC = total iron binding capacity). Reduction in iron stores stimulates the synthesis of transferrin. The decrease in absolute iron concentration, coupled with the increase in transferrin, causes a marked reduction in percentage saturation of iron by transferring as TIBC. With continued iron depletion there follows a decrease in RBC count, Hb, Hct followed by microcytosis and hypochromasia.
24. The classic case of iron deficiency anemia is a microcytic (decreased MCV), hypochromic (decreased MCH & MCHC) anemia with anisocytosis (increased RDW). Serum iron, % Iron saturation/TIBC, and ferritin are markedly decreased.
25. Anemia may manifest as a symptom, sign, or laboratory finding. The cause or etiology of the anemia must always be established. Chronic blood loss is the most common cause of iron deficiency anemia. For premenopausal females, it is usually due to metrorrhagia or dysfunctional uterine bleeding. In postmenopausal females and in males, anemia is usually due to GI bleeding. Iron deficiency in a male and in a postmenopausal female is malignancy till proven otherwise. Anemia during infancy and pregnancy reflects poor medical care delivery and nutrition.
26. The most common symptoms of iron deficiency are nonspecific such as fatigue and decreased exercise tolerance. The other findings are rarely seen today.
27. Koilonychia is thickened fingernails seen in iron deficiency anemia is thought to be secondary to chronic hypoxia.
28. This image is the classic peripheral blood picture in iron deficiency anemia. Most of the red cells are smaller than the nucleus of the small lymphocyte (7u) and are microcytic (decreased MCV). The area of central pallor is increased in these microcytic erythrocytes, indicating decreased hemoglobin concentration (decreased MCH/MCHC). Notice how the hemoglobin is irregularly distributed in these cells. There are a few cells that appear to be well hemoglobinated; these are transfused red cells. Also, notice the variation in red cell size or what is termed anisocytosis (increased RDW). There is also poikilocytosis or variation in red cell shape. See Robbins fig. 12-10.
29. Megaloblastic anemias (MCV > 100) are usually caused either by impaired absorption of Vitamin B12 (cobalamin) and/or folic acid deficiency. Since both of these vitamins are needed for DNA synthesis, the effects on erythropoiesis are similar. Megaloblastic anemia caused by vitamin B12 deficiency is also known as pernicious anemia (PA). Vitamin B12 deficiency is most often due to malabsorption. Gastric acid is needed to release B12 complexes from food. Intrinsic factor (IF) secreted by gastric parietal cells is necessary for the absorption of Vitamin B12. The Intrinsic Factor-B12 complexes are then absorbed in the distal ileum. Any derangement of these steps can lead to malabsorption of vitamin B12.
30. Deficiency of vitamin B12 in the Western world is rare and body stores can last for decades. The most common cause of B12 deficiency or pernicious anemia in the Western world is the presence of autoantibodies, which block B12 absorption. These autoantibodies can be of several different types. Blocking antibodies, which block the binding of B12 to intrinsic factor (IF), binding antibodies which react with IF-B12 complexes and prevent them from binding to the ileal receptor, and parietal cell antibodies which bind to the parietal cells of the gastric mucosa.
31. Both B12 and folate deficiency impair DNA synthesis and affect rapidly dividing cells, such of those of the hematopoietic and GI systems.
32. Megaloblastic anemia (whether due to B12 or folate deficiency) has similar laboratory findings. All myeloid cell lines (red cells, granulocytes, and platelets) can be affected. The morphologic hallmark of megaloblastic anemia is the production of megaloblasts in the bone marrow and peripheral blood macrocytes (MCV >100). There is accumulation of the deranged megaloblasts in the bone marrow with destruction in the bone marrow (autohemolysis). In the peripheral blood, macrocytes are destroyed by the reticuloendothelial system. Granulocytes become enlarged and are hypersegmented. There may be decreased platelets (thrombocytopenia) and a decreased granulocyte count (neutropenia).
33. The classical findings in the peripheral blood are the presence of macrocytic ovalocytes and hypersegmented polymorphonuclear leukocytes. With B12 deficiency, there is also associated neurological defects as a consequence of demyelination of the posterior and lateral columns of the spinal cord. Compare the normoblasts with the megaloblasts in Robbins fig. 12-11.
34. Although autoantibodies are the most common cause of pernicious anemia (PA), other causes include atrophic gastritis (failure to secrete intrinsic factor) and achlorhydria (absent gastric acid).
35. Other causes of B12 deficiency are listed. Don’t ever forget the fish tapeworm.
36. The erythroid series is most often affected by megaloblastic anemia whether from B12 or folate deficiency.
37. In this image, normal erythroid cells are depicted on the left and megaloblastic erythroids are depicted on the right. One can not tell morphologically from either the bone marrow or peripheral blood the etiology of megaloblastic anemia. You have to measure B12, folate, and get a good clinical history. In the image on the left, the largest cell is the proerythroblast and you can see the orderly maturation as the nucleus progressively condenses and the cytoplasm takes on its pink color. In the megaloblasts on the right, the nucleus has a fine reticulated chromatin reflecting poor DNA synthesis and the cells are large. A neurological history and examination are most important since B12 deficiency causes degeneration of the posterior columns of the spinal cord.
38. Tetrahydrofolate (F4) is necessary as an acceptor and donor in the formation of thymine which is used in DNA synthesis. Deficiency of folate slows DNA synthesis. B12 deficiency leads to an internal folate deficiency by reducing the availability of tetrahydrofolate.
39. Although the anemias of B12 and folate deficiency are similar, B12 deficiency also leads to degeneration of the posterior and lateral tracts of the spinal columns. Patients with B12 deficiency have both a motor and sensory neuropathy such as burning in the hands and feet, loss of gait, and loss of proprioception. In fact, neuropathy can occur without anemia. With parenteral B12 there is brisk reticulocytosis within a few days.
40. The laboratory findings of isolated B12 deficiency are summarized.
41. Folate Deficiency causes a similar megaloblastic anemia as B12 deficiency. There are no neurological complications in folate deficiency. Folate deficiency is more common than B12 deficiency. Folate deficiency is common in alcoholics, the elderly, indigent, and those with malabsorption.
42. There is increased need for folate during pregnancy, and there is impaired utilization of folate with chemotherapy. The best test to detect folate deficiency is red blood cell folate. The serum folate can be “normal” where whole blood (red cell) folate can be actually decreased.


43. The anemia of chronic disease is a common form of anemia associated with variety of different chronic diseases and is relatively common in hospitalized patients. Although the patient has anemia (decreased Hb/Hct), the patient usually, but not always, has normochromic (MCH is normal) normocytic (MCV is normal) indices. The presence of increased storage iron in bone marrow macrophages, increased serum ferritin level, and reduced iron binding capacity rule out iron deficiency. Common causes of anemia of chronic disease include renal failure, lung and breast cancer, rheumatoid arthritis, and chronic infections.
44. Chronic inflammation or malignancy triggers secretion of various cytokines which impede the transfer of iron from the storage pool to the erythroid precursors. Anemia of chronic disease is also known as the anemia of poor iron reutilization.
45. This is a bone marrow aspirate from a patient with a normochromic, normocytic anemia who had rheumatoid arthritis. Serum iron was mildly decreased, as was the TIBC. Serum ferritin was increased. There is an overabundance of iron in the bone marrow macrophages (Prussian blue stain). Treatment of the underlying condition corrects the anemia; treatment with erythropoietin will improve the anemia. Iron supplements will not improve normochromic normocytic anemia.
46. Aplastic anemia is characterized by the suppression/failure of pleuipotential stem cells, with resultant anemia, thrombocytopenia, and leukopenia (collectively known as pancytopenia).
47. The bone marrow biopsy in aplastic anemia shows a markedly hypocellular bone marrow (<10%). Marrow fat is relatively increased with scattered foci of lymphocytes and plasma cells.
48. Causes of aplastic anemia (stem cell failure) are as follows:

Primary/Idiopathic >50%
Secondary
Drugs/Chemicals
Idiosyncratic-not known to be myelotoxic
Chloramphenicol, Phenylbutazone
Dose Related-Chemotherapy, Benzene
Total Body Irradiation
Viral Infections-HIV, non-A, non-B Hepatitis

Even when the cause of the aplastic anemia is known, the actual pathophysiology is not. Leading contenders are T-cell suppression or genetically damaged stem cells.
49. Myelophthisic anemia is bone marrow failure due to marrow replacement by metastatic carcinoma (breast, prostate, thyroid), fibrosis, and other hematopoietic diseases. The patient will have an anemia, thrombocytopenia (decreased platelet count), and misshapen (tear drop cells, schistocytes), and immature (nucleated) red cells.
50. This patient’s bone marrow biopsy shows compete replacement of the bone marrow by metastatic breast cancer. The cancer forms nests of cells. Compare this bone marrow biopsy with the normal bone marrow in Image # 10. [/right]