L3. Pathogenesis and Morphology of Anemia PDF

03 Pathology Hamda Al-Thawadi Pathogenesis and Morphology of Anemia 10th March 2020 Amira Gamal Abdelaziz M Tawengi This document resorted to: 1. “Anemia” Lecture Slides, Dr. Hamda Al-Thawadi. 2. “Basic Pathology, Robbins 10th Edition” Chapter 12. 3. “First Aid, USMLE step 1, 30th Edition” Section 3...

03 Pathology Hamda Al-Thawadi Pathogenesis and Morphology of Anemia 10th March 2020 Amira Gamal Abdelaziz M Tawengi This document resorted to: 1. “Anemia” Lecture Slides, Dr. Hamda Al-Thawadi. 2. “Basic Pathology, Robbins 10th Edition” Chapter 12. 3. “First Aid, USMLE step 1, 30th Edition” Section 3. Normal RBC Normal RBC has a biconcave shape (thin centre and thick edges) to squeeze easily in capillaries No nucleus to give more space for haemoglobin Cell membrane is composed of proteins and lipids, if their genes are abnormal; abnormal shape of RBC results The normal RBC diameter is about 7.5 micro µm Normal Hemoglobin Hemoglobin composed of 4 subunits proteins: 2α and 2β Each subunit composed of group of amino acids Each amino acid is encoded by a gene, which, if mutated results in abnormal haemoglobin (hemoglobinopathies) Descriptive Terms In this lecture we will discuss the causes and morphology of different types of anemia. So, in order to describe the morphology of abnormal RBC, we need to know the main descriptive terms that we use for that seek. Term Description Anisocytosis Variation in RBCS size Poikilocytosis Anisochromia Variation in RBCS shape Variation on RBCS color Indices* MCV (mean corpuscular volume) RDW (Red blood cell distribution width) The variation in RBC shape ≥ 10 % MCH (mean corpuscular haemoglobin) Now, let’s talk about each descriptive term individually. Anisocytosis Descriptive term Diameter MCV 6.7-8 µm (average 7.5 µm) 80-100 fl less than 6.7 µm MCV< 80 fl 8.2-12 µm MCV > 100 fl Normal Microcyte Macrocyte Mean cell (Corpuscular) volume (MCV): the average volume per red cell, expressed in femtoliters (cubic microns) Mean cell hemoglobin (MCH): the average mass of hemoglobin per red cell, expressed in picograms Mean cell hemoglobin concentration (MCHC): the average concentration of hemoglobin in a given volume of packed red cells, exprssed in grams per decilitre Red cell distribution width (RDW): the coefficient of variation of red cell volume Megaloblast 12-25 µm - Poikilocytosis Descriptive term Description Morphology Target Cell (Codocyte) RBC resembles a shooting target Dark centre with Hb surrounded by a white ring Can happen in thalassemia RBC Spherocyte Stomatocyte Sickle Cell with smaller diameter and without biconcave shape Small, spherical cells without central pallor RBC has a slit-like or coffee bean shape (elongated fade in the centre) RBC has a crescent-shape Sickling occurs with low O2 conditions (e.g., high altitude, acidosis) Acanthocyte (spur cell) RBC has thorny spike-like projections Projections of varying size at irregular intervals. Elliptocyte RBC is elongated, cigar or sausage shape Caused by mutation in genes encoding RBC membrane proteins (e.g., spectrin) Schistocyte (e.g., helmet cell) Teardrop (Dacrocytes) Burr Cell (Echinocytes) RBC is fragmented RBC has teardrop shape RBC “sheds a tear” because it’s mechanically squeezed out of its home in the bone marrow Can happen in thalassemia RBC has spine projections Smaller and more uniform projections than acanthocytes Happens due to alterations in proteins and lipids of cell membrane Anisochromia Descriptive term Normochromia (Normal) Description MCH RBC has normal Hb concentration 27-33 Central pallor exceeds ⅓ of cell diameter MCH < 27 Chromatin is higher looks darker MCH > 33 Hypochromia Hyperchromia Variation in Inclusion Inclusion means the appearance of microparticles -after staining- within the cell due to abnormalities happening during maturation steps. Type Description Normal Cabot’s ring Morphology RBC has no inclusions (no dots, rings, blue staining or precipitation) RBC has ring shape or figure-8 structure Inclusion = Remnants of nuclear membrane Basophilic stippling RBC has deep blue granulations of variable size Inclusion = precipitated RNA (do not contain iron) Howell-Jolly bodies RBC has round solid dark inclusion Inclusion = DNA fragment Basophilic nuclear remnants, normally removed by splenic macrophages Variation in Distribution (size and shape are normal) Type* Description Agglutination RBCs aggregate due to presence of Abs Can be a feature of immune system disease Rouleaux RBCs appears as a stack of coins *both types cannot happen at the same time Morphology Diagnostic Tests 1- Peripheral Blood Smear (blood film) (PBS) A thin film of blood which is examined under a microscope. It provides information on the number, size and shapes of normal and abnormal RBCs, WBCs and platelets in circulation. Steps: Ø 1. A drop of blood is placed on the specimen slide (2-3 mm in diameter). 2. A spreader slide is placed on the specimen slide, in front of the blood drop. 3. The spreader slide is bent at a 45° angle, pulled to the back and then forward. A monolayer of blood will be formed in order to examine the size and shape of each RBC individually. 4. Patient name and date are labelled on one edge. 5. The slide rapidly dried. 6. Ready for fixation, staining and examination. 2- Bone Marrow Biopsy (BMB) Procedure of taking sample of bone marrow (soft tissue) to examine the normal production of the cells in the histopathology lab. This procedure is done in order to identify the cause behind the abnormal RBCs in the PBS. Indicated as a: Ø Diagnostic procedure e.g. aplastic anemia or leukemia Ø Therapeutic purpose e.g. bone marrow transplantation Contraindicated in patient with hemorrhagic or bone disorders. Ø 1. 2. 3. Steps: Consent is taken Patient placed in lateral decubitus position The area is palpated to detect the posterior iliac crest (BMB can be taken from the sternum, however it is very painful. That is why we take it from iliac crest instead) 4. The site is sterilized 5. Local aesthesia administered Ø Tools: The sample is placed in a container with fixed solution to prevent infection/ decay and to prepare it for slicing and staining. Sent to histopathology for slicing, staining and examination under the microscope Anemia Anemia is defined as a reduction in the oxygentransporting capacity of blood, resulting from a decrease in the red cell mass to subnormal levels. In other words, it a reduction in the total circulating erythrocyte mass. It is diagnosed when hemoglobin conc., hematocrit or erythrocyte count are below normal. Classification of Anemia (based on the cause) Decreased Production of Erythrocytes Blood Loss Trauma Surgery GI Bleeding (e.g., bloody stools) Menstrual Bleeding; menorrhagia (heavy menstrual bleeding) Aplastic Anemia Anemia with chronic renal failure Anemia with chronic disease Megaloblastic Anemia Pernicious Anemia Folic Acid Deficiency Iron Deficiency Anemia Increased Destruction of Erythrocytes Thalassemia Sickle Cell Disease Hereditary Spherocytosis Hereditary Elliptocytosis G6PD Deficiency In this lecture we will focus on the anemias that are caused by decreased production of erythrocytes. Decreased Production of erythrocytes (based on the cause) Disturbed proliferation and maturation of erythroblasts Disturbed proliferation and differentiation of stem cells Defective DNA synthesis: Aplastic anemia Pure red cell aplasia Deficiency or impaired use of vitamin B12 and folic acid (megaloblastic anemias) Defective hemoglobin synthesis: Deficient heme synthesis: iron deficiency, sideroblastic anemias Deficient globin synthesis: thalassemias Anemia of renal failure (erythropoietin deficiency) Anemia of chronic disease (iron sequestration, relative erythropoietin deficiency) Anemia of endocrine disorders Decreased Production of erythrocytes (based on the size) Microcytic (MCV < 80 fL) Anemia of chronic disease Iron deficiency (late) Normocytic (MCV 80–100 fL) Macrocytic (MCV > 100 fL) Nonhemolytic (low reticulocyte index)* Megaloblastic Iron deficiency (early) Anemia of chronic disease Aplastic anemia Chronic kidney disease Folate deficiency Vitamin B12 deficiency *Reticulocyte index Also called corrected reticulocyte count. Used to correct falsely elevated reticulocyte count in anemia. Measures appropriate bone marrow response to anemic conditions (effective erythropoiesis). High reticulocyte index (RI) indicates compensatory RBC production; low RI indicates inadequate response to correct anemia. Anemia Pathogenesis Aplastic Anemia Chronic disorder of hemopoietic stem cells Caused by bone marrow failure (hypocellularity) resulting from including: o 65% cases are idiopathic o Inherited disease e.g. Fanconi Anemia, defects in telomerase and DNA repair o Drugs or chemicals o Infections o Irradiation Clinical findings Results in cellular depletion of the bone marrow (Pancytopenia) ↓ reticulocyte count, ↑ EPO. Pancytopenia characterized by anemia, leukopenia, and thrombocytopenia (not to be confused with Pure red cell aplasia which causes anemia only). Normal cell morphology, but hypocellular bone marrow with fatty infiltration Symptoms: fatigue, malaise, pallor, purpura, mucosal bleeding, petechiae, infection. Morphology A. Bone marrow biopsy showing normal cells. B. Bone marrow biopsy of a patient with aplastic Anemia, there is decrease in the number of cells (pancytopenia) and the bone marrow replaced by fat. Anemia A. Peripheral blood film showing normal number of cells. B. Peripheral blood film of a patient with aplastic Anemia, there is decrease in the number of cells (pancytopenia). Pathogenesis Clinical features Anemia With Chronic Renal Failure Normally, the decrease in tissue oxygen tension that accompanies anemia triggers increased production of the growth factor erythropoietin from specialized cells in the kidney à compensatory hyperplasia of erythroid precursors in the bone marrow. However, in case of chronic renal failure, EPO will not be released à retaining low RBCs production. Bone marrow is normal Normocytic normochromic anemia Burr cells due to alteration in lipid composition of red cell membrane Morphology Anemia Anemia With Chronic Disease Pathogenesis Clinical findings Common in chronic inflammatory disorders (rheumatoid arthritis and SLE) and in chronic infectious diseases (TB, AIDS) It occurs in a variety of disorders associated with sustained inflammation: o Chronic microbial infections, such as osteomyelitis and bacterial endocarditis o Chronic immune disorders, such as rheumatoid ↓ Iron, ↓ TIBC, ↑ Ferritin – %TS. arthritis o Neoplasms, such as Hodgkin lymphoma Mild to moderate normocytic (but can become microcytic) Chronic inflammation à proinflammatory cytokines normochromic anemia. such as IL-6 à increase hepatic hepcidin synthesis à elevated hepcidin levels à downregulating ferroportin in macrophages & intestinal mucosa à blocking the transfer of iron to erythroid precursors & absorption in gut. In addition, chronic inflammation blunts erythropoietin synthesis by the kidney, lowering red cell production by the marrow. Megaloblastic Anemias Caused by impaired DNA synthesis as a result of deficiency due to either: o Vitamin B12 (Pernicious Anemia) o Folate Ø Megaloblastic anemia arrises from metabolic defects that lead to inadequate biosynthesis of thymidine, a building block of DNA. Folate and vitamin B12 are both essential for the synthesis of thymidylate, that is required for DNA replication. Ø Thymidine deficiency causes abnormalities in rapidly dividing cells throughout the body, but the hematopoietic marrow is most severely affected. Ø Many red cell progenitors are so defective in DNA synthesis that they undergo apoptosis in the marrow (ineffective hematopoiesis). Others mature into red cells but do so after fewer cell divisions, further diminishing the output of red cells. Morphology Ø When DNA synthesis is impaired, the cell cycle cannot progress from the G2 growth stage to the mitosis (M) stage à continuing cell growth without division à Megaloblastic cell (large unfunctional cell) Ø Megaloblastic anemia is a state of macrocytic anemia, in which the developing RBCs remain immature. Some additional abnormal morphologies may be seen such as marcoovalocytes, hypersegmented neutrophils (≥5 nuclei) and anisocytosis A blood film is showing the circulating erythrocytes with prominent poikilocytosis having large oval (Megaloblast) shape. Neutrophils with 6 or more nuclear lobes (hyper-segmentation). Hypersegmentation can be seen also in megakaryocytes. Bone marrow biopsy: Erythroid hyperplasia and megaloblast (Sieve-like) nuclear chromatin that is not completely condensed. Bone marrow biopsy: Large metamyelocytes with horseshoeshaped nuclei (Arrow). Anemia Pernicious Anemia/ Vitamin B12 (Cobalamin) Deficiency Anemia Pathogenesis Vitamin B12 absorption: B12 found in meat, fish, liver, egg and milk B12 binds to a gastric glycoprotein (intrinsic factor IF) that is secreted by parietal cells IF protects B12 from degradation by the intestinal enzymes until B12-IF complex reaches receptors in the ileum Clinical findings 1. Neurologic symptoms; reversible dementia, numbness, tingling in feet and hands etc. 2. Pallor 3. Easy fatigability 4. In severe cases, dyspnea and even congestive heart Causes of Vitamin B12 deficiency anemia: failure Ø Parietal cells destructed by autoimmune Abs, resulting in increased atrophic gastritis à impaired IF production à Vitamin B12 5. The destruction of deficiency erythroid progenitors Ø Poor diet (e.g., veganism) because of ineffective Ø Low absorption: erythropoiesis may Gastrectomy (due to loss of IF–producing cells) give rise to mild Defective intestinal absorption jaundice Neoplasm in ileum The diagnosis of a megaloblastic anemia is readily made from the examination of smears of peripheral blood and bone marrow. The anemia of folate deficiency is best distinguished from that of vitamin B12 deficiency by measuring serum and red cell folate and vitamin B12 levels. Anemia Folic Acid Deficiency Pathogenesis Clinical findings Folate is present in leafy vegetable, meat and egg Folic acid absorbed in jejunum Common cause: Ø Inadequate dietary intake, sometimes complicated by increased metabolic demands (hemolytic anemia, pregnant women) Ø Pregnancy No neurologic Ø Acoholism (interfere with folic acid metabolism) symptoms (as Ø Malabsorptive disorders, such as celiac disease compared to Dihydrofolate Reductase B12 deficiency) Dihydrofolate Tetrahydrofolate* *Tetrahydrofolate acts as an acceptor and donor of one-carbon units in several reactions that are required for the synthesis of deoxythymidine monophosphate (dTMP) Intracellular stores of folate fall à Insufficient dTMP is synthesized à DNA replication is blocked à Megaloblastic anemia. Anemia Iron Deficiency Anemia Pathogenesis Causes: Chronic blood loss: GIT (e.g., peptic ulcers, colon cancer, hemorrhoids) and the female genital tract (e.g., menorrhagia, endometrial cancer). Poor diet Increased demands not met by normal dietary intake occur during pregnancy and infancy. Malabsorption can occur with celiac disease or after gastrectomy Clinical findings ↓ iron, ↑ TIBC, ↓ ferritin, ↓↓%TS, ↑ RDW Symptoms: In severe iron deficiency, depletion of essential iron-containing enzymes that utilize oxygen, give rise to changes: Alopecia, hair loss Koilonychia: Due to ridges formation in nail beds (spooning of the nails) Iron Deficiency à Iron stores are depleted à decline in serum ferritin and the absence of stainable iron in the bone marrow à decrease in Pagophagia (Pica) Craving serum iron and a rise in the serum ice/ glass/ paintings transferrin à the capacity to (non-food stuffs) synthesize hemoglobin, myoglobin, and other iron-containing proteins is diminished à microcytic anemia Atrophic Glossitis; Loss of papilla Angular Stomatitis Morphology In peripheral blood smear: RBCs are hypochromic microcytic. In bone marrow, mild hyperplasia. Further clinical features Ø Plummer-Vinson syndrome can develop in iron deficiency anemia. Ø Thinning of part in the esophagus due to mucosal damage. Ø Can develop to carcinoma. Ø Characterized by dysphagia, atrophic glossitis and angular stomatitis. Review Pathological change: Hypersegmented neutrophils Underlying cause: Impaired DNA synthesis as a result of B12 and folate deficiency. Shape of the nucleus in bone marrow biopsy: horseshoeshaped nuclei of neutrophils, while sieve-like nucleus of erythroblasts. Pathological change: Koilonychia (spooing of the nails). Type of anemia associated with this pathological change: Iron deficiency anemia. Don’t hesitate to contact Amira Gamal/Abdelaziz M Tawengi regarding any clarification, concern or suggestion!