3L - Compre Hema 1 Lec PDF

 CHAPTER 1: OVERVIEW OF CLINICAL RBC Indices can be computed using the 3 RBC LABORATORY HEMATOLOGY parameters (hgb, hct, RBC count) o MCV mean cell voluma...

 CHAPTER 1: OVERVIEW OF CLINICAL RBC Indices can be computed using the 3 RBC LABORATORY HEMATOLOGY parameters (hgb, hct, RBC count) o MCV mean cell voluma GENERALITIES Based on the hematocrit of the patient How many mL of blood do a normal human body has? together with the RBC count 5000-6000 mL or 5-6 L o MCH mean cell hemoglobin What are the functions of blood? Hemoglobin and RBC count of the patient Depends on the blood cell. Usually carries o MCHC mean cell hemoglobin concentration oxygen to the different parts or tissues of our hemoglobin/hematocrit (hgb/hct) body and it protects us from inflammation and o RDW red cell distribution and width infection Use or function Aids in hemostasis o To detect and diagnose the disease What are the three families of blood cells? By knowing hgb, hct, and RBC count Erythrocytes (Red Blood Cells) along with others, could help Leukocytes (White Blood Cells) doctors/clinician to diagnose a disease Thrombocytes (Platelets) o To assess and monitor the disease What is the definition of hematology? For example, a patient suffering from Hema = blood anemia is given a medicine to cure that Logy = study anemia, after some time you can repeat Hematology = study of blood cells the RBC parameter by doing laboratory test so that you can monitor if there is an HISTORY improvement of the disease or none, if Athanasius Kircher (1657) the drug is effective or not. This is how to assess the usefulness of the RBC worms in the blood parameters when assessing the status of No proper identification whether RBC, WBC, or the patient. platelets o To differentiate anemia, polycythemia, and systemic conditions that affects red blood cells Anton van Leeuwenhook (1674) Anemia - RBC parameters are very low Discovered the presence of RBCs Polycythemia - RBC parameters are excessively high Giulio Bizzozero (1800) Anemia is related to systemic conditions Platelets (petites plaques) Ex. A patient suffering from Lupus Erythematosus (LE) wherein the kidneys James Homer Wright (1902) are affected. Kidneys are helpful for the production of RBCs. If there is an o is used to differentiate different cells underlying condition that would affect the found in the body according to the red cell, then it could be related to cytoplasm and nucleus systemic conditions. o the description of the cytoplasm and Reticulocytes nucleus according to the affinity to the o young RBCs, an RBC parameter stain o polychromatophilic erythrocytes o Indicative of bone marrow regeneration RED BLOOD CELL PARAMETERS during blood loss and certain types of Hemoglobin (hb, hgb) anemia o Oxygen is attached to the hemoglobin In case of blood loss, these erythrocytes Hematocrit (hct)/PCV (pack cell volume) will come out in the peripheral blood in o Consist of packed cell RBC count ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 2  order to add more RBC in the periphery, color of the granules present in their circulation. cytoplasm These reticulocytes are in the bone shape of the nucleus marrow and when observed under the microscope there are still remnants of RNA. Supravital staining (BCB, NMB) o BCB Brilliant Cresyl Blue o NMB New Methylene Blue o Chromatin bodies are observed by this stain o Live red cells are utilized in order the RNA would be stained. Granular Cells Color of the Granules Basophil Blue course granules obscuring the nucleus (if the granules are not dissolved) Eosinophil Reddish orange course granules in the cytoplasm Neutrophil Fine pink granules Nucleus Basophil Rare; has globulations on its nucleus (when stained, the granules in the Reticulocytes basophils are soluble in water thus losing its granules) WHITE BLOOD CELLS Eosinophil Bilobed found in the periphery of the Function mainly to protect us from infection/injury cell Hemocytometry = counting 5,000 Neutrophil 3 to 5 lobes 10,000/cumm (band neutrophil: young neutrophils o Similar to RBC, involved in counting due to the nucleus of the cell manually possessing C-shaped or S-shaped; no Leukopenia = low WBC count lobes are present and these are Leukocytosis = high WBCs count connect by a thin filament) Differential count Agranular Cells o Granular there are granules found in Nucleus the cytoplasm of the cell Lymphocytes Compact and almost occupied the Basophils whole cytoplasm Eosinophils Monocytes Kidney-shaped nucleus or inverse Neutrophils brain-like convolutions o Agranular without granules in the cytoplasm of the cell Cytoplasm Lymphocytes Lymphocytes Scanty cytoplasm (small) Monocytes Monocytes Smooth cytoplasm; vacuolations are o These cells can be identified according to sometimes present in the cytoplasm of the: morphology ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 3 Comparisons: Specimen quality Eosinophil and Neutrophil o Before working on a specimen, the o Eosinophil: course granules quality must be inspected o Neutrophil: fine granules o All examinations that are requested in the laboratory should be inspected or PLATELETS/THROMBOCYTES examined prior running the test. Maintains vascular integrity o CBC specimen should not have Controls hemostasis presence of fibrin o Controls bleeding Formulation of fibrin indicates o Primary hemostasis the activation of coagulation involves platelets factors o Secondary hemostasis Specimen accession involves coagulation factors o Numbering of specimens received in the Characteristics: laboratory o Very small (2-4 mu), round, anucleated, o Assigned a definite number slightly granular o Ability to aggregate and adhere with one BLOOD FILM EXAMINATION another Smear preparation/characteristics Once a person is injured, platelets immediately o Characteristics of a good smear go to the site of bleeding and form a platelet plug Proper amount of blood in the During surgery, platelets activates clotting factors slide to have a good quality of to form the secondary hemostasis blood smears Staining procedures o Blood smear should be stained before examining under the microscope o Cells are observed under oil immersion objective Examine the morphology of the RBCs o Identify whether: Normocytic normal size cell Macrocytic large cell size Platelets Microcytic small cell size Normal RBC size 6-8 COMPLETE BLOOD COUNT (RBC, WBC, Platelets) micrometer in diameter Types of specimen: Normochromic normal color; o Capillary blood RBC possess normal quantity of o Venous blood hgb Phlebotomist Hypochromic decreased o In charge of collecting blood from the concentration of hgb patient to be examined Hyperchromic increase o In the Philippines, he/she must be a concentration of hgb graduate of medical technology because The chromocity will depend on the they are the ones that are trained to hemoglobin content of the RBC become phlebotomist. WBC percent distribution is obtained o In other countries, only 6 months training (differential count) of phlebotomy is required. o 100 leukocytes are counted ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 4  ENDOTHELIAL CELLS Veins are composed of simple squamous cells Maintains vascular integrity o Bumpy obstacles are passed through by platelets which tend to bring out their pseudopod adhering to one another. It has the ability to adhere and aggregate. Maintenance of normal blood flow Storage site of some cytokines involved in hemostasis o Example: Von Willebrand Factors stored in the endothelial cells COAGULATION Factors involve = platelets, coagulation factors Hemostasis = primary, secondary Laboratory test = PT, APTT, Fibrinogen Assay, D-dimer o Determines if there is a problem in hemostasis of an individual ADVANCED HEMATOLOGY PROCEDURES 1. Bone Marrow Examination Bone marrow aspirate or one marrow biopsy Considered as an advanced morphological procedure because it determines whether the patient is suffering from blood diseases Production of RBC is more on the flat bones o Sternum In adults, done on the posterior/anterior portion of the iliac crest May be done in fetal position What can be seen in the bone marrow aspirate? Aspirate Erythroid Islands (to become erythrocytes) Megakaryocytes Megakaryoblasts (thrombocytes and myelocytic series) ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 5 2. Special Stains (Cytochemical) Used to identify immature and predominant cells present in the bone marrow aspirate o Predominant cells found are the erythroid islands o Problems in the blood would result to more immature leukocytes than erythroid islands o In cases of aplastic leukemia, 5. Cytogenetics predominant cells would be fat deals with chromosomes also known as cells karyotyping used for diagnosis of Myeloperoxidase oncologic and hematologic disorders Sudan Black Nonspecific and specific esterase 6. Molecular Cytogenetics Periodic Acid Schiff primarily defines a large set of the Tartrate Resistant Acid Phosphatase techniques that operate either with the Alkaline phosphatase entire genome or with specific targeted DNA sequences 3. Flow Cytometry most modern technique of cell studies identifies and quantifies populations of cells in a heterogeneous sample usually blood, bone marrow or lymph Principle of blood count in the laboratory 4. Immunophenotyping QUALITY CONTROL Specimen integrity surface, nucleus, or cytoplasm that helps o Whether the specimen is fit for identify the lineage of cells using examination antibodies o For CBC and examination of coagulation factors, there should be no presence of clots Responsibility for accuracy ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 6  o Run standards and controls in order to have accurate results Proper judgment Timeliness Internal controls o All examinations done in the laboratory should be practiced with quality control ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 7  CHAPTER 4: HEMATOPOIESIS PRENATAL HEMATOPOIESIS DEFINITION A process through which the blood manufactures 1. MESOBLASTIC PERIOD blood cells. (PRIMITIVE ERYTHROPOIESIS) Continuous, regulated process of renewal, Also known as yolk sac period. proliferation, differentiation, and maturation of all Begins as early as the nineteenth day after blood cell lines. fertilization in the yolk sac of the embryo Prenatal and postnatal hematopoiesis o Early in embryonic development, cells o Prenatal life - where blood cell production from the mesoderm migrate to the yolk begins sac. o The original pool of hematopoietic stem cells is o Some of these mesodermal cells form formed during embryogenesis in a complex primitive erythroblasts in the central part developmental process that involves that of the yolk sac and other fraction of cells several anatomic sites such as the yolk sac, become angioblasts. aorta-gonad-mesonephros (AGM) and liver. o Angioblasts will surround the cavity of the o Shortly before birth, the hematopoietic stem yolk sac and eventually form blood cells colonize the bone marrow. During vessels. postnatal life, a steady state is established in Only erythrocytes are made (primitive which hematopoietic stem cells pool size is erythroblasts) maintained by the regulation of self-renewal and The RBCs contain unique fetal hemoglobins differentiation processes. (Gower-1, Gower-2, and Portland) ERYTHROPOIESIS Occurs intravascularly Process that give rise to mature erythrocytes in the o Type of hematopoiesis that occurs in the blood circulation yolk sac cavity. o Later in the period, the yolk sac will LEUKOPOIESIS diminish and mesodermal cells will Produces mature WBCs migrate to aorta-gonad-mesonephros region in preparation for definitive MYELOPOIESIS hematopoiesis. Process wherein myelocytic cells are produced, developed, and mature LYMPHOIESIS Process wherein lymphocytic cells are produced, developed, and mature MEGAKORYOPOIESIS Refer to the production of megakaryocytes Megakaryocytes are giant cells inside the bone marrow In the process of maturation, it will break up and release fragments called platelets or thrombocytes ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 8  2. HEPATIC PERIOD Also known as liver period Begins around 5-7 gestational weeks (1-2 months of gestation) The fetal liver becomes the chief site of blood cell production The spleen, kidney, thymus and lymph nodes contribute to hematopoiesis Occurs extravascularly Beginning of definitive hematopoiesis o Due to production of lymphoid stem cells, which will later on become lymphocytes, will commence on this period The yolk sac period is the start of fetal blood cell Megakaryopoiesis begins production. Hematopoiesis in the liver reaches its peak by Hgb F and adult hemoglobins o Erythrocytes that are produced in this the 3rd month of fetal development then gradually period have fetal hemoglobin and adult declines after the 6th month. The spleen starts to participate in blood cell hemoglobin production at about 2 ½ months of fetal life and 3. MYELOID/MEDULLARY PERIOD ends at about 7th month. The bone marrow, although it begins to generate Begins at the 4th to 5th month of fetal blood cells during fetal life, will continuously development produce blood cells postnatal up to adulthood. Occurs in the medulla of the bone marrow o Hence called the Myeloid/Medullary MEDULLARY VS EXTRAMEDULLARY Period HEMATOPOIESIS By the end of the 24th week (6th month) of Hematopoiesis in the bone marrow is called gestation: Bone marrow becomes the primary medullary hematopoiesis site of blood cell production. Hematopoiesis in areas other than the bone Detectable levels of EPO, G-CSF, GM-CSF marrow is called extramedullary o EPO Erythropoietin hematopoiesis o G-CSF Granulocyte colony-stimulating o Ex. Hematopoiesis in the yolk sac and factor liver. o GM-CSF Granulocyte-macrophage colony-stimulating factor Notes from sir: Fetal Hgb and adult Hgb are detectable during Why do we have extramedullary hematopoiesis? this phase Our body may need an increased level of RBCs, an increased supply of oxygen Notes from sir: During this phase, some cytokines can now be seen. HEMATOPOIETIC TISSUES They are essential for specific lineage production, e.g. Involved in the production, proliferation, EPO for RBC production, G-CSF for granulocyte maturation and destruction of blood cells. production, and GM-CSF (M may stand for monocyte or macrophage as the former is the predecessor of the o Bone marrow latter) for macrophage. o Liver o Lymph nodes As we age, the number of Hgb F in our bodies o Spleen decrease while Hgb A increases. o Thymus ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO | 3L-MT 9  BONE MARROW The bone marrow contains hematopoietic cells, stromal cells, and blood vessels. Located inside the spongy bone Stromal cells originate from mesenchymal cells In a normal adult, ½ of the bone marrow is that migrate into the central cavity of the bone hematopoietically active (red marrow) and ½ is marrow. This include endothelial cells, inactive, fatty marrow (yellow marrow) adipocytes, macrophages, lymphocytes, The marrow contains both Erythroid (RBC) and osteoblasts, osteoclasts, and reticular adventitial leukocyte (WBC) precursors as well as platelet cells. precursors Endothelial cells are broad, flat cells that Early in life most of the marrow is red marrow and forms single continuous layer along the inner it gradually decreases with age to the adult level surface of the arteries, veins, and vascular of 50% sinuses. In certain pathologic states the bone marrow can Megakaryocytes are easily spotted in the picture increase its activity to 5-10X its normal rate. due to their big size. When this happens, the bone marrow is said to o Cellularity: the ratio of marrow cells to fat be hyperplastic because it replaces the yellow o Normocellular: 30-70% Hematopoietic marrow with red marrow. Stem Cells (HSCs) This occurs in conditions where there is o Hypercellular / Hyperplastic: >70% HSCs increased or ineffective hematopoiesis. o Hypocellular / Hypoplastic: 3.4 M:E Ratio: 1:1 (normal ratio is 1.5-3.3:1) Bilirubin and Lactate Dehydrogenase M:E ratio is Levels decreased due to o Hemolytic anemia due to ineffective increased hematopoiesis erythropoietic activity o Elevated unconjugated bilirubin, total bilirubin, and lactate dehydrogenase bands (RBC-derived) No consistent changes in o Release of hemoglobin from RBC megakarocytes released to increased bilirubin and There is increased lactate dehydrogenase levels erythropoietic activity but o In cases of megaloblastic anemia, maturation is defective there can be cases of hemolytic resulting to apoptosis anemia as well because precursors WBC precursors also undergo more apoptosis due to increase leading to ineffective erythropoiesis hypersegmented neutrophils o Protoporphyrin IX will be converted to Megakaryocytes are normally bilirubin increased in size so the Unconjugated bilirubin is change is not as significant; it increased is the largest hematopoietic Lactate dehydrogenase cell known to be a nonspecific This results to pancytopenia enzyme but it is known as the only hematological marker because it is greatly increased SEQUENCE OF DEVELOPMENT OF in hematological diseases MEGALOBLASTIC ANEMIA Increases in cases of leukemia, blood cell 1. Decrease in vitamin levels malignancies, 2. Hypersegmentation of neutrophils in peripheral hemolytic anemia and blood (hallmark) o We take note of it and their distribution pernicious anemia in the differential count of 100 WBCs Also a marker for 3. Oval macrocytes in peripheral blood acute myocardial 4. Megaloblastosis in bone marrow infarction 5. Anemia Highest elevation is due to megaloblastic ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 19  ASSAYS FOR FOLATE, VITAMIN B12, Transcobalamin I deficiency METHYLMALONIC ACID, AND HOMOCYSTEINE Megadose vitamin C therapy Although bone marrow aspiration is confirmatory for False Increases in Folate Assay Results megaloblastosis, the invasiveness of the procedure Recent meal and its expense mean that other testing is performed Alcoholism more often than a bone marrow examination False Decreases in Folate Assay Results The confirmation of megaloblastic morphology in the Severe anorexia requiring hospitalization marrow does not identify its cause Acute alcohol consumption Tests for serum levels of folate and vitamin B12 are Normal pregnancy readily available using immunoassay Anticonvulsant therapy o serum vitamin B12 may also be assayed by competitive binding chemiluminescence Some laboratories conduct a reflexive assay for MMA o however, there are a number of interferences if vitamin B12 levels are low with these assays that can cause false Vitamin B12 increased and decreased results o plays a role in folate metabolism o reflexive testing to MMA and homocysteine o a cofactor in the conversion of methylmalonyl (discussed later) can increase diagnostic CoA to succinyl CoA by the enzyme accuracy methylmalonyl CoA mutase RBC folate levels may also be measured o if deficient, methylmalonyl CoA accumulates Unlike serum folate levels, which fluctuate with diet, some of it hydrolyzes to methylmalonic RBC folate values are stable and may be a more acid, and the increase can be detected accurate reflection of true folate status in serum and urine o however, current RBC folate tests have less Because MMA is also elevated in patients with than optimal sensitivity and specificity and impaired renal function, the test is not specific, and have not been validated in actual patients thus increased levels cannot be definitively related to with normal and deficient folate levels vitamin B12 deficiency Thus the serum folate level is preferred over RBC Methylmalonic acid: folate level in the United States as the initial test for o assayed by gas chromatography-tandem evaluation of folate deficiency mass spectrometry Homocysteine levels: BOX 18.5 Causes of False Increases and o affected by deficiencies in either folate or Decreases of Vitamin B12 and Folate Assays vitamin B12 False Increases in Vitamin B12 Assay Results o 5-Methyl THF donates a methyl group to Assay technical failure homocysteine in the generation of Occult malignancy methionine Alcoholic liver disease this reaction uses vitamin B12 as a Renal disease Increased transcobalamin I and II binders (e.g., coenzyme myeloproliferative states, o a deficiency in either folate or vitamin B12 hepatomas, and fibrolamellar hepatic tumors) results in elevated levels of homocysteine Activated transcobalamin II-producing Total homocysteine: macrophages (e.g., autoimmune diseases, o measured in either plasma or serum monoblastic leukemias, and lymphomas) Homocysteine: Release of cobalamin from hepatocytes (e.g., o may be assayed by gas chromatography- active liver disease) mass spectrometry, high-performance liquid High serum anti-intrinsic factor antibody titer chromatography, or fluorescence False Decreases in Vitamin B12 Assay Results polarization immunoassay Haptocorrin deficiency o Homocysteine levels: are also elevated in Folate deficiency patients with renal failure and dehydration Plasma cell myeloma Human immunodeficiency virus Pregnancy Plasma cell myeloma ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 20 ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 21  TABLE 18.1 Laboratory Tests Used to Diagnose Vitamin B12 and Folate Deficiency FOLATE DEFICIENCY VITAMIN B12 DEFICIENCY Screening Tests Complete blood count Same as Folate Deficiency Manual differential count Same as Folate Deficiency Hypersegmented neutrophils, oval macrocytes, anisocytosis, poikilocytosis, RBC inclusions Absolute reticulocyte count Serum total and indirect bilirubin Serum lactate dehydrogenase Specific Bone marrow examination Erythroid hyperplasia (ineffective) Same as Folate Deficiency diagnostic tests Presence of megaloblasts Serum vitamin B12 N Serum folate RBC folate Serum methylmalonic acid N Serum plasma homocysteine Antibodies to intrinsic factor and Absent Present in pernicious anemia gastric parietal cells Serum gastrin N Can be markedly elevated in pernicious anemia Gastric analysis N Achlorhydria in pernicious anemia Holotranscobalamin assay N Stool analysis for parasites Negative Diphyllobotrium latum may be the cause of deficiency , Increased; , decreased; HCT, hematocrit; HGB, hemoglobin; MCH, mean cell hemoglobin; MCV, mean cell volume; N, within reference interval; PLT, platelet; RBC, red blood cell; WBC, white blood cell. *Bone marrow examination and gastric analysis are not usually required for diagnosis. Without vitamin B12, the cell is unable to produce intracellular polyglutamated tetrahydrofolate; therefore 5-methyltetrahydrofolate leaks out of the cell, which results in a decreased level of intracellular folate. Holotranscobalamin level is also decreased in transcobalamin deficiency. GASTRIC ANALYSIS AND SERUM GASTRIN Antibodies to intrinsic factor and parietal cells can be Gastric analysis: detected in the serum of most patients with pernicious o used to confirm achlorhydria, an expected anemia finding in pernicious anemia Various immunoassays can detect intrinsic factor- Achlorhydria: blocking antibodies o occurs in other conditions, however, o parietal cell antibodies can be detected by including natural aging indirect fluorescent antibody techniques or When other causes of vitamin B12 deficiency have enzyme-linked immunosorbent assays been eliminated, a finding of achlorhydria is Anti-intrinsic factor antibodies are highly specific and supportive, although not diagnostic, of pernicious confirmatory for pernicious anemia, but their absence anemia does not rule out the condition H1 concentration: The test for parietal cell antibodies is nonspecific and o determined by pH measurement not clinically useful for the diagnosis of pernicious As a result of the gastric achlorhydria, serum gastrin anemia levelscan be markedly elevated Serum gastrin: HOLOTRANSCOBALAMIN ASSAY o measured by immunoassay, including Holotranscobalamin is the metabolically active form chemiluminescent immunometric assays of vitamin B12 ANTIBODY ASSAYS ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 22  o until recently, methods for measuring o Detection of eggs or proglottids of D. holotranscobalamin were manual and not latum suitable for use in clinical laboratories o D. latum is a pseudophyllidian, and is non- Newer, more rapid immunoassays using monoclonal o Only egg is applicable for detection antibodies specific for holotranscobalamin have been developed in the past several years that are both sensitive and specific. Recent studies support the use of holotranscobalamin to detect vitamin B12 deficiency and recommend its use in screening for metabolic vitamin B12 deficiency STOOL ANALYSIS FOR PARASITES When vitamin B12 is found to be deficient, a stool analysis for eggs or proglottids of the fish tapeworm D. latum may be part of the diagnostic workup Confirmatory: Bone marrow aspiration MACROCYTIC NONMEGALOBLASTIC ANEMIAS Serum vitamin B12 Macrocytic nonmegaloblastic anemia: o Competitive chemiluminescence o macrocytic anemias in which DNA synthesis Reflexive testing to MMA and homocysteine is unimpaired o MMA: GC-MS (Gas Chromatography o macrocytosis tends to be mild Mass Spectrophotometry) o MCV: ranges from 100 to 110 fL and rarely o Homocysteine: GC-MS, HPLC (High Performance Liquid Chromatography) exceeds 120 fL o Both MMA and homocysteine are Patients with nonmegaloblastic, macrocytic anemia increased during deficiency of vitamin lack hypersegmented neutrophils and oval B12 and folate macrocytes in the peripheral blood and megaloblasts in the bone marrow OTHER SPECIFIC DIAGNOSTIC TESTS Macrocytosis: may be physiologically normal, as in Gastric Analysis and Serum Gastrin the newborn or the result of a pathologic condition, as o Confirmation of achlorhydria Supportive of pernicious in liver disease, chronic alcoholism, or bone marrow anemia failure Increased serum gastrin Reticulocytosis: a common cause of macrocytosis Due to decreased HCl due to achlorhydria; Unimpaired DNA synthesis Common cause: Reticulocytosis feedback to increase gastrin production to MCV: 100-110 fL (mild macrocytosis) produce more HCl Lack hypersegmented neutrophils and oval macrocytes in the peripheral blood Antibody Assays o Detection of Lack megaloblasts in bone marrow Anti-IF antibodies (specifically attacking intrinsic factors), Physiologic: Newborns anti-parietal cells antibodies Pathologic: o Methods: IFA, ELISA o Liver disease o Usually for vitamin B12 deficiency o Chronic alcoholism o Bone marrow failure Holotranscobalamin Assay o Holotranscobalamin metabolically active form of vitamin B12 TREATMENT o Recommended: screening for Treatment should be directed at the specific vitamin metabolic vitamin B12 deficiency deficiency established by the diagnostic tests and should include addressing the cause of the deficiency Stool Analysis for Parasites ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 23 (e.g., better nutrition, treatment for D. latum) if When proper trea possible response is prompt and brisk and can be used to Vitamin B12: confirm the accuracy of the diagnosis o may be administered intramuscularly to treat o bone marrow morphology will begin to revert pernicious anemia to bypass the need for to a normoblastic appearance within a few intrinsic factor hours of treatment o high-dose oral vitamin B12 treatment is o a substantial reticulocyte response is increasingly popular in the treatment of apparent at about 1 week pernicious anemia o hemoglobin increasing toward normal levels Regardless of the treatment modality, those with in about 3 weeks pernicious anemia or malabsorption must have o hypersegmented neutrophils disappear from lifelong vitamin replacement therapy the peripheral blood within 2 weeks of Folic acid: can be administered orally initiation of treatment The inappropriate treatment of vitamin B12 deficiency Thus with proper treatment, hematologic parameters with folic acid improves the anemia but does not may return to normal within 3 to 6 weeks and correct or stop the progress of the neurologic correction of the megaloblastic anemia may occur in damage, which may advance to an irreversible state 6 to 8 weeks Thus proper diagnosis before treatment is important Iron: often supplemented concurrently to support the rapid cell production that accompanies effective treatment ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 24 CHAPTER SUMMARY: TIBC, decreased or normal transferrin saturation, Impaired iron kinetics or heme metabolism can and normal or increased ferritin level. result in microcytic, hypochromic anemias. Sideroblastic anemias develop when the Three conditions affecting iron kinetics can result synthesis of protoporphyrin or the incorporation of in microcytic, hypochromic anemias: iron iron into protoporphyrin is blocked. The result is deficiency anemia, anemia of chronic accumulation of iron in the mitochondria of inflammation, and sideroblastic anemias, developing erythroblasts. When stained with especially lead poisoning. The red blood cells Prussian blue, the iron appears in deposits around (RBCs) in thalassemias also may be microcytic the nucleus of the developing erythroblasts in the and hypochromic, and this condition must be bone marrow. These cells are called ring differentiated from the anemias of disordered iron sideroblasts. metabolism. Protoporphyrin synthesis and iron incorporation Iron deficiency results from inadequate iron into protoporphyrin can be blocked when any of intake, increased need, decreased absorption, or the enzymes of the heme synthetic pathway are excessive loss. All four of these situations create deficient or impaired. Deficiencies of these a relative deficit of body iron that over time results enzymes may be hereditary, as in the porphyrias, in a microcytic, hypochromic anemia. or acquired, as in heavy metal poisoning. The Infants, children, and women of childbearing age most common of the latter conditions is lead are at greatest risk for iron deficiency anemia. If poisoning. iron deficiency anemia is present in men and Iron studies in sideroblastic anemias show postmenopausal women, the possibility of elevated levels of serum iron and serum ferritin, gastrointestinal bleeding should be investigated decreased or normal TIBC, and increased because it is the primary, although not the only, transferrin saturation. Test values for the cause of iron loss. accumulating products of the heme synthetic Iron deficiency is suspected when the complete pathway, such as zinc protoporphyrin (ZPP) or blood count (CBC) shows microcytic, free erythrocyte protoporphyrin (FEP), are also hypochromic RBCs and elevated RBC distribution elevated. width (RDW) but no consistent morphologic Lead interferes with several steps in heme abnormality. The diagnosis is confirmed with iron synthesis, preventing iron incorporation into heme studies showing low levels of serum iron and and resulting in a normocytic, normochromic serum ferritin, elevated total iron-binding capacity anemia, although with long-term exposure it can (TIBC), and decreased transferrin saturation. be microcytic and hypochromic. Lead also impairs Inadequate dietary iron is treated by oral the pentose-phosphate shunt, which adds a supplementation, and with good patient hemolytic component to the anemia. adherence, the anemia should be corrected within Children are especially vulnerable to the effects of 3 months. Gastrointestinal distress resulting from lead on the central nervous system, which may iron supplements can make patient adherence a result in irreversible brain damage. Treatment significant concern. Other causes of iron consists of removing the source of lead from the deficiency must be treated by eliminating the necessary, chelating underlying cause or with intravenous iron drug therapy to facilitate excretion of lead in the administration. urine. Central nervous system effects are typically Anemia of chronic inflammation is associated with not reversed with treatment. chronic infections such as tuberculosis, chronic Porphyrias are diseases in which porphyrin inflammatory conditions such as rheumatoid production is impaired. They can be acquired, arthritis, and malignancies. It may be a microcytic, such as lead poisoning, or inherited with mutations hypochromic anemia, but most often it is a mild affecting enzymes in the heme synthetic pathway. normocytic, normochromic anemia. Three hereditary porphyrias have hematologic In anemia of chronic inflammation, increased manifestations including anemia and fluorescent levels of hepcidin, an acute phase reactant, erythroblasts caused by accumulated porphyrins. decrease iron absorption in the intestines and Because the body has no mechanism for iron sequester iron in macrophages and hepatocytes. excretion, iron overload can occur when Bone marrow macrophages show abundant transfusions are used to sustain patients with stainable iron, whereas developing erythroblasts chronic anemias such as b-thalassemia major show inadequate iron (iron-restricted (called transfusion-related hemosiderosis). erythropoiesis). Inflammatory cellular products Iron-loading anemias are those with erythroid also impair the production and action of hyperplasia that increases levels of erythroferrone erythropoietin.RBC life span is shortened. leading to decreased hepcidin, increased Iron studies in the anemia of chronic inflammation ferroportin activity, and increased iron absorption. show decreased serum iron level, decreased A defective HFE gene can lead to hereditary hemochromatosis by decreased hepcidin ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 25  production. Affected men develop symptoms earlier in life than women; homozygotes develop more severe disease than heterozygotes. Mutations of other genes affecting iron regulation can produce a phenotype similar to that of hereditary hemochromatosis. When the hepcidin or hemojuvelin gene is mutated, the disease develops early in life, affecting even teenagers. Free iron becomes available in cells when ferritin and hemosiderin become saturated. Free iron causes tissue damage by creating free radicals that lead to cell membrane damage and perhaps mutations. The liver, pancreas, skin, and heart muscle are especially vulnerable to damage by excess iron deposition. Elevated transferrin saturation or serum ferritin can be an indicator of hemochromatosis that can be diagnosed fully using genetic testing to identify mutated genes. Hereditary hemochromatosis and similar diseases are treated by lifelong periodic phlebotomy to induce a mild iron deficiency anemia and keep body iron levels low. Transfusionrelated hemosiderosis must be treated with iron-chelating drugs. ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 26 References Anemia Caused by Defects of DNA Metabolism: University of Santo Tomas powerpoint presentation: Anemia Caused by Defects of DNA Metabolism Notes from the synchronous discussion by Mr. Clarenz Sarit M. Concepcion Notes from the asynchronous discussion by Mr. Ron Christian G. Sison es and Applications (6th ed.). Saunders. https://doi.org/10.1016/C2013-0-19483-4 ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 27  CHAPTER 19: BONE MARROW FAILURE PATHOPHYSIOLOGY OF BONE MARROW FAILURE Bone marrow failure is the reduction or cessation of o decreased production of hematopoietic blood cell production affecting one or more cell lines. growth factors or related hormones; and Pancytopenia, or decreased numbers of circulating o loss of normal hematopoietic tissue as a red blood cells (RBCs), white blood cells (WBCs), and result of infiltration of the marrow space with platelets, is seen in most cases of bone marrow abnormal cells. failure, particularly in severe or advanced stages. Clinical consequences of bone marrow failure vary, The pathophysiology of bone marrow failure depending on the extent and duration of the includes: cytopenias. o destruction of hematopoietic stem cells Severe pancytopenia can be rapidly fatal if as a result of injury by drugs, untreated. Some patients may initially be chemicals, radiation, viruses, or asymptomatic, and their cytopenia may be detected autoimmune mechanisms during a routine blood examination. o premature senescence and apoptosis of Thrombocytopenia can result in bleeding and hematopoietic stem cells increased bruising. as a result of genetic mutations; Decreased RBCs and hemoglobin can result in o ineffective hematopoiesis fatigue, pallor, and cardiovascular complications. caused by stem cell mutations or Sustained neutropenia increases the risk of life- vitamin B12 or folate deficiency threatening bacterial or fungal infections. o disruption of the bone marrow microenvironment that supports hematopoiesis Recorded Lecture: PATHOPHYSIOLOGY OF BONE MARROW Bone marrow failure causes rare symptoms characterized by the inability of the bone marrow to produce enough one or more cell lines. o Pancytomenia is often due to problems of the bone marrow but there might be other underlying causes. It occurs when all three cell lines (RBCs, WBCs, and platelets) are low in number. If only a single line is affected, it is always the erythrocytes. Bone marrow failure can be the following mechanisms: o Destruction of HSC (drugs, chemicals, radiation, viruses, autoimmune mechanisms or AIM) o Premature senescence or apoptosis of HSC (mutation) o Ineffective hematopoiesis (Stem cell mutation, vitamin deficiency) o Disruption of bone marrow microenvironment BM microenvironment is composed of the supporting cells: Osteoblasts Osteoclasts Osteocytes Mesenchymal stem cells Endothelial cells Fibroblasts Macrophages Reticular cells Sympathetic nerve cells, etc. o Decreased production of hematopoietic growth factors (HGF) or hormones Interleukins: IL-1, IL-3, IL-6 for RBCs, granulocyte and monocyte, IL-5 for eosinophil, and IL-2, IL- 4, IL-6, IL-7, IL-12 for lymphocyte ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 1  Colony stimulating factors: GM-CSF, G-CSF, M-CSF Ligands or stem cell factors: Flt3 ligands, alpha tumor necrosis factor, beta-1 transforming growth factor for lymphocyte o Loss of normal hematopoietic tissues (infiltration with abnormal cells) EXTENT AND DURATION OF CYTOPENIAS There are varying conditions of bone marrow failure that depend on both the extent and duration of pancytomenia. Initially, the condition can be undetected, thereby increasing the cases of fatality. Pancytomenias are often undetected during routine blood examination. Severe o Fatal if untreated o Bleeding and increased bruising caused by thrombocytopenia o Fatigue, pallor, CV complications caused by anemia: low RBC and hemoglobin levels o Bacterial infection caused by leukocytopenia, which is more often than not accompanied by neutropenia) APLASTIC ANEMIA Aplastic anemia is a rare but potentially fatal bone Chemicals marrow failure syndrome. In 1888, Ehrlich provided Insecticides the first case report of aplastic anemia involving a Cutting/lubricating oils patient with severe anemia, neutropenia, and a o Viruses Epstein-Barr virus hypocellular marrow on postmortem examination. Hepatitis virus (non-A, non-B, non- The name aplastic anemia was given to the disease C, non-G) by Vaquez and Aubertin in 1904. Human immunodeficiency virus The characteristic features of aplastic anemia o Miscellaneous conditions include pancytopenia, reticulocytopenia, bone Paroxysmal nocturnal marrow hypocellularity, and depletion of hemoglobinuria Autoimmune disease hematopoietic stem cells (Box 19.1). Pregnancy Approximately 80% to 85% of aplastic anemia cases Inherited/Congenital Bone Marrow Failure are acquired, whereas 15% to 20% are Syndromes (15% to 20% of Cases) inherited/congenital. Box 19.2 provides an etiologic Fanconi anemia classification. Dyskeratosis congenita Shwachman-Bodian-Diamond syndrome BOX 19.1 Characteristics Features of Aplastic Anemia Pancytopenia Reticulocytopenia Bone marrow hypocellularity Depletion of hematopoietic stem cells BOX 19.2 Etiologic Classification of Bone Marrow Failure Acquired Aplastic Anemia Idiopathic (70% of cases) Secondary (10%–15% of cases) o Dose dependent/predictable Cytotoxic drugs Benzene Radiation o Idiosyncratic Drugs (Box 19.3) ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 2  ACQUIRED APLASTIC ANEMIA Acquired aplastic anemia is classified into two Generally, idiosyncratic secondary aplastic major categories: idiopathic and secondary. anemia is a rare event and likely is due to a Idiopathic acquired aplastic anemia has no known combination of genetic and environmental factors in cause. susceptible individuals. Secondary acquired aplastic anemia is associated There are no readily available tests that predict with an identified cause. Approximately 70% of all individual susceptibility to these idiosyncratic aplastic anemia cases are idiopathic, whereas 10% reactions. However, genetic variations in immune to 15% are secondary. response pathways or metabolic enzymes may play Idiopathic and secondary acquired aplastic anemia a role. have similar clinical and laboratory findings. Patients may initially present with macrocytic or normocytic anemia and reticulocytopenia. BOX 19.3 Selected Drugs Reported to Have a Rare Pancytopenia may develop slowly or progress at a Association With Idiosyncratic Secondary rapid rate, with complete cessation of Aplastic Anemia Antiarthritics hematopoiesis. Gold compounds Penicillamine INCIDENCE In North America and Europe, the annual incidence Antibiotics is approximately 1 in 500,000. Chloramphenicol In Asia and East Asia, the incidence is two to three Sulfonamides times higher than in North America or Europe, which Anticonvulsants may be due to environmental and/or genetic Carbamazepine differences. Hydantoins Aplastic anemia can occur at any age, with peak Phenacemide incidence at 15 to 25 years and the second highest frequency at older than 60 years. There is no Antidepressants gender predisposition. Dothiepin Phenothiazine ETIOLOGY Antidiabetic Agents As the name indicates, the cause of idiopathic Chlorpropamide aplastic anemia is unknown. Tolbutamide Secondary aplastic anemia is associated with Carbutamide exposure to certain drugs, chemicals, radiation, or infections. Anti-inflammatories (Nonsteroidal) Cytotoxic drugs, radiation, and benzenes are Diclofenac responsible for 10% of secondary aplastic anemia Fenbufen cases and suppress the bone marrow in a Fenoprofen predictable, dose-dependent manner. Ibuprofen Indomethacin Depending on the dose and exposure duration, the Naproxen bone marrow generally recovers after withdrawal of Phenylbutazone the agent. Alternatively, approximately 70% of cases Piroxicam of secondary aplastic anemia occur as a result of Sulindac idiosyncratic reactions to drugs or chemicals. In idiosyncratic reactions the bone marrow failure is Antiprotozoals unpredictable and unrelated to dose. Chloroquine Documentation of a responsible factor or agent in Quinacrine these cases is difficult because evidence is primarily Antithyroidals circumstantial, and symptoms may occur months or Methimazole years after exposure. Methylthiouracil Some drugs associated with idiosyncratic secondary aplastic anemia are listed in Box 19.3. Carbonic Anhydrase Inhibitors ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 3  Methazolamide PATHOPHYSIOLOGY Mesalazine The primary lesion in acquired aplastic anemia is Acetazolamide a quantitative and qualitative deficiency of hematopoietic stem cells. There is an approximately twofold higher Stem cells of patients with acquired aplastic anemia incidence of human leukocyte antigen-DR2 (HLA- have diminished colony formation in methylcellulose DR2) and its major serologic split, HLA-DR15, in cultures. The hematopoietic stem and early aplastic anemia patients compared with the general progenitor cell compartment is identified by population, but the relationship of this finding to expression of CD34 surface antigens. disease pathophysiology has not been elucidated. The CD34+ cell population in the bone marrow of There are also reports that genetic polymorphisms patients with acquired aplastic anemia can be 10% in enzymes that metabolize benzene increase or lower than that seen in healthy individuals. In susceptibility to toxicity, even at low exposure levels. addition, these CD34+ cells have increased These include polymorphisms in glutathione S- expression of Fas receptors that mediate apoptosis transferase (GST) enzymes (GSTT1 and GSTM1), and increased expression of apoptosis-related myeloperoxidase, nicotinamide adenine dinucleotide genes. phosphate (reduced form, NADPH), quinine Bone marrow stromal cells are functionally normal oxidoreductase 1, and cytochrome oxidase P450 in acquired aplastic anemia. They produce normal or 2E1. even increased quantities of growth factors and are A deficiency in GST as a result of the GSTT1 null able to support the growth of CD34+ cells from genotype is overrepresented in Caucasians, healthy donors in culture and in vivo after Hispanics, and Asians with aplastic anemia, with a transplantation. frequency of 30%, 28%, and 75%, respectively. Individuals with aplastic anemia also have elevated Caucasian patients with aplastic anemia also have serum levels of erythropoietin, thrombopoietin, a higher frequency (22%) of the GSTM1/GSTT1 null granulocyte colony-stimulating factor (G-CSF), genotype than the general population. and granulocytemacrophage colony-stimulating GST is important for metabolism and factor (GM-CSF). In addition, serum levels of FLT3 neutralization of chemical toxins, and deficiencies ligand, a growth factor that stimulates proliferation of of this enzyme may increase the risk of aplastic stem and progenitor cells, is up to 200 time higher in anemia. Further study is required to assess how patients with severe aplastic anemia compared with these genetic variations, and other yet undiscovered healthy controls. factors, contribute to aplastic anemia. However, despite their elevated levels, growth Acquired aplastic anemia occurs occasionally as a factors are generally unsuccessful in correcting the complication of infection with Epstein-Barr virus, cytopenias found in acquired aplastic anemia. human immunodeficiency virus (HIV), hepatitis The severe depletion of hematopoietic stem and virus, and human parvovirus B19. progenitor cells from the bone marrow may be due In 2% to 10% of patients with acquired aplastic to direct damage to stem cells, immune damage to anemia, there is a history of acute non-typable stem cells, or other unknown mechanisms. hepatitis (non-A, non-B, etc.) occurring 1 to 3 Direct damage to stem and progenitor cells months before the onset of pancytopenia; this is results from deoxyribonucleic acid (DNA) injury after thought to represent autoimmune hepatitis. The exposure to cytotoxic drugs, chemicals, radiation, or acquired aplastic anemia in these cases may be viruses mediated by such mechanisms as interferon- Immune damage to stem cells results from (IFN- ) and c tokine release. exposure to drugs, chemicals, viruses, or other Aplastic anemia associated with pregnancy is a agents that cause an autoimmune cytotoxic T rare occurrence, with fewer than 100 cases lymphocytic destruction of stem and progenitor cells. reported in the literature. Approximately 10% of An autoimmune pathophysiology was first suggested individuals with acquired aplastic anemia have a in the 1970s when aplastic anemia patients concomitant autoimmune disease and approximately undergoing pretransplant immunosuppressive 10% develop hemolytic or thrombotic manifestations conditioning had an improvement in cell counts. of paroxysmal nocturnal hemoglobinuria (PNH). is Further evidence supporting an autoimmune discussed later. pathophysiology include (1) elevated blood and bone ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 4  marrow cytotoxic (CD8+) T lymphocytes with an telomeres undergo proliferation arrest and oligoclonal expansion of specific T cell clones; (2) premature apoptosis. increased T cell production of such cytokines as IFN- Telomerase is an enzyme complex that repairs and g and tumor necrosis factor-a (TNF-a), which inhibit maintains telomeres. Approximately 10% of patients hematopoiesis and induce apoptosis; (3) with acquired aplastic anemia and shortened upregulation of T-bet, a transcription factor that binds telomeres have a mutation in the telomerase to the promoter of the IFN-g gene30; (4) increased complex gene for either the ribonucleic acid (RNA) TNF-a receptors on CD34+ cells; and (5) template (TERC) or the reverse transcriptase improvement in cytopenias after immunosuppressive (TERT). therapy (IST). The cause for shortened telomeres in the other 90% Approximately two-thirds of patients with acquired of patients may be due to stress hematopoiesis or aplastic anemia respond to IST. The non- other yet unidentified mutations. In stress responders may have a severely depleted stem hematopoiesis, there is an increase in progenitor cell cell compartment or other pathophysiologic turnover, and the telomeres become shorter with factors contributing to their cytopenias. each cell division. Possible autoimmune mechanisms include Approximately 4% of patients with acquired aplastic mutation of stem cell antigens and disruption of anemia and shortened telomeres have mutations in immune regulation. Young and colleagues showed the Shwachman-Bodian-Diamond syndrome that environmental exposures may alter self- (SBDS) gene. proteins, induce expression of abnormal or novel The SBDS gene product is involved in ribosome antigens, or induce an immune response that cross- biogenesis, and its relationship to telomere reacts with self-antigens. maintenance is currently unknown. TERT/TERC and Solomou and colleagues. demonstrated that SBDS mutations also occur in the inherited bone CD4+CD25+FOXP3+ regulatory T cells are marrow failure syndromes dyskeratosis congenita decreased in aplastic anemia. These regulatory T (DC) and SBDS, respectively, and some patients cells normally suppress autoreactive T cells, and a diagnosed with acquired aplastic anemia who have deficit of these cells may facilitate an autoimmune these mutations may actually have DC or SBDS. reaction. Correct differentiation between acquired aplastic Furthermore, a number of individuals with aplastic anemia and inherited bone marrow failure anemia have single nucleotide polymorphisms in syndromes has important implications for IFN- /+874 TT, TNF- /–308 AA, transforming growth appropriate treatment and prognosis. factor- 1/–509 TT, and interleukin-6/–174 GG. Immunosuppressive therapy is not nearly as These polymorphisms result in cytokine effective in inherited bone marrow failure compared overproduction and may impart a genetic with acquired aplastic anemia. Furthermore, susceptibility to aplastic anemia as well as hematopoietic stem cell transplantation, the only contribute to its severity. known curative treatment for DC and SBDS and a The specific antigens responsible for triggering and treatment option for acquired aplastic anemia, sustaining the autoimmune attack on stem cells are should not be performed with HLA-matched siblings unknown. Candidate antigens have been identified who test positive for the same genetic mutation. from aplastic anemia patient sera, including Shortened telomeres occur more often in patients kinectin, diazepam-binding inhibitor-related whose pancytopenia does not respond to protein 1, and moesin. These proteins are immunosuppressive therapy. expressed in hematopoietic progenitor cells, but their Defective telomere maintenance may be another role in the pathogenesis of aplastic anemia requires pathophysiologic mechanism of stem cell injury, further investigation. imparting susceptibility to aplastic anemia after an Approximately one-third of patients with acquired environmental insult. aplastic anemia have shortened telomeres in their peripheral blood granulocytes compared with age- matched controls. Telomeres protect the ends of chromosomes from damage and erosion, and cells with abnormally short ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 5  CLINICAL FINDINGS and percent transferrin saturation may be increased, Symptoms vary in acquired aplastic anemia, which reflects decreased iron use for erythropoiesis. ranging from asymptomatic to severe. Liver function test results may be abnormal in Patients usually present with symptoms of cases of hepatitis-associated aplastic anemia. insidious-onset anemia, with pallor, fatigue, and Approximately two-thirds of patients have small weakness. numbers (less than 25%) of PNH clones in the Severe and prolonged anemia can result in serious peripheral blood, but only 10% of patients develop a cardiovascular complications, including tachycardia, sufficient number of PNH cells to have the clinical hypotension, cardiac failure, and death. and biochemical manifestations of PNH disease. Symptoms of thrombocytopenia are also varied PNH is characterized by an acquired stem cell and include petechiae, bruising, epistaxis, mucosal mutation resulting in lack of the bleeding, menorrhagia, retinal hemorrhages, glycosylphosphatidylinositol (GPI)-linked proteins intestinal bleeding, and intracranial hemorrhage. CD55 and CD59. Fever and bacterial or fungal infections are unusual at initial presentation but may occur after prolonged periods of neutropenia. Splenomegaly and hepatomegaly are typically absent. LABORATORY FINDINGS Pancytopenia is typical, although initially only one or two cell lines may be decreased. The absolute neutrophil count is decreased, and the absolute lymphocyte count may be normal or decreased. The hemoglobin is usually less than 10 g/dL, the mean cell volume (MCV) is increased or normal, and the percent and absolute reticulocyte counts are decreased. Table 19.1 lists the diagnostic criteria for aplastic anemia by degree of severity. The absence of CD55 and CD59 on the surface of the RBCs renders them more susceptible to TABLE 19.1 Diagnostic Criteria for Aplastic complement-mediated cell lysis. It is important to test Anemia for PNH in acquired aplastic anemia because of the MAA SAA VSAA increased risk of hemolytic or thrombotic Bone Hypocellular Bone marrow Same as SAA Marrow bone marrow Cellularity < complications. plus at least two 25%* plus at Historically, PNH diagnosis depended on the Ham of the least two of following the following: acid hemolysis test: Patients cells were placed in Neutrophils 0.5-1.5 0.2-0.5 3 mg/dL, Reticulocyte count >10 o Require regular transfusions ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 12  PERIPHERAL BLOOD FILM/SMEAR: o Spherocytes: Hallmark of HS o Microspherocytes: small, round, dense RBCs filled with Hgb, no central pallor. o Anisocytosis, Poikilocytosis are present. Presence of variability in size and shape o RBC Morphology variations depending on the mutated gene: B-spectrin: Acanthocytes (irregular projections on the red blood cells) Band 3: Pincered or Mushroom shaped cells EPB4.2: Ovalostomatocytes o ***Spherocytes are NOT specific for HS. It can also be observed in other types of hemolytic anemia Spherocytes (refer to pointed arrows) RBC INDICES: o MCHC: > 36 mg/dL (for moderate to severe) o MCV: WIR (within reference range) to slightly below Serum haptoglobin: Decreased o Due to increased extravascular hemolysis Serum indirect Bilirubin: Increased Serum lactate dehydrogenase: Increased Bone marrow exam: o Erythroid Hyperplasia due to increased demand for RBC to replace prematurely destroyed spherocytes o Usually not required for diagnosis OSMOTIC FRAGILITY TEST Also known as EOFT (Erythrocyte Osmotic Fragility Test) Demonstrates Increased RBC Fragility on RBCs with decreased surface area-to-volume ratios. RBCs are subjected to increasing hypotonic solutions. Shift to the LEFT, Increased Osmotic Fragility o Initial hemolysis starts at concentration > 0.5 Shift to the RIGHT, Decreased Osmotic Fragility In cases of increased surface area to volume ratio such as in cases of thalassemia Disadvantages: Time consuming, Needs Fresh Heparinized sample collected without trauma, accurately prepared NaCl solutions. o Hemoconcentration should not be observed Refer to graph: o In 0.85 NaCl, the amount of water entering the cell is equivalent to water leaving the cell o As solution becomes hypotonic, more water will enter the cell causing it to burst o In HS, they would lyse more easily in less hypotonic solutions due to its decreased surface area to volume ratio ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 13  Osmotic fragility test graph Cartoon about principle of OFT Spherocytes have decreased surface area to volume ratio, medyo mataba na sila so kapag sinubject sa hypotonic solution, ayaw nila ng water Spherocytes cannot retain water anymore so they would burst Normal discoid RBCs have enough room to expand as water enters the cells Spherocytes would have earlier hemolysis EOSIN-5’-MALEIMIDE BINDING TEST Confirmatory test for HS. More sensitive alternative to EOFT. EMA binds to transmembrane proteins (Band3, Rh, RHAg and CD47) Measures Mean Fluorescence Intensity. HS: decreased intensity The fluorescent dye would bound to transmembrane proteins, the intensity of fluorescence would decrease in absence of transmembrane proteins OTHER TESTS D SDS-PAGE o Electrophoresis that would identify the membrane protein deficiencies RADIOIMMUNOASSAY o For quantification OSMOTIC GRADIENT EKTACYTOMETRY o Used to check for the variation in membrane surface area and cell water content ACIDIFIED GLYCEROL TEST o The amount of hemolysis is also measured AUTOHEMOLYSIS TEST o Hemolysis is measured; samples are incubated with and without glucose HYPERTONIC CRYOHEMOLYSIS TEST ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 14  o HS patients are particularly sensitive to cooling lalo na at 0 degrees Celsius and hypertonic solutions MOLECULAR STUDIES o Detection of genetic mutations COMPLICATIONS HEMOLYTIC CRISIS o rare associated with viral syndromes i.e. Infectious Mononucleosis. Spleen enlarges, sluggish blood flow and destroys RBCs. APLASTIC CRISIS o Marked decrease in reticulocytes. Associated with Parvovirus B19 infection, destroys progenitor cells in BM and suppresses erythropoiesis. o Leads to anemia and eventually to decreased reticulocytes MEGALOBLASTIC CRISIS o Increased Folate utilization. Particularly acute during pregnancy, and recovery from aplastic crisis CHOLETHIASIS o Due to increase in bilirubin o There will be bilirubin stones in the gallbladder CHRONIC ULCERATION o When spheroc tes cannot go to capillaries, the o ld form clots (also termed as dermatitis of the legs ) o Rare complication TREATMENT Walang treatment sa mild HS, sa moderate and severe lang Regular Transfusion o To replace blood with normal discoid RBCs Splenectomy o Without the spleen, life of spherocytes can be prolonged o There is increased risk of infection since spleen is in charge in helping to protect from infection DIFFERENTIAL DIAGNOSIS HS: Negative DAT (Direct Antiglobulin Test) o DAT would be positive in immune disorders EMA Binding Test ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 15  HEREDITARY ELLIPTOCYTOSIS Hereditary elliptocytosis (HE) is a heterogeneous damaged RBCs become trapped or acquire further group of hemolytic anemias caused by defects in damage in the spleen, which results in extravascular proteins that disrupt the horizontal or lateral hemolysis and anemia. interactions in the protein cytoskeleton. o In general, patients who are heterozygous It reportedly exists in all of its forms in 1 in 2000 to 1 for a mutation are asymptomatic, and their in 4000 individuals in the United States, but because RBCs have a normal life span; those who the majority of cases are asymptomatic, the actual are homozygous for a mutation or prevalence is not known. compound heterozygous for two mutations The disease is more common in Africa and can have moderate to severe anemia that Mediterranean regions, where there is a high can be life threatening. prevalence of malaria. Elliptocytosis can also be seen in patients with the o The prevalence in West Africa of certain Leach phenotype who lack Gerbich antigens and spectrin mutations associated with HE is glycoprotein C (GPC). between 0.6% and 1.6%.13 o The phenotype is due to a mutation in the o The molecular basis for the association of genes for GPC and results in the absence of elliptocytosis and malaria is unknown. the glycoprotein in the RBC membrane. The inheritance pattern in HE is mainly autosomal o The Gerbich antigens are normally dominant. expressed on the extracellular domains of GPC and thus are absent in this condition. PATHOPHYSIOLOGY Heterozygotes have normal RBC morphology, and HE results from gene mutations in which the homozygotes have mild elliptocytosis but no anemia. defective proteins disrupt the horizontal linkages in The reason for the elliptocyte morphology may be a the protein cytoskeleton and weaken the mechanical defect in the interaction between GPC and protein stability of the membrane. 4.1 in the actin junctional complex. The HE phenotype can result from various The RBCs in HE patients all show some degree of mutations in at least three genes: decreased thermal stability. Cases in which the o SPTA1 RBCs show marked RBC fragmentation on heating which codes for α-spectrin were previously classified as HPP. 65% of cases o HPP is now considered a severe form of HE o SPTB that exists in either the homozygous or which codes for -spectrin compound heterozygous state. 30% of cases o EPB41 CLINICAL AND LABORATORY FINDINGS which codes for protein 4.1 The vast majority of patients with HE are 5% of cases asymptomatic, and only about 10% have moderate A mutation database for HE is available and lists to severe anemia. 46 different mutations. The spectrin mutations Some may have a mild compensated hemolytic disrupt spectrin dimer interactions and the EPB41 anemia, as evidenced by a slight increase in the mutations result in weakened spectrin interactions reticulocyte count and a decrease in haptoglobin with actin junctional complexes. level, or develop transient hemolysis in response to RBCs are biconcave and discoid at first but become other conditions such as viral infections, pregnancy, elliptical over time after repeated exposure to the hypersplenism, or vitamin B12 deficiency. shear stresses in the peripheral circulation. The Often an asymptomatic patient is diagnosed after extent of the disruption of the spectrin dimer a peripheral blood film is examined for another interactions seems to be associated with the severity condition. of the clinical manifestations. Rarely, heterozygous parents with undiagnosed, In severe cases the protein cytoskeleton is asymptomatic HE have offspring who are weakened to such a point that cell fragmentation homozygous or compound heterozygous for their occurs. As a result, there is membrane loss and a mutations and have moderate to very severe decrease in surface area-to-volume ratio that hemolysis. Some of these asymptomatic parents reduces the deformability of the RBCs. The have normal RBC morphology and laboratory tests. ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 16  The characteristic finding in HE is elliptical or o Normal RBCs do not fragment until cigarshaped RBCs on the peripheral blood film in reaching a temperature of 49° C. numbers that can vary from a few to 100% (Figure Thermal sensitivity is not specific for HPP, 21.6). however; it also occurs in cases of HE with spectrin mutations. RBCs with the HPP phenotype show a lower fluorescence than RBCs in HS when incubated with eosin-5’-maleimide and analyzed by flow cytometry. Mutation screening using molecular tests or quantification of membrane proteins by SDS-PAGE may be also be performed. As in HS, patients with moderate or severe hemolytic anemia as a result of HE can develop cholelithiasis because of bilirubin gallstones, and hemolytic, aplastic, and megaloblastic crises can occur. The number of elliptocytes does not correlate with disease severity. Investigation of elliptocytosis begins by taking a thorough: o patient and family history, o performing a physical examination, o and examining the peripheral blood films of the parents Other laboratory tests may be needed to rule out other conditions in which elliptocytes may be present, such as: o iron deficiency o anemia o thalassemia o megaloblastic anemia o myelodysplastic syndrome, and o primary myelofibrosis In these cases the elliptocytes usually comprise less than one-third of the RBCs. An acquired defect in the gene for protein 4.1 may be found in myelodysplastic syndrome. In the homozygous or compound heterozygous states the anemia is moderate to severe, the osmotic fragility is increased, and biochemical evidence of excessive hemolysis is present. TREATMENT The peripheral blood film in patients with the HPP Asymptomatic HE patients require no phenotype shows extreme poikilocytosis with treatment. HE patients who are significantly fragmentation, microspherocytosis, and anemic and show signs of hemolysis respond elliptocytosis similar to that in patients with thermal well to splenectomy. Transfusions are burns. occasionally needed for life-threatening anemia. o The MCV is very low (50 to 65 fL) because of the RBC fragments. RBCs in the HPP phenotype show marked thermal sensitivity. After incubation of a blood specimen at 41° C to 45° C, the RBCs fragment. ENCISCO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 17  Recorded Lecture: HEREDITARY ELLIPTOCYTOSIS Mainly Autosomal dominant trait. Deficient Protein/ Mutated Gene: a- Spectrin, B-Spectrin, Protein 4.1 Weaken the Mechanical Stability of the membrane More common in Africa and Mediterranean regions Leads to easily fragmented RBCs PATHOPHYSIOLOGY Defects/ Mutations in proteins that disrupt the horizontal/ lateral interactions in the protein cytoskeleton. Normal, discoid RBCs become elliptical from repeated exposure to shear stress in the peripheral circulation = ELLIPTOCYTES (Elliptical/ Cigar Shaped RBCs) o Decreased surface area-to-volume ratio. Reduced deformability. o Survival in spleen is decreased. (membrane loss or become trapped) Elliptocytes Elliptocytosis in patients with Leach Phenotype o Lacks Gerbich Antigens and GPC o Mutations in Glycoprotein C = defect in GPC and protein 4.1 interactions. o Defect in linking between protein 4.1 and the actin junctional complex, leading to elongation and elliptocyte morphology Hereditary Pyropoikilocytosis o Severe form of Heredita