Hema Interview Week 2 PDF

MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 ERYTHROCYTE PRODUCTION AND FUNCTION 1. What are RBCs? Recognize and identify the cells in the erythrocytic series. The red blood cell (RBC), o...

MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 ERYTHROCYTE PRODUCTION AND FUNCTION 1. What are RBCs? Recognize and identify the cells in the erythrocytic series. The red blood cell (RBC), or erythrocyte, provides a classic example of the biological principle that cells have specialized functions and that their structures are specific for those functions. The erythrocyte has one true function: to carry oxygen from the lung to the tissues, where the oxygen is re- leased. This is accomplished by the attachment of the oxygen to hemoglobin, the major cytoplasmic component of mature RBCs. The role of the RBC in returning carbon dioxide to the lungs and buffering the pH of the blood is important but is quite secondary to its oxygen-carrying function. To pro- tect this essential life function, the mechanisms controlling development, production, and normal destruction of RBCs are fine tuned to avoid interruptions in oxygen delivery, even under adverse conditions such as blood loss with hemorrhage. — Rodaks Hematology Clinical Principles and Applications 1. PRONORMOBLAST (RUBRIBLAST) - Earliest precursor identifiable by light microscopy - N:C ratio of 8:1 - Cytoplasm is dark blue because of the concentration of ribosomes and RNA - Undergoes mitosis and give rise to 2 daughter pronormoblasts - Nucleus is round to oval, containing 1-2 nucleoli - Located in BM - Globin production begins 2. BASOPHILIC PRONORMOBLAST - N:C ratio of 6:1 - The chromatin begins to condense 16 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 - Cytoplasm may be a deeper richer blue than in the pronormoblast, hence the name basophilic - Undergoes mitosis - Located in BM - Detectable Hemoglobin synthesis occurs 3. POLYCHROMATIC NORMOBLAST - N:C ratio 4:1 to about 1:1 by the end of the stage - Murky gray blue - Polychromatophilic means “many color loving” - Last stage of cell division - Located in BM - Hemoglobin synthesis increases 4. ORTHOCHROMATIC - Nucleus is completely condense (pyknotic) - N:C ratio of 1:2 - Salmon pink due to nearly complete hemoglobin production. - Not capable of division because of the condensation of the chromatin 5. RETICULOCYTE - No nucleus - Residual of RNA and ribosomes that why may bluish tinge pa sya - Cannot divide - Retained in the BM for 1-2 days and then move to peripheral blood about 1 day. 6. MATURE ERYTHROCYTE - No nucleus - 7-8 microns - Life span 120 days - Biconcave shape - Salmon pink (Wright stain) Identify the areas of RBC metabolism crucial for normal erythrocyte survival and function. GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS) Three alternate pathways, called diversions or shunts, branch from the glycolytic pathway. Hexose Monophosphate Pathway the HMP is a vital metabolic pathway in RBCs that plays a crucial role in protecting the cells from oxidative stress. A deficiency in G6PD, the rate-limiting enzyme of the HMP, can lead to hemolytic anemia. Understanding the HMP and its role in RBC function is essential for diagnosing and managing various hematological disorders. 17 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Methemoglobin Reductase Pathway is essential for maintaining the oxygen-carrying capacity of RBCs. If this pathway is impaired, methemoglobin levels can rise, leading to methemoglobinemia. This condition can cause symptoms such as cyanosis, fatigue, headache, and shortness of breath. In summary, the methemoglobin reductase pathway is a critical metabolic process that ensures the proper functioning of hemoglobin and the delivery of oxygen to tissues. Rapoport-Leubering Pathway also known as the 2,3-bisphosphoglycerate (2,3-BPG) shunt, is a metabolic pathway that occurs exclusively in red blood cells (RBCs). It is a variant of glycolysis that bypasses the 1,3-bisphosphoglycerate kinase step. It is a vital metabolic pathway in RBCs that regulates hemoglobin's oxygen affinity, ensuring efficient oxygen delivery to tissues. 3. Describe the function of biochemical substances that compose the RBC membrane. BIOCHEMIC PERCENTA FUNCTIONS AL GE SUBSTANC E Proteins 52% Structural, functional and transport roles, ensuring cell stability, shape and gas exchange Lipids 40% RBC membrane with flexibility and fluidity forming a semi-permeable barrier Carbohydra 8% Immune recognition, negative (-) surface charge and blood tes group antigenicity 4. List the steps in extravascular and intravascular breakdown of RBCs. EXTRAVASCULAR DESTRUCTION Macrophages-mediate hemolysis Occurs in mononuclear phagocytic cells of the spleen, liver and bone marrow Phagocytosis and removal of aged RBCs Release of hemoglobin Majority of RBC follow this type of destruction Steps in Extravascular Destruction 1. RBC lysis within macrophage 18 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 2. Major components are catabolized 3. Iron is removed from the heme, stored as ferritin until transported out. The iron will be recycled and removed from the heme. This will be stored as ferritin until it will be transported out and will be used for the formation of hemoglobin 4. Globin is degraded and returned to the metabolic amino acid pool. Usually, the metabolic amino acid pool would be in the liver 5. Protoporphyrin is degraded to bilirubin- released into plasma and excreted as bile. INTRAVASCULAR DESTRUCTION Mechanical hemolysis or fragmentation Small portion of RBCs rupture within the lumen of blood vessels Steps in Intravascular Destruction: 1. Hemoglobin dissociate into alpha-B dimer bound to haptoglobin 2. Carried to the liver and undergo extravascular RBC destruction 3. Filtered and reabsorbed by PCT 4. Reached renal threshold and appear in the urine as free Hb - The kidney tubules have threshold for certain substances to be reabsorbed 5. Iron in the tubular cell complex with hemosiderin and excreted in the urine. 6. Cleared directly by hepatic uptake or oxidized to methemoglobin: heme bound to hemopexin HEMOGLOBIN DETERMINATION 1. What is hemoglobin? What is its importance? Hemoglobin Serves as the heart of red blood cell physiology Occupies 95% of the RBC cytoplasm Its main function is to transport oxygen to tissues and transport CO2 to the lungs for exhalation. Conc in RBC: 34g/dL Molecular weight: 64,000 D or 64 KD It is a complex protein found within red blood cells that plays a crucial role in oxygen transport throughout the body. It consists of four subunits, each containing a heme group with an iron atom at its center. This iron atom is responsible for binding to oxygen molecules in the lungs and releasing them to tissues that require oxygen for cellular respiration. 19 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Conjugated globular protein: ○ 4 heme groups ○ 2 heterogeneous pair of polypeptide (globin) chains ○ Each heme molecule is attached to each globin chain forming a tetramer ○ 2-alpha & 2 beta surrounding a globin group, tetrahedral Hemoglobin's primary function is to efficiently transport oxygen from the lungs to the body's tissues. It also plays a role in transporting carbon dioxide, a waste product of cellular respiration, from the tissues back to the lungs for exhalation. Hemoglobin helps maintain the acid-base balance of the blood by binding to hydrogen ions. It contributes to the biconcave shape of red blood cells, which maximizes their surface area for gas exchange. 2. How is hemoglobin formed? HEME BIOSYNTHESIS Mitochondria and Cytoplasm of the bone marrow erythrocyte precursors, heme and porphyrin synthesis occurs. Erythrocyte precursors: Pronormoblast through the circulating reticulocytes (polychromatophilic erythrocyte) Assembly of iron and porphyrin: Mitochondria Assembly of heme group and globin chain: Cytoplasm Steps: 1. Mitochondria Glycine and succinyl coenzyme A (CoA) assembly in the catalyzed by aminolevulinate synthase to form aminolevulinic acid (ALA) ; Glycine + CoA = ALA 2. Cytoplasm ALA undergoes several transformations from porphobilinogen (PBG) to coproporphyrinogen III catalyzed by coproporphyrinogen oxidase,becomes protoporphyrinogen IX ○ PBG → coproporphyrinogen III + coproporphyrinogen oxidase → protoporphyrinogen IX 3. Mitochondria Protoporphyrinogen IX is converted to protoporphyrin IX by protoporphyrinogen oxidase ○ i. Protoporphyrinogen IX + protoporphyrinogen oxidase → protoporphyrin IX 20 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Ferrous (Fe2+) ion is added, catalyzed by ferrochelatase (heme synthase) to form heme Protoporphyrin IX + ferrous ion + ferrochelatase → heme Iron Transport from Plasma to Erythroid Precursors Apo-Tf - a transferrin that does not carry or bind iron Transferrin - carries or bind iron Steps: 1. Transferrin carries iron (ferric Fe3+) from the plasma to developing erythroid cells Transferrin molecule has 2 binding sites for iron. 1 Transferrin molecule can carry 2 molecules ferric type of iron 2. Fe3+ binds to transferrin receptors on erythroid precursor cell membranes Transferrin receptor can bind 4 iron molecules and 2 transferrin 3. The receptors and transferrin (with bound iron) are brought into the cell in an endosome 4. Acidification of the endosome releases the iron from transferrin Usually, the acidified endosome would need H+ to acidify until pH 5.5. 5. Iron is transported out of the endosome and into the mitochondria where it is reduced to the Ferrous state, and is united with protoporphyrin IX to make heme. Transferrin receptor will go back to the cell membrane. Transferring will go back to plasma and called as “Apo-Tf”, and will become transferrin again once there is a need to carry iron. 6. Heme leaves the mitochondria and is joined to the globin chains in the cytoplasm GLOBIN SYNTHESIS Globin biosynthesis happens in the Nucleus of erythroid precursor: ○ Transcription of the globin genes to messenger ribonucleic acid (mRNA) happens in the nucleus ○ Transcription occurs to make a globin polypeptide chain ○ The chains are released in the Ribosomes Cytoplasmic ribosomes: Translation of mRNA to the globin polypeptide chain ○ Chains are released from the ribosomes in the cytoplasm, and they would be assembled to be incorporated with heme molecule (to make the heme structure complete) 3. What are the normal and abnormal derivatives of hemoglobin? Normal Hemoglobin Derivatives 1. Oxyhemoglobin (HbO2): ○ The oxygenated form of hemoglobin. 21 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 ○ Predominant form in arterial blood. ○ Responsible for transporting oxygen from the lungs to tissues. 2. Deoxyhemoglobin (Hb): ○ The deoxygenated form of hemoglobin. ○ Predominant form in venous blood. ○ Releases oxygen to tissues. Abnormal Hemoglobin Derivatives 1. Carboxyhemoglobin (COHb): ○ Formed when hemoglobin binds to carbon monoxide (CO). ○ CO has a higher affinity for hemoglobin than oxygen, impairing oxygen transport. ○ Can lead to carbon monoxide poisoning, especially in environments with high CO levels (e.g., smoke inhalation, industrial exposure). ○ Cherry red color ○ Carboxyhemoglobin may be detected by spectral absorption instruments at 540 nm 2. Methemoglobin (MetHb): ○ Occurs when the iron in hemoglobin is oxidized from Fe2+ to Fe3+. ○ Methemoglobin cannot bind oxygen effectively. ○ Cannot carry oxygen because oxidized ferric ion cannot bind it ○ Can be caused by certain drugs, toxins, or genetic disorders. ○ Chocolate brown ○ Methemoglobin is assayed by spectral absorption analysis instruments such as CO-oximeter 3. Sulfhemoglobin: ○ Formed when hemoglobin reacts with sulfur compounds. ○ Cannot transport oxygen. ○ Formed by the addition of sulfur atom to the pyrrole ring of heme and has a greenish pigment ○ Can be caused by exposure to certain drugs or chemicals. ○ Mauve lavender 4. What are hemoglobinopathies? Enumerate and explain each. Hemoglobinopathies are a group of inherited blood disorders that affect the structure or production of hemoglobin, the protein in red blood cells that carries oxygen. They are primarily caused by mutations in the genes that code for hemoglobin. Types of Hemoglobinopathies 22 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 1. Structural Hemoglobinopathies (Qualitative): ○ Hemoglobin S (HbS) Disease: This is the most common form of structural hemoglobinopathy, particularly prevalent in individuals of African descent. It results from a single amino acid substitution in the beta-globin chain. The abnormal hemoglobin molecules polymerize, causing red blood cells to become sickle-shaped, leading to hemolytic anemia, pain crises, and organ damage. ○ Hemoglobin C Disease: This is another common structural hemoglobinopathy, particularly prevalent in individuals of African and Mediterranean descent. It results from a single amino acid substitution in the beta-globin chain. Individuals with HbC disease typically have a milder form of hemolytic anemia compared to those with sickle cell disease. ○ Hemoglobin E Disease: This is a common hemoglobinopathy in Southeast Asia. It results from a single amino acid substitution in the beta-globin chain. Individuals with HbE disease usually have a mild microcytic anemia. 2. Thalassemias (Quantitative): ○ Alpha-thalassemia: This disorder results from a decreased production of alpha-globin chains. The severity of the disease depends on the number of affected genes. ○ Beta-thalassemia: This disorder results from a decreased production of beta-globin chains. It can range from mild to severe, with severe forms requiring lifelong blood transfusions. Laboratory Diagnosis of Hemoglobinopathies Complete Blood Count (CBC) Peripheral Blood Smear Hemoglobin Electrophoresis High-Performance Liquid Chromatography (HPLC) DNA Analysis 5. Explain the oxygen dissociation curve. Hemoglobin readily bind O2 molecules in the lungs ○ Lungs: ↑affinity ○ Tissues: ↓affinity 1 gram hemoglobin : 1.34 mL O2 O2 affinity relates with partial pressure of O2 (PO2) Relationship is described by the Oxygen Dissociation Curve 23 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 X-axis: O2 partial pressure Y-axis: %O2 saturation Normally, PO2 of approximately 27 mm Hg results in 50% O2 saturation of the hemoglobin molecule Curve can shift from left to right ↓ %O2 saturation ↓ O2 partial pressure Shift to the left Shift to the right Conditions in the lungs Conditions in the tissues ↑ O2 tension and saturation ↓ O2 tension and saturation ↑ hemoglobin-O2 affinity ↓ hemoglobin-O2 affinity ↓ blood pH ↑ blood pH ↑ pCO2 ↓ pCO2 ↑ 2,3-BPG ↓ 2,3-BPG ↑ H + ions ↓ H + ions ↑ temperature ↓ temperature ↑ abnormal hemoglobins with lower ↑ Hb F (high affinity to O2 but is not affinity to O2 readily released causing hypoxia) ↑ myoglobin (tighter affinity with O2) ↑ abnormal hemoglobins with higher affinity to O2 Bohr Effect 24 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Shift to the curve due to a change in pH of H+ concentration In the lungs, pH is high and O2 affinity is high, which favors uptake or binding of O2 In the tissues, pH is low and O2 affinity is low, which favors release of O2 6. Describe the procedure in performing the Sahli-Adams and cyanmethemoglobin methods. Identify the appropriate reagents, their composition and function. Sahli Adams Method Direct visual colorimetric method Known hemoglobin is converted into a brown colored solution of acid hematin in the presence of a dilute acid Acid hematin solution is diluted with distilled water drop by drop until the color matches that of the standard in the comparator block Reagents Used 1. 0.1 N Hydrochloric Acid (HCl) - used to convert hemoglobin to soluble acid hematin 2. Distilled water - diluent used for matching color with the standard Steps of Sahli Adams Method: 1. Place 0.1 N hydrochloric acid up to the 2 mark on the gram scale of a graduated Sahli-Adams hemometer. 2. Draw a well-mixed anticoagulated whole blood into a Sahli-Adams pipette up to the 0.02 mL (20 µL) mark. Most preferred tube is EDTA 3. Wipe the outside portion of the pipette. Always remember not to wipe the tip of the pipette so that blood would not be absorbed and the volume will not change. If not done, results will falsely increase 4. Transfer the blood sample into the hemometer tube containing 0.1 N HCl making sure that the pipette is rinsed with the mixture at least 3 times. 3 times: to ensure that the sample is incorporated 5. Let it stand for at least 10 minutes. Hemoglobin will be converted into acid hematin 6. Place the hemometer tube into the comparator block and carefully add distilled water drop by drop. In this step, hemoglobin is already converted to soluble acid hematin 7. Mix the contents using a stirrer and compare the color of the diluted sample with the color of the standard. 8. When the color of the diluted sample matches that of the standard, read the height of the diluted sample directly on the gram scale of the hemometer tube. 25 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 End point: when the solution in the hemometer matches with the standard comparator block The height of the volume as it matches with the comparator block is directly proportional to the quantity of hemoglobin in the sample. 9. Report the result in grams per 100 mL of blood or grams %. The height corresponds to the volume of hemoglobin. Ex. when you have the 13th mark or 13 in the calibration mark, therefore it is 13.0 grams per 100 mL of blood or 13 grams % of hemoglobin Cyanohemoglobin Method (Hemoglobin Cyanide Method/ Ferricyanide Method) Not performed in the laboratory Indirect method Uses spectrophotometer: more accurate A mix of cyanine method and Ferricyanide method Recommended manual procedure for hemoglobin determination Utilizes the Drabkin’s solution which is sensitive to light (Thus Cyanmethemoglobin is stored in amber bottles) Principle: Blood is diluted in an alkaline Drabkin’s solution of potassium ferricyanide, sodium bicarbonate and a surfactant. The ferricyanide converts the hemoglobin iron from ferrous to ferric state to form methemoglobin which then combines with potassium cyanide to form the stable pigment,cyanmethemoglobin. The color intensity of the mixture is directly proportional to the hemoglobin concentration Final color of solution: cyan or blue Drabkin’s Solution Components 1. (Potassium ferricyanide) K3Fe(CN)3 – oxidizes hemoglobin to methemoglobin 2. (Potassium cyanide) KCN – converts methemoglobin to cyanohemoglobin 3. Surfactant & Detergent – minimizes turbidity (from lipoproteins) & enhances lysis of methemoglobin 4. (Sodium bicarbonate/ Dihydrogen potassium phosphate) KH2PO4 – allows test to be read after 3 minutes instead of 15 minutes Modified Drabkin’s Solution is the other name of Dihydrogen Potassium Phosphate. This maintains stability of reactions 7. Give the standard reference values for hemoglobin. Male: 13.5-18.0 g/dL (2.09-2.71 mmol/L) Female: 12.0-16.0 g/dL (1.86-2.48 mmol/L) 26 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 RETICULOCYTE COUNT 1. What are reticulocytes? What is the clinical value or reticulocyte count? State the principle of reticulocyte count. - Also known as polychromatophilic erythrocytes - Young RBCs that contain RNA - Resides in bone marrow for 1 to 2 days and 1 day in the circulation 1. Reticulocytes are immature red blood cells (RBCs) that have recently been released from the bone marrow. They contain remnants of RNA and other cellular organelles, which give them a distinctive appearance under a microscope when stained with specific dyes. 2. It is a laboratory test that measures the percentage of reticulocytes in your blood. It provides valuable information about the bone marrow's ability to produce new red blood cells. 3. Reticulocyte counting involves staining the blood sample with a supravital dye, such as new methylene blue. This dye stains the residual RNA in reticulocytes, making them appear as reticulated cells under a microscope. The number of reticulocytes is then counted and expressed as a percentage of the total number of red blood cells. Key Points: Reticulocytes are immature red blood cells. A reticulocyte count helps assess bone marrow function. A low reticulocyte count may indicate decreased bone marrow activity. A high reticulocyte count may suggest increased bone marrow activity. Supravital staining is used to identify reticulocytes. 2. Describe the procedure in performing the manual reticulocyte count. Identify the appropriate stains used and their compositions. Principle: Supravital stains (New Methylene Blue, Brilliant Cresyl Blue) bind, neutralize, and cross-link RNA. They cause the ribosomal and residual RNA to co-precipitate with few remaining mitochondria and ferritin masses in living young erythrocytes to form microscopically visible, dark-blue clusters and filaments. Procedure; 27 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 1. Mix equal amounts of blood and New Methylene Blue (2-3 drops or 50 ul each) in a clean, dry test tube. 2. Incubate the tube at room temperature for 10 to 15 minutes or in water bath at 37 degree celsius for 5 to 10 minutes 3. Remix the preparation after incubation 4. Prepare blood smear 5. Count reticulocytes seen within 1000 RBCs under OIO in areas where cells are close together but not touching each other 6. Calculate the reticulocyte count 3. Explain how to calculate for the following: relative value, absolute value, CRC, and RPI and identify its standard reference values. Relative Value Definition: The percentage of a specific cell type in relation to the total number of cells counted. Calculation: Relative Value (%) = (Number of specific cells / Total number of cells) x 100 Example: If you count 10 neutrophils in a total of 100 white blood cells, the relative value of neutrophils is 10%. Absolute Value Definition: The actual number of a specific cell type per unit volume of blood. Calculation: Absolute Value = Relative Value (%) x Total WBC count Example: If the relative value of neutrophils is 10% and the total WBC count is 10,000 cells/µL, the absolute neutrophil count is 1,000 cells/µL. Corrected Reticulocyte Count (CRC) Definition: A corrected measure of reticulocyte production, adjusted for the patient's hematocrit level. 28 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Calculation: CRC = (Reticulocyte count (%) x Patient's hematocrit) / Normal hematocrit Standard Reference Value: Typically, a CRC of 1-3% is considered normal. Reticulocyte Production Index (RPI) Definition: A more refined measure of reticulocyte production, considering the maturation time of reticulocytes. Calculation: RPI = CRC / Maturation time (days) Standard Reference Value: An RPI of 1 indicates normal bone marrow production. A value greater than 1 suggests increased production, while a value less than 1 indicates decreased production. 4. What is a calibrated Miller Disc? How is it used? A calibrated Miller disc is a specialized tool used in microscopy, particularly in hematology, to facilitate the accurate measurement of small objects, such as cells. It is a disc marked with a series of calibrated circles, allowing for easy size comparison of microscopic specimens. In the context of reticulocyte count, the calibrated Miller disc helps standardize the process of counting reticulocytes—immature red blood cells that contain residual RNA and can be identified using specific stains. By using the Miller disc, a technician can ensure that the area being observed under the microscope is consistent, leading to more accurate counts of reticulocytes within a defined volume of blood. This method is important for assessing bone marrow function and the body's response to anemia. In summary, the calibrated Miller disc aids in obtaining precise measurements and counts in various hematological assessments, including reticulocyte evaluation. Miller Disk An ocular device to facilitate counting of reticulocyte Reduces labor-intensive process of Reticulocyte count Usually mounted into eyepiece of microscope 29 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 RBCs counted in smaller square (square B - 1/9 of large square) ○ Minimum: 112 cells Reticulocyte is counted in larger square (square A) ○ Minimum: 1008 cells Formula: ○ Reticulocytes % = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶𝑒𝑙𝑙𝑠 𝑖𝑛 𝑆𝑞𝑢𝑎𝑟𝑒 𝐴 (𝐿𝑎𝑟𝑔𝑒𝑟) 𝑥 100 / 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶𝑒𝑙𝑙𝑠 𝑖𝑛 𝑆𝑞𝑢𝑎𝑟𝑒 𝐵 (𝑆𝑚𝑎𝑙𝑙𝑒𝑟) 𝑥 9 ○ 100 and 9 are constant 5. Identify the potential sources of error when performing manual reticulocyte count. Subjective Interpretation: The identification of reticulocytes can be subjective, especially for less experienced technicians. Counting Accuracy: Inaccurate counting of reticulocytes can lead to significant errors in the final result. Dye Quality: The quality of the supravital stain, such as new methylene blue, can affect the staining intensity of reticulocytes. Staining Time: Improper staining time can lead to under- or overstaining, affecting visibility. Slide Preparation: Inadequate slide preparation, including poor smear quality or excessive drying, can compromise cell morphology and staining. Hemolysis: Hemolysis can interfere with staining and cell morphology. Platelet Clumping: Platelet clumps can be mistaken for reticulocytes, leading to falsely elevated counts. Errors in Calculations: Mistakes in calculations, especially when determining the dilution factor and calculating the final percentage, can significantly affect the result. 30 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Temperature and Humidity: Extreme temperature and humidity can affect the staining process and cell morphology. 6. Identify the conditions that may influence the increase and decrease of reticulocyte values. Conditions that Increase Reticulocyte Values Hemolytic Anemias: Conditions where red blood cells are destroyed faster than they can be replaced, such as sickle cell anemia, autoimmune hemolytic anemia, or drug-induced hemolysis. Acute Blood Loss: Significant blood loss triggers the bone marrow to increase red blood cell production. Iron Deficiency Anemia (Early Stage): In the early stages of iron deficiency anemia, the bone marrow may compensate by increasing reticulocyte production. Treatment Response: Effective treatment for anemia, such as iron supplementation or vitamin B12 therapy, can lead to a rise in reticulocyte count. Conditions that Decrease Reticulocyte Values Aplastic Anemia: A condition where the bone marrow fails to produce enough blood cells. Chronic Kidney Disease: Kidney disease can impair the production of erythropoietin, a hormone that stimulates red blood cell production. Severe Anemia: In severe anemia, the bone marrow may become overwhelmed and unable to increase reticulocyte production. Bone Marrow Failure Syndromes: Conditions like myelodysplastic syndromes can affect bone marrow function and reduce reticulocyte production. ANEMIA 1. How is anemia define.Discuss the laboratory diagnosis of anemia Anemia is a condition characterized by a decrease in the number of red blood cells or hemoglobin in the blood. This reduction in oxygen-carrying capacity can lead to various symptoms, including fatigue, weakness, and shortness of breath. 31 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 1. Red Blood Cell Indices: ○ Hemoglobin (Hb): Measures the oxygen-carrying capacity of blood. ○ Hematocrit (Hct): Represents the percentage of red blood cells in blood volume. ○ Mean Corpuscular Volume (MCV): Indicates the average size of red blood cells. ○ Mean Corpuscular Hemoglobin (MCH): Measures the average amount of hemoglobin in a single red blood cell. ○ Mean Corpuscular Hemoglobin Concentration (MCHC): Reflects the average concentration of hemoglobin in a given volume of red blood cells. 2. Reticulocyte Count: ○ Evaluates the bone marrow's response to anemia by measuring the number of immature red blood cells. 3. Peripheral Blood Smear: ○ Visualizes the morphology of red blood cells, white blood cells, and platelets. ○ Helps identify specific types of anemia, such as iron deficiency anemia, megaloblastic anemia, or hemolytic anemia. Interpretation of Results: Microcytic Anemia: Characterized by small, pale red blood cells. ○ Common causes: Iron deficiency anemia, thalassemia, and lead poisoning. Macrocytic Anemia: Characterized by large red blood cells. ○ Common causes: Vitamin B12 deficiency, folate deficiency, and certain liver diseases. Normocytic Anemia: Characterized by normal-sized red blood cells. ○ Common causes: Chronic kidney disease, chronic inflammation, and acute blood loss. Additional Tests: Iron Studies: To assess iron status, including serum iron, ferritin, and total iron-binding capacity (TIBC). Vitamin B12 and Folate Levels: To evaluate for deficiencies in these essential nutrients. Coombs' Test: To detect autoimmune hemolytic anemia. Bone Marrow Examination: To assess bone marrow function and identify specific abnormalities. 32 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 2. What are the mechanisms of anemia? What are the different approaches to evaluating anemia? Decreased Red Blood Cell Production: Deficiency Anemias: ○ Iron deficiency anemia ○ Vitamin B12 deficiency anemia ○ Folate deficiency anemia Bone Marrow Failure: ○ Aplastic anemia ○ Myelodysplastic syndromes Chronic Disease Anemia: ○ Chronic inflammation ○ Chronic kidney disease Increased Red Blood Cell Destruction (Hemolytic Anemias): Intrinsic Defects: ○ Hereditary spherocytosis ○ Sickle cell anemia ○ Thalassemia Extrinsic Defects: ○ Immune hemolytic anemia ○ Drug-induced hemolytic anemia ○ Microangiopathic hemolytic anemia Blood Loss: Acute blood loss (e.g., trauma, surgery) Chronic blood loss (e.g., gastrointestinal bleeding, menorrhagia) Approaches to evaluate anemia Clinical History: Assess symptoms like fatigue, weakness, shortness of breath, and pallor. Inquire about factors such as diet, medications, and medical history. Identify potential causes, like recent blood loss, chronic diseases, or family history of anemia. Physical Examination: Look for signs of anemia, including pallor, tachycardia, and splenomegaly. 33 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Assess vital signs, such as blood pressure and heart rate. Laboratory Tests: Complete Blood Count (CBC): Evaluate red blood cell indices, hemoglobin, hematocrit, and white blood cell count. Reticulocyte Count: Assess bone marrow response to anemia. Peripheral Blood Smear: Examine red blood cell morphology, including size, shape, and color. Iron Studies: Measure serum iron, ferritin, and total iron-binding capacity to assess iron status. Vitamin B12 and Folate Levels: Evaluate for deficiencies in these nutrients. Coombs' Test: Detects antibodies that may be causing hemolytic anemia. Bone Marrow Examination: In some cases, a bone marrow biopsy may be necessary to identify specific causes of anemia. 3. Discuss the disorders of iron kinetics and heme metabolism. A. Iron-restricted anemias Impaired production resulting from the lack of raw materials for hemoglobin assembly Iron is the rate limiting factor Microcytic, Hypochromic, Insufficient erythropoiesis, Cytoplasmic maturation abnormality Iron deficiency anemia: ○ Etiology Anemia of chronic inflammation B. Sideroblastic anemias C. Iron overload 7. Describe the instrinsic defects leading to increased erythrocyte descrtruction A. Red cell membrane abnormalities B. Red blood cell enzymopathie Intrinsic defects are abnormalities within the red blood cell itself that lead to its premature destruction. 34 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 1. Membrane Defects: Hereditary Spherocytosis: A genetic disorder characterized by a deficiency in spectrin, a protein essential for maintaining the red blood cell's biconcave shape. This deficiency leads to the formation of spherocytes, which are more fragile and susceptible to destruction. Hereditary Elliptocytosis: Another genetic disorder resulting from defects in spectrin, band 3, or protein 4.1. This causes red blood cells to become elliptical or oval-shaped, making them more rigid and prone to hemolysis. 2. Hemoglobinopathies: Sickle Cell Disease: A genetic disorder caused by a point mutation in the hemoglobin gene, resulting in the production of abnormal hemoglobin S. Under low-oxygen conditions, hemoglobin S polymerizes, causing red blood cells to become sickle-shaped and fragile. Thalassemia: A group of genetic disorders characterized by reduced or absent production of hemoglobin chains. This imbalance leads to the formation of unstable hemoglobin molecules and the production of abnormal red blood cells. 3. Enzyme Deficiencies: Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency: A common enzyme deficiency that affects the red blood cell's ability to maintain its reduced glutathione level. This deficiency makes red blood cells more susceptible to oxidative stress, leading to hemolysis. Pyruvate Kinase Deficiency: A less common enzyme deficiency that impairs the red blood cell's ability to generate ATP. This can lead to a decreased cell membrane flexibility and increased susceptibility to hemolysis. 8. Describe the intrinsic defects leading to increased erythrocyte destruction caused by nonimmune causes A. Macroangiopathic by nonimmune causes B. Microangiopathic hemolytic anemias C. Other non-immune causes of erythrocyte destruction 35 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Intrinsic defects refer to abnormalities within the red blood cell itself that make it susceptible to premature destruction. 1. Membrane Defects: Hereditary spherocytosis: A genetic disorder characterized by a deficiency in spectrin or ankyrin, leading to a loss of membrane surface area and the formation of spherocytes. These spherocytes are more rigid and fragile, making them prone to premature destruction in the spleen. Hereditary elliptocytosis: A genetic disorder resulting from defects in spectrin, protein 4.1, or glycophorin C, causing the red cells to become elliptically shaped. While these cells are generally less fragile than spherocytes, they can be more susceptible to mechanical stress and hemolysis. Hereditary stomatocytosis: A rare disorder characterized by a defect in cation permeability, leading to increased sodium and calcium influx into the red cell. This can result in cell dehydration, increased membrane rigidity, and hemolysis. Abetalipoproteinemia: A rare inherited disorder characterized by a deficiency in lipoproteins, including cholesterol and phospholipids, essential for maintaining red cell membrane integrity. This can lead to acanthocytosis, a condition where red cells have irregularly shaped projections, and increased hemolysis. 2. Hemoglobinopathies: Sickle cell disease: A genetic disorder caused by a point mutation in the beta-globin gene, resulting in the production of abnormal hemoglobin S. Under conditions of low oxygen tension, hemoglobin S polymerizes, causing red cells to become sickle-shaped. Sickle cells are rigid and fragile, leading to hemolysis and vascular occlusion. Hemoglobin C disease: A less severe hemoglobinopathy caused by a point mutation in the beta-globin gene, resulting in the production of hemoglobin C. Hemoglobin C can form crystals within red cells, leading to their deformation and destruction. Unstable hemoglobin disorders: A group of disorders characterized by the production of unstable hemoglobin variants that are prone to denaturation and precipitation. This can lead to hemolysis and the formation of Heinz bodies, which are inclusions within red cells. Thalassemia: A group of genetic disorders characterized by a decreased production of one or more globin chains, leading to a relative excess of the other chain. This imbalance can result in the formation of ineffective erythropoiesis and hemolytic anemia. 36 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 9. Describe the extrinsic defects leading to increased erythrocyte destruction caused by immune causes A. Autoimmune hemolytic anemia Four major categories: ○ Warm Autoimmune Hemolytic Anemia ○ Cold Agglutinin Disease ○ Paroxysmal Cold Hemoglobinuria ○ Mixed-Type Autoimmune Hemolytic Anemia B. Alloimmune hemolytic anemia C. Drug-induced hemolytic anemia Immune-mediated hemolytic anemias (IMHAs) are a group of disorders characterized by the premature destruction of red blood cells (RBCs) due to autoimmune processes. The immune system mistakenly identifies RBC antigens as foreign, leading to the production of autoantibodies. These autoantibodies bind to the RBC surface, triggering their destruction through various mechanisms: 1. Complement-Mediated Hemolysis: Classical Pathway: Autoantibodies bind to the RBC surface, activating the classical complement pathway. Membrane Attack Complex (MAC) Formation: This leads to the formation of MAC, which inserts into the RBC membrane, causing cell lysis. 2. Phagocytosis: Opsonization: Autoantibodies and complement components coat the RBCs, making them attractive targets for phagocytosis by macrophages in the spleen and liver. Ingestion and Degradation: Phagocytes engulf and degrade the opsonized RBCs. 3. Antibody-Dependent Cellular Cytotoxicity (ADCC): NK Cell Activation: Autoantibodies bind to the RBC surface, activating natural killer (NK) cells. Cytotoxic Granule Release: NK cells release cytotoxic granules containing perforin and granzymes, which induce cell death. Clinical Manifestations of IMHAs: Anemia: Reduced RBC count due to increased destruction. 37 MLS INTERNSHIP: HEMATOLOGY ORAL ASSESSMENTS BS MEDICAL LABORATORY SCIENCE 1st SEMESTER A.Y. 2024-2025 Hemoglobinuria: Presence of free hemoglobin in the urine, resulting from intravascular hemolysis. Jaundice: Increased bilirubin levels due to the breakdown of hemoglobin. Splenomegaly: Enlargement of the spleen due to increased phagocytic activity. Laboratory Findings in IMHAs: Peripheral Blood Smear: ○ Spherocytes: Small, dense RBCs without central pallor. ○ Schistocytes: Fragmented RBCs. ○ Polychromasia: Increased number of immature RBCs. ○ Reticulocytosis: Increased number of reticulocytes, indicating bone marrow response to anemia. Direct Antiglobulin Test (DAT): Positive test indicates the presence of antibodies or complement components on the RBC surface. Coombs' Test: Another name for the DAT. Indirect Antiglobulin Test (IAT): Detects the presence of circulating autoantibodies in the patient's serum. Treatment of IMHAs: Corticosteroids: Suppress the immune system and reduce autoantibody production. Immunosuppressive Drugs: Used in severe cases to further suppress the immune system. Splenectomy: Removal of the spleen, a major site of RBC destruction. Intravenous Immunoglobulin (IVIG): Provides passive immunity and may block autoantibody binding to RBCs. Rituximab: A monoclonal antibody targeting CD20-positive B cells, may be used in refractory cases. 38