HEMA LEC 🩸 2 - Hematology Lecture Notes PDF

| Topic # 1| [MLS 417-LEC] Hematology 2 - Lecture P1: NON MALIGNANT LEUKOCYTE DISORDERS Professor: Chessa Louise L. Belandres, RMT, MD | Prof. Lee-An Anayon, RMT Date: January 22, 2024 MORPHOLOGIC CHANGES Often the body's response to various diseases and toxic challenges ○ If there are infection/mal...

| Topic # 1| [MLS 417-LEC] Hematology 2 - Lecture P1: NON MALIGNANT LEUKOCYTE DISORDERS Professor: Chessa Louise L. Belandres, RMT, MD | Prof. Lee-An Anayon, RMT Date: January 22, 2024 MORPHOLOGIC CHANGES Often the body's response to various diseases and toxic challenges ○ If there are infection/malignancies → there are changes in cell morphology to attack foreign materials going inside the body The type of cell affected depends on its function Automated hema analyzers do not detect specific abnormalities ○ The medical technologist will be the one to countercheck MORPHOLOGIC ABNORMALITIES OF LEUKOCYTE Toxic Granulation Neutrophil Hypersegmentation Dohle Bodies May-Hegglin Anomaly Lysosomal Storage Diseases Alder-Reilly Anomaly Pelger-Huët Anomaly TOXIC GRANULATION Prominent dark granulation, either fine or heavy, can be observed in the band and segmented neutrophils or monocytes ○ represent the precipitation of ribosomal protein (RNA) caused by metabolic toxicity within the cells Present granules: Azurophilic (primary) granules that are peroxidase-positive. Associated with infectious states, in conditions such as burns and malignant disorders, or as the result of drug therapy. Represent precipitation of ribosomal protein (RNA) due to metabolic toxicity within the cells Found in burns, severe infections, cancer (malignancy), hematoma, tissue undergoing necrosis, or as a result of drug therapy Extent of toxic granulation is usually graded on a scale of 1+ to 4+ being the most severe. Grading depends on the coarseness and amount of granulation within cytoplasm Number of affected neutrophils also correlates w/ C-Reactive Protein which is a marker if there is inflammation. DOHLE BODIES seen near the periphery of the cytoplasm of neutrophils, although they may be seen in monocytes or lymphocytes. represent aggregates of rough endoplasmic reticulum (RNA) Remnants are arranged in a parallel manner. These are the intracytoplasmic pale blue, round inclusion, seen near the periphery of the cytoplasm of neutrophils but may also be seen in monocytes or lymphocytes ○ Delay in preparation of smears make the inclusion more grey than blue or may not be visible at all aggregates of RER (remnants of rRNA) may be seen in conjunction with toxic granuation may also be seen normally but in small amounts associated with burns, infectious disease, scarlet fever, aplastic anemia, but nonspecific because it can also be seen in pregnancy Nonspecific Conditions where Döhle bodies are seen: 1. Bacterial infections 2. Sepsis 3. Pregnancy 4. Viral infections 5. Burns 6. Intake of certain drugs HYPERSEGMENTATION Autosomal dominant (Steininger) → asymptomatic More than five lobes or nuclear segments most frequently seen in segmented neutrophils with more than five lobes or nuclear segments frequently associated with deficiencies of vitamin B12 or folic acid and exists along with abnormally enlarged, oval-shaped erythrocytes ○ ACQUIRED HYPERSEGEMENTATION: associated w/ Vit. B12 deficiency Visible abnormalities in RBC Undritz Anomaly HYPERsegmentation of Neutrophils (>5 lobes) if Acquired hypersegmentation: most often associated with MegaloblasFc Anemia (Vit B9 or B12 deficiency) Hereditary Neutrophil Hypersegmentation non megaloblastic anemia no clinical problems; non-pathologic PELGER HUËT ANOMALY (PHA) / NUCLEAR HYPOSEGMENTATION Benign, Autosomal dominant disorder decreased nuclear segmentation (Hyposegementation) ○ distinctive coarse chromatin clumping pattern mutation in the lamin β-receptor gene ○ plays a major role in leukocyte nuclear shape changes Neutrophils in PHA appear to function normally In heterozygous PHS, individuals are clinically normal, while in homozygous PHS, cognitive impairment, heart defects, and skeletal abnormalities may occur. Pelger-Huët (PH) nuclei may appear round, ovoid, or peanut-shaped HYPOlobulation / HYPOsegmentation (2 lobes) of neutrophils: failure of segmentation of granulocytic nuclei ○ Pince-nez (Spectacle-like) ○ Peanut ○ Dumbbell ○ cells have normal function @mlstranses | 1 Pelger-Huët Anomaly TRUE PHA VS PSEUDO-PHA Myelocyte/Metamyelocyte Cell size is smaller Relatively bigger N:C ratio is lower N:C ratio is higher # of Affected Clls Chromatin is darker, more coarse, more densely clumped Colorless cytoplasm TRUE PHA PSEUDO-PHA 63 - 93% myeloid bias —> Pelger-Huët anomaly (peanut shaped) - One of the important characteristics we have to observe for in the Pelger-Huët Anomaly is the “Pince-nez” morphology (Spectacle like) Nuclear chromatin condenses —> segments disappear —> dark-staining spheres Apopotopic nucleus Dying neutrophils Pyknotic Nucleus - X or Sex Chromatin Darkly stained structure in the nucleus most often found in the periphery of the nucleus, sometimes in other parts AKA: X or Sex Chromatin Deactivated or inactive X chromosome → also known as Barr bodies ○ Usually involved in normal female cells Since the female contains two x chromosomes – the other chromosome is inactivated – sometimes it forms like a barr body In males, barr bodies are not that visible since they only have one X chromosome – it is always active Some disorders, where there is an excess X chromosome in males – barr bodies will be present, because the other X will be deactivate @mlstranses | 2 For three X chromosomes, the other two will be inactivated AUER RODS Pink or red stained needle like crystals in the cytoplasm of myeloid cells Agglomeration of primary granules Positive for: Myeloperoxidase, esterase, acid phosphatase Diagnostic of a myeloid neoplasm (Acute myeloid leukemia) FAGGOT CELLS Usually larger and rounder in shape (vs. Dohle bodies in yellow) Auer rods form Clusters Suggestive of Acute promyelocytic leukemia Usually forms in the cytoplasm of promyelocytes or blast MAY HEGGLIN ANOMALY autosomal dominant disorder variable thrombocytopenia, giant platelets, and large Döhle body-like inclusions in neutrophils, eosinophils, basophils, and monocytes mutation in the MYH9 gene on chromosome 22q12-13 ○ Disorderly production in the Myosin Heavy Chain Type IIA affects megakaryocyte maturation and platelet fragmentation when shedding from megakaryocytes Most individuals with May Hegglin anomaly are asymptomatic, but a few have mild bleeding tendencies related to the degree of thrombocytopenia Large, hypogranular platelets. Thrombocytopenia (approx 40- 80 x 10^9/L) Prolonged BT The inclusion in the red arrow is thought to be made up of mRNA, but it is actually the precipitation/accumulation of Myosin in the heavy chain that is present. How to differentiate Döhle bodies from the May-Hegglin Anomalies? Döhle-body like inclusion in MHA is usually larger, and rounder in shape vs. Döhle LYSOSOMAL STORAGE DISEASE group of more than 50 inherited enzyme deficiencies resulting from mutations in genes that code for the production of lysosomal enzymes. flawed degradation of phagocytized material and buildup of undigested substrates within lysosomes All cells containing lysosomes can be affected LSD include 1. sphingolipidoses, 2. oligosaccharidoses, 3. mucolipidoses, 4. mucopolysaccharidoses (MPS), 5. lipoprotein storage disorders, and 6. lysosomal transport defects MUCOPOLYSACCHARIDOSES caused by deficient activity of an enzyme necessary for the degradation of dermatan sulfate, heparan sulfate, keratan sulfate, and/or chondroitin sulfate Function is not affected Results in serious physical and cognitive problems and shortened survival @mlstranses | 3 SPHINGOLIPIDOSES Gaucher’s disease Niemann pick’s disease GAUCHER’S DISEASE defect or deficiency in the catabolic enzyme b-glucocerebrosidase (gene located at 1q21-q22) ○ Enzyme is necessary for glycolipid metabolism accumulation of unmetabolized substrate sphingolipid glucocerebroside in macrophages throughout the body, including osteoclasts in bone and microglia in the brain. (lungs,liver, spleen briain) b-glucosidase (glucocerebrosidase) is available to confirm diagnosis Treatment of Gaucher disease includes enzyme replacement therapy with recombinant glucocerebrosidase GAUCHER CELLS – abundant fibrillar blue-gray cytoplasm with a striated or wrinkled appearance (sometimes described as onion skin-like) STAINS POSITIVE WITH 1. Trichome 2. Aldehyde function 3. Periodic Acid Schiff 4. Acid phosphatase STAINS POSITIVE WITH 1. Sudan Black B 2. Oil Red O FOAM CELL – macrophage with lipid-filled lysosomes that appear as small vacuoles; nucleus is eccentric autosomal recessive deficiency of the enzyme sphingomyelinase recessive mutaFons in the SMPD1 gene ○ A deficiency of acid Sphingomyelinase (ASM) and a subsequent buildup of the sphingomyelin in the liver, spleen, lungs Defective NPC1 and NPC2 genes ○ Regulate intracellular processing and transport of LDL-derived cholesterol large cells filled with lipid droplets Accumulation of sphingomyelin in lysosomes Foam cells: macrophages whose cytoplasm is swollen by numerous lipid droplets in the bone marrow SEA BLUE HISTIOCYTOSIS adult form of niemann pick disease and chronic granulocytic leukemia histiocytes filled with lipid rich granules that stain blue-green with polychrome stain such as giemsa or wright Accumulation of phosphoshingolipids in cytoplasm autosomal recessive most common lysosomal lipid storage diseases abundant fibrillary blue-gray cytoplasm with striated or wrinkled appearance (“onion skin-like”) B-glucosidase (glucocerebrosidase) is available to confirm diagnosis treatment: Enzyme replacement therapy with recombinant glucocerebrosidase NIEMANN-PICK DISEASE (FOAM CELLS) accumulation of fat in cellular lysosomes of vital organs recessive mutations in the SMPD1 gene — a deficiency of acid sphingomyelinase (ASM) and a subsequent buildup of the sphingomyelin in the liver, spleen, and lungs @mlstranses | 4 1. 2. 3. Gaucher’s Disease – if there is a lack of enzyme, it cannot be converted into CERAMIDE Accumulation happens - forming Gaucher cell If there is no sphingomyelinase, Sphingomyelin accumulates forming foam cell (Neimann-Pick Disease) TOXIC GRANULATION VS ALDER REILLY ANOMALY Wbc Affected Neutroph ilia With Left Shift Dohle Bodies Toxic granulation Neutrophil only +/- +/- Alder Reilly Anomaly Neutrophil, monocyte, lymphocyte - - ALDER REILLY ANOMALY Autosomal recessive (Steininger) Characterized by granulocytes (monocytes and lymphocytes less often) with large, darkly staining metachromatic cytoplasmic granules The characteristic granulation, called Reilly bodies, is also found in the mucopolysaccharidoses (MPSs). ○ However, Reilly bodies can also be seen in Neutrophils/Monocytes/lymphocytes granules: Mucopolysaccharides resemble toxic granulation ○ Toxic granulation occurs only on Neutrophils. seen in Gargoylism and other mucopolysaccharidosis @mlstranses | 5 FUNCTIONAL LEUKOCYTE ALTERATIONS INHERITED ABNORMALITIES IN GRANULOCYTE FUNCTION Job’s syndrome Lazy leukocyte syndrome JOB’S SYNDROME AKA: autosomal dominant hyperimmunoglobulin e syndrome familial disorder; mutation in the STAT 3 gene ○ STAT3 gene is invovled in the intracellular signalling cascade ○ Helpful in the TH17 cell differentiation random movement of phagocytes are normal, but directional motility is impaired - cells response to chemotactic factors is slow ○ resulting to more time for bacteria to multiply before attacked Hyper-IgE Syndrome (abnormally increased) defect in cellular response to chemotaxis familial disorder Mutation in the STAT3 gene deficits in phagocyte function ○ Ineffective killing ○ Decrease activation ○ Lack of IL-12 production ○ random movement is okay but no directional movement - T helper cells cannot differentiate into Th1 cells (so high Th2 cells) ○ High serum IgE levels ○ Eosinophilia Deficits in delayed T helper responses Autosomal Dominant Hyperimmunoglobulin E (IgE) syndrome (ADHIES) Clinical Triad ○ Atopic dermatitis ○ Recurrent skin staphylococcal infections ○ Recurrent pulmonary infectons Other Manifestations: ○ Failure to lose primary teeth ○ Connective tissue abnormalities ○ Skeletal abnormalities DEFECTS IN MICROBICIDAL ACTIVITY Chronic Granulomatous Disease Myeloperoxidase Activity Chediak Higashi Syndrome CHRONIC GRANULOMATOUS DISEASE group of disorders involving inheritance or either X-linked or autosomal recessive gene that affects neutrophil microbicidal function. X-linked - accounts 70% and more severe Symptoms: ○ recurrent suppurative infection ○ Pneumonia ○ Osteomyelitis ○ draining adenopathy ○ Liver abscesses ○ Dermatitis ○ Hypergammaglobulinemia MAIN PROBLEM: decreased ability of phagocytes to produce superoxide and reactive oxygen species. (ROS) PATHOPHYSIOLOGY: one or more mutations in genes responsible for proteins that make up a complex known as NADPH oxidase ○ A genetic defect in any of the several components of the NADPH oxidase system can result in the CGD phenotype by making the neutrophil incapable of generating an oxidative burst. - In a normal setting If a microbe is engulfed by the phagocytic cell, NADPH oxidase… If there is oxygen, it forms Hydrogen peroxide w/ addition of water – and NADPH will be reduced to NADP with the help of NADPH oxidase enzyme No NADPH oxidase enzyme= cannot form reactive oxygen species (hydrogen peroxide) HOW TO CHECK IF WBC IS NORMAL OR HAS CGD NITROBLUE TETRAZOLIUM REDUCTION TEST LAZY LEUKOCYTE SYNDROME rare condition both random and direct movement are impaired release of cells from the bone marrow - POOR ○ neutropenia is a consistent finding ○ cells fail to respond to inflammatory stimuli but otherwise appear to have normal phagocytic and bactericidal activity. Problems in releasing of neutrophil to the peripheral blood ○ clinical features: low-grade fever and recurrent infections (involving the gums, mouth, and ears) ○ it also contains defective actin filaments aka Schwachman Syndrome defective locomotion/mobility actin filaments in the neutrophil is defective ○ Chemotaxis response defective ○ Defective mobility (random and directed) ○ Neutropenia altered structure and function of microfilament leads to altered deformability normal phagocytic and bactericidal activities @mlstranses | 6 Normal PMN - Reduce the Yellow Water-Soluble Nitroblue Tetrazolium to a Dark Blue Insoluble Formazan – NORMAL RESULT But since it is abnormal - it will not- reduce the Yellow Substance and will retain as Yellow – ABNORMAL RESULT FLOW CYTOMETRIC ASSAY labelled with dihydrorhodamine (DHR) DHR will fluoresce - reduced Defective locomotion/mobility rare, majority X-linked but some AR or point mutations Mutations in any of 4 genes for NADPH OXIDASE most serious disorder related to a defect in microbicidal activity → recurrent life-threatening disorder composed of a group of genetic disorders in which neutrophils and monocytes CAN INGEST but CANNOT KILL catalase-positive microorganisms Main problem: decrease ability of phagocytes to produce superoxide and reactive oxygen species Pathophysiology: ○ Mutation in genes responsible for proteins that make NADPH Oxidase ○ This results to neutrophil incapable of generating an oxidative burst ○ NADPH oxidase: produce ROS (H2O2) that kill engulf microorganisms Catalase-positive orgs: ○ S. au ○ Burkholderia cepacian ○ Chromobacterium violaceum ○ G pos enteric bacteria ○ various fungi (Aspergillus, Nocardia) Stimulated phagocytes → X Respiratory burst → X free radicals Associated with: ○ recurrent supparative infection ○ Pneumonia ○ Osteomyelitis ○ draining adenopathy ○ liver abscesses ○ Dermatitis ○ hypergammaglobulinemia Negatuve Nitroblue Tetrazolium (NBT) screening: due to abnormal oxidase acFvity ○ indirect test for respiratory burst power ○ normal PMN: reduce the yellow water soluble nitroblue tetrazolium to a dark blue insoluble formazan MYELOPEROXIDASE DEFICIENCY AKA Alius-Grignaschi anomaly benign inherited disorder that is usually transmitted by autosomal recessive genes. MAIN PROBLEM: absence of MPO enzyme from neutrophils and monocytes, but not eosinophils ○ Bacterial killing is slowed but complete. ○ COMPENSATION: Respiratory burst activity increased. Seen in patients with acute and chronic leukemias, myelodysplastic syndromes, Hodgkin disease, and carcinoma. LIPIDOSES Chediak Higashi Syndrome Niemann Pick Syndrome Tay Sach Syndrome CHEDIAK-HIGASHI SYNDROME INHERITED, AUTOSOMAL RECESSIVE TRAIT mutation in the CHS1 LYST gene on chromosome 1q42.1-2 ○ CHS1 LYST gene is responsible for encoding protein that regulates the morphology and function of lyososome related urganelles There is now an abnormal functioning of the lysosomal trafficking regulator protein which affects the size and the function of lysosomes abnormally large lysosomes, which contain fused dysfunctional granules ○ Can now engulf the microorganism, but DOES NOT have the capacity to digest the microorganism inside Why? → Dysfunctional granules fuse (primary, secondary, and even specific) Impairing its original function CLINICAL MANIFESTATIONS OF CHS: All because of the large granules in a variety of cells: HYPOPIGMENTATION OR PARTIAL ALBINISM ○ Impaired packaging of melanin into giant melanosome granules disturb melanin traffic ○ Fair skin, pale retina, and light blond frosted or siverly hair (Oculocutaneous Albinism) Severed Immunodeficiency Impaired release of lytic secretory granules by NK cells, neutrophil defects Neurologic Abnormalities cytoplasmic inclusions, resembling large lysosomes were present in all types of neurons; peripheral and cranial neuropathy. Mild Bleeding Techniques Absent or reduced number and irregular morphology of platelet-dense bodies w/c are required for the second wave of platelet aggregation MPO deficiency / Alius-Grignaschi Anomaly benign inherited disorder AR Main problem: absence of MPO enzyme in neutrophils and monocytes but not in Eosinophils MPO mediates oxidative destruction of microbes by H2O2 bacterial killing is slowed but complete compensation: Respiratory burst activity increased partial deficiency of MPO seen in: ○ Acute and Chronic Leukemia ○ Myelodysplastic syndromes ○ Hodgkin Disease ○ Carcinoma @mlstranses | 7 From transes of Dr. Belandres: rare, autosomal recessive disease of immune dysregulation Mutation in the CHS1 LYST gene on chr 1q42.1-2 ○ Encodes for protein that regulates the morphology and function of lysosomerelated organelles giant cytoplasmic granule (peroxidase positive) in all granule containing cells abnormal lysosomal development in neutrophils, monocytes, lymphocytes abnormal lysosomes cannot fuse with phagosomes neutrophils display impaired chemotaxis and delayed killing of ingested bacteria patients suffer from frequent infections Patients often have bleeding issues clinical manifestations begin in infancy with partial albinism (oculocutaneous) and severe recurrent life-threatening bacterial infections TAY SACH SYNDROME not part of Morphologic and functional Abnormalities (but discussed in the book) deficiency of hexosaminidase A occurs when harmful quantities of a fatty acid derivative called a ganglioside accumulate in the nerve cells of the brain @mlstranses | 8 Topic 2 | [MLS 417-LAB] Hematology 2 - Lecture P2: Introduction to RBC Abnormalities Professor: Lee-An Anayon, RMT Date: February 11, 2024 IMPORTANT TERMINOLOGIES ABSOLUTE VS. RELATIVE ABSOLUTE In absolute anemia or polycythemia there is a TRUE DECREASE or INCREASE in the Red Cell Mass (RCM), respectively. ○ If True anemia – there is really a problem in the RBC; Lysis occurs; rapid destruction of RBC ○ RCM refers to the volume of the plasma RELATIVE Changes in plasma volume causing changes in cellular components to cellular components ○ There is a shift of fluid from extracellular to intracellular – The blood will be diluted ○ Decreased RBC count due to the dilution phenomenon that happened during the changes in plasma volume ○ It does not mean that the patient has true anemia Relative anemia – pregnancy and diseases associated with hyperproteinemia ○ Relative anemia occurs in pregnancy due to plasma volume expansion, making RBC concentration appear low without a true RBC deficiency. ○ Diseases with hyperproteinemia involve excessive blood protein levels, potentially affecting blood viscosity and composition, but don't necessarily decrease RBC count. Relative erythrocytosis – dehydration ○ In this state (dehydrated state), the concentration of RBCs appears elevated because there's less plasma to dilute them, even though the actual number of RBCs may remain normal. ERYTHROCYTOSIS AND POLYCYTHEMIA (Increased RBC) (↓) Decreased Hemoglobin ○ Due to the dilution effect of the increased RBCs (↑) Increased Hematocrit (↑) Increased Number of Red Cells ERYTHROCYTE ABNORMALITIES A. Decreased Concentration Ineffective/Insufficient erythrocyte production ○ Hypoproliferative disorders Precursor cells inside the bone marrow has low count; cannot produce now enough RBC ○ Maturation disorders Bone marrow is incapable of producing mature RBC Our RBCs are only released in the peripheral blood only when it matures – except in cases of blood loss where reticulocytes are released early Increased RBC destruction/ Loss Nothing wrong with the bone marrow but RBC destruction is rapid such in cases of: ○ Hemolytic disorders ○ Blood loss – menstruation That is why in cases when we will have a reticulocyte count in the laboratory our sample should come from our classmates who is in her period ANEMIA, ERYTHROCYTOSIS AND POLYCYTHEMIA ANEMIA (Decreased RBC) (↓) Decreased Hemoglobin ○ Due to increased destruction of RBC the Hgb are released (↓) Decreased Number of Red Cells ○ Still due to increased destruction of RBC Why is it important to diagnose the cause of anemia? Why must it be prompt? Because sometimes the presence of anemia is not alone in itself, it may have underlying diseases, that’s why we have anemia; It must be prompt, for the doctors to give the final diagnosis and for the immediate resolution of the disease Anemia can be categorized into 4 as mentioned in the table above @mlstranses | 1 B. Increased Concentration SECONDARY (High altitude) When you live in a high altitude area your hematocrit is increased to compensate the oxygen that the body needs RELATIVE When you smoke tobacco you inhale carbon dioxide and not oxygen, so your body needs to compensate to balance the carbon dioxide and the oxygen ANEMIA Anemia comes from the greek word ANAIMA which means without blood; take note that anemia IS NOT A DISEASE, we only have anemia because of the underlying disease FUNCTIONALLY: Decrease in the oxygen carrying capacity of the blood ○ Because of the decreased hgb present hence the decrease in oxygen carrying capacity OPERATIONALLY: there is reduction in the hemoglobin content of blood ○ May lead to hypoxia and eventual tissue necrosis PATIENT HISTORY AND CLINICAL FINDINGS History and Physical examination are important Obtaining detailed history ○ Includes the hereditary make up of the patient ○ What you ate last time ○ What is your occupation ○ What chemicals you have been exposed CLUES TO HEMATOLOGIC DISORDER: ○ Skin — if petechiae is observed it might indicate that you have a hematologic disorder ○ Eyes — there is a pallor or yellowish eyes; specially if there is jaundice there is increased RBC destruction, if not RBC destruction it might signal that there might be something wrong with the liver ○ Mouth — mucosal bleeding is observed; sometimes if you have anemia RBCs are not the only one affected, other cells might be affected as well specially if the precursor cells are the one that is defective ○ Sternal tenderness ○ Lymphadenopathy ○ Cardiac Murmurs or arrhythmias ○ Splenomegaly and Hepatomegaly Both spleen and liver is ENLARGED JAUNDICE – most important for the assessment of anemia MODERATE ANEMIA Hemoglobin concentration of 7-10 g/dL May cause pallor of conjunctiva and nail beds SEVERE ANEMIA Hemoglobin concentration of 8 um in diameter), MCV > 100 fL Megaloblastic anemia Myelodysplastic syndromes Chronic liver disease Bone marrow failure Reticulocytosis Oval macrocyte Large oval RBC Megaloblastic anemia Microcyte Small RBC (