02 - Pathology and Diagnostics - Blood Cell Analysis PDF

Pathology and Diagnostics C. Garlanda – Clinical Pathology – Lecture 2 Blood Cell Analysis 21/11/2024 – Group 38 (Theofilopoulou & Surendran) The topic is to understand the type of information we can get from blood cell...

Pathology and Diagnostics C. Garlanda – Clinical Pathology – Lecture 2 Blood Cell Analysis 21/11/2024 – Group 38 (Theofilopoulou & Surendran) The topic is to understand the type of information we can get from blood cell analysis. Reminder on blood components: After centrifuging the blood sample, you get two components: plasma and the cellular elements. Blood cell analysis focuses on the cellular elements: platelets, WBCs, and RBCs (refer to the diagram for percentages). Collection of blood You choose the tube type depending on the aim. Each tube has different characteristics. You will use a different one for serum, erythrocyte sedimentation rate, etc. The most common tube used to collect plasma is EDTA. Orange-capped vial: consists of blood coagulation accelerator - thrombin - so we end up with serum faster. Brownish-red-capped vial: no additives, coagulation is performed at normal speed for collection of serum. Purple-capped vial: contains EDTA which prevents the coagulation process and enables us to collect all blood cells to analyze. 1  Black-capped tube: used for erythrocyte sedimentation rate (ESR) tests. Reminder of cellular elements: - Erythrocytes are the highest number: 4-6 million/µL, and their function is to transport oxygen and carbon dioxide. - Leukocytes (WBCs) are around 4,800-10,800/µL. You can collect leukocytes from the lymphatic system, but for today’s discussion we will be collecting them from blood. - Platelets/thrombocytes are pieces of megakaryocytes (bone marrow cells). Their function is to stop bleeding by forming a plug. In smears they appear as very small dots along RBCs. These cells originate from HSCs which reside in the bone marrow. So a possible reason to have abnormal values is due to the production at the level of the bone marrow. The HSCs divide into two types of stem cells: - Myeloid stem cells which form all the lymphocytes. - Lymphoid stem cells which give rise to erythrocytes, platelets, and granulocytes. 2 Complete Blood Count (CBC) CBC is a panel of tests that evaluate the 3 types of cells that circulate in the blood: RBC, WBC and platelets. After centrifugation, you have the separation of the cells depending on their dimension. They will be eliminated through apertures of different sizes: Platelets → smallest aperture RBCs → medium aperture ○ Some undergo lysis and release haemoglobin so it is important to measure it WBCs → large aperture Their dimension is measured while they pass through the aperture. The thickness of the cells are important in conditions that alter their size. The CBC is often used as a broad screening test to determine an individual’s general health status. It can be as a routine health examination or to screen a wide range of conditions. They help diagnose if you have a suspicion of a condition such as: - Anemia - Infections - Inflammation - Bleeding disorders - Leukaemia and more. Cases in which you ask for a CBC: 1. There are signs and symptoms that may be related to disorders that affect the blood cells: Fatigue or weakness, pallor, dyspnea, chest pain (poor cardiac oxygenation), altered mental status (poor cerebral oxygenation), Fever: an infection, Inflammation (auto-immune conditions), Bruising or bleeding (coagulation disorder) 2. When there is already a diagnosis and you want to follow the severity of the condition 3. Monitor the effectiveness and/or response to treatment - monitor toxicity and the condition of the bone marrow. a. Some therapies, such as chemotherapy, target cancer cells but also target sites with high rates of replication like the bone marrow which produces many cells every day. So a drug that targets cell replication, will also affect the bone marrow. 3  WBCs are therefore followed to see if the toxicity is tolerated or not and this way you might skip a treatment if toxicity is too high. Red Blood Cells (RBC) or Erythrocytes The cytoplasm is rich in haemoglobin, an iron-containing biomolecule that binds oxygen and is responsible for the blood's red colour. In humans, mature red blood cells are flexible biconcave disks that lack a cell nucleus and most organelles. The cells develop in the bone marrow and circulate for about 100– 120 days in the body before the macrophages remove them. Approximately a quarter of the cells in the human body are red blood cells. When RBCs are normal, they have a zone of central pallor (“clear part” in the middle) about ⅓ the size of the RBC. RBC analysis can show: Variation in size = anisocytosis Variation in shape = poikilocytosis In the image on the right, we see a few small blue platelets. There is an immature neutrophil on the left and a segmented neutrophil on the right, the mature form. Steps in Erythropoiesis Erythropoiesis occurs in the bone marrow and we see the specific names in the image above. The change in color is because, initially the cytoplasm is full of ribosomes and it progressively 4 reduces because it is replaced by the hemoglobin. The bone marrow releases the reticulocytes into the circulation so we can also find those in the blood. Reticulocyte Count Reticulocytes are immature non-nucleated red cells that contain RNA and continue to synthesize hemoglobin after the loss of the nucleus. Because they lose their RNA 1-3 days after reaching the blood from the marrow, a reticulocyte count provides an estimate of the rate of red cell production (erythropoiesis). In conditions of anaemia not due to production or maturation defect, the bone marrow responds with increased erythropoiesis stimulated by an increase in renal production of erythropoietin (EPO) in response to hypoxia. An absolute reticulocyte count is more helpful than a percentage. This is because the HCT (hematocrit: space occupied by the cells) can be influenced by the state of hydration of the patient so you cannot only measure the percentage because it can be affected by the HCT. Normally, reticulocytes comprise 0.5 - 2.5% of all RBCs. Reticulocyte index (RI) = (reticulocyte %) * (patient’s HCT/normal HCT) A normal RI is < 3%. Increased RI (>3%) indicates reticulocytosis, a normal response to blood loss or anaemia. Since reticulocytes are larger than RBCs, the MCV and RDW are elevated. The combination of anaemia with a low or normal reticulocyte count indicates that the bone marrow is unable to respond normally. This may be either due to a lack of essential ingredients (iron deficiency, vitamin B12 or folate deficiency), bone marrow disease, or chronic disease. Reticulocyte counting can be performed manually or automatically: Manually: blood smear stained with a supravital dye (e.g. methylene blue) staining the precipitated ribosomes. 5  Automated (most common): The blood sample is stained with supravital dye, is passed through an automated counter (laser) and the light scatter is used to enumerate reticulocytes. Haemoglobin (Hb) Measurement of haemoglobin (Hb) content is the parameter most widely used to diagnose anaemia. Hb is a red pigment molecule which is responsible for the red colour of RBCs. Hb contains 4 molecules of heme and 4 of globin (2 ɑ chains and 2 β chains). Each molecule of heme contains one iron ion (Fe2+), so there are a total of 4 iron ions per Hb molecule. The Hb test measures the concentration of Hb expressed as grams of haemoglobin per deciliter of whole blood. Normal ranges differ depending on age and gender. Normal range for women: 12-16 g/dL Normal range for men: 13.5-17.5 g/dL Due to the menstrual cycle, women have lower haemoglobin levels but after menopause, the levels come back to normal. Hematocrit (HCT) The packed cell volume (HCT) is the percentage of total volume occupied by packed RBCs when a given volume of whole blood is centrifuged at a constant speed for a constant period of time. You withdraw a blood sample from a small capillary (ie. from the tip of the finger) and then centrifuge it. Normal percentage in men is 48% Normal percentage in women is 38% Dehydrated individuals can have higher than normal HCT. HCT is an important parameter used as it is one of the most precise methods of determining the degree of anaemia or polycythemia (the opposite of anaemia, an abnormal increase in RBC production). 6 RBC Indices These are calculations that provide information on the physical characteristics of the RBCs: 1. Mean Corpuscular Volume (MCV) 2. Mean Corpuscular Hemoglobin (MCH) 3. Mean Corpuscular Hemoglobin Concentration (MCHC) 4. Red Cell Distribution Width (RDW) 1. Mean Corpuscular Volume (MCV): a measurement that indicates the average volume of RBCs and is calculated from the HCT and RBC count. It is expressed in femtoliters (1^(-15) L) or cubic micrometers (µm^3). MCV = HCT * 1000/RBC(millions/µL) In patients with anaemia, the MCV measurement allows classification as either microcytic anaemia (MCV < normal range) or macrocytic anaemia (MCV > normal range). The yellow arrows point to some RBCs that are smaller than normal in the case of microcytic anaemia. This helps guide the treatment of the patient. 2. Mean Corpuscular Hemoglobin (MCH): indicates the content (weight) of Hb of the average RBC. It is calculated from the Hb concentration and the RBC count. It is expressed in picograms. MCH = Hb(g/dL) / RBC (millions/µL) MCH value is decreased in hypochromic anaemias (a form of anaemia with a low amount of haemoglobin inside the RBCs). MCV and MCH are measured together because they change in parallel in the same way. If there is an increase in MCV and MCH, then there is a need for folic acid and/or vitamin B12, nutrients to produce RBCs. This means that the cells are not replicating enough in the bone marrow so cells are larger and contain more Hb. 7  If there is a decrease in MCV and MCH, then there is a need for iron, copper or vitamin B6. 3. Mean Cell Hemoglobin Concentration (MCHC): the average concentration of Hb in a given volume of packed red cells. It is calculated from the Hb concentration and HCT. It is expressed in g/dL. MCHC = Hb (g/dL) / HCT 4. Red Cell Distribution Width (RDW): measures how dishomogeneous the cells are, via variation in RBC size or RBC volume. RDW is elevated in accordance with anisocytosis (size variation). For instance, when elevated RDW is reported on complete blood count then marked anisocytosis is expected to be found on the peripheral blood smear review. CBC components and their normal ranges [suggested to remember these] 1. WBC: 4.5-11.0 X 103/µL 2. RBC: Male: 4.5-5.5 X 106 /µL, Female: 4.0-5.0 X 106 /µL 3. HGB: Male: 14-17.4 g/dL. Female: 12.0-16.0 g/dL 4. HCT: Male: 42-52%, Female: 36-46% 5. MCV: 80-100 fl 6. MCH: 28-34 pg 7. MCHC: 32-36 g/dL or % 8. RDW: 12.0-14.6% 9. PLT: 150-450 X 103 /µL 8 RBC Evaluation Prof read the tables from the slides 9 10 Blood Smear/Film Blood smears are often used as a follow-up test to abnormal results on a CBC to evaluate the different types of blood cells. It helps to diagnose and/or monitor numerous conditions that affect blood cell populations. A drop of blood is spread thinly onto a glass slide that is then stained. There are automated digital systems that analyze these smears. Clinical Significance of Blood Smear Analysis You analyze blood smears in cases of: - Anemia, and unexplained jaundice suggesting lysis of RBCs - Sickle cell disease - Petechiae (skin lesions due to thrombi) suggesting thrombocytopenia or neutropenia - Lymphoma diagnosis - Myeloproliferative disease - Acute or recent-onset renal failure - Bacterial or parasitic diseases like malaria - Disseminated nonhematopoietic cancer - General ill health with fever etc. 11 Descriptive terms used on peripheral smears: Anisocytosis: marked variation in RBC sizes (visual counterpart of increased RDW) Hypochromia or hypochromasia: RBCs are paler than normal because they contain less hemoglobin (visual counterpart of decreased MCH) Macrocytosis: increased number of large RBCs (visual counterpart of increased MCV) Microcytosis: increased number of small RBCs (visual counterpart of decreased MCV) Poikilocytosis: marked variation in the shape of RBCs For example, you may have normo-hypochromic anaemia and/or normo-microcytic anaemia (paler and smaller than normal). They can be due to: 1. Disorders of iron utilization: - Iron deficiency - Anaemia of chronic disease 2. Disorders of globin synthesis: - Thalassemias - Other hemoglobinopathies Another example: megaloblastic anaemia (larger cells) may be due to: 1. Vitamin B12/folic acid deficiency 2. Patient receiving antimetabolites of DNA synthesis like chemotherapy or antimicrobial agents This is the second most common type of anemia. It can be associated with macrocytic anemia and pancytopenia but also hypersegmented neutrophils (mentioned later on) suggesting that the bone marrow is not producing enough cells. Anemia with high reticulocyte counts Differential diagnosis: - Bleeding - blood loss internal and external - Hemolysis - immune, mechanical, toxic, infections Laboratory evaluation: You may ask for more tests to determine the cause of increased RBCs. Blood film, RBC, spherocytes, Parasites, Reticulocytes. Tests to detect bilirubin and haptoglobin (removes free toxic Hb) which would indicate hemolysis Direct and indirect Coombs test - to test for autoimmune hemolytic anemia Hemoglobin electrophoresis, G6PD (Glucose-6- phosphate dehydrogenase) screen etc. 12 Coombs test In cases of suspected autoimmune hemolytic anemia, you can perform a direct or indirect Coombs test. Patients with autoimmune hemolytic anemia have autoimmune antibodies binding to RBCs (gray in the diagram). Direct Coombs test: You collect their blood and add secondary antibodies (blue) to recognise the auto-antibodies on the RBCs. If there is agglutination, it is positive. Indirect Coombs test: performed to assess the transfusion compatibility for surgeries. It checks if the patient's serum contains antibodies against specific blood factors, such as ABO. Mixing the patient's serum with different groups of red blood cells and observing clumping after adding anti-immunoglobulins indicates incompatibility. Anemia with low MCV and low reticulocytes Differential diagnosis: - Iron deficiency - Anemia of chronic disease Laboratory evaluation: - Serum iron levels, iron-binding proteins, ferritin 13  - However the iron may be normal because the patient may have problems with the proteins that bind and transport iron so you must ask for both. - Blood smear - microcytic/hypochromic RBCs Hematopoietic stem cells and inflammatory signals Hematopoietic stem cells typically reside in a specialized niche, where they adhere to it through cell adhesion molecules. Spindle-shaped osteoblastic cells constitute the HSC niche. Activated HSCs generate HSCs (self-renewal) and progenitor cells (differentiation) by asymmetric cell division. HSCs lose the abilities of multi-potency and self-renewal, following their cell division and differentiation. Inflammatory signals regulate the fate of HSC trigger differentiation of the cells. The effect of inflammatory signals is described as a ‘push and pull’ on HSCs. - The ‘push’: HSCs divide in direct response to stimuli associated with infections (PAMPs) or in response to pro-inflammatory cytokines that are induced during infection. - The ‘pull’: HSCs divide following the depletion of committed progenitor populations from the bone marrow. Essentially, they are being pushed by the impact of cytokines and mediators on one side and pulled by the reduced numbers of progenitors on the other side. 14 Under homeostatic conditions: the system is regulated by BM niche signals and basal levels of proinflammatory cytokines, which maintains a balance between HSC dormancy and lineage priming. In response to proinflammatory signals: HSCs undergo distinct fate changes, including expansion of Meg/E and myeloid-biased precursors, resulting in rapid production of platelets and myeloid populations. Meanwhile, lymphoid output is suppressed. White blood cells Neutrophils A band neutrophil is an immature neutrophil. Its presence indicates a robust proliferation rate in the bone marrow and is typically elevated during infections. Unlike mature, segmented 15 neutrophils, band neutrophils have a nucleus that is less divided. Segmented neutrophils represent the mature form of these immune cells There are thousands of neutrophils per microliter of blood, and their developmental stage can provide valuable diagnostic information. To classify and analyze neutrophils, a system based on the number and arrangement of nuclear lobes was developed, known as the Arneth formula. This formula assesses bone marrow activity and the immune response by evaluating the "shift" in neutrophil nuclear segmentation: A shift to the left occurs when more immature neutrophils (such as band cells) are present. This suggests an active bone marrow response, often due to infections, inflammation or an hemolytic crisis. A shift to the right occurs when more hypersegmented neutrophils are present. This is associated with conditions like nutrient deficiencies (vitamins B12 an folate), megaloblastic anemia or the presence of aging neutrophils in chronic illnesses. Eosinophils Eosinophils have traditionally been recognized for their role in combating parasitic infections but now their primary function is more commonly associated with allergic reactions and asthma. When interpreting lab results, it is crucial to consider the context. Elevated eosinophil counts may indicate a parasitic infection in regions where such infections are prevalent, whereas in other areas, allergies or asthma are more likely explanations. Eosinophils are generated in the bone marrow, circulate in the blood for around 18 hours, and then migrate to tissues, skin or GI tract, typically in response to stress. Basophils Basophils have a similar function, as they are granular cells packed with histamine. They become activated when they encounter IgG antibodies bound to antigens, triggering degranulation and the release of histamine and other inflammatory mediators. During degranulation, they release mediators such as histamine, platelet-activating factor (PAF), and heparin. These mediators are known to be associated with hypersensitivity reactions. Monocytes Monocytes are the largest white blood cells, with a large nucleus, and serve as precursors to macrophages in the bloodstream. They play a crucial role in patrolling tissues, searching for pathogens and any particulate matter. After this patrol, they migrate to the tissues, where they 16 differentiate into macrophages. The function and characteristics of macrophages vary depending on the specific tissue in which they reside. However, their primary role is to help restore and maintain tissue homeostasis. Lymphocytes Lymphocytes can be divided into T lymphocytes and plasma cells. These lymphocytes originate not only in the bone marrow but also in secondary lymphoid tissues such as the spleen, lymph nodes, and tonsils. WBC Count Variations in white blood cell (WBC) count can manifest as either a decrease, known as leukopenia, or an increase, referred to as leukocytosis. Leukopenia can result from various conditions, including: ○ Bone marrow disorders that impair WBC production. ○ Autoimmune diseases that target and destroy WBCs. ○ Severe infections and sepsis, where the bone marrow cannot produce sufficient cells to meet the body's demands. ○ Certain cancers, such as lymphoma or metastatic tumors infiltrating the bone marrow. ○ Diseases of the immune system like HIV, which are linked to the destruction of WBCs by autoimmune or viral mechanisms. Leukocytosis can result from: ○ Infections: common during the acute phase of bacterial or viral infections, as the bone marrow increases WBC production in response to microbial components and cytokines. ○ Inflammatory processes: trigger WBC production as part of the immune response to inflammation. ○ Leukemia: leads to leukocytosis with abnormal and immature WBCs. ○ Allergies and asthma: may cause leukocytosis with a specific increase in eosinophils. ○ Tissue damage: trauma, burns, or myocardial infarction stimulate the bone marrow to produce more WBCs to aid in tissue repair and recovery, even intense exercise may be sufficient for leukocytes to migrate into the circulation. 17 Regulation of Circulating Leukocytes The number of white blood cells (WBCs) in circulation, particularly neutrophils, is influenced by three main factors: bone marrow production, demargination from the marginal pool, and migration to tissues. Neutrophils, the most abundant WBC subtype, are distributed between the circulating pool and the marginal pool, the latter consisting of cells adhered to the endothelial layer of blood vessels. Blood tests assess only the circulating pool. In response to infections or inflammation, increased bone marrow production and demarginalization elevate circulating neutrophils. However, during severe infections, many neutrophils migrate to tissues, reducing their presence in circulation despite increased production. Absolute neutrophil count To determine the absolute count, we multiply the percentage of neutrophils by the total white blood cell count. 𝐴𝑁𝐶 = 𝑊𝐵𝐶 (𝑐𝑒𝑙𝑙𝑠/μ𝐿) × %𝑁𝑒𝑢𝑡𝑟𝑜𝑝ℎ𝑖𝑙𝑠 The absolute neutrophil count in circulation is influenced by several factors. We discussed how the hematopoietic stem cells differentiate towards the myeloid lineage, impacting the inflow from the bone marrow. Additionally, there's a proportion of neutrophils in the marginal granulocyte pool and the circulating granulocyte pool. Normally, these two pools are in equilibrium, with the marginal pool adhering or rolling along the vessel walls, making it challenging to capture in a blood sample. However, in certain conditions, such as hormonal stimuli like cortisol, the marginal pool may be released into circulation. Thus, when counting neutrophils, we must account for three factors: generation, loss in tissues, and movement from the marginal to the circulating pool. The marginal pool consists of neutrophils adhered to the endothelium, which can mobilize into circulation under specific physiological or pathological stimuli. Neutropenia indicates a reduction in the count, which is typically associated with an increased risk of infections due to compromised immune defence. Common causes include: ○ Severe infections such as sepsis, where neutrophils rapidly migrate to infected tissues. ○ Autoimmune disorders, in which neutrophils are targeted and destroyed. 18  ○ Bone marrow suppression, resulting from chemotherapy, radiation therapy, or certain toxins. ○ Immunodeficiency states, such as HIV. ○ Hematologic malignancies like leukemia or pre-neoplastic conditions affecting marrow function. Neutrophilia suggests an elevated neutrophil count, which typically reflects an active immune response. It may occur in: ○ Infections, particularly bacterial, where neutrophil production increases to combat pathogens. ○ Inflammatory states, such as rheumatoid arthritis or acute gout. ○ Tissue necrosis, caused by trauma, burns, or myocardial infarction. ○ Physiological conditions, including stress or intense physical activity. ○ Malignancies, including certain leukemias and tumors producing inflammatory cytokines. Eosinophil and Basophil count Changes in eosinophil and basophil counts are useful diagnostic indicators for specific conditions, though reductions in these cell types generally lack clinical significance. Eosinophilia, an increase in eosinophil count, is most commonly associated with: Allergic disorders, such as asthma, allergic rhinitis, or atopic dermatitis, where eosinophils play a key role in the hypersensitivity response. Parasitic infections, particularly helminthic infections, which are more prevalent in regions such as Africa, Asia, and South America. Eosinophils are recruited in response to parasitic antigens and aid in host defence. Inflammatory conditions, including autoimmune or gastrointestinal disorders like celiac disease or inflammatory bowel diseases (IBD) such as Crohn's disease or ulcerative colitis. Hematologic malignancies, including certain leukaemias, lymphomas, or hypereosinophilic syndromes, where eosinophil proliferation is driven by abnormal cytokine signalling. Basophilia, an increase in basophil count, is less common but can overlap etiologically with eosinophilia. 19 Monocyte count Monocytopenia observed over time may indicate underlying bone marrow dysfunction or hematologic disorders such as leukemia, where monocyte production is impaired. Monocytosis, an elevated monocyte count, is typically associated with chronic infections, such as tuberculosis or fungal infections, where monocytes are recruited to sites of persistent infection. In these conditions, monocytes play a key role in chronic inflammation and immune response. During the course of acute inflammation or infection, there is often an initial increase in neutrophils, followed by a subsequent rise in monocytes. Neutrophils, which are the first responders to bacterial infections, dominate the early immune response. As the infection progresses or the body shifts towards resolution, monocytes increase, both in relative percentage and absolute number, marking the transition to the recovery phase. In this phase, neutrophils return to baseline levels, and monocytes continue to increase, facilitating tissue repair and the resolution of inflammation. Lymphocyte count Lymphocytopenia - Autoimmune disorders like Lupus - Infections such as HIV and influenza - Chemotherapy (affects all blood cell lineages) - Corticosteroids Lymphocytosis Reactive Lymphocytosis: the bone marrow is actively responding to the infective stimulus. Physiological although it reflects a problem. ○ Acute viral infections ○ Certain bacterial infections ○ Chronic inflammation disorders ○ Trauma ○ Surgery Primary Lymphocytosis: usually linked to neoplastic transformations ○ May reflect a tumor ○ Leukemia ○ Lymphoma 20 Lymphocyte Count in SARS-CoV-2 Infection In SARS-CoV-2 infection, the number of lymphocytes, particularly CD4+ and CD8+ T lymphocytes, is significantly affected by the severity of the disease. In more severe cases, increased cytokine production occurs, which can affect bone marrow function. This leads to the migration of lymphocytes from the circulating pool to the tissues, resulting in a mild reduction in circulating lymphocyte numbers. Cytokine Storm In severe COVID-19, a cytokine storm may occur, characterized by an excessive release of pro-inflammatory cytokines. This overwhelming cytokine release has a profound impact on the immune system and bone marrow, resulting in an increase in white blood cells, particularly neutrophils and monocytes, while lymphocytes are reduced. Despite the overall increase in the total white blood cell count, the number of lymphocytes remains diminished due to their redistribution and the bone marrow's response to the inflammatory signals. This phenomenon is not exclusive to COVID-19 and can also be observed in other severe viral infections, where an imbalanced cytokine response results in lymphopenia or lymphocytopenia. A similar immune dysregulation is often seen in sepsis, where cytokine storms contribute to the suppression of lymphocyte levels, impairing the body’s ability to mount an effective immune response. Atypical Lymphocytosis In specific conditions like infectious mononucleosis, you might encounter what's known as atypical lymphocytosis. These are not your typical lymphocytes; they're larger than usual and have a unique morphology, often influenced by their interaction with surrounding red blood cells. This atypical lymphocytosis is associated with the infection. 21