Interpretation of CBC in Practice PDF

Summary

A presentation about the interpretation of complete blood counts (CBCs) in practice. It covers the basic concepts, components, automation methods of CBC, advantages of automation, principles of automated blood cell analyzers, light scattering, automated differential counts, and more. It gives a good overview of CBC analysis for hematology students or professionals.

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Interpretation of CBC in practice Supervised by: Dr. Ihsan Mardan Humood Presented by : Ban Kadhim Qasim 2nd year hematology resident Complete blood count (CBC): The most common test used in clinical medicine. Determine type and severity of blood cell abnormalities. Nowad...

Interpretation of CBC in practice Supervised by: Dr. Ihsan Mardan Humood Presented by : Ban Kadhim Qasim 2nd year hematology resident Complete blood count (CBC): The most common test used in clinical medicine. Determine type and severity of blood cell abnormalities. Nowadays, CBC is fully automated and highly reproducible. Correct interpretation of automated CBC can reduce rate of unnecessary blood smear examination and provide useful information for provisional diagnosis of RBC and WBC diseases. Component of automated CBC: Blood count basic parameters: Hb, Hct, RBC, WBC, platelet. Red cell indices: MCV, MCH, MCHC, RDW WBC differentials. Platelets count and indices. Reticulocyte count. Advantages of automation Increase efficiency; streamline workflow Increase productivity Increase test volumes with no additional Decrease turnaround time (TAT); improve service Decrease costs Basic principles of automated blood cell analyzer: Electrical impedance: o Depends on the fact that red cells are poor conductors of electricity, whereas certain diluents are good conductors; this difference forms the basis of the counting systems. o It is the traditional method for counting cells is electrical impedance, also known as the Coulter Principle. It is used in almost every hematologyanalyzer.. o Whole blood is passed between two electrodes through an aperture so narrow that only one cell can pass through at a time. o The impedance changes as a cell passes through. The change in impedance is proportional to cell volume, resulting in a cell count and measure of volume. o Impedance analysis returns CBCs and three-part WBC differentials (granulocytes, lymphocytes, and monocytes) but cannot distinguish between the similarly sized granular leukocytes: eosinophils, basophils, and neutrophils. o Counting rates of up to 10,000 cells per second can be achieved and a typical impedance analysis can be carried out in less than a minute Light scattering: o The light‐scattering principle of cell counting is based on the observation that microscopic particles, such as blood cells, scatter into small (0–15°) angles, most of the visible light incident upon them. This principle is used to count red blood cells, white blood cells, and platelets. o By correlating different optical signals generated by a single blood cell, detailed information is collected that allows for classifying the cell in a multi-dimensional space. For example, forward scatter (very low angle) mainly depends on cell size; sideward scatter (90 degrees) predominantly reflects nuclear segmentation; and intermediate-angle scatter carriesinformation on the presence and number of cytoplasmic granules. schematic illustrating light-scatter method of cell counting Automated differential count: Most automated differential counters that are now available use flow cytometry incorporated into a full blood counter rather than being stand- alone differential counters. Increasingly, automated blood cell counters have a differential counting capacity, providing either a three-part or a five- to seven-part differential count. Counts are performed on diluted whole blood in which red cells are either lysed or are rendered transparent. Analysis may be dependent only on volume and other physical characteristics of the cell or also on binding of certain dyes to granules or activity of cellular enzymes such as peroxidase Three-part differential: A three-part differential count assigns cells to categories usually designated (1)‘granulocytes’ or ‘large cells’; (2)‘lymphocytes’ or ‘small cells’; and (3)‘monocytes’, ‘mononuclear cells’, or ‘middle cells. In theory, the granulocyte category includes eosinophils and basophils, but in practice it is common for an appreciable proportion of cells of these types to be excluded from the granulocyte category and to be counted instead in the monocyte category. Some other three-part differentials categorize leucocytes as WBC-small cell ratio (equivalent to lymphocytes), WBC-middle cell ratio (equivalent to monocytes, eosinophils and basophils) and WBC-large cell ratio (equivalent to neutrophils). instruments incorporating a three-part differential count, although not capable of enumerating eosinophils or basophils as individual categories of cell, are able to flag a significant proportion of samples that have an increased number of one of these cell types. Five and Seven-part differentials: Five- to seven-part differential counts classify cells as neutrophils, eosinophils, basophils, lymphocytes and monocytes and in an extended differential count may also include immature granulocytes or large immature cells (composed of blasts and immature granulocytes) and atypical lymphocytes (including small blasts). Automated instruments performing differential counts (that do not enumerate immature granulocytes or NRBC separately) are able to flag or reject counts from the majority of samples with NRBC, myelocytes, promyelocytes, blasts or atypical lymphocytes. The automated nucleated red blood cell count: Enumeration of NRBC is important because their presence can have a direct effect on the accuracy of the WBC on some blood cell counters. The correct WBC was previously only obtained by examination of a peripheral blood film. The NRBCs are reported as the number per 100 white blood cells and subtraction of the number of NRBCs from the total nucleated count gives the correct WBC. The morphological correction of the WBC can be inaccurate since if the nuclear size of an NRBC falls below the white blood cell threshold of the instrument, these cells are not included in the automated WBC in the first place. Instruments determine NRBCs by staining them with a nuclear dye and using either fluorescence laser light scatter or flow cytometry to separate them from WBC or a combination of impedance and cell volume. Flagging of automated blood counts: Flagging is defined as a system of signaling or communicating the message with a “flag.” In the hematology laboratory, a flag is the signal to the operator that the analyzed data may have a significant abnormality during analyzing blood samples. It thus necessitates confirmation of the results by microscopic examination of a Hematology analyzers generate suspect flags in the presence of abnormal cells. Measurement of hemoglobin : Some automated counters still measure Hb by a modification of the manual HiCN method with cyanide reagent; The basis of the method is dilution of blood in a solution containing potassium cyanide and potassium ferricyanide. Hemoglobin, Hi and HbCO, but not SHb, are converted to HiCN. The absorbance of the solution is then measured in a spectrometer at a wavelength of 540 nm or in a photoelectric colorimeter with a yellow-green filter. Erythrocyte Sedimentation Rate (ESR) The ESR is a measurement of the rate at which the erythrocytes settle from the plasma. The sedimentation process consists of three phases. Phase 1 occurs within the first 5–10 minutes and represents the aggregation phase when erythrocytes form rouleaux. The second phase is the sedimentation phase when erythrocytes aggregate and the aggregates fall out of solution. The third phase is the packing phase in which the erythrocyte aggregates pack closely together at the bottom of the tube. The rate of erythrocyte settling depends on (1) the plasma’s protein composition, (2) the erythrocytes’ size and shape (3) the erythrocyte concentration. The effects of plasma proteins are most profound during the APR. Plasma proteins that are positively charged can neutralize and reduce the negative surface charge on erythrocytes, thereby decreasing the repulsive force and prompting erythrocytes to aggregate. In inflammatory states, acute-phase proteins in plasma, particularly fibrinogen, may efficiently facilitate aggregation of erythrocytes and hence increase the ESR. In contrast, patients with hypoalbuminemia, seen in many chronic diseases, may have a decreased ESR. erythrocytes is significantly influenced by the sizes, shapes and numbers of those cells, hematological abnormalities may also alter ESR values. For example, patients with anemia generally have an elevated ESR. These patients have fewer erythrocytes and the resulting reduced friction between those cells can lead to faster sedimentation rates in vitro. However, patients with macrocytic (large erythrocyte) anemia usually have a higher ESR than do patients with microcytic (small erythrocyte) anemia, because large cells tend to sediment more rapidly than do small cells. Moreover, because irregularly shaped or rounded erythrocytes do not aggregate as well as normal disc-shaped erythrocytes, patients with diseases such as sickle cell anemia may have low (even zero) ESR values. Conditions Associated with an ElevatedESR Acute and chronic infections Acute coronary syndrome Acute ischemic stroke Multiple myeloma Osteomyelitis Pelvic inflammatory disease Pregnancy Pulmonary tuberculosis Rheumatic fever Rheumatoid arthritis Systemic lupus erythematosus Subacute bacterial endocarditis Waldenstrom’s macroglobulinemia For ESR, the CLSI recommends the Westergren methodin which EDTA- anticoagulated whole blood is diluted with 0.85%NaCl The diluted blood is aspirated into a calibrated Westergren pipet, and the cells are allowed to settle for aperiod of exactly 1 hour (at the end of which the distance in millimeters between the meniscus of the plasma and the top of the sedimented erythrocyte column is read. Thereference interval for ESR varies with age and sex. Automated methods for determining ESR have been available since the 1990s. The principle of measurement for each instrument is an adaptation of the manual WestergrenESR method that allows determination of ESR in a shorter20-30minutes. ] CBC automation and interpretation RBC indices: RBC count: Is a count of the actual number of red blood cells in your blood sample. Normal value in adult: male = 5 ± 0.5 million/mm3 Female = 4.3 ± 0.5 million/mm3 Low RBC count (erythrocytopenia) usually reflect anemia due to: o Blood loss: Trauma, surgery, GI bleed and gynecological disturbance. o Impaired production: Pure red cell aplasia, pernicious anemia, megaloblastic anemia, iron deficiency anemia, thalassemia, anemia of prematurity and anemia of chronic disorder o Increased destruction: 1-Intra-corpuscular causes: Hereditary spherocytosis, sickle cell anemia, G6PD, pyruvate kinase deficiency and PNH. 2-Extra-corpuscular causes: Autoimmune, hemolytic disease of newborn, mismatch transfusion, microangiopathic hemolytic anemia TTP, HUS, DIC and infections. High RBC count (erythrocytosis): occurs in polycythemia Vera, smoking, thalassemia trait and high attitude Hematocrit (HCT): The ratio of the volume of red blood cells to the total volume of blood. The hematocrit is expressed as a percentage. For example, a hematocrit of 30% means that there are 30 milliliters of red blood cells in 100 milliliters of blood. Normal range: Adult male= 38-54 %. Adult female = 34-46.5 %. Can be calculated by multiplying RBC count x 3. Therefore, high or low level of HCT correlate with same causes of high and low RBC count A higher than normal hematocrit may indicate: Abnormal increase in red blood cells (erythrocytosis) A disorder, such as polycythemia vera that causes your body to produce too many red blood cells (in polycythemia it may rise to as high as 70 %). At higher altitudes, there is a lower oxygen supply in the air and thus hematocrit levels may increase over time. Low blood oxygen levels (hypoxia) Lung or heart disease if the body senses low oxygen levels, it will make more red blood cells in an effort to increase the amount of oxygen in the blood Dehydration and Burn( due to loss of plasma) A lower than normal hematocrit may indicate: An insufficient supply of healthy red blood cells (anemia) A large number of white blood cells — usually a very small portion of your blood due to long-term illness, infection, leukemia, lymphoma or other disorders of white blood cells Acute kidney disease (lower Erythropoietin production lead to less RBCs production by the bone marrow). Pregnancy may lead to women having additional fluid in blood. This could potentially lead to a small drop in hematocrit levels Hemoglobin (Hb): Is the protein molecule that carries oxygen in the red blood cells. Normal range: different according to age and sex: o Adult Male = 13.0-18.0 g/dl. o Adult Female = 11.5-16.5 g/dl. Low Hb level= anemia. High Hb level= polycythemia Mean corpuscular volume (MCV): Is a measure of the average volume of a red blood corpuscle (or red blood cell). MCV = HCT x 10/RBC count. Normal range differs according to the patient age, usually higher in neonatal period, in adult it is usually (78-98 femtoliter FL). Plays an important role in classification of anemia: o MCV < 78 → microcytic anemia as in IDA, Thalassemia and ACD MCV 78-98 with low Hb value → normocytic anemia: acute blood loss, leukemias and ACD. o MCV > 98 → macrocytic anemia as in megaloblastic anemia, hypothyroidism, MDS and alcohol intake Mean cell hemoglobin (MCH): Is the average mass of hemoglobin (Hb) per red blood cell (RBC) in a sample of blood. MCH = Hb x 10 / RBC count. Normal range = 27-31 picograms/cell. The amount of hemoglobin per RBC depends on hemoglobin synthesis and the size of theRBC The weight of the red cell is determined by the iron (as part of the hemoglobin molecule), thus MCH in picogram is the weight of one red cell. In iron deficiency anemia the cell weight becomes lighter, thus a MCH < 27pg is an indication of iron deficiency. The MCH decreases when Hb synthesis is reduced, or when RBCs are smaller than normal, such as in cases of iron-deficiency anemia. Mean corpuscular hemoglobin concentration (MCHC): Is a measure of the concentration of hemoglobin in a given volume of packed red blood cell. MCHC = Hb/HCT. Normal range = 32-36 gm/dl. Low MCHC correlate with low Hb value and the anemia is called hypochromic as in IDA. High MCHC value means the RBCs are hyperchromatic and there is a high concentration of hemoglobin in the red blood cell as in AIHA, HS and in megaloblastic anemia. Spurious increase in MCHC (>36g/l) can occur due to cold agglutinin (RBC aggregates). And hypertriglyceridemia (>2000mg/dl). Red cell distribution width (RDW): Is a measure of the range of variation of red blood cell (RBC) volume (confession variation of cells). Normal range: RDW-CV 11.5-14.5% RDW-SD 42.5 ± 3.5 fl. If anemia is observed, RDW results are often used together with mean corpuscular volume (MCV) results to determine the possible causes of the anemia. It is mainly used to differentiate an anemia of mixed causes from an anemia of a single cause. Normal RDW in presence of anemia may indicate thalassemia trait or anemia of chronic disease.Higher RDW values indicate greater variation in size of cells usually seen in IDA, MA, mixed deficiency and in microcytic anemia received blood or hematinics WBC/differential count: Normal WBC count and its differential count differs according to patient age, usually higher pediatric age group than in adults. Normal count in adults: 4-11 x109/l Low WBC count called leucopenia, due to many causes: Infection. Inflammation. Drug administration (chemotherapy). Malignancy. Aplastic anemia. Severe nutritional deficiency. High WBC count called leukocytosis, due to: Infection. Inflammation. Drug administration like steroid. Malignancy like leukemias, myelo and lympho-proliferative disorders. Leukemoid reaction. Spurious WBC count occurs in presence of nucleated red cells, lysis resistant RBC (Hb-C and Hb-S) and lipids Two types of WBC: Granulocytes consist of: o Neutrophils. o Eosinophils. o Basophils. Agranulocytes consist of: o Lymphocytes. o Monocytes. Neutrophil: Neutrophils are the most abundant type of granulocytes and make up 40% to 70% of all white blood cells in adult humans. Normal count: varies according to age, generally: 2- 7x109/l Eosinophil: Normal count: 0.2-0.5x109/l Basophil: Usually found in low count and not always seen in peripheral blood smear. Normal count: varies according to age, generally: 0.02-0.1x109/l Lymphocyte: The second most abundant type of WBC and the normal value differs according to the patient age. Normal count in: children: 2-9 x109/l, adults: 1-3 x109/l Monocyte: The largest type of WBC that can differentiate into macrophages and myeloid linage dendritic cells. Normal count: 0.2-91 x109/l PLT Count: Generally, the normal platelets count in adults ranges between 150-450x109/l, higher counts are seen in children. Elevated platelets count, thrombocytosis, can be reactive secondary to blood loss infection, inflammation malignancy, splenectomy and IDA or can be as a part of myeloproliferative disorder. Thrombocytopenia can be due to peripheral consumption and destruction(splenomegaly, immune, infection, drugs, TTP) or failure of production secondary to BM failure (aplastic anemia, leukemia, MDS) or infiltration by fibrosis, carcinoma, lymphoma Pseudo thrombocytopenia can occur due to presence of platelets clumps (EDTA induced). Spurious higher platelets count can be observed in presence of severe RBC microcytosis/ fragmentation where micro-red cells are miscounted as platelets Platelet indices: 2 1- Plateletcrit (PCT): represents the Volume occupied by platelets in the blood and expressed as a percentage o PCT = platelet count × MPV / 10,000 o The normal range for PCT is 0.22–0.24% 2- Mean platelet volume (MPV): a calculated measure of thrombocyte volume measured in FL. o Typically, the normal range: 7.2–11.7 fL. o When platelet production is decreased, young platelets become bigger and more active, and MPV levels increase. o Increased MPV indicates increased platelet diameter, which can be used as a marker of production rate and platelet activation. o Thrombocytopenia with MPV is associated with ITP and Hereditary platelet disorders (May Hegglin anomaly, Bernard Soulier syndrome) while htormbocytopenia with low MPV can be found in Wiskott Aldrich syndrome. 3- Platelet volume distribution width (PDW): is an indicator of volume variability in platelets size again expressed as a percentage o Increased in the presence of platelet anisocytosis o Normal range: 8.3 to 56.6% 4- Platelet larger cell ratio (P-LCR): Indicator of larger (> 12 fL) circulating platelets, again expressed as a percentage o The normal percentage range is 15–35%. o It has also been used to monitor platelet activity 5- Other indices include: Mean platelet component (MPC), Mean platelet mass (MPM), and Immature platelet fraction (IPF, early index of thrombopoiesis Histogram CBC histogram analysis , has a good potential to provide diagnostically relevant information about many disease process even before higher level investigations are ordered. It is a universal, economical and simple method to narrow down the differential diagnosis at early stages of patient evaluation. WBC Histogram A normal WBC histogram is shown in the Lymphocytes are distributed between 50-100 fL, mixed cell population (monocytes, basophils and eosinophils) between 100-150 fL, neutrophils between 150-300 fL A normal WBC histogram. Lymphocytes are distributed between 50-100 fL, mixed cell population (monocytes, basophils and eosinophils) between 100-150 fL, and neutrophils between 150-300 fL RBC and platelet distribution curve: RBCs and platelets are counted in the same chamber and plotted in a same graph An arbitrary line is drawn through the trough of the curve which is usually in the 25-30 fL regions. All the cells towards the left of this line are counted as platelets and towards the right as RBCs Normal platelet and RBC histogram REFERANCE 1. Dacie and Lewis PRACTICAL HAEMATOLOGY chapter3 2. Fujimoto, K. 1999. Principles of measurement in hematology analyzers manufactured by Sysmex Corporation. Sysmex J. 3. Roy M, Akshay A. M2G1G2 white blood cell flag by three-part automated hematology analyzer: A hint to dengue infection in appropriate clinical context J Lab Physicians. 2019;11:103– 4.Brown, Barbara A. Haematology: Principles and Procedures. Sixth Edition. 4 Hoffbrand AV, Moss PAH, Pettit JE, editors. Essential Haematology. 5th ed.

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