The document discusses various topics related to CBC morphological assessment including red blood cell analysis, indices calculations, classification of anemia, and microscopic examination of blood films. Red blood cell analysis involves quantitative and qualitative characteristics to evaluate red blood cell concentration, size, and hemoglobin content. Anemia can be classified morphologically based on red blood cell size as macrocytic, microcytic, or normocytic, or etiologically based on causes related to reduced production, increased destruction, or blood loss.
The document discusses various topics related to CBC morphological assessment including red blood cell analysis, indices calculations, classification of anemia, and microscopic examination of blood films. Red blood cell analysis involves quantitative and qualitative characteristics to evaluate red blood cell concentration, size, and hemoglobin content. Anemia can be classified morphologically based on red blood cell size as macrocytic, microcytic, or normocytic, or etiologically based on causes related to reduced production, increased destruction, or blood loss.
The document discusses various topics related to CBC morphological assessment including red blood cell analysis, indices calculations, classification of anemia, and microscopic examination of blood films. Red blood cell analysis involves quantitative and qualitative characteristics to evaluate red blood cell concentration, size, and hemoglobin content. Anemia can be classified morphologically based on red blood cell size as macrocytic, microcytic, or normocytic, or etiologically based on causes related to reduced production, increased destruction, or blood loss.
The document discusses various topics related to CBC morphological assessment including red blood cell analysis, indices calculations, classification of anemia, and microscopic examination of blood films. Red blood cell analysis involves quantitative and qualitative characteristics to evaluate red blood cell concentration, size, and hemoglobin content. Anemia can be classified morphologically based on red blood cell size as macrocytic, microcytic, or normocytic, or etiologically based on causes related to reduced production, increased destruction, or blood loss.
BSC - OMDURMAN AHLIA HIGH DOPLOMA DGREE - ELZAEM EL-AZHARY FORMER HEAD OF HEMATOLOGY & BLOOD BANK MINISTRY OF HEALTH – LABORATORY ADMINISTRATION KHARTOUM STATE MARKETING MANAGER-LAB EQP –DIVISION ALGAM COMPANY FOR DRUGS & CHEMICAL LTD
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Learning Objective *1- TO KNOW Red Blood Cell Analysis . *2- TO KNOW THE IMPORTANCE OF INDICES CALCULATIONS IN BLOOD TESTING. *3- TO Know THE Classification Of Anemia. *4- TO KNOW How To Check Leishman Stain. *5- TO KNOW The differential white cell count.
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Learning Objective *6- TO KNOW The Value of Good Morphological Assessment. *7- TO KNOW Microscopic Examination of Blood Films. *8- TO KNOW PROCEDURE FOR EXAMINATION OF STAINED BLOOD SMEAR. *9- TO KNOW Sources of error OF STAINED BLOOD SMEAR.
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Red Blood Cell Analysis
*Red Blood Cell Are Define :
* 1-Three Quantative Value: 1- Volume of packed red cell (VPRC) or hematocrite. 2- Hb concentration . 3- The red cell concentration per unite of volume.
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Red Blood Cell Analysis • *2- Three Qualative Characteristic Of The Red Cell Population. • 1- MCV (Mean Cell Volume) • 2- MCH (Mean Cell Hemoglobin) • 3-MCHC (Mean Cell Hemoglobin Concentration)
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CONTENUE • *1- volume of the red cell hematocrite : • *It is the proportion of the volume of blood sample that is occupied by red blood cell. • *determine manually by centrifugation of blood at agiving speed and time in standardized glass tube with uniform bore. • *The height of the column of red cell compared with that of the total column of blood following centrifugation yields the hematocrite .
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Source Of Inherent Error
• *Hematocrite measure the red cell
concentration not red cell mass there for: • *1-Pateiont in shock or with volume depletion may have normal or high hematocrite measurement due to hemoconcentration despite a decreased red cell mass.
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Source Of Inherent Error
• *2- Trapping of plasma in the red cell column
• *The plasma may trapped in: • *Sickle cell –microcytic cell- macrocytic cell- spherocyte cell at a high level because of increase cellular rigidity.
THE IMPORTANCE OF INDICES CALCULATIONS IN BLOOD TESTING • *The RBC indices is a mathematical calculation to yield three clinical parameters useful in diagnosing anemia. • *1- Mean Corpuscular Volume (MCV): • average RBC volume is calculated by dividing the hematocrit value by the RBC count value. • *MCV = Hematocrit (L/L) x 1000 Red cell count 10/L)
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* Normal values :are 76 to 96 femtoliters (fL). *value is below 76 fL, then this indicates microcytic anemia. value greater than 96 would suggest macrocytic anemia. * falsely elevated: *1-Agglutination of RBC as in cold agglutinin disease . *2- Sever hyperglassemia may cause somatic swelling of RBC during analysis
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• *2-Mean Corpuscular Hemoglobin (MCH). This describes the hemoglobin content of each RBC . • *obtained by dividing the hemoglobin value by the RBC count value. • *The normal value ranges :from 27 to 32 picograms (pg). • *Values below 27 pg are suggestive of hypochromic anemia. • * falsely elevated by hyperlibidemia or leukocytosis.
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• *3-Mean Corpuscular Hemoglobin Concentration (MCHC): • *describing the hemoglobin concentration in RBC’s. • *MCHC := Hb (g/dl)/ hematocrit(L/L) • *Normal values range : 31 to 36 g/dL. • * Low value suggest hypochromic anemias. • * MCHC affected by factor effect the PCV &Hb g/dl • *Note: Indices normal values may vary slightly from region to region or lab to lab.
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Red Cell Distribution Width • *Reflects the range of red cell sizes measured within a sample. • *RDW has been proposed to be useful in early classification of anemia • *1- become abnormal earlier in nutritional deficiency anemia than any of the other red cell parameter especially in case of IDA .
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Red Cell Distribution Width • *2- useful in characterizing microcytic anemia particularly distinguishing between IDA (high RDW low MCV )and uncomplicated heterozygous thalassemia (normal RDW low MCV). • *3-useful to identified the following (red cell fragmentation –agglutination –dimorphic cell populations). • *4-Cold agglutinin disease increase MCV &RDW volume .
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ANEMIAS MAY BE CLASSIFIED BY RDW AND MCV RDW = N MCV = N RDW = I MCV = N Normocytic: acute hemorrhage sideroblastic chronic liver disease early IDA
RDW = N MCV = D RDW = I MCV = D
Microcytic: heterozygous thalassemia iron deficiency anemia anemia of chronic disease homozygous thalassemia
of circulating RBC’s and/or hemoglobin concentration and\ or PCV below the normal range according to age & sex. • *This means that the hemoglobin value will drop below 13 g/dL in the adult male and below 11 g/dL in the female.
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NAZAR AHMED SANGOOR 2008 18 Classification Of Anemia
• *A e ia’s may be classified in more than one
way. • *1- morphologically in which there are three general types of anemia: *a. macrocytic *b. microcytic *c. normocytic .
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Classification Of Anemia
• *2- pathophysiological or etiological classification
*two general groups of anemias appear. * A-Those anemias that are caused by a reduction in the number of RBC’s due to a maturation defect. *B-The other group of anemias are those due to increased RBC destruction
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Classification Of Anemia • *Morphological Classification Of Anemia • *Groups anemia by red blood cell size. • *1- Macrocytic Normochromic Red Blood Cell • *A- vitamin B12 deficiency. Folic acid deficiency: • 1- pernicious anemia 2- sprue • 3- following gastrectomy 4- dietary • 5- abnormal intrinsic factor 6- tape worm infection • *B- disease of the liver:
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Classification Of Anemia
• *2- Normocytic Normochromic Red Blood Cell:
• *A- defective formation of the blood cell or the presence of tumor cell in the bone marrow • 1- a plastic anemia . 2- leukemia . • 3- Hodgkin's disease . 4- multiple myeloma.
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Classification Of Anemia
• 5-leukoerythroblastosis. 6- metstatic cancer
• 7- anemia associated with renal and endocrine disease. • 8- anemia associated with inflammatory disease (chronic disorder).
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CONTENUE • *B- Abnormal hemoglobin increased destruction of the red blood cell • 1-certain acquired hemolytic anemia. • 2- paroxysmal nocturnal hemoglobinuria. • 3- sickle cell anemia. • 4- hemolytic disease of the new born. • 5- anemia of chronic renal insufficiency.
• 4-Miscellaneous. 1- Anemia Of Liver Disease • 2- Sulfhemoglobinemia • 3- Porphyries • 4- Methemoglobinemia • 5- Acute Blood Loss (e.g. car accident) • 6-Proximal Nocturnal Hemoglobinurea
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*How To Check Leishman Stain • *Check the nuclose of wbc & the cytoplasm • *Check if there is eosinophil to see the pinkish color of granules. • *Check the reddish brown color of RBC. • *To obtain good staining film avoid the air which well evaporate the glycerol. • *Buffer the stain by use buffered DW • PH 7.2-7.4. NAZAR AHMED SANGOOR 2008 35 Staining characteristics of a correctly stained normal film Nuclei Purple Cytoplasm Erythrocytes Deep pink Reticulocytes Grey-blue Neutrophils Orange-pink Lymphocytes Blue ; some small lymphocytes deep blue Monocytes Grey-blue
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36 *Staining characteristics of a correctly stained normal film Basophils Blue Granules Neutrophils Fine purple Eosinophils Red-orange Basophils Purple-black Monocytes Fine reddish (azurophil) Platelets Purple
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Staining faults Too blue Smear too thick Inadequate time in buffer Buffer pH too high Staining time too long Diluted stain overused, requires replenishment Stock MGG solution incorrectly made or made from impure dye Stock solution left exposed to bright daylight NAZAR AHMED SANGOOR 2008 38 Staining faults Too pink Excessive time in buffer
Buffer pH too low
Stock MGG solution
incorrectly made or made from impure dye Cover slip mounted before film is completely dry Too faint Staining time too short
Excessive washing after
staining NAZAR AHMED SANGOOR 2008 39 Staining faults Stain deposit Stain solution left in uncovered jar or tray Stain solution not filtered
Dirty slides
Blue background Inadequate fixation
Prolonged storage before
fixation Blood anticoagulated with heparin NAZAR AHMED SANGOOR 2008 40 *Making a blood film *A blood film may be made from non- anticoagulated (native) blood, obtained either from a vein or capillary. * from EDTA-anticoagulated blood Chelation of calcium by EDTA hinders platelets aggregation so that platelets are evenly spread and their numbers can be assessed more easily.
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*Films prepared from capillary blood usually show prominent platelet aggregation . *films from native venous blood often show small Aggregates . *Films prepared from native venous or capillary blood are free of artifacts due to storage or the effects of the anticoagulant.
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*Good laboratory practice includes: *recording the date and time the specimen is received in the laboratory. *making a film shortly after receipt of the specimen. *In this way the length of any delay in transit is known and attribution of morphological changes to prolonged storage of EDTA-anticoagulated blood.
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*A drop of blood (either native or anticoagulated) is placed near one end of the slide. *Anticoagulated blood from screw-top containers can be applied to the slide using a capillary tube, which is then discarded.
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*A drop of blood from specimen containers with penetrable lids can be applied to the slide by means of a special device that perforates the lid. *The spreader is applied at an angle of 25–30°, in front of the drop of blood, and is drawn back into it. *Once the blood has run along its back edge, the spreader is advanced with a smooth steady motion so that a thin film of blood is spread over the slide.
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*If the angle of the spreader is too obtuse or the speed of spreading is too fast, the film will be too short. *An experienced operator learns to recognize blood with a higher than normal haematocrit (Hct), which is more viscous and requires a more acute angle to make a satisfactory film and, conversely, blood with a lower than normal Hct, which requires a more obtuse angle.
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*The differential white cell count *A differential white cell count is the assigning of leucocytes to their individual categories, this categorization being expressed as a percentage or, when the WBC is available, as an absolute count. *The ICSH recommends that the differential leucocyte count be expressed in absolute numbers.
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*Cells that are normally present in the peripheral blood can be assigned to five or six categories, depending on whether non-segmented or band forms of neutrophils are separated from segmented neutrophils or are counted with them. *The differential count also includes any abnormal cells that may be present. *NRBC can be included as separate category in the differential count or, alternatively,their number can be expressed per 100 white cells.
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The Value of Good Morphological Assessment *1. Many blood diseases can confidently be diagnosed, or at least strongly suspected, on the morphology. *This can save a great deal of time, money, and even discomfort for the patient . *2. There are some diseases where the FBC can be completely normal and only the morphology will show abnormalities, such as lead poisoning.
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*3. It has been pointed out that the absolute amount of plasma water affects the counts and many of the measurements in the FBC, which may produce spuriously abnormal results. * Plasma water does not affect the morphology, and this may be of assistance in such cases. *4. The presence in a report of an obviously careful morphological assessment indicates that the FBC as a whole was looked at by a professional.
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*Blood films is essential in cases of 1. Leukopenia. 2. Leukocytosis. 3. Thrombocytosis. 4. Thrombocytopenia. 5. Anemia of unknown etiology. 6. A moderately to severely raised RDW even in an otherwise normal count. 7. A moderate to severe left shift with a normal WCC. 8. The presence of nucleated RBCs.
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*Microscopic Examination of Blood Films
*1-Survey the film at x10 magnification to get a general
impression of its quality. *2- Find an area where the red cells are evenly distributed, just touching but not overlapping, and study their morphology at x 40. *3-Scan the film to get an impression whether the leucocytes are increased or decreased.
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*Microscopic Examination of Blood Films
*4- Identify any unusual or abnormal
cells. *5-Estimate the relative proportion of platelets and note the presence of abnormally large platelets. *6-Use the x100 lens for studying the fine details of the cell morphology.
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*PROCEDURE FOR EXAMINATION OF STAINED BLOOD SMEAR
**1- Check the blood smear to ensure that it is well
made . *The tail of the film should be smooth **2- Examine the blood smear using the low power (10x) objective. *A- the red cell and white blood cell and platelets must be correctly stained
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The Smear
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PROCEDURE FOR EXAMINATION OF STAINED BLOOD SMEAR • *B- check that there is even distribution of the WBC on the smear. • *C- Estimate the WBC count by noting the number of WBC / field and the number of WBC in relation to the RBC count it should agree with the test result obtained
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CONTENUE
• *D- when scanning the blood smear it is
important to note any thing unusual or irregular such as large abnormal looking cell or rouleaux formation of the RBC. • *E- choose the area of the blood smear where the differential count is to begin place a drop of oil on the slid and carefully change the oil immersion to objective (100x ). • **3- perform the differential cell count and at the same time examine the morphology of the WBC.
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* type of examination can be choose *1- using the cross – sectional or crenellation technique . *The wbc are counted in consecutive fields as the blood film moved from side to side . *Counting should begin in the thin area of the smear where the red blood cell are slightly over lapping and procedure into the thicker area.
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*2-longitudinal method • *The wbc are counted in consecutive field from tail toward the head of the smear. • *This is the ideal method if the smear is thin enough so that the wbc may be identified all the way to the beginning of the smear.
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*3- The battlement field *uses a pattern of consecutive field beginning near the tail on a horizontal edge count three consecutive horizontal edge field ( moving away from the tail count two fields toward the center of the smear count this fields horizontal moving away from the tail count two fields vertically to the edge.
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Recommended Method For Differential
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CONTENUE
• **4- Identify each wbc seen and record on
differential cell counted until 100 wbc have been counted. • *If nucleated red cell have been counted enumerate them on a separate counter these cell are not to be included in the 100 cell differential count but are reported as # of NRBC /100 WBC
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CONTENUE
• *If any megakaryocytic cell or fragments
smudge cell or epithelial cell are seen these cell should also be enumerated in the same manner as the NRBC and reported as the • # /100 wbc
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CONTENUE
*2- Examine the RBC morphology in a thin area
of the slide where only a few of the RBC slight over lap not any variation from normal and classify these irregularities as slight, moderate, marked ,or sever
ERYTHROCYTE MORPHOLOGY EVALUATION CRITERIA Morphology Normal SL MO MAR SEV Characteristics Limits Poikilocytosis 0-2 3-10 11-20 21-50 >50 Polychromatophilia (adult) 0-1 2-5 6-10 11-20 >50 Polychromatophilia (infant) 1-6 7-15 16-20 21-50 >50 Schistocytes None 1-5 6-10 11-20 >20 Sickle Cells None (if present in any number, report as positive) Spherocytes 0-2 3-10 11-20 21-50 >50 Stomatocytes 0-2 3-10 11-20 21-50 >50 Target Cells (Codocytes) 0-2 3-10 11-20 21-50 >50 Tear Drop Cells 0-2 2-5 6-10 11-20 >20 Rouleaux Formation Aggregates of 3 to 4 RBC’s = 1+ Aggregates of 5 to 10 RBC’s = 2+ Aggregates of >10 RBC’s = 3+ only a few free RBC’s noted
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CONTENUE *3- Examine the platelets on the smear for morphology and number present. * Determine the approximate number of platelets per field a normal blood smear (normal RBC & normal platelets count )should show approximately 8-20 platelets per field . One method for reporting platelets estimates is to determine the average number of platelets per field 5 to 10 different field and multiply this result by 20.000 .
*1- when reporting manual differential Wbcc the result are
reported as a percentage of each cell type present. *The most accurate and also the most preferable method of reporting is the absolute count because the result expressed in percent is relative number and can be misleading to the clinician. *The absolute count is calculated as: Absolute# of cell/L=% of cell type in deferential X Wbc/L
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• *2- when counting a deferential the following outline may be followed • *A- WBC : • *- check for even distribution and estimate the number present also look for any gross abnormalities present on the smear • *-perform the differential count • *-examine for morphology abnormality
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*Relative And Absolute Normal Adult Values
Normal Range: WBC Count = 5000 µL to 10,000/µL
Relative Values Absolute Values Bands = 0 to 6% Bands = 0 to 600/µL Neutrophils = 55 to 68% Neutrophils = 2,000 to 6800/µL Lymphocytes = 20 to 40% Lymphocytes = 1000 to 4000/µ Monocytes = 2 to 8% Monocytes = 200 to 800/µL Eosinophils = 0 to 3% Eosinophils = 0 to 300/µL Basophils = 0 to 1% Basophils = 0 to 100/µL
• *C- Platelets • - Estimate number present • -Examine for morphologic abnormalities • 3-When studding stain smear do not examine in the thick area always look for the cell where it well distributed
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*4- when the white count below 1.000 it may be difficult to find many wbc on stained smear so differential may be perform by counting 50 wbc annotation on the report must be made that only 50 wbc were counted (alternatively Buffy coat smear may be perform
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*5- when the differential show an abnormal distribution of cell type as a- over 10%eosinophils b- over 2% basophile • c- over 11%monocytes d- more lymphocyte than neutrophil (except for children) • *A 200 cell differential may performed then the result are average (divided by 2)and notation is giving that 200 cell where counted NAZAR AHMED SANGOOR 2008 75 • *6- before reporting the platelets is decrease • *Scan the film for clump on low power especially the feathered edge also recheck the tube for clot • *7- if the differential count show the present of immature granulocyte cell this is termed a shift to left (leukemia, bacterial infection) • A shift to right refer to an increase number of hyper segmented neutrophils.
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*Sources of error • *Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide • *Holes in film: Slide contaminated with fat or grease • *Irregular leucocyte and platelet distribution, especially in tail: poor film-making technique • *Film too short and too thick: spreader held at incorrect angle
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*Sources of error • *Film extending to end of slide: blood drop too large • *Short thin film: blood drop too small • *Film extends to edge of slide: spreader too wide or not positioned correctly • *Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water
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ANY QUESTION ?
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Learning Outcome *1- KNOWING Red Blood Cell Analysis . *2- KNOWING THE IMPORTANCE OF INDICES CALCULATIONS IN BLOOD TESTING. *3- KNOWING THE Classification Of Anemia. *4- KNOWING How To Check Leishman Stain. *5- KNOWING The differential white cell count.
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Learning Outcome *6- KNOWING The Value of Good Morphological Assessment. *7- KNOWING Microscopic Examination of Blood Films. *8- KNOWING PROCEDURE FOR EXAMINATION OF STAINED BLOOD SMEAR. *9- KNOWING Sources of error OF STAINED BLOOD SMEAR.
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Summary
*1- CBC morphological assessment is very
important diagnostic tool need to be done professionally by expert person who followed good laboratory practices and get enough knowledge to examine the smear.
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Summary
*2- person who examine the smear
should have background knowledge about normal and abnormal blood cell morphology. *3- interpretation of result obtain is very important.