Routine Hematology Procedures – Flashcards
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Classic method to measure Hb-
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Cyanmethemoglobin method- specimen EDTA whole blood. Hemoglobincyanide reagent changes iron from ferrous state to ferric state and forms cyanmethemoglobin (measured at 540 nm on spectrophometer)
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All forms of Hgb are measured except...
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Sulfhemoglobin
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Factors that affect hemoglobin-
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1. High white count 2. Lipemic specimen(cloudy cause high fat content) 3. Hemoglobin C crystals(incomplete lysis) 4. Scratched/dirty curvettes
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Abnormal hemoglobin pigments-
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Not capable of transporting oxygen, if you get too much in system causes hypoxia(decreased oxygen) and cyanosis(blue skin)
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3 most significant abnormal hemoglobin pigments-
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1.Carboxyhemoglobin 2. Methemoglobin 3. Sulfhemoglobin
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Carboxyhemoglobin
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Formed when carbon monoxide binds with hemoglobin giving cherry red color characteristic( CO affinity for Hb is 200X that of O) smokers have 1-10% chance
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Methemoglobin
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Formed when ferrous iron oxidized to ferric state, is reversible, and normal at 1-2%
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Sulfhemoglobin
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Not normally found in blood at any level and its formation is not reversible for life of RBC, formed by certain drugs/chemicals
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Hematocrit(Hct)-
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Space occupied by packed RBCs of centrifuges while blood. Expressed as %- also called PCV-packed cell volume(taken at top of RBC layer)
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Normal values for hematocrit for adult males-
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40-55%
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Normal values for hematocrit for adult females-
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36-48%
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Hematocrit is smaller in
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Anemia
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Microhematocrit method-
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Collected in capillary tubes or transferred from EDTA tube. Blue ringed capillary tube=no anticoagulant , red ring=heparin. Centrifuge 3-5 min at 10000-15000 g of force
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Blood cell counts-
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Volume reported in Liters usually
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1 cu mm=
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1 uL= 10^-6 Liters
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1 x 10^6 uL =
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1 L = 1000000 uL
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White blood cell count normal value-
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4000-11000 /uL (4.0-11.0 x10^9 /L)
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White blood cell count-
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Expressed as # per Liter
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WBC good indicator of...
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Infection- elevated in bacterial infections, appendicitis, leukemia a, pregnancy, etc. (low in viral diseases)
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Leukocytosis-
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When WBC is over 11000/uL
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Leukopenia-
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When WBC is under 4000/ uL
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Manual white blood cell count-
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WBC,RBC, platelet,& eosinophils counts all use same techniques/method for dilution and counting cells
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Acceptable diluting fluids for WBC count-
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2% acetic acid, 1% HCL, 1%ammonium oxalate(unopette) or Turks diluting fluid(glacial acid, gentian violet and H2O)
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Neubauer hemocytometer-
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Slide like device allows manually count cells in microscopic grid
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Of 9 large squares 1 is equal to...
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.1 uL (1mm x 1mm)
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Thoma white count pipette-
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3 number markings on it- .5,1,& 11. Marking used to make whole blood dilutions with dilution fluids
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When drawing blood to .5 mark on white and diluting to 11 results in-
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1:20 dilution
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Drawing blood to 1 mark on white and diluting to 11-
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1:10 dilution
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Thoma red count pipette-
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3 numbers consisting of .5,1,& 101
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Drawing blood to .5 mark on red count to 101 mark makes
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1:200 dilution
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Drawing blood to 1 mark on red count to 101 dilute is a
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1:100 dilution
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Manual calculation of blood cell counts-
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# of cells counted X dilution divided by #of squares counted X the volume of that size square = cells/uL
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2 WBC unopette system-
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1. Uses 1:20 dilution 2. Makes 1:100 dilution and can be used to count platelets as well
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Blood smears-
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Take drop of blood and spreading onto slide for purpose of identifying cells/determining morphology- differential(requires 100 WBC to be identified)
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3 types of slides that can be made:
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1. Wedge smear(most common) 2. Spun smear 3. Cover glass smear
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Wedge smears-
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Stained with romanowsky stains either wright stain or wright-giesma stain(polychromatic stains) contain 2 ingredients- methylene blue(basic dye)& Eosin Y or B(acidic dye)
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Should be stained..
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Within 2 hours and stained 2-4 hours for best results
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pH of romanowsky stains-
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Must be between 6.4-6.7 to have base and acid stain evenly
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If stain is too alkaline..
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Is will stain too blue
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If stain is too acid...(low)
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Will stain too red
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Differential cell count-
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Performed to determine relative number/% of each type of white blood cell present in blood
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For platelet estimation...
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Approximately 8-20 platelets/oil iteration field(1000X) is considered normal
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Shift to the left
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Increase in immature granulocytic cells(bands,metas,myelocytes)
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Shift to right
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Increase in hyper segmented neutrophils(more mature)
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Absolute WBC count
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% of each WBC determined by performing differential called relative cell count- determined by multiplying total RBC count by percent(decimal) of each WBC count
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Relative cell count normals %-
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1. Neutrophils: 35-70% 2. Lymphocytes: 25-45% 3. Monocytes: 1-10% 4. Bands: 0-6% 5.eosinophils: 0-4% 6. Basophils: 0-2%
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Absolute cell count normals x 10^9/L -
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1. Neutrophils: 1.5-7.5 2. Lymphocytes: 1-4.5 3. Monocytes:0.1-1.0 4. Bands:0-0.6 5. Eosinophils:0-0.4 6. Basophils:0-0.2
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Red blood cell indices-
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Used to define size and hemoglobin content of RBC &differentiation of anemias- 3 main: 1. Mean corpuscular volume(MCV) 2. Mean corpuscular hemoglobin(MCH) 3. Mean corpuscular hemoglobin concentration(MCHC)
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Mean Corpuscular Volume(MCV)-
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Hct/ RBC/L)x 10. Indicates average size of RBC. Elevated value=macrocytic RBCs, decreases value=microcytic
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Normal range for corpuscular volume-
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80-100 fL
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Mean Corpuscular Hemoglobin(MCH)-
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Hgb g/dL / RBC/L x 10. Indicates average weight of hemoglobin in erythrocyte. Normal range=27-32 pg
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Mean Corpuscular Hemoglobin Concentration(MCHC)-
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Hgb g/dL / Hct% x 100. Is average conc. of hemoglobin per unit volume of RBC- normal range 32-37%
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Erythrocyte sedimentation rate(ESR/sedrate)-
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Distance RBCs fall in anticoagulated whole blood for 1 hour. Affected by 3 factors: 1. Erythrocytes(sickle cells, spherocytes) 2. Plasma(>viscosity=<ESR) 3. Mechanical & technical factors(ESR tube be perpendicular- a 3 degree tilt can cause 30% error
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Larger the particle, the....
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Faster is falls
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Altered plasma proteins m...
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Causes rouleaux formation and increases ESR
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Sickle cells& spherocytes...
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Decrease ESR
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In polycythemia...
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ESR will be normal
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When is ESR increased?
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Pregnancy(3rd month), acute/chronic infection, rheumatic fever, rheumatoid arthritis,menstrations, TB, etc.
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Modified westergren method-
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3 mL EDTA whole blood set within 2 hours. Mixed whole blood diluted with .85% NaCl in Westergren pipet
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Normal values for modified westergren method for women-
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0-15 mm/hr
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Normal values for modified westergren method for men/children-
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0-10 mm/hr
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Reticulocyte count-
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RBCs go through 6 stages of development
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Normal adult reticulocyte count-
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0.5-1.5%
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Normals infant reticulocyte count-
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2-6%
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Decreased reticulocyte count caused by-
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Aplastic anemia, chemotherapy
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Increased reticulocyte count caused by-
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Acute/chronic blood loss, iron therapy for IDA, thalassemia, etc.
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retics to be stained with supravital stain..
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New Methylene Blue
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Principle-
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Retic in RBC which has lost nucleus but has small amnt RNA present
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Count approximately _____ RBC for reticulocyte count
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1000
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Retic count formula-
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#of retics counted/ # of RBCs X 100= % of retics (use miller disc)
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RBC inclusion bodies stained with New Methylene Blue and can be confused with retics-
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Howell-Jolly bodies, Heinz bodies, hemoglobin H,& most difficult is Pappenheimer bodies (look on Wright stained smear)
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Corrected retic count-
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Patient Hct / normal Hct(45) X retic count%
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Platelet count-
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Normal range 150-400 x 10^9 /L
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Thrombocytosis-
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Increase in platelets (polycythemia, idiopathic thrombocythemia, splenectomy)
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Thrombocytopenia-
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Decrease in platelets(chemotherapy, radiation, aplastic anemia, acute leukemia)
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Methods for platelet count-
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Counted on hemocytometer using regular microscope of phase contrast microscope
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Unopette method-
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1:100 dilution with 1% ammonium oxalate, let stand for 10 min, discard 3-4 drops from mixture/charge hemocytometer
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Rees-Ecker method-
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Reference method utilizes brilliant cresyl blue as combination stain does not hemolysis RBC but stains platelets light bluish color making them highly refractive( must be done in 30 min)
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Eisinophil count-
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1. Indirect method- %Eos from diff. X WBC/L=Eos/L 2. Direct method-same as WBC&RBC count reference range 50-350 x 10^6/L
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Eosinopenia-
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Low Eos count (Cushing disease, shock)
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Eosinophilia-
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High Eos count (allergic reaction, parasitic infections)
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Diluting fluids-
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Phyloxine diluting fluids, Pilots solution, Randolphs stain
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Counting chambers-
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1. Newbauer hemocytometer-0.9uL total volume on one side 2. Fuchs-Rosenthal-3.2uL one side
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3. Speirs-Levy-
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2.0uL,4 sides =8.0
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Eos/L=
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Eos counted X dilution / volume of chamber x 10^6
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Sickle cell disease and test-
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1. Sodium Metabisulfate method 2. Tube solubility test(sodium dithinoite tube test)
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Concentration of Hb S with sickle cell anemia-
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80-100% slight oxygen decrease cause RBC to sickle
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Concentration of Hb S with sickle trait -
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20-40% much go much lower for cells to sickle
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Sodium Metabisulfate method-
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Sodium Metabisulfate deoxygenate RBC causing cells to sickle wig those that can sickle
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Sodium dithinoite tube test(tube solubility test)-
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Sodium glhydrosulfate regent containing saponin mixed with specimen causes turbid mixture when Hb S present tube placed in from of whit paper with lines on it
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Hemoglobin electrophoresis-
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Preformed as confirmation test on positive sickle cell tests since Hb Barrs and Hb C Harlem cause false positives
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