Erythrocyte Sedimentation Rate (ESR)

Erythrocyte Sedimentation Rate (ESR)  The rate at which RBCs sediment in a period of 1h:  If anti-coagulated blood is allowed to stand vertically in a tube : the red cells will settle progressively to the bottom & leaving clear plasma above (RBC and plasma will be separated). Equipment:  Westergren tube  Westergren stand  Anticoagulated blood Westergren Method For Estimation Of ESR 1) Pipette sodium citrate to the Westergren tube (0.2 ml) 4) Add 1.8 ml whole blood to the Westergren tube: 5) 6) Place the Westergren tube into its stand. In one hour, record the distance in millimeters between the: meniscus of the plasma and the top of the erythrocyte column. Increased ESR may be due to : Pregnancy / Inflammation / Cancer Normal values : Men 2-10 mm/hr Women 2-15 mm/hr Decreased ESR may be due to : Polycythemia / Sickle cell anemia Hemoglobin A globular protein present in the human blood It is composed of : the protein globin & the pigment heme (combine with oxygen is due to: 4 iron atoms). Hb measurement with : Sahli's Hemoglobinometer consists of:  2 Standard color tube.  1 graduated tube (Sahli column)  A capillary measuring tube (Sahli Pipette) Procedure Sahli's test 1) By Pasteur pipette , Place 0.02 ml of HCL, into the graduated tube. 2) Suck up the blood from the finger by the Sahli Pipette till the 0.02 ml and transfer the blood into the mixing tube 5) Add drop by drop water, mixing well by a glass rod and compare the color of this tube with the standard tube. 6) Continue adding water until reaching the standard color. 7) After 2min, read the Hb content from the scale on the graduated tube. The principle in Sahli's method involves:  the conversion of Hb to acid hematien by the action of HCl. Normal range of Hb content : In male 14- 16 / In female 12- 14 gm/100ml of blood What is Blood Pressure ?? The cardiovascular system is overfilled with blood: (contents more than capacity): the blood exerts outward force against the vessel walls as it flows through them. The term BP : refers to the force exerted by the blood as it presses and attempts to stretch the walls of blood vessels. The BP is not steady (unchanging) throughout the cardiac cycle but fluctuating, i.e. it is pulsatile: It rises and falls; When reaching its maximum during systole, it is called the Systolic BP (SBP). and falling to its minimum during diastole of the heart ,when it is called Diastolic BP (DBP). Apparatuses: 1. Stethoscope 2. Sphygmomanometer ( “BP apparatus”) 1. Place the cuff over the upper arm. (The lower edge of the cuff should be about 2.5 cm above the elbow joint.) 2. Place the chest-piece of the stethoscope on the place of detecting the pulse ,and keep it in position with your fingers . 3. Inflate the cuff until the pressure reaches about 160 mmHg. 4. The artery is compressed by inflating a rubber bag. 5. The pressure is then slowly released(Slowly reduce the pressure in the cuff (by deflate the cuff. 6. & the flow of blood through the obstructed segment (turbulent blood flow) can be detected by listening or feeling the pulse. Auscultatory method:  requires the stethoscope  Ordinarily no sound are heard when a stethoscope is applied over the brachial: Laminar flow is silent.  if the cuff pressure is raised above the systolic pressure and then gradually lowered, a series of sounds, called Korotkoff sounds, are heard over the artery , below the cuff.  The Systolic Pressure is the pressure at which sharp, tapping sounds are first heard. 6) Continue slowly reducing cuff pressure   The Diastolic Pressure is defined as the pressure at which the sounds disappear. laminar flow again is held: (silent) Palpatory method: 1. Palpate the radial pulse: the index and middle fingers, near the base of the thumb. 2. While palpating the radial pulse, rapidly inflate the cuff until there is no detectable pulse & then add another 30 mm ( value in the range of 160–180) 3.  Deflate the rubber bulb by opening the valve , so pressure release slowly . When you detect the pulse again by palpation,This is Systolic pressure. Disadvantages : only the systolic pressure, the diastolic pressure cannot be measured. Advantages: The BP can be measured in noisy environment. Mean arterial pressure (MAP) • useful measure of the adequacy of tissue perfusion, • is not a simple average of systolic and diastolic BP s. (diastole continues for twice as long as systole.) MAP = [(2 x diastolic)+systolic] / 3 Blood Group testing Blood Group Ag on cell (RBC type) Ab in plasma A A Anti-B B B Anti-A AB A&B none O None Anti-A & B 1) Put 3 blood drops on the slide 2) Add 1 drop of anti-A , anti- B, anti-R on them. 3) Mix with a stick & Leave the test for 2-3 min at room temp. Rh (D) Antigen  Rh (Rhesus) is a blood group system with many antigens: C,D,E  Rh refers to the presence or absence of the D antigen on the RBC.  Makes the Blood Type “Positive” or “Negative”  Positive= Agglutination with RhD Serum  Negative = No Agglutination with RhD Serum Blood group compatibility for Blood transfusion Blood Group Ag on cell Ab in plasma A A Anti-B A or O B B Anti-A B or O AB A&B none AB, A, B or O O None Anti-A & B O Transfuse with group In blood transfusion :  Rh⁻ person cannot receive blood from Rh⁺ person,  whereas Rh⁺ person can receive blood from both Rh⁻ & Rh⁺ person. Rh incompatibility 1) blood transfusion: If a Rh⁻ person receive blood from Rh⁺ person for first time: there will be formation antibodies (antiRhD) in the plasma So, if a second transfusion is done again with Rh⁺ blood, the antibodies which are already present causes clumping. 2) Rh incompatibility in pregnancy Work With Microscope Body Tube Nosepiece Objective Lens Eyepiece: Ocular lens Arm Stage Stage Clips Diaphragm Light Coarse adjustment screw Fine Focus Base To determine the Magnification 1. Each objective will have written the magnification. 2. In addition, the ocular lens (eyepiece) has a magnification (10). 3. The total magnification is the ocular x objective.  light microscope has 3 magnifications: Scanning (4X), Low(10) and High(40) Using a Microscope 1) Plug your microscope in to the outlet. 2) Place slide on stage and lock clips and Adjust light source 3) Start on the high magnification (40x) . Use the Coarse screw : only contact the objective lens with the slide, don't touch .then the lowest power (4x) is clicked into position, and Look through the eyepiece. 4. Use fine adjustment knob until the image comes into focus (make the image clear.) Cell count with hemocytometer Neubauer chamber The counting chamber : a thick glass microscope slide with the size of a glass slide. Hemocytometer Neubauer chamber: Cover slip: Squared glass , is placed on the top of the Neubabuer chamber. the chamber is designed so that the distance between the bottom of the chamber and the cover is 0.1 mm (1 μl) The counting area (Under the microscope )  It’s engraved with ruled grid of the lines (with a laser-etched) By 4X magnification: 9 large squares . Each square is 1mm long and 1mm wide. The central large square is divided into 25 squares (By 10X magnification). Each of them is further divided into 16 small squares. Each group of 16 small squares is separated by closely ruled triple lines. (By 40X magnification) The 4corner squares are further divided into 16 small squares. (By 10X magnification) 2) Mélangeur Pipette (Thoma) A glass capillary pipette used for diluting the blood. A volumetric pipette (with bulb) allows extremely : accurate solution volume measurement A) RBC pipette: B) it has 3 markings: 0.5, 1.0, and 101 (B) WBC pipette: it has 3 markings: 0.5, 1.0, and 11 Cont. Equipments: Mélangeur Pipette A) RBC pipette: it has 3 markings: 0.5, 1.0, and 101 (B) WBC pipette: it has 3 markings: 0.5, 1.0, and 11 ( Differences Between the Two Pipettes RBC pipette WBC pipette Calibrations 0.5 & 1 0.5 & 1.0 below the bulb/11 below the bulb/ 101 above above. Capillary bore Narrow: a slow-speed pipette. Wider: fast-speed pipette. Bulb larger ,with red bead. smaller , with white bead. Volume of the bulb 100 times the stem volume 10 times the stem volume The dilution 1 in 100 / 1 in 200 1 in 1 0 / 1 in 20. Total Red Blood Cell Count Principle : Since the number of blood cells is very high difficult to count, even under the microscope.  overcome the difficulty by : diluting the blood to a known degree with suitable diluting fluids. Diluting Fluid Hayem’s solution: For RBC must be:  Isotonic fluid: Preserve RBC shape (Maintain the normal disk shape ):  anti-hemolysis  due to high Hb content, RBCs can be visualized in the microscope without staining. Steps in Hemocytometry: process of cell counting 1) Pipetting (filling the pipette ) the blood and diluting it: Draw the blood up to exactly 0.5 mark and dilute to the 101 mark. 2) Mix (rotate) continuously for 2-3 minutes: 3) Load the cleaned hemocytometer: (The capillary effect will draw the mixture solution into the chamber) 5) The cells spread out in a single layer which makes their counting easy. 6) Using Conventional light microscope:, Normal ranges of the number of erythrocytes per µL of blood Male 5.2 /Female 4.7 W W The large center square :Observed with the 10X objective), 25 middle squares, (with triple lines) each divided to 16 small squares Depth = 0.1 mm Grids W W RBC use : 5 small squares in the center large square WBC use: 4 corner large squares using 10x objective 1) Using the 10X objective lens , on the large central square. 2) RBCs counting is performed in the small squares, using 40x objective The square are used to count RBCs: five designated squares (of the 25 squares, only the four corner squares, and one middle) Total RBC Count N x Dilution Factor x = Volume Correction Factor N = the total number of red cells counted in the 5 medium squares We need to consider all 25 medium squares in the centrally square Dilution Factor= 200 Draw the blood up to exactly 0.5 mark & dilute to the 101 mark: Dilution 1 : 200 Volume Correction Factor= X 10 the volume underside of each coverslip is 0.1 mm3 N in five medium squares X 5 / X 200 X 1 0 = N in five medium squares X 10,000 so in 1 mm3: X 10 WBC manual Count To determine dilution factor RBC pipette: We fill the pipette with blood to mark : “0.5” & reach with dilution fluid to mark : “ 101” : This gives a dilution factor of 200. WBC Pipette: We fill the pipette with blood to mark : “1” & reach with dilution fluid to mark : “ 10” : This gives a dilution factor of 10.  Diluting Solution For WBC count: Turk‘s solution Glacial acetic acid = 1.5ml → lyses RBC crystal violet (stain WBC) 1% = 1ml→ stain nuclei of WBC For WBC count: use 4 corner large squares using 10x objective . make an average of them. Calculations: Total cell Count Total WBC Count = N x Diluting Factor x Volume Correction Factor Mean number of cells in 1 big square x 10 x 10 = N X 100 N in 4 big corner squares 4 Normal ranges of Leukocytes: Adult 4,000 – 11,000 cell/mm³ Leukocytosis Leukopenia  Leukocytosis • as an indicator of body defense against the bacteria, parasites, toxins • Leukemia (high immature WBCs)  Leukopenia ◦ Typhoid infection,TB , Measles ◦ Result from X-ray therapy ◦ Alcoholism Differential Leukocyte Count A total white blood cell count is not an indicative of the severity of the infection, inflammation,..  We need to determine the number of different types of white blood cells. Slide Method for Differential Leukocyte Count Procedure: 1. Preparation of blood smear: a thin layer of blood smeared on a slide 2. Fixation of blood smear. 3. Staining of blood smear. Prepare a blood smear:  Note: As soon as the drop of blood is placed on the glass slide, the smear should be made without delay. Any delay results in an abnormal distribution of the cells, with many of the large white cells accumulating at the thin edge of the smear. Characteristics of a Good Smear 2) Slide fixation (with Methyl alcohol (methanol) )  To preserve the morphology and chemical status of the cells: slide must be fixed as soon as possible after preparation. act as a ‘glue’ which fixes the blood cells to the slide : so they are not washed away during staining. 1. dig in the slide to the jar containing methanol (1 minute) 2. Empty the excess amount 3) Staining of blood smear: GIEMSA 1) Place slides into the staining counter: (Flood it) 2) Leave it in stain box for 20 minutes 3) Wash off by tap water directly to the center of the slide to : prevent a residue of stain. 4) Stand slide on end, and let dry in air. The combinations have two essential ingredients : 1. Eosin : (a pink color). 2. Methylene-blue: ( a blue-violet color) Observing direction : using the zigzag method  Observe one field and record the number of each type WBC  Then turn to another field in the snake-liked direction  *avoid repeat or miss some cells  The diff. count takes place under 100X lens.  Using the oil immersion lens (correcting the refractive index & have more clear images)  Count 100 WBCs including all cell lines from immature to mature. Reporting results: % of each cell type in differential The diff. count takes place under 100X lens. Using the immersion oil lens (correcting the refractive index & have more clear images)  Count 100 WBCs including all cell lines from immature to mature. Reporting results: % of each cell type in differential 5 types of WBCs: % of total WBC diameter (µm ) Neutrophil 62% 10-16 Lymphocyte 30% 7-12 Monocytes 5% Eosinophil 2% 14-18 0.4% 12-16 Cell type Basophil Smallest 14-20 Largest Granulocytes 1) Neutrophils : most numerous Nucleus:2-5 lobes dark purple / dense chromatin 2) Eosinophils ( Acidophils): Cytoplasm : full of red granules Nucleus: 2 lobes (like a pair of glass). 3) Basophils: Cytoplasm : with dark blue granules  Nucleus: blue, obscure Segmented neutrophil Band neutrophil: An immature cell Hyper-segmented neutrophil:  Increased bands mean acute infection (usually bacterial)  Hyper-segmented neutrophil:very old cells Agranulocytes Lymphocytes (30%) Smallest WBC Nucleus: dark blue \round/ dense chromatin A big nucleus occupies most of the cell. Monocytes (5%) Largest of WBCs Nucleus: light purple, large kidney shaped small amount of cytoplasm Monocyte: Lymphocyte lymphob last lymphocyte Diagnostic importance ↑Neutrophils : inflammation/body's primary defense against bacterial infection. ↑Eosinophils : allergy, parasitic infections ↓ Eozinophils – stress ↑ Lymphocytes : cancer (leukemias –cancerous production of lymphoid cells) An alteration in BM function (leukemia) may cause an increase in them .