A RBC count is used to evaluate any type of decrease or increase in red blood cells. These changes must be interpreted in conjunction with other parameters, such as hemoglobin and/or hematocrit. Normal Value RBC: 1. adult female: 4.2 to 5.4 million/mm 3
2. adult male: 4.6 to 6.2 million/mm 3
Below N may indicate anemia, bleeding, kidney disease, bone marrow failure Above N may indicate congenital heart disease, dehydration, obstructive lung disease, or bone marrow over-production , beta thalassemia trait , deficiencies of iron, folate, vitamin B12, and vitamin B6.
Leukocyte Cell contain nucleus with various morphology and various size Leukocyte Function of counting The WBC count is used to suggest the presence of an infection or leukemia. It is also used to help monitor the bodys response to various treatments and to monitor bone marrow function.
Normal Value WBC: adult: 4,500 to 11,000/mL
Below N (leukopenia). It can result from many different situations, such as chemotherapy, radiation therapy, or diseases of the immune system.
Above N (leukocytosis). This can result from bacterial infections, inflammation, leukemia, trauma, or stress.
Function of counting Bleeding disorders or other bone marrow diseases, such as leukemia, require the determination of the number of platelets present and/or their ability to function correctly.
0,5 vol of blood goes into bulb first 101 1 = 100 vol of diluted blood, of which 0,5 vol is undiluted blood. Dilution of blood is 0,5 : 100 or 1:200 1 vol diluting fluid remains in the stem and does not dilute blood Leukocyte Cell Counting Pipette (Thoma) 0 , 5
1
1 1
11 vol = Total capacity of pipet
0,5 vol of blood goes into bulb first 11 1 = 10 vol of diluted blood, of which 0,5 vol is undiluted blood. Dilution of blood is 0,5 : 10 or 1:20 1 vol diluting fluid remains in the stem and does not dilute blood Diluting solution
Manual counting Tool : Haemacytometer Negative : tedious Time consuming Coefficient of variation 8-20%
R R R R R
1 mm
1 mm
1 mm
1
m m
1
m m
1
m m
0,2 mm 1 mm 2
0,0025 mm 2
R 0,04 mm 2
Automatic COUNTING Positive : speed precision Automated calculation of Ht and hematologic constants printouts Coefficient of variation 2-4% Example procedure to counting leukocyte with Thoma pipette: draw EDTA blood to exactly 0,5 wipe the tip of pipet (without touching the opening capillary) immerse pipet in the diluting fluid suction by mouth gently until exactly 101 (beyond 101 discard; air bubbles discard) rotate the pipet, shake about 1 min discard the first 3 or 4 drops of diluted blood from pipet fill the counting chamber via capillary attraction if overfilling (presence of fluid in the moat surrounding) clean & refill if insufficient adding dilution if air bubbles are present (indicate moisture / dirty hemocytometer) clean & refill making the count
Manual Procedure No. of cells/mm 3 undiluted blood = counted cell x 10000
No. of cells/mm 3 undiluted blood = total no. of counted cells in 5 squares surface area counted x height of counting chamber x dilution of blood
~ counted cells (0,2x0,2)x5 x 1 / 10 x 1 / 200
area which erythrocyte can be counted : A, B, C, D, E counting begin in the left upper corner to the right, drop down to the second row and count to the left. - do not count cells those are touching lower and right hands lines Formula for erythrocyte count Dihitung
Tidak dihitung Formula for leukocyte count Leukocytes/mm 3 blood = cells counted in 4 large squarer area counted x height of chamber x dilution
counted cells = counted cells x 50 (1x1)x 4 x 1 / 10 x 1 / 20
Hematocrit (packed cell volume) Hct : the percentage of red blood cells volume to the whole blood volume A measure of both the number and the size of red blood cell Hct reflects the concentration of red blood cell, not the total of red blood cell mass Hct value : Male 40-48 Female 37-43 Hematocrit Hct test is used to evaluate : Anemia Polycythemia Response to treatment of anemia and polycythemia Decision to undergo blood transfusion and the effectiveness of those transfusions Principle of Hct test Blood + anticoagulant
Centrifuge
Blood cells will precipitate
Erythrocyte platelet+ (bottom) leukocyte
Technique to do Hct test Macromethod of wintrobe Venous blood +anticoagulant
Wintrobe tube
centrifuge 3000 rpm 30
Read the scale from bottom Hct = L1/L2 L1 = the height of erythrocyte column, exluded buffy coat L2 = the height of the whole blood specimen Can be done after ESR test Interpretation of Hct result Increased hematocrit : Dehydration resolved with adequate hydration Polycythemia vera could be due to problem with bone marrow Early indication of dengue shock syndrome Polycythemia secondary to chronic hipoxia i.e COPD Living in high altitude Interpretation of Hct result Decreased hematocrit : Indication of anemia Further testing may be necessary to determine the exact cause of anemia ( i.e deficiency of iron, folate, vit B12) Recent bleeding Liver cirrhosis Bone marrow suppressant effect of drugs Sickle cell anemia Hemolysis Pregnancy due to extra fluid in the blood Erythrocyte Sedimentation Rate (ESR) The rate at which erythrocytes settle to the bottom of a test tube through the forces of gravity in a certain period of time, usually 60 minutes It is directly proportional to the weight of cell aggregates and inversely to the surface area and plasma viscosity (microcyte < macrocyte) ESR is a sensitive but nonspecific (the earliest indicator when other chemical and physical signs are normal) Function of ESR test : monitor inflammatory or malignant disease Aid detection and diagnosis of occult disease i.e tb, tissue necrosis, chronic infection
ESR is governed by the balance between pro- sedimentation factors,mainly plasma protein (fibrinogen, IgG, CRP) and factors resisteng sedimentation (negative charge of erythrocyte called zeta potential and ratio of surface area to volume)
When an inflammatory process occurs, the high proportion of plasma proteins causes erythrocyte to stick to each other to form roleaux
Roleaux will settle faster
sedimentation Methods to determine ESR Principle : Anticoagulated blood is placed in vertical tube
Erythrocytes will tend to fall toward the bottom
The length of fall of the column of erythrocytes in 60 minutes =ESR ESR test Westergren Wintrobe Comparison of ESR method Westergren Wintrobe Anticoagulant Na sitrat 3.8% (0.4 ml/1.6 ml blood) Balanced oxalate Dried EDTA Dilution 5/4 - Length of equipment 300 mm, pipet 110 mm, tube Diameter 2.5 mm 2.5 mm Scale 0-200 mm 0-100 mm Normal value Male Female
0-10 mm/hr 0-15 mm/hr
0-10 mm/hr 0-20 mm/hr
Advantage easy Reliable disadvantage If ESR>50 mm/hr, the result is unreliable More difficult Westergren wintrobe Peripheral Blood Smear (Blood Film) Blood Film : Overview A blood test that gives information about the number and shape of blood cells It is still the best method for definitely evaluating and identifying immature and abnormal cells (if examined by trained eyes) The results of blood film evaluation are not always diagnostic and more often indicate the presence of an underlying condition further diagnostic testing Sample : capillary blood venous blood + EDTA/heparin (not oxalate!!!) Film Staining Staining : technique used in microscopy to enhance contrast in the microscopic image Principle procedure Fixation
Staining
Mounting Fixation To preserve the shape of cells/tissues due to difference in tonicity Saline concentration inside cells > staining fluid hypotonic shock Fixation will hinder inflation of cells when stainning Fixation can be done by immersing the film in a jar containing fixative solution or covering horizontally supported film Fixative solution : formaldehyde, methanol, ethanol, citric acid
Staining To observe and recognize different kind of cells by using certain dyes
Mounting To keep the film for a long time After drying the slide, place a drop of Canada balsam or another medium mountant on the smear, then mount the coverslip.
Acidophilic : structures that take up only acid dye Basophilic : structures that take up only basic dye Neutrophilic : structures stained by combination of acid and basic dye Dyes Acid i.e eosin Basic i.e methylene blue and its analogues (azure A.B,C) Romanowsky principle A staining technique based on electrostatic interaction between dye and target molecules Acid dyes carry negative charges and bind to basic component of cells i.e RBC and granules of eosinophil Basic dyes carry positive charges and bind to acidic component of cells i.e nuclei of WBC, granules of basophil, RNA molecule of WBC cytoplasm Some staining methods following romanowsky principle : Wright stain Giemsa Stain Leishmann Stain Jenner Stain Wright Stain It is used to stain peripheral blood film and bone marrow aspirates Dye : methyl alcoholic solution of eosin and a complex mixture of thiazine (methylene blue 50-75% and azure B 10-25%) Function of wright stain to distinguish blood cell types to perform differential WBC count to stain chromosome to facilitate diagnosing of diseases Procedure of Wright Stain
Slide is exposed to undiluted stain solution for 2 minutes. An equal amount of buffer (pH 6.4) is added and mixed gently for 10 minutes The stain is flushed gently to coarsely from the slide with aquadest Air dry the slide in a tilted position
Cell Figure with Wright Stain (films stained well with Wright stain have a pink color when viewed with naked eye) Basophils has dark purple granules (which contain histamines) in their cytoplasm
Eosinophils has red- orange granules and each distinctly discernible Neutrophils (50-70% WBC) Diameter 12 m The cytoplasm is pale and often contains small pink to purple granule Erythrocytes are pink platelet erythrocyte Monocytes (6% WBC) The largest WBC (12-20 m) Characterized by blue-grey cytoplasm and have a folded nucleus( a great reniform or horseshoe-shaped nucleus, in some cases even bi-lobed.)
Lymphocytes Accounts for 20-40% WBC Round in shape Nucleus is generally large in relation to cytoplasm Cytoplasm appears pale blue and generally doesnt contain any granules Type B and T cannot be distinguished