Al-Azhar University- Gaza
Faculty of Applied Medical Sciences
Laboratory Medicine Department
Practical
Hematology
Manual #1
Prepared by: Ashraf Shaqalaih BSc(MT), MSc(MT), CLS(H), CLSp(H)
Clinical Laboratory Specialist in Hematology
Clinical Immunohematologist Technologist (Lic#238, State of California, USA)
Anticoagulants Used In The Hematology Laboratory
Anticoagulantsare defined as substances which prevent blood clotting / coagulation, and allow separation of the blood into cellular and liquid (plasma) components. Generally plasma contains coagulation factors. The three anticoagulants commonly used in hematology laboratory are:
1]Ethylene Di-Amine Tetra-Acetic Acid (EDTA):
EDTA can be found in three salt forms:
1-Tri-Potassium EDTA
2-Di-Sodium EDTA
3-Di-Lithium EDTA
Also, EDTA can be crystalline or liquid. Liquid EDTA tubes requires specific filling volume to avoid dilution effect. So, blood : anticoagulant ratio must be maintained (this is applicable to all anticoagulants). EDTA is also known as Versene or Sequestrene. EDTA acts by chelating / removing ionized calcium (calcium is required for blood to clot, so when it is removed blood will not clot). Generally tri-Potassium EDTA is better than di-Sodium EDTA and di-Lithium EDTA.
Always, be sure to mix blood with anticoagulant in a manner that guarantee proper complete mixing, by gentle repeated inversion of the tube, in figure of 8 inversion for at least 20 times, do not shake or use vigorous inversion, since this may cause hemolysis, and disintegration of cells, and the final effect will be erroneous low results for cellular components of blood, which are our hematology laboratory interest.
EDTA is the most commonly used anticoagulant in the hematology laboratory, and is the anticoagulant of choice for the CBC.
Excess EDTA (i.e. more EDTA, you fill less blood volume, so EDTA is in excess), causes shrinkage of RBC’s, causing falsely / erroneously reduced hematocrit (HCT), and subsequent increase in MCHC and decrease in MCV (MCV and MCHC are RBC indices that will be studied later). Platelets are also affected, they will swell and subsequently disintegrate, causing erroneously high platelet count, since platelets will be disintegrated into more than one fragment, each fragment will be counted as one platelet (for example if one platelet will be disintegrated into 4 fragments, the 4 fragments will be counted as 4 platelets, but actually they represent one platelet, causing erroneously high platelet count).
From the previous discussion we conclude that correct ratio of blood to anticoagulant is very important, to rule out these in vitro effects.
EDTA can induce platelet aggregation and clumping, causing falsely decreased platelet count, because these platelet clumps will not be counted as platelets, they may counted as red blood cells (causing low platelet count and high red blood cells counts). This technical problem can be solved by (1) repeated measurements, (2) extraction of new sample and repeat measurements, (3) study the automated cell histograms, and (4) by visualizing blood film, looking for these platelet clumps. Also, Aggregated and clumped platelets interferes with WBC counting zone in automated hematology counters that use electrical impedance technology.
2]Sodium Citrate
Is the anticoagulant of choice for coagulation and platelet function tests, also is used for ESR (erythrocyte sedimentation rate test). It acts by precipitating calcium, thus it will not be available for clotting process. It came in a liquid form, as 3.8% tri-sodium citrate. For coagulation testing, the ratio of 9 volumes of blood to one volume of anticoagulant (9 volumes blood:1 volume anticoagulant) is very critical (very important), as variation from this ratio may cause errors. For ESR (4) volumes of blood to one volume of anticoagulant is used (4 : 1).Generally, this anticoagulant is not suitable for routine hematology testing. From this we conclude that sodium citrate acts as anticoagulant and as diluent (as in the case of ESR). Because of its dilution effect it can’t be used for CBC.
3]Heparin
Heparin is an acid mucopolysaccharide, it acts by complexing with anti-thrombin to prevent blood clotting (antithrombin is one of the natural/physiological inhibitors of blood coagulation, which is found in vivo, this will be studied later in coagulation and hemostasis modules). It is not suitable for blood films staining, since it gives too blue coloration to the background, when films are stained with Romanovsky stains, also, heparin may cause leukocyte and platelet clumping , this is why heparin is not suitable for routine hematology tests. It is the preferred anticoagulant for osmotic fragility test ( a special hematology procedure, that will be studied in this course). Heparin also is used in capillary tubes for spun hematocrit (HCT) (heparin cover the entire capillary tube glass), these capillary tubes are also called microhematocrit capillary tubes. Heparin is also used for L.E. cell preparation (L.E.= Lupus Erythromatosus).
- Heparin is found in basophil and mast cell granules.
- Heparin is used therapeutically as an in vivo anticoagulant.
Anticoagulants commonly Used in the Hematology Laboratory and their use:
No. / Anticoagulant / Hematology Laboratory Use / Universal Color Code1 / EDTA / Routine Hematology Procedures. / Lavender, Pink
2 / Sodium citrate / Coagulation , Platelets Tests, ESR. / Blue
3 / Heparin / Osmotic Fragility, Spun Hematocrit / Green, Brown
HEMOCYTOMETRY
Improved Neubauer Hemacytometer
Hemacytometry
Hemacytometry means the use of the hemacytometer counting chamber to count blood cells (to count WBC, RBC, and Platelets, as will as, counting cells in other body fluids, e.g. CSF and semen analysis). Hemacytometer is a counting chamber device made of heavy glass with strict specifications, it resemble a glass slide. Also, the hemacytometer have a special glass slide manufactured to strict specifications, it is very thick and non-flexible. There are many types of hemacytometers, in which they differ in rulings, but the commonest and the easiest one is the Improved Neubauer Chamber, brightline type. When viewing the hemacytometer from the top (figure below), it has 2 raised platforms surrounded by depressions on three sides, each raised platform has a ruled counting area marked off by precise lines etched into the glass. The raised areas and depression form H letter, this “H” has two coverglass supports on each side which are exactly 0.1 mm higher than the raised platforms. The coverglass is placed on top of the coverglass supports so it covers both ruled areas. The depth between the bottom of the ruled area and the coverglass is exactly 0.1 mm. So, coverglass function is to confines the fluid and regulates the depth of the fluid to be applied.
Figure1: Top view of the hemacytometer
Figure 2: Coverglass position on the hemacytometer
Hemacytometer Counting Areas
Hemacytometer has 2 identical ruled counting areas, each composed of etched area consists of a large square, with a diameter of 3 mm. This large square is subdivided to 9 small squares, each with a diameter of 1 mm. So, each 1mm square can accommodate a volume of 1 mm x 1mm x 0.1 mm (depth) = 0.1 mm³ (cubic millimeter). WBC cells are counted in the entire 9 squares. The central square is further subdivided into 25 smaller squares each with a diameter of 0.2 mm, so the volume accommodated within this square will be 0.2 mm x 0.2 mm x 0.1 mm(depth) = 0.004 mm³ (cubic millimeter). Red blood cells are counted in the large central square (1 from 9 squares), in which only the four corner squares and the center square (look figure 3 , in which “R” denotes for red blood cells). Platelets are counted in the entire large center squares (the 25 small squares).
Figure 3 - Red Blood Cells Counting Area
Using The Hemacytometer
1-Position a clean, dust free, coverslip so it covers the ruled counting areas of a clean hemacytometer.
2-Fill the hemacytometer with the fluid containing cells to be counted, by touching the tip of the capillary tube or micropipette tip to the point where the coverslip and raised platform meet on one side, the fluid will drawn under the coverslip and over the counting area by capillary action, this requires about 10 l.
3-Repeat on opposite side of the chamber.
4-The chamber must not be overfilled or underfilled, if accurate results are needed!.
5-Place the hemacytometer on the microscope stage, so one of the ruled counting areas is aligned directly above the light source (condenser); rotate the low power objective (x10) into place; using the coarse focus knob, move the low power objective very near the coverslip; rotate coarse focus knob to increase the distance between the low power objective (X10) and the hemacytometer until etched/ruled lines come into focus; all nine large squares must be viewable; very carefully, rotate the high power objective (X40) into place, with the aid of fine focus knob, adjust the focus until the etched lines come into focus, you can now carefully move the hemacytometer by using the mechanical stage, so that the ruled area on the other side can be viewed.
The Counting Pattern
Either left to right or right to left counting pattern can be used ( fig.4); but with the insurance that each cell is counted only once, to accomplish this, cells that touch the right boundary lines or the bottom boundary lines are not counted, because they will be counted with the other squares (look figure). After cells are counted on one side, the hemacytometer is moved and the cells are counted on the other side. Results for each side are recorded, then are totaled and the average is calculated.
Figure 4 Counting Pattern
Figure 5: Cells touching the right and bottom boundaries are not counted
Calculating The Cell Counts
1st.The total number of cells per cubic millimeter of sample can be calculated from:
- The average number of cells counted.
- The ruled areas contain an exact volume of diluted sample.
- The dilution of the sample.
2nd.The hemacytometer Formula:
N x D (mm) x DF
= C/mm³
A (mm ²)
Where:
A-C/mm³ = number of cells/ mm³
B-N= Total number of cells counted in the counting chamber.
C-D (mm) = Depth factor in mm
D-DF = Dilution Factor
E-A (mm²) = Area counted (mm²)
- The dilution factor is determined by the blood dilution made by you as a laboratory technologist..
- The depth factor is always = 10 (1/0.1).
- The area counted will vary for each type of cell count and is calculated using the dimensions of the ruled area.
Comments:
Although some specialists still considers hemacytometry is the standard method of cell counting, but its C.V. is high, which indicates impression and sometimes inaccuracy, especially when counting red blood cells . In cases of leukopenia (low WBC count, below normal ranges ), still hemacytometry the method of choice for cell counting.
WBC (Leukocyte) Manual Counting
Principle:
Blood sample is mixed and diluted with weak concentration of hydrochloric acid (HCl), or acetic acid (in specified known volumes). Weak acids will lyse red blood cells, and will darken WBC’s to facilitate counting by the hemacytometer.
Manual WBC counting is used in cases of very low WBC count (leukopenia) with automated hematology cell counters, and when automated cell counters are not available.
Sample:
EDTA anticoagulated whole venous blood.
Reagent and Supplies To Prepare Diluting Fluid:
1-Volumetric Flask 100 cc.
2-Serological pipettes.
3-Concentrated HCL
4-Glacial Acetic Acid
Preparation of Diluting Fluid:
Diluting fluid is either:
1% hydrochloric acid in distilled water ( 1 ml Conc. HCL + 99 ml Dist. water).
2% Acetic Acid in distilled water ( Turk’s solution) (2 ml glacial acetic acid
+ 98 ml distilled water).
Glassware, Apparatus, Equipment :
1-Neubauer improved hemacytometer.
2-Clean cover slip slide (especially made for the hemacytometer).
3-Automatic micropipette (20 l, 380 l are the required volumes).
4-Gauze 10 x 10 cm
5-Glass/Plastic tubes- (12x75 mm).
6-Handy tally counter.
7-Conventional light microscope.
Procedure:
1-Mix the blood sample gently but thoroughly by inversion, manually or by mechanical rocking mixer.
2-Pipette 0.38 ml (380 l) of diluting fluid into a 12x75 mm tube.
3-Pipette 0.02 ml (20 l) of well mixed blood to be counted and wipe the tip with gauze into the tube containing diluting fluid and mix the tube.
4-Let the tube stand for 2-3 minutes to ensure complete RBC lyses, then mix well.
5-Prepare the clean hemacytometer and cover it with the designed coverslip.
6-Load one side of the hemacytometer with the aid of a capillary tube or micropipette, do not attempt to overload or underload the hemacytometer.
7-Allow the hemacytometer to sit for several minutes to allow the WBC’s to settle in the counting chamber, to avoid drying effect, place the loaded hemacytometer in a covered Petri dish with a moist gauze, until counting.
8-Place the hemacytometer in the microscope stage.
9-Focus with x10 objective lens (low power), with lowering the condenser.
10-The WBC’s are counted in the 9 corner large squares, with the aid of hand tally counter.
11-Follow the counting pattern shown in the figure below. During counting, do not count cells that touch the right or bottom boundaries to ensure unduplicated counting.
12- The total counted WBC’s in the 9 squares are added together.
Fig. WBC’s are counted in the 9 hemacytometer squares
If the number of cells in a square varies from any other square by more than 9 cells, the count must be repeated, because this represents an uneven distribution of cells, which is may be caused by improper mixing of the dilution or improperly filled hemacytometer.
Calculations:
N x D (mm) x DF
Total WBC Count =
A (mm²)
Where:
N = Total WBC counted by the counting chamber.
Depth factor in mm = 10
DF = Dilution Factor = 20
A (mm²) = Area counted = 3 mm x 3 mm = 9 mm²
So,
N x 10 mm x 20
Total WBC Count =
9 mm²
Example:
20 / 19 / 18
21 / 14 / 16
19 / 16 / 15
N= 20 +19 +18 +21 +14 +16 +19 +16 +15 = Tallied 158 Counted WBC Cell
158 x 10 x 20
Total WBC Count / cumm = = 3500 / cumm = 3.5 x 109/L
9
ReferenceRange
Adults : 4.5 – 11.0 x 109 /L
Six years: 4.5 – 12.0 x 109 /L
One year: 6.0 – 14.0 x 109 /L
Newborn: 9.0 – 30.0 x 109 /L
WBC count varies according to age but not to sex.
Sources of Error:
1-Contaminated diluting fluid.
2-Incorrect dilution.
3-Uncalibrated Micropipettes.
4-Uneven distribution of WBC’s.
5-Presence of clumped WBC’s.
6-Unclean hemacytometer or cover slips.
7-Presence of air bubbles.
8-Incompletely filled hemacytometer.
9-Over flow.
10-Presence of debris.
11-Drying of the dilution in the hemacytometer.
RBC Manual Count
Principle:
A specified volume of blood is diluted with a specified volume of isotonic fluid. This isotonic diluting fluid will not lyse RBC’s, and will facilitate counting with the aid of the hemacytometer.
Sample:
EDTA anticoagulated whole venous blood.
Diluting Fluid:
- Isotonic saline:0.85% sodium chloride (NaCl) in distilled water.
OR
- 10 ml of 40% Formalin made up to 1 liter with 32 g/l Tri-sodium Citrate.
OR
- 6.25 g of crystalline Sodium Sulfate. Transfer to 100 cc volumetric flask, and add approximately 50 cc distilled water. Then add 16.7 ml of Glacial Acetic Acid. Finally add distilled water up to the mark.
Apparatus and Equipment:
1-Micropipette – 20 l is the desired volume.
2-Serological Pipette, 5ml.
3-Handy Tally counter.
4-Improved Neubauer counting chamber with the cover slips.
5-Conventional light microscope.
Procedure:
1-Pipette 4.0 ml of diluting fluid into a tube.
2-Pipette 20 l of will mixed anticoagulated whole blood to the tube.
3-Mix continuously for 2-3 minutes.
4-Load the cleaned hemacytometer.
5-Place the hemacytometer on the microscope stage, lower the condenser.
6-Focus with x10 objective lens on the large central square. This square is ruled into 25 small squares, each of which is further divided into 16 smaller squares, of the 25 squares, only the four corner squares, and one middle square are used to count RBC’s.
7-Switch to x40 objective lens, and start counting in the five designated squares.
Calculations:
N x Dilution Factor x Depth Factor
Total RBC Count =
Area Counted (mm²)
Where:
N= Total number of red cells counted in the counting chamber.
Dil. Factor = 0.02 : 4 = 2 : 400 = 1:200, Dilution Factor = 200.
Depth Factor = 10
Area Counted = 0.2 x 0.2 x 5 = 0.2 mm²
So,
N x 200 x 10
Total RBC count = = N x 10,000
0.2
NormalReferenceRange:
Males : 4.6 – 6.2 x 1012/L
Females : 4.2 – 5.4 x 1012/L
Children: 4.5 – 5.1 x 1012/L
Sources of Error:
Same as WBC manual Counting, refer to WBC manual Counting.
Hemoglobinometry
Hemoglobin Determination
Decrease in hemoglobin concentration beyond established normal ranges for age and sex is called “ anemia”, whereas increase in hemoglobin concentration beyond established normal ranges for age , sex, and geographical distribution is called “polycythemia”. So that, for correct diagnosis it is important to determine accurately and precisely hemoglobin concentration.
Many methods are available for the determination of hemoglobin, but among them the relevant, and the recommended one is the Modified Drabkin’s Method. ICSH (International Committee for Standardization in Hematology) consider this method as the reference method for hemoglobin determination.
Drabkin’s solution contains the following:-
1-Potassium Ferricyanide
2-Potassium Cyanide.
3-Non- ionic Detergent
4-Dihydrogen Potassium Phosphate.
Well mixed EDTA anticoagulated blood is diluted in Drabkin’s solution; non-ionic detergent will lyse the red cells to (1) liberate hemoglobin, and to (2) decrease the turbidity caused by red cell membrane fragments by dissolving them. Then, hemoglobin is oxidized and converted to methemoglobin (Hi) by potassium ferricyanide, this step is accelerated by the dihydrogen potassium phosphate, and requires approximately 3 minutes for total conversion. Potassium cyanide will provide cyanide ions to form cyanomethemoglobin (HiCN), which have a broad spectrum of absorption at 540 nm. The absorption can then be compared with a hemoglobin standard with a known hemoglobin concentration, and by applying Beer’s law extract the hemoglobin concentration of the unknown (i.e. the patient).