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 Hematology Laboratory

Anticoagulants Used In The Hematology Laboratory Anticoagulants are 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: 123Tri-Potassium EDTA Di-Sodium EDTA 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 diSodium 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 RBCs, 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
3

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 cant 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
4

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 . 1 2 3

Anticoagulant EDTA Sodium citrate Heparin

Hematology Laboratory Use Routine Hematology Procedures. Coagulation , Platelets Tests, ESR. Osmotic Fragility, Spun Hematocrit

Universal Color Code Lavender, Pink Blue Green, Brown

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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.
6

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, bright line 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
r regulates

the depth of the fluid to be applied.

HTIW MROFTALP NOISSERPED DEPAHS-H AERA DELUR

RAHZA LA

SSALGREVOCCOVERGLASS GNITNUOC COUNTING REBMAHC REBMAHC STROPPUS SSALGREVOC

Figure1: Top view of the hemacytometer

SSALGREVOC

SSALGREVOC TROPPUS MROFTALP RETNEC

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 mm (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 mm (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 1Position a clean, dust free, coverslip so it covers the Fill the hemacytometer with the fluid containing cells to ruled counting areas of a clean hemacytometer.
2-

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. 34Repeat on opposite side of the chamber. The chamber must not be overfilled or underfilled, if Place the hemacytometer on the microscope stage, so

accurate results are needed!.

5-

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
9

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.
Begin

Figure 4 Counting Pattern

Figure 5: Cells touching the right and bottom boundaries are not counted

Calculating The Cell Counts 1. The total number of cells per cubic millimeter of sample can be calculated from: 1. 2. 3. The average number of cells counted. The ruled areas contain an exact volume of diluted The dilution of the sample. N x D (mm) x DF = C/mm
10

sample. 2. The hemacytometer Formula:

Cells tou ching rig ht, and botto m boundari es counted! are not

A (mm ) Where:
A-

C/mm = number of cells/ mm N= Total number of cells counted in the counting D (mm) = Depth factor in mm DF = Dilution Factor A (mm) = Area counted (mm)

BCDE-

chamber.

1. The dilution factor is determined by the blood dilution made by you as a laboratory technologist.. 2. The depth factor is always = 10 (1/0.1). 3. 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.

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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 WBCs 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 2% Acetic Acid in distilled water ( Turks solution) (2 ml + 98 ml distilled water). Glassware, Apparatus, Equipment : 1- Neubauer improved hemacytometer. 2- Clean cover slip slide (especially made for the hemacytometer).
3-

ml Dist. water). glacial acetic acid

Automatic micropipette (20 l, 380 l are the required

volumes).
12

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.
23-

Pipette 0.38 ml (380 l) of diluting fluid into a 12x75 mm tube. 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 WBCs 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 WBCs 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.
13

Begin

12- The total counted WBCs in the 9 squares are added together.

Fig. WBCs 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 =
14

A (mm) Where:

N = Total WBC counted by the counting chamber. Depth factor in mm = 10 DF = Dilution Factor = 20 A (mm) = Area counted = 3 mm x 3 mm = 9 mm N x 10 mm x 20

So, Total WBC Count = 9 mm Example:

20 21 19 19 14 16 18 16 15
Hemacytometer Squares

N= 20 +19 +18 +21 +14 +16 +19 +16 +15 = Tallied 158 Counted WBC Cell 158 x 10 x 20 Total WBC Count / cumm = 9 = 3500 / cumm = 3.5 x 109/L

Reference Range

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.

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3- Uncalibrated Micropipettes. 4- Uneven distribution of WBCs. 5- Presence of clumped WBCs. 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.
1 ml of gentian violet can be added to the diluent to color the white blood cells, thus counting will be easier.

In leukopenia ( decreased WBC count), with a total WBC count below 2500/cumm, the blood is diluted 1:10, whereas in leukocytosis (increased count), the dilution may be 1:100 or even 1:200.

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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 RBCs, and will facilitate counting with the aid of the hemacytometer. Sample: EDTA anticoagulated whole venous blood. Isotonic saline:0.85% sodium chloride (NaCl) in distilled OR 10 ml of 40% Formalin made up to 1 liter with 32 g/l TriOR 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-

Diluting Fluid: water.

sodium Citrate.

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.
17

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 RBCs. 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 (mm) Where:

N= Total number of red cells counted in the counting 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 mm N x 200 x 10 Total RBC count = 0.2 = N x 10,000

chamber.

So,

Normal Reference Range: 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:
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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 Drabkins Method. ICSH (International Committee for Standardization in Hematology) consider this method as the reference method for hemoglobin determination. Drabkins solution contains the following:1234Potassium Ferricyanide Potassium Cyanide. Non- ionic Detergent Dihydrogen Potassium Phosphate. Well mixed EDTA anticoagulated blood is diluted in Drabkins 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
19

with a hemoglobin standard with a known hemoglobin concentration, and by applying Beers law extract the hemoglobin concentration of the unknown (i.e. the patient).

Hemoglobin + Potassium Ferricyanide Methemoglobin + Potassium Cyanide

Methemoglobin (Hi) Cyanomethemoglobin

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Hemoglobin Concentration Determination Modified Drabkins Method

Principle: Whole blood is diluted in a solution containing Potassium Ferricyanide and Potassium Cyanide. Hemoglobin will be oxidized by the action of Potassium Ferricyanide to form Methemoglobin (Hemiglobin, Hi). Potassium Cyanide will provide Cyanide ions to form Cyanomethemoglobin (HiCN). This solution can be measured spectrophotometrically and compared to known hemoglobin standards. This procedure is applicable in diagnosing and monitoring therapy in cases of hemoglobin deficiency anemias.

Sample: EDTA anticoagulated venous whole blood .

Apparatus: 1- Brown bottle- 1 liter 2- Volumetric flask 1 liter 3- Balance 4- Spectrophotometer adjusted at 540 nm 5- Spectrophotometer Cuvettes 6- Glass or plastic centrifuge tubes
7-

Micropipette (adjusted at 20 l)

8- 5 ml volumetric pipette or graduated pipette (or you can use bottle top dispenser) Reagents:
1- Potassium Ferricyanide K3Fe (CN)6 2- Potassium Cyanide (KCN) 3- Dihydrogen Potassium Phosphate KH2PO4 0.200 g (200 mg) 0.050 g (50 mg) 0.140 g (140 mg)

4- Distilled Water (laboratory grade 1) 5- Non-Ionic Detergent:

- Sterox S.E.
21

0.5 ml

or - Trinton X-100 or - Quolac Nic 218

1.0 ml 1.0 ml

6- Standard Cyanomethemoglobin solution/ solutions. Working Drabkins Solution Preparation: Add the reagents to the volumetric flask and add distilled water up to the mark (avoid bubble formation), with continuous mixing as you are adding the distilled water. Transfer to a stoppered brown bottle, and label with name, date of preparation, and the name of the technologist who prepared the solution. Ready made commercial preparations are available in the market, just dilution with distilled water is required, such that preparations are for example available from Randox company. Procedure:
1-

Add 20 l of whole anticoagulated blood to 5 ml of Drabkins

solution. 2- Mix well. 3- Allow the mixture to stand at room temperature for at least 3 minutes. 4- Measure the absorbance at 540 nm against a diluent Drabkins solution (blank). 5- Measure the absorbance of the standard HiCN solution in the same manner. 6- Extract the unknown hemoglobin concentration, using the following equation:

Abs. of Unknown Unknown Hb concentration = Abs. of STD x Conc. Of STD

22

Or read directly from the hemoglobin standard curve. See below, how to prepare the hemoglobin standard curve. Hemoglobin Standard Curve Preparation: A standard curve must be made each time new Drabkins solution is prepared. A commercially prepared standard kit with hemoglobin concentrations of 20, 15, 10, 5 g/dl is available, read each corresponding absorbance, and plot the results on a linear graph paper (absorbance versus Hb concentration). Or, you can use a stock standard solution of 20 g/d, and dilute it to various Hb concentrations with Drabkins solution.
Absorbance 0.3 0.2 0.1

10

15

20

Hb-Concentration- g/dl

Hemoglobin Standard Curve Notes: 1- Drabkins method is the recommended method by the ICSH. 2- Drabkins solution should be clear and have a pH of 7.0 to 7.4, discard if turbid. 3- The Drabkins solution is the blank, and should read zero (0) absorption. 4- Take care when preparing the solution, as cyanide is fatal, and toxic, although the amount of cyanide in the prepared solution is less than the human lethal dose. 5- Do not expose the solution to acids, because cyanide will be released.
23

6- Keep the solution in a dark bottle at room temperature, but discard after a month. 7- All types of hemoglobin which include hemoglobin, oxyhemoglobin, carboxy hemoglobin, methemoglobin are measured by this method, only Sulfhemoglobin (S-Hb) is not measured by Drabkins method.. 8- Sulfhemoglobin is found at increased amounts in cases of Clostridium septicemia, as a result of drug intake, and in severe cases of constipation. At increased amounts blood sample may color as lavender to green. 9- Increased methemoglobin concentration occurs as a result of inherited conditions or more commonly as acquired as a result of drug intake or exposure to chemicals. 10-Turbidity due to leukocytosis , lipemia, or high protein levels as seen in para proteinemias ( e. g. multiple myeloma ) may interfere and cause erroneously high hemoglobin concentration. Prepare sample blank to overcome erroneous readings). 11-Try as possible as you can to obtain venous whole blood. 12- Use automatic micropipettes, but not Sahli pipettes, to reduce technical errors. 13- Panic values for hemoglobin is less than 6.0 g/dl. 14- Hemoglobin concentration is unrelated to patient eating status. 15- Blood obtained from heavy smokers requires 3 minutes more incubation time for full conversion of hemoglobin to methemoglobin. 16- Do not expose Drabkins solution and standards to light, and sunlight. 17- People living at high attitudes tend to have polycythemic hemoglobin concentrations, because of the low oxygen tension at high attitudes, causing tissue hypoxia,
24

so that body

compensate and adapt for this by increasing hemoglobin. 18- Pregnant females tend to have lower hemoglobin because their concentrations than normal for their age,

fetuses compete with them for iron, vitamins and other essential substances for hemoglobin synthesis, this is why pregnant females are of great needs for iron and vitamin supplements during their pregnancies. Preparation Of Sample Blank Turbidity can cause erroneously increased hemoglobin concentration due to increased light scattering, to overcome this you can prepare a sample blank. To prepare a sample blank use the formula: 5 ml N= (1- Hct)
Where N = ml of Drabkins to be added to 20 l of patient plasma.

So, when you have a turbid sample, take a portion from it, and centrifuge it, take 20 l of its turbid plasma and add it to the calculated Drabkins solution amount according to the above formula . This is considered the zero blank, adjust the spectrophotometer absorbance reading to zero, then read the absorbance of the patient hemoglobin according to the procedure above. The sample blank will correct the erroneous hemoglobin result caused by turbidity. Example: If a patient has a hematocrit of 50, then: 5 ml N
25

5 = = 10 ml

(1- 0.50)

0.50

So, the sample blank is prepared by adding 20 l of patients plasma to 10 ml Drabkins solution.

Hemoglobin concentration is expressed in g/dl (gram per deciliter)

Reference Hemoglobin Concentration Ranges: Males : 13.5 18.0 g/dl Females: 12.0 16.0 g/dl Newborn: 16.5 19.5 g/dl Children : 11.2 16.5 g/dl (varies with age)

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Spun Microhematocrit
Principle Hematocrit is the ratio of the total volume of RBCs to that of whole blood expressed as percentage(%) (whole blood = total volume of cells + plasma). The second synonym for hematocrit is PCV (Packed Cell Volume). The procedure is easy to perform, whole blood is centrifuged in a narrow tube (capillary tube), cellular elements will be separated from the plasma, after centrifugation blood will be separated into 3 layers : (1) Bottom layer contains packed RBCs, (2) Middle layer contains WBCs and Platelets (on top of RBCs), (3) Upper plasma layer. The hematocrit value is determined by comparing the volume of RBCs to the total volume of the whole blood sample, it is usually reported as a %.

27

Sample: EDTA anticoagulated whole venous blood (correct volume is highly required. When EDTA is in excess, cell shrinkage occurs, and as a result a falsely low hematocrit is obtained, with corresponding increase in MCHC, and decrease in MCV). tube. Apparatus and Materials: 1- Microhematocrit centrifuge. 2- Modeling clay (seal material). 3- Capillary tubes (7 cm long, 1mm diameter) 4- Hematocrit measuring device reader or a conventional ruler. Procedure: 1- Fill the capillary tube with blood by capillary attraction. Either from free flowing finger punctured by a sterile lancet/ or from a well mixed anticoagulated whole venous blood (this requires only few microliters of blood). 2- Seal with the modeling clay the empty end of the capillary tube. 3- Place and position the capillary tube in the radial grooves of the microhematocrit centrifuge with the sealed end away from the center (pointed toward the outside). 4- Centrifuge for 5 minutes at 12000 g, so that additional centrifugation does not pack the red blood cells more. 5- The height of the RBC column, and the total column should be measured with the aid of a ruler in cm and mm, then divide the RBC column height over the total column height (total height = RBC column + buffy coat + plasma column), or simply with the aid of a special HCT reader device.
28

Heparin Or directly from a finger prick, to a heparin coated capillary

6- Express the results in percentage (%).

Buffy coat is the layer where WBCs and Platelets are collected to, after centrifuging a whole blood sample, this is the middle whitish-tan colored layer.

Plasma Layer Buffy Coat Layer Red Blood Cell Layer

Buffy coat layer will contain all nucleated cells, including the nucleated red cells, which are not normally found in the peripheral blood, but are seen in pathological conditions. Also, all abnormal cells, including leukemic cells are found in this layer, i.e. the buffy coat layer.

Reference intervals:Males : 40 - 53% Females : 37 - 47% Newborns: 51 - 60% Children : 34 - 49% Notes:
1-

Higher values than the reference intervals is called Lower values than the reference intervals is called anemia.

polycythemia.
2-

3- In cases of very high HCT, additional centrifugation for 5 minutes is recommended to reduce plasma trapping. In general the higher the hematocrit, the greater the centrifugal force required. 4- Adequate centrifugation time and speed are important for accurate hematocrit.

29

5- Cells should be packed so that additional centrifugation does not alter or reduce HCT reading. 6- Plasma trapping is slightly more in macrocytic anemias, spherocytosis, hypochromic anemias, and in sickle cell anemia. 7- Errors may occur as a result of: Inadequate mixing of the blood. Improper reading of the column lengths. Inclusion of buffy coat height with RBC column height (in

leukocytosis or in thrombocytosis, the buffy coat column height will be increased). Plasma trapping is still one of the causes of erroneously Hemolysis of blood sample (due to improper collection, increased HCT results. delay in processing) will cause erroneously decreased HCT. 8- Increased anticoagulant to red cell ratio (short EDTA sample), will cause red cell shrinkage and the hematocrit will be erroneously decreased. Clinical Significance: HCT is used to detect anemias, polycythemias, the MCV, and the MCHC manually.
If you direct the capillary tube towards the microhematocrit centrifuge center, the sealed material will be removed, and at the end of centrifugation you will find an empty capillary tube, blood will go out from the tube!!

hemodilution,

hemo-concentration, and also is used in the laboratory to calculate

Nowadays, Hct is supplied by the widely used automated hematology analyzers. But this Hct is calculated rather than measured, these analyzers are not equipped with centrifuges, Hct is calculated from the MCV, and RBC count, by using the following formula: MCV x RBC Hct= 10

30

The sealed end of the capillary tube should be directed to the outside. The microhematocrit should be read at the top of red cell layer not at the top of the buffy coat.

There are two types of capillary tubes, red banded and blue banded capillary tubes. The red banded are heparin coated, and use it when doing finger prick hematocrit. Blue banded capillary tubes are plain, and use it with EDTA blood samples.

Manual hematocrit is slightly more than the calculated automated hematocrit, because of the trapped plasma which will be included with manual hematocrit, and excluded with automated hematocrit (because it is calculated not measured).

31

Red Blood Cell Indices

Red blood indices are calculated parameters which determine red blood cell size, hemoglobin content of red cells, and hemoglobin concentration of red cells. These parameters are useful in classifying anemias into microcytic, normocytic, or macrocytic; and hypochromic or normochromic. These parameters are calculated from total red cell count, hematocrit and hemoglobin. 1MCV Mean Cell (Corpuscular) Volume, is the average volume of red cells. This parameter is useful in classifying anemias into: Microcytic, normocytic, and macrocytic. MCV is calculated from the hematocrit (HCT), and the Red Blood Cells Count (RBC count).

HCT MCV = RBC x 10

The results of MCV are expressed in femtoliters (fl). 1 fl = 1 x 10-15 L. In automated hematology analyzers measure (not calculating) MCV from the area under the RBC histogram, and then calculating the HCT from MCV and Total RBC count. MCV Normal Range: 80 96 fl

If results are less than 80 fl, the red cells are said to be Slight Microcytosis Marked Microcytosis 75-79 fl <70 fl

Microcytic: a. b. c.
32

Moderate Microcytosis 70-74 fl

If results are within 80-96 fl, the red cells are said to be If results are higher than 96 fl, the red cells are said to be Slight Macrocytosis Marked Macrocytosis MCH 96-105 fl > 110 fl

Normocytic.

Macrocytic: a. b. c. 2Moderate Macrocytosis 106-110 fl

Mean Cell Hemoglobin, is the hemoglobin content in the average red blood cell, or in other words, the average weight of hemoglobin per RBC. It is calculated from the hemoglobin concentration (Hb), and the total RBC count.

Hb g/dl MCH = x 10

RBC Results of MCH are expressed in picograms (pg). 1 pg = 1 g = 1012

g.

MCH Normal Range: 27 32 pg 3Macrocytic red cells have higher MCH, because they are Microcytic red cells have lower MCH, because they are MCHC larger and contain more hemoglobin. smaller and contain less hemoglobin. Mean Cell Hemoglobin Concentration, is the average hemoglobin concentration in 100 cc red blood cells. It indicates the average weight of hemoglobin as compared to the cell size. It correlates with the degree of hemoglobinization of
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the red cells on the peripheral blood film. MCHC is calculated from the hematocrit and hemoglobin.

Hb g/dl MCHC = HCT x 100

OR
MCH in picograms MCHC = MCV in femtoliters

Results of MCHC are expressed in percentage (%) or gm/dl.

Normal Range: 32 36 g/dl (%)

If results are within this range, it is said that red cells are If results are less than normal, red cells are said to be

Normochromic.

Hypochromic, which is seen in microcytic hypochromic anemias e.g. iron deficiency anemia. Notes: The only highly comparable red cell parameter between automated cell counters and manual hematology tests is the MCHC, because MCHC needs hemoglobin, and hematocrit in order to calculate it , which are easy to perform manually with high reproducibility and accuracy. Red cells cant accommodate more than 37 g/dl of hemoglobin, which is seen only in cases associated with spherocytosis. Macrocytic anemias have normal MCHC. If you have a case with high MCHC, and you checked the blood film and you didnt find spherocytes, this may indicate an error in hemoglobin
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and/or Hct. Since Hct is a calculated parameter, it is derived from RBC and MCV, so may also indicate an error in RBC count and / or MCV. Solutions to resolve this error include: retesting the specimen, perform a spun microhematocrit, performing a manual hemoglobin determination, and checking the quality control and other patients results.
Hematology Automated Analyzers nowadays can perform all of the following: 1- Count RBC 2- Measure hemoglobin spectrophotometrically. 3- Directly measure MCV, from the area under the RBC histogram. 4- Calculate Hematocrit, which is derived from MCV and RBC count. 5- Calculate MCH, which is derived from Hb, and RBC count. 6- Calculate MCHC, which is derived from Hct, and Hb.

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Preparation of Blood Films

Principle: Blood film enables us to evaluate WBC, RBC, and PLT morphology, also, allows us to perform differential WBC count, furthermore estimation of WBC and platelets counts can be done on blood films. Blood films are made on glass microscopic slides. Sample: Finger stick blood or EDTA anticoagulated venous whole blood may be used. Films of peripheral blood must be made immediately. Films may be made from EDTA anticoagulated blood as long as two to three hours after collection. All specimens should be free of clots. Procedure: 12Use clean standard size glass slides (3 inch x 1 inch = Place a small drop of well mixed anticoagulated whole 7.5 cm x 2.5 cm), wiped from dust just immediately before use. blood, in the center line of the slide, about 1.5 to 2 cm from one end, with the aid of a capillary tube. 3Immediately, without delay, with the aid of a second clean slide with uniform smooth edges (spreader slide), with a 30 40 degrees angle, move back so blood drop will spread along the edge of the spreader slide, when this occurs, spread, or smear the film by a quick, unhazizating, uniform forward motion of the spreader.

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Notes:

Before preparing the films, you must check that blood

samples are free from clots, and this is done with two wooden applicator sticks. If clots are present the specimen is unsatisfactory.

Films can be labeled with patients name and /or Lab. No. on With anemia (low Hct, reduced viscosity), the spreading With polycythemia (high Hct, increased viscosity),the With large blood drops, increase the spreading angle. With small blood drops, decrease the spreading angle. If the anemia is too severe, let the blood specimen

the thick end of the film itself, after being dried, by using a pencil.

angle should be greater, to avoid running off the slide.

spreading angle should be less, to avoid short, too thick films.

settle, so that blood is divided into two layers, plasma layer and red cell layer, then discard part of the plasma layer, then
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mix the blood specimen, by doing this you have increased the viscosity of blood, by this you will be able to prepare a nice blood film.

DO NOT ATTEMPT TO CENTRIFUGE TO DISCARD PLASMA, THIS MAY DISTORT AND DISINTEGRATE THE CELLS, WHICH ARE OUR INTEREST!

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Staining Blood Films With Romanovsky Stains

Blood films are stained so that morphology of blood cells become more easily viewed, identified, and evaluated. In addition, blood films may be examined for the presence of blood parasites (Malaria, Trypanosoma, Babesia). Furthermore, stained blood films can provide important information about a patients health, they may lead to a diagnosis or verify a diagnosis, or they may rule out a diagnosis. Evaluation of stained blood films also may lead to the decision of performing other hematology special blood stain procedures in order to identify specific cell components. As soon blood films are air dried , it is best to stain them as soon as possible. Blood films are stained with one of the Romanovsky stains, which are universally used for staining blood films. There remarkable property is creating distinctions in shades of staining granules differentially and this is dependent on two staining components: Azure B (the basic dye) and Eosin Y ( the acidic dye). Other factors which affects the staining results include : 1) Staining time, 2) Ratio of Azure B to Eosin Y, 3) pH of the staining solution . Azure B will stain the acidic cell Components (e.g. nucleus, because it contains nucleic acids; basophilic granules also take the Azure B staining because they contain heparin, which is acidic in origin), while Eosin Y will stain the alkaline basic components ( e.g. Eosinophilic granules in eosinophils, because these granules contain spermine derivatives, which are basic in origin). Red cells have affinity for acidic Eosin Y dye, because it contain hemoglobin which is basic in origin.

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Romanovsky stains include:

Giemsa Stain Wrights Stain Leishman Stain May-Grnwald Stain Leishman Stain Wrights Stain Let the films be air dried. Put the films on a staining trough rack. Flood the slides with the stain. After 2 minutes ( or more, if the stain in newly

The widely and popular used Romanovsky stains are: 1234-

Leishman Stain Procedure:

prepared), add double volume of water, and blow to mix the stain with water, until a shiny layer is seen. 5678After 5-7 minutes, wash with a stream of water. Wipe the back of the slides with gauze. Set the films in upright position on a filter paper to dry . Read the blood films microscopically.
If delay in staining blood films may occur, fix the films in absolute methanol, for 1-2 minutes, but do not stain the slides until completely dried.

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Romanovsky Stain Blood Cell Characteristics No . 1 2 3 4 5 6 7 8 9 Cell Structure Red cells Nuclei of all cell types Lymphocyte cytoplasm Monocyte cytoplasm Platelets cytoplasm Neutrophilic granules Eosinophilic granules Basophilic granules Platelets granules Staining characteristics Red or pinkish red Purple/violet Blue Grayish blue Light blue Violet-pink Orange-red Purplish black/ Deep blue Purple

Sources Of Errors In Staining 1- Stain Precipitate: May obscure cell details, and may cause confusion with inclusion bodies. Filter the stain before use. 2- pH of the buffer or water: colors: morphology. 5- Non-stain related errors: 123EDTA causes crenation of the cells after blood collection. Severely anemic blood samples causes slower drying Old blood specimens may cause disintegration in WBCs Too long staining time causes too blue slides (overstaining). Too short staining time causes too red slides. 4- Forced drying may alter color intensities and/or distort cell Too acidic pH causes too pinkish slides. Too basic pH causes too bluish slides.

3- Improper stain timing may result in faded staining or altered

(before staining) due to excessive plasma. and decrease in their numbers.

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4-

Collection of blood in heparin causes blue staining of also heparin

RBCs with bluish background, which makes heparin unsatisfactory for routine hematology testing, induces platelet aggregation and clumping , with subsequent erroneous platelet count with automated counters.

Always filter the stain before each use, to eliminate stain precipitates.

Automatic stainers are available in the market, in which slides are moved automatically and they are stained as they move.

WBC Differential Count

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Principle: Testing a Romanovsky stained blood films in order to determine and assess the percentage of various classes of WBCs present, and to assess red blood cells and platelets morphology. Increased or decreased normal WBCs class/ subpopulation counts or the presence of immature precursors of WBCs or RBCs in the peripheral blood film are of diagnostic importance in various inflammatory and disease states. Morphological red blood cells abnormality are important in various anemias (spherocytes, sickle cells, acanthocytes, burr cells, microcytes, macrocytes, target cells, .. etc). Platelet morphology, distribution, and size abnormality are suggesting a platelet disorder. Sample: EDTA anticoagulated whole venous blood film, bone marrow film, and body fluid sediments (e.g. CSF). Reagents, Supplies, and Apparatus: 1- Differential Tally Counter. 2- Conventional Binocular Microscope. 3- Oil immersion. 4- Well Stained Blood Film/s. Procedure: 1- Focus the film under x10 lens, and scan the film to check cell distribution. 2- Add a drop of oil, and move to the x100 oil immersion lens. 3- Choose a suitable area, where cells are evenly distributed without appreciable overlapping- the monolayer cell zone. 4- Count the WBCs using tracking pattern. 5- Each cell identified should be immediately tallied as:
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Neutrophil- segmented. Neutrophil band Lymphocyte Monocyte Eosinophil Basophil Immature cells: blast, promyelocyte, myelocyte, metamyelocyte, Variant atypical lymphocyte.

promonocyte. 6- Morphological abnormalities of WBCs, RBCs and platelets should be noted.

7-

Nucleated red blood cell precursors (nucleated red blood cells-

NRBC) are not included in the differential count, but are counted per 100 WBCs, and if they are more than 10 NRBC/100 WBCs, a corrected WBC count should be made (because as discussed before that NRBCs are counted as WBCs, and will be included in the total WBC count erroneously) by applying the following formula:

Uncorrected WBC X 100 Corrected WBC count = NRBC + 100

NRBC is the number of NRBC seen per 100 WBC during

differential process. 8- Express the results as percentage for each cell class/ subpopulation.
! Count in the monolayer zone

Method of Differential Counting Pattern- Tracking Pattern

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If total WBC count is high (more than 20 x 109/L), a 200 or 300 cell differential is advisable.

Blood film made for WBC differential counting should be

evaluated for red cells. Red cells are evaluated for variation in red cell volume/size, variation in shape, variation in staining properties, alteration in distribution, presence of intracellular inclusions and the presence of extracellular or intracellular parasites. Blood film for WBC differential counting should be evaluated for variation in platelet size (large, giant), presence of megakaryocyte fragments, dwarf megakaryocytes, or megakaryocytes. Also, platelet distribution(satellitism, aggregation, clumping) which produce erroneous platelets count results with impedance technology electronic counters, should be noted (causing thrombocytopenia, and an increase in WBC count and/or RBC count, and affects RBC and WBC histograms).

Blood film is evaluated for abnormal WBCs inclusions, toxic

granulation, Alder-Reilly granules , Chediak Higashi granules, Dhle bodies, and toxic vacuolization. Immature WBC cells ( left shift) should be included in the differential. Noting hypersegmentation (right shift), and hyposegmentation (Pelger Huet anomaly, or pseudopelger Huet cells). All of the above may indicate specific disease process of acquired or inherited origin.
Most of abnormal / immature cells tend to be accumulated at the blood film edges, do not forget to scan these areas. All nucleated red cells, especially megaloblasts also tend to be accumulated at these edges. Scan the blood film edges !!!

Normal Values for Differential WBC count in Adults:

Cell Type/ Population
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%Normal Differential

Normal Absolute Count * x 109

Neutrophils- Segmented Neutrophil- Band Lymphocytes Monocytes Eosinophils Basophils

50-70% 0-10% 15-45% 0-10% 0-6% 0-1%

2.0-7.0 x 109/L 0.0-1.0 x 109/L 0.6-4.0 x 109/L 0.0-1.0 x 109/L 0.2-0.7 x 109/L 0.0-0.2 x 109/L

*Absolute count is obtained by multiplying the % differential of the cell type in concern by the total WBC count, obtained either by manual or automated methods. automated CBC counters supply us with this absolute count, for the main three cell types (i.e. the neutrophils, the lymphocytes, and the monocytes). So, differential count can be expressed as percentage and also as absolute count. Automated Differential Counting Nowadays, hematology laboratory is equipped with automated hematology analyzers capable of automated differential counting, which are more quicker, precise, and accurate than the manual time consuming differential count, but this is true when the sample contains normal cell populations. When the sample contains abnormal or immature cell populations, your eyes are not substituted. Although current laser cell counters identify abnormal and immature cell populations in a sample, but this does not substitute looking to a nice looking blood film to identify these abnormal cell populations or subpopulation. Clinical Significance Of Increased Normal WBC Counts: Neutrophilia: Bacterial Infections, Inflammation , Stress, Chronic Myelocytic Leukemia. Lymphocytosis : Viral Infections, Whooping cough, Chronic of Lymphocytic Leukemia. Monocytosis: Tuberculosis, Rheumatoid Arthritis, Pyrexia unknown origin. Eosinophilia: Invasive parasite, Active Allergy, Myeloproliferative
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diseases/disorders.. Basophilia: Ulcerative Colitis , Myeloproliferative Diseases, Hyperlipidemia. Blood Film WBC and Platelet Quantitative Estimation Total WBC count can be quantitatively estimated from blood films (under x 40 objective lens), also this estimation may be used for quality control purposes, and as delta check for manual and automated WBC counts, follow the table below for estimating WBC count in blood films:
Number of WBC cells seen per x40 field 24 46 6 10 10 13 Estimated total WBC Count 4,000 6,000 /cumm 6,000 10,000 /cumm 10,000 13,000 /cumm 13,000 20,000 /cumm

Also WBC count can be estimated quantitatively from blood film,

by the following formula : Average number of nucleated cells per field at x 100 magnification = nucleated cell count x 109/L.

Platelets can be estimated quantitatively from blood films, each

platelet seen under oil immersion lens approximately equals to 20,000 PLT/cumm. Normally, blood film from healthy individuals usually shows 7-22 platelets per oil immersion field. When platelet aggregates or clumps are present in the blood film, then platelet estimation would be absolutely unreliable.

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Differential Tally Counter

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