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

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:

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.

ANTICOAGULANTS Used in Hematology Laboratory

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 Code

1 EDTA Routine Hematology Procedures. Lavender, Pink

2 Sodium citrate Coagulation , Platelets Tests, ESR. Blue

3 Heparin Osmotic Fragility, Spun Hematocrit Green, Brown

I am a tube containing

specified volume of anticoagulant, please only

fill me with the correct

required volume of blood

from the patient, do not attempt to overfill or under

fill me, your results may be

negatively affected, so help

me and help your self.

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, 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

regulates the depth of the fluid to be applied.

‏

Figure1: Top view of the hemacytometer

Figure 2: Coverglass position on the hemacytometer

COVERGLASS

COUNTING CHAMBER COVERGLASS SUPPORTS

AL AZHAR

H-SHAPED DEPRESSIONPLATFORM WITH

RULED AREA

COVERGLASS

COVERGLASS SUPPORTS

AL AZHAR

COUNTING

CHAMBER

COVERGLASS

CENTER PLATFORM

COVERGLASS SUPPORT

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:

1. The average number of cells counted.

2. The ruled areas contain an exact volume of diluted sample.

3. The dilution of the sample.

2nd. The hemacytometer Formula:

N x D (mm) x DF = C/mm³ A (mm ²)

Cells

touching

right, and

bottom

boundaries

are not

counted!

Begin

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)

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.

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)

Begin

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

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.

3- Uncalibrated Micropipettes.

4- Uneven distribution of WBC’s.

Hemacytometer Squares

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.

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.

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 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:

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).

Hemoglobin + Potassium Ferricyanide Methemoglobin (Hi)

Methemoglobin + Potassium Cyanide Cyanomethemoglobin

Hemoglobin Concentration Determination

Modified Drabkin’s 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 anemia’s.

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 0.200 g (200 mg)

2- Potassium Cyanide (KCN) 0.050 g (50 mg)

3- Dihydrogen Potassium Phosphate KH2PO4 0.140 g (140 mg)

4- Distilled Water (laboratory grade 1)

5- Non-Ionic Detergent:

- Sterox S.E. 0.5 ml

or - Trinton X-100 1.0 ml

or - Quolac Nic 218 1.0 ml

6- Standard Cyanomethemoglobin solution/ solutions.

Working Drabkin’s 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 Drabkin’s

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 Drabkin’s

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 = x Conc. Of STD Abs. of STD

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 Drabkin’s

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 Drabkin’s solution.

Absorbance 0.3

0.2

0.1

5 10 15 20 Hb-Concentration- g/dl

Hemoglobin Standard Curve

Notes:

1- Drabkin’s method is the recommended method by the ICSH.

2- Drabkin’s solution should be clear and have a pH of 7.0 to 7.4,

discard if turbid.

3- The Drabkin’s 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.

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 Drabkin’s 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 smoker’s requires 3 minutes more

incubation time for full conversion of hemoglobin to

methemoglobin.

16- Do not expose Drabkin’s 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, so that body

compensate and adapt for this by increasing hemoglobin.

18- Pregnant females tend to have lower hemoglobin

concentrations than normal for their age, because their

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 Drabkin’s 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 it’s turbid plasma and add it to the

calculated Drabkin’s 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 5 N = = = 10 ml (1- 0.50) 0.50

So, the sample blank is prepared by adding 20 l of patient’s

plasma to 10 ml Drabkin’s solution.

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)

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

Spun Microhematocrit

Principle

Hematocrit is the ratio of the total volume of RBC’s 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 RBC’s, (2) Middle layer contains WBC’s and Platelets (on

top of RBC’s), (3) Upper plasma layer. The hematocrit value is

determined by comparing the volume of RBC’s to the total volume of

the whole blood sample, it is usually reported as a %.

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).

Heparin

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

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.

6- Express the results in percentage (%).

Reference intervals:-

Males : 40 - 53%

Females : 37 - 47%

Newborns: 51 - 60%

Children : 34 - 49%

Notes:

1- Higher values than the reference intervals is called

polycythemia.

2- Lower values than the reference intervals is called anemia.

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.

5- Cells should be packed so that additional centrifugation does

not alter or reduce HCT reading.

Buffy coat is the layer where WBC’s and Platelets are collected to, after centrifuging a whole blood sample, this is the middle whitish-tan

colored 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.

Red Blood Cell Layer

Buffy Coat Layer

Plasma Layer

6- Plasma trapping is slightly more in macrocytic anemia’s,

spherocytosis, hypochromic anemia’s, 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

increased HCT results.

Hemolysis of blood sample (due to improper collection,

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 anemia’s, polycythemias, hemodilution,

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

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!!

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

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).

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 anemia’s into microcytic, normocytic, or

macrocytic; and hypochromic or normochromic. These parameters

are calculated from total red cell count, hematocrit and hemoglobin.

1- MCV

Mean Cell (Corpuscular) Volume, is the average volume of

red cells. This parameter is useful in classifying anemia’s

into: Microcytic, normocytic, and macrocytic. MCV is calculated

from the hematocrit (HCT), and the Red Blood Cells Count (RBC

count).

HCT MCV = x 10 RBC

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

Microcytic:

a. Slight Microcytosis 75-79 fl

b. Moderate Microcytosis 70-74 fl

c. Marked Microcytosis <70 fl

If results are within 80-96 fl, the red cells are said to be

Normocytic.

If results are higher than 96 fl, the red cells are said to be

Macrocytic:

a. Slight Macrocytosis 96-105 fl

b. Moderate Macrocytosis 106-110 fl

c. Marked Macrocytosis > 110 fl

2- MCH

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 = 10-

12 g.

MCH Normal Range:

27 – 32 pg

Macrocytic red cells have higher MCH, because they are

larger and contain more hemoglobin.

Microcytic red cells have lower MCH, because they are

smaller and contain less hemoglobin.

3- MCHC

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

the red cells on the peripheral blood film. MCHC is calculated from

the hematocrit and hemoglobin.

Hb g/dl MCHC = x 100 HCT

OR

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

Normochromic.

If results are less than normal, red cells are said to be

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 can’t 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

didn’t find spherocytes, this may indicate an error in hemoglobin

MCH in picograms MCHC = MCV in femtoliters

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.

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:

1- Use clean standard size glass slides (3 inch x 1 inch =

7.5 cm x 2.5 cm), wiped from dust just immediately before use.

2- Place a small drop of well mixed anticoagulated whole

blood, in the center line of the slide, about 1.5 to 2 cm from one

end, with the aid of a capillary tube.

3- Immediately, 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.

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 patient’s name and /or Lab. No. on

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

With anemia (low Hct, reduced viscosity), the spreading

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

With polycythemia (high Hct, increased viscosity),the

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

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

settle, so that blood is divided into two layers, plasma layer

and red cell layer, then discard part of the plasma layer, then

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!

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.

Romanovsky stains include:

Giemsa Stain

Wright’s Stain

Leishman Stain

May-Grünwald Stain

The widely and popular used Romanovsky stains are:

Leishman Stain

Wright’s Stain

Leishman Stain Procedure:

1- Let the films be air dried.

2- Put the films on a staining trough rack.

3- Flood the slides with the stain.

4- After 2 minutes ( or more, if the stain in newly

prepared), add double volume of water, and blow to mix the

stain with water, until a shiny layer is seen.

5- After 5-7 minutes, wash with a stream of water.

6- Wipe the back of the slides with gauze.

7- Set the films in upright position on a filter paper to dry .

8- 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.

Romanovsky Stain Blood Cell Characteristics

No. Cell Structure Staining characteristics

1 Red cells Red or pinkish red

2 Nuclei of all cell types Purple/violet

3 Lymphocyte cytoplasm Blue

4 Monocyte cytoplasm Grayish blue

5 Platelets cytoplasm Light blue

6 Neutrophilic granules Violet-pink

7 Eosinophilic granules Orange-red

8 Basophilic granules Purplish black/ Deep blue

9 Platelets granules 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:

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

colors:

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

morphology.

5- Non-stain related errors:

1st- EDTA causes crenation of the cells after blood collection.

2nd- Severely anemic blood samples causes slower drying

(before staining) due to excessive plasma.

3rd- Old blood specimens may cause disintegration in WBC’s

and decrease in their numbers.

4th- Collection of blood in heparin causes blue staining of

RBC’s with bluish background, which makes heparin

unsatisfactory for routine hematology testing, also heparin

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

Principle:

Testing a Romanovsky stained blood films in order to

determine and assess the percentage of various classes of

WBC’s present, and to assess red blood cells and platelets

morphology.

Increased or decreased normal WBC’s class/ subpopulation

counts or the presence of immature precursors of WBC’s or

RBC’s in the peripheral blood film are of diagnostic importance

in various inflammatory and disease states.

Morphological red blood cells abnormality are important in

various anemia’s (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 WBC’s using tracking pattern.

5- Each cell identified should be immediately tallied as:

Neutrophil- segmented.

Neutrophil – band

Lymphocyte

Monocyte

Eosinophil

Basophil

Immature cells: blast, promyelocyte, myelocyte, metamyelocyte,

promonocyte.

Variant atypical lymphocyte.

6- Morphological abnormalities of WBC’s, RBC’s 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 WBC’s, and if they are more than 10 NRBC/100 WBC’s, a

corrected WBC count should be made (because as discussed

before that NRBC’s are counted as WBC’s, 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

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 !!!

Method of Differential Counting Pattern- Tracking Pattern

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 WBC’s inclusions, toxic

granulation, Alder-Reilly granules , Chediak Higashi granules, Döhle

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.

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

Normal Values for Differential WBC count in Adults:

Cell Type/ Population %Normal Differential Normal Absolute Count * x 109

Neutrophils- Segmented 50-70% 2.0-7.0 x 109/L

Neutrophil- Band 0-10% 0.0-1.0 x 109/L

Lymphocytes 15-45% 0.6-4.0 x 109/L

Monocytes 0-10% 0.0-1.0 x 109/L

Eosinophils 0-6% 0.2-0.7 x 109/L

Basophils 0-1% 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

Lymphocytic Leukemia.

Monocytosis: Tuberculosis, Rheumatoid Arthritis, Pyrexia of

unknown origin.

Eosinophilia: Invasive parasite, Active Allergy, Myeloproliferative

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 Estimated total WBC Count

2 – 4 4,000 – 6,000 /cumm

4 – 6 6,000 – 10,000 /cumm

6 – 10 10,000 – 13,000 /cumm

10 –13 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.

Differential Tally Counter