[Hematocrit (HIT) or Packed Cell Volume (PCV) test] Manually – the method only

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Estimation of the erythrocyte volume fraction [Hematocrit (HIT) or Packed Cell Volume (PCV)] Manually – the method only.

We will talk about the interpretation of results in another sections

A) Introduction

In this test, we want to measure the volume that RBCs occupy in the whole blood…….. To measure this, we have 2 ways:

1) measure the volume of each Red Blood Cell (RBC) individually and calculate the sum of total volume of all RBCs

2) Pack all the RBCs together and measure the total volume that occupied by them

Diagram shows the blood after centrifugaiton

Diagram shows the blood after centrifugaiton

Of course, the way (no.1) is impossible and the way (no.2) is possible and we use the way no.2  in this test and hence the name Packed Cell Volume (PCV) as we will see in the next sections.

But how can we pack all the RBCs together?

Of course by a centrifuge

And in this case we will separate the whole blood into 3 regions, Plasma, a buffy coat layer (i.e. White blood cells) and the RBCs layer …….examination of each of them has an important value as we see in the following sections

 1) Value of the test 

The packed cell volume (PCV), also referred to as hematocrit is used to screen for anemia when it is not possible to measure hemoglobin accurately and mains electricity is available to operate a microhematocrit centrifuge (you will see it soon).

Terminology: The International Council for Standardization in Hematology (ICSH) recommends that PCV be used when blood is centrifuged in a capillary tube, and the word haematocrit be used when an autoanalyzer is used to compute the value

Value of PCV (i.e. the RBCs region)

==> The PCV is also used in the investigation of dehydration, burns, and polycythemia (i.e. a condition in which the plasma volume is decreased or the RBCs number increased).

Value of examining plasma following centrifugation

Plasma from normal blood appears straw-coloured.

In severe iron deficiency the plasma appears colourless.

When the blood contains an increased amount of bilirubin, as in haemolytic anaemia, the plasma will appear abnormally yellow.

Trypanosoma_cruzi_crithidiaWhen the plasma is pink-red this indicates a haemolyzed sample (less commonly, haemoglobinaemia). A further blood sample should be tested.

When lipids are raised, the plasma appears white and cloudy.

In African trypanosomiasis endemic areas, examination of the plasma above the buffy coat layer following centrifugation can help to detect trypanosomes which causes the sleeping sickness and which caused by the  parasitic protozoan trypanosomes of the genus Trypanosoma.


Examination of the buffy coat layer:

When white cell numbers are significantly increased, this will be shown by an increase in the volume of buffy coat layer. When this is seen, perform a total white cell count and differential count.

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 2) Principle of the test 

The packed cell volume is that proportion of whole blood occupied by RBCs when it is packed together, expressed as a ratio (litre/litre).

(RCF) = Relative Centrifugal Force and (xg) = times of gravity

Anticoagulated blood in a glass capillary of specified length, bore size, and wall-thickness is centrifuged in a microhaematocrit centrifuge at RCF 12 000–15 000 xg for 3–5 minutes to obtain constant packing of the red cells. A small amount of plasma remains trapped between the packed red cells.

The PCV value is read from the scale of a microhaematocrit reader (we will see it later) or calculated by dividing the height of the red cell column by the height of the total column of blood.

Note: Due to trapped plasma, PCV values using a centrifugation technique are 1–3% higher than those obtained when using an electronic cell analyzer which computes the value from the MCV and red cell count.

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 3) ٍSpecimen 

To measure the PCV, either well mixed well oxygenated EDTA anticoagulated blood can be used or capillary blood collected directly for finger prick using lancet into a heparinized capillary tube.

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 4) Measuring the volume of RBCs and Measuring units 

There are no measuring units for PCV >>>WHY?<<<<<

We said that in this test we measure the volume of the total RBCs when they are packed together without any spacing between them

Of course that  is right,

But how we can measure their volume???

The volume is 3 dimensions parameter [(length × width = area) × height], but in this test we use a capillary tube whose volume equals area of the circle of tube diameter × length of the tube

Vol of tube = Area of the circle (Πr2)  × Length of tube

But volume in this way can’t help us, because we don’t know that RBCs occupy certain volume from 6 liters or 5 liters??? Actually, you will don’t know this if you get the RBCs volume in this way ……

The aim of this test, is to know the dehydration cases and lose or increase of RBCs in the circulation …. And to know this we should to know the percentage of the whole blood volume (i.e. Vol%) that occupied by the RBCs ……. So, we want to compare the RBCs volume with the total volume of the whole blood but what is the object that we will get its volume?.

That means,

PCV or HIT = [Volume of RBCs × 100] / total volume = …. vol%

Or it can be just a fraction

HIT = [Volume of RBCs] / total volume = ….

But in the test we measure the length of both RBCs column and the total column not a volume?

Yes, that is right …. I will show you

Vol of tube = Area of the circle × Length of tube

HIT = [area of the circle × height of RBCs column × 100] / [area of the circle × height of total blood colmun]

HIT = [area of the circle × height of RBCs column × 100] / [area of the circle × height of total blood colmun]

HIT = [height of RBCs column × 100] / height of total blood column

For example, if the volume of erythrocytes in 1 litre (1000 ml) of blood is 450ml, the erythrocyte volume fraction is 450ml/1000ml = 0.45 or  [450ml × 100]/1000 ml = 45% and since the fraction is millilitres divided by millilitres, the unit “ml” cancels out, and the result is a simple decimal fraction or a percentage with no unit.

Thus, there are NO MEASURING UNITS for Hematocrit.

The remainder of the blood is made up almost entirely of plasma, together with a small volume of leukocytes. If the latter are ignored, the plasma volume fraction in the above example would be 550ml/1000ml = 0.55 (note that 0.45 + 0.55 = 1.0; i.e. erythrocyte volume fraction plus plasma volume fraction = 1). The erythrocyte volume fraction or hematocrit (i.e. PCV) is therefore a measure of the proportion of erythrocytes to plasma

In the traditional system, the “packed cell volume” in the example given would be 45%. Note that, in using SI units, the numerical value does not change, but becomes 0.45 instead of 45%.

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 5) Methods of meaasurments 

There are 2 methods:

–  Micro-scale method

–  Macro-scale method

If you have the microhemtatocrit centrifuge, then you will use the micro-scale method and if you don’t have it, you will use the macro-scale method.

But the Micro-scale is more preferable because it is quicker and a blood from a finger can be used.

B) Micro-scale method

 1) Principle 

Capillary tubes

Capillary tubes

The blood (mixed with anticoagulant) is placed in a long capillary tube and centrifuged in a microhaematocrit centrifuge. The level reached by the column of erythrocytes is read with a scale reader. This method is preferable to that using a macro-scale: it is quicker, and blood from the finger can be used.

 2) Materials and reagents 

How to prepare EDTA and Wintrobes reagent?? CLICK HERE

Microhaematocrit centrifuge

Scale reader (usually provided with the centrifuge)

Soft wax

Soft wax

Capillary tubes, 75mm long with a 1.5-mm bore, containing dried heparin (if capillary blood is used; if venous blood mixed with EDTA dipotassium salt, 10% solution (reagent no. 22) is used, “heparinized” tubes are not required)

Filter-paper

Soft wax or plastic modelling clay (or a Bunsen burner or spirit lamp)

Sterile blood lancet

70% Ethanol.

Microhaematocrit centrifuge

The micro-hematocrit centifuge

The micro-hematocrit centifuge

The main 2 parts in the microhematocrit centrifuge are:

1)  The Hematocrit Rotor: which held the capillary tubes in  microhematocrit centrifuge:

–  The places that we put the capillary tubes in are numbered and you should write down the number of each tube on the cheat of the corresponding sample to avoid writing the wrong result in the wrong sample cheat

 

The rotor of the micro-hematocrit centrifuge

The rotor of the micro-hematocrit centrifuge

 

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2)  The hematocrit reader:

–  It is the scalar by which we measure the percentage of RBCs column to the total column.

– It measures the percentage of the fraction directly not the length of the columns

– There are 3 major types of scalar; hematocrit reader card, spiral micro-hematocrit reader and the circular micro-hematocrit reader but we will talk about how to use each one in the procedure.

Circular Hematocrit reader

Circular Hematocrit reader

Hematocrit reader card

Hematocrit reader card

Spiral hematocrit reader

Spiral hematocrit reader

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The simplest one – in my opinion – is the micro-hematocrit reader card because you can made it manually on a graph paper as follow:

1)    Bring a Graph paper, 15–20cm wide, ruled in millimetres.

2)    On the left-hand vertical edge, starting at the bottom, make a series of 10 marks at intervals of 4mm.

3)    On the right-hand vertical edge, in the same manner, make 10 marks at intervals of 6mm.

4)    Using a ruler, draw 10 sloping lines connecting each mark on the left margin to the corresponding mark on the right margin.

5)    In the left margin, opposite the bottom horizontal line of the graph paper, write “0”. Continue up the left margin, marking each sloping line you have drawn as follows: 0.1, 0.2, 0.3, etc.; the top sloping line will be marked 1.0.

6)    In the right margin, write the same numbers opposite the other ends of the sloping lines.

7)    Now, again using a ruler, draw a second series of sloping lines, but make them much lighter than the first set of lines.

8)    Each light line should be drawn exactly in the middle of the space between each pair of heavy lines.

9)    Finally, following the printed lines of the graph paper, draw a series of heavy vertical lines at intervals of about 3cm. Your scale should look like the one in the following picture.

10)     Instead of making your own scale, you could use the one printed here for reading erythrocyte volume fractions. (Cover it with a sheet of plastic.)

 

Hematocrit reader card made manually by Dr. Kyrolus K. F

Hematocrit reader card made manually by Dr. Kyrolus K. F

 

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 3) Specimens 

Collection of specimens

a) Capillary blood specimens

Diagram shows the right sites for pricking with lancet

Diagram shows the right sites for pricking with lancet

1) Using a blood lancet, draw blood by pricking either:

–  the third or fourth finger

–  the lobe of the ear

– the heel (infants)

after sterilizing the chosen area with ethanol.

The blood should flow freely or with very little pressure to the area. Wipe away the first drop with filter-paper.

Pricking  finger with lancet

Pricking finger with lancet

Pricking finger with lancet

Pricking finger with lancet

 

 

 

 

Pricking infant heel

Pricking infant heel

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2. Apply the tip capillary tube containing dried heparin to the drop of blood.

–  The blood flows into the tube by capillarity

–  Fill about three-quarters of the tube.

Collecting blood from finger puncturing by lancet

Collecting blood from finger puncturing by lancet

Collecting blood from infant heel for hematocrit test

Collecting blood from infant heel for hematocrit test

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Filling capillary tube from anticoagulated blood by capillary tube

Filling capillary tube from anticoagulated blood by capillary tube

b) Venous blood specimens

1) Collect a venous blood specimen and add it to a test-tube containing EDTA dipotassium salt solution (or in a ready EDTA tubes).

2) Remove the cap of the whole blood tube and carefully angle the whole blood then insert one end of the capillary into the blood.

–         The capillary tube automatically aspirate the blood by the capillary action.

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 4) Plugging or sealing the capillary tube 

Plug the end of the tube that has not come into contact with the blood with soft wax or plastic modeling clay.

–  Check that it is completely plugged to a depth of about 2mm.

Plugging the capillary tube

Plugging the capillary tube

 

sealing the capillary tube with clay

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clay

clay

 

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Alternatively, seal the end of the tube by heating it carefully over a Bunsen burner or spirit lamp.

– Leave it to cool in a horizontal position.

Sealing the capillary tube by the flame from a spirit lamp or pilot flame from a Bunsen burner can distort the glass, causing breakage when the internal lid is screwed down on the rotor. Red cells may also be lyzed by the heated glass. Use of an open flame is also a fire hazard.

It is useful to have ready a numbered stand containing plastic modelling clay, so that each patient’s tube can be stuck upright next to the corresponding number.

Capillary tube stand with soft wax

Capillary tube stand with soft wax

 

sealing the capillary tube by flaming

 

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 5) Measurement technique 

(1) Centrifugation:

1. Place the capillary tubes in the numbered slots in the centrifuge head.

–  Making sure that the number on the slot corresponds to the specimen number.

–  The sealed end of the tube should point outwards, away from the centre to avoid blood leaking out during centrifugation

–   Make sure that you balanced the microcentrifuge by putting another capillary tube opposite to that of the sample.

Microcentifuge balance

Microcentifuge balance

The clay end outside away  from the center

The clay end outside away from the center

Diagram shows the blood after centrifugaiton

Diagram shows the blood after centrifugaiton

2. Centrifuge at 3000 g (for the period of time recommended by the manufacturer of the centrifuge — usually 10 minutes).

After centrifugation, the tubes will show three layers:

— at the top, a column of plasma;

— in the middle, a very thin layer of leukocytes and platelets ;

— at the bottom, a column of erythrocytes.

Now we get ready to read the results on the hematocrit reader

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(2) Reading the results on the hematocrit reader:-

The erythrocyte volume fraction (i.e. reading the PCV) reading is made exactly at the top of the column of erythrocytes.

Using the hematocrit card:

Advanced reader card:

There are many types of the manufactured reader cad but we will get one type for example

Hold the tube against the scale so that the bottom of the column of erythrocytes (not the bottom of the tube) is aligned with the horizontal zero line.

Move the tube across the scale until the line marked 1.0 passes through the top of the plasma column.

Check to make sure that the bottom of the column of erythrocytes is still on the zero line; also check (by means of the heavy vertical lines) that the tube is vertical.

Move the reader bar until the its line passes through the top of the RBCs column

 

Reading Hematocrit value by reader card

Reading Hematocrit value by reader card

Simple reader card:

Hold the tube against the scale so that the bottom of the column of erythrocytes (not the bottom of the tube) is aligned with the horizontal zero line.

Move the tube across the scale until the line marked 1.0 passes through the top of the plasma column.

Check to make sure that the bottom of the column of erythrocytes is still on the zero line; also check (by means of the heavy vertical lines) that the tube is vertical.

PCV_card_hemolytic_anemia

micro-hematocrit-manually-(2)

The line that passes through the top of the column of erythrocytes gives the erythrocyte volume fraction (0.5 in the previous image). The light intermediate lines represent intervals of 0.05;

If the top of the column of erythrocytes is not on a line, but between a heavy line and a light line, its position can be estimated to the nearest 0.01.

If your laboratory has not yet changed to SI units and is still using the traditional system, the same chart can be used. Simply read the numbers as percentages instead of fractions. For example, instead of “erythrocyte volume fraction, 0.5” report the result as “packed cell volume, 40%”.

Note the inner lid is the scalar

Note the inner lid is the scalar

Using the spiral reader:

Spiral reader is found on the inner lid itself

1)    Place the inner lid on a clean white paper to make the scale sharper

2)    Just move the capillary tube until the top of the plasma column hit the line marked  100

3)    Make sure the bottom of RBCs column is at the line marked zero

Then the PCV ot HIT is at the line that passes through the top of the RBCs column

 

Spiral hematocrit reader

Spiral hematocrit reader

 

hct

Using the circular reader:

This is the more preferred in my opinion because it is the easiest and the most accurate

1)    Place the tube in groove of the plastic holder where the clay end towards the center.

2)    Rotate assembly so that the pin stops, lining up with the 100 percent mark.

 

Rotate assembly so that the pin stops, lining up with the 100 percent mark

Rotate assembly so that the pin stops, lining up with the 100 percent mark

 

3)    Rotate the upper disk to move the curved line with the top of the plasma.

 

The curved line at the top of the plasma

The curved line at the top of the plasma

 

4)    Rotate the entire assembly until the curved line is lined up with the boundary between the packed cells and the plasma.  Read the percent packed cells off the disc at the right.  (It reads 44.9 %)

 

Rotate the entire assembly until the curved line passes trough the top of the RBCs column (PCV = 44.9%)

Rotate the entire assembly until the curved line passes trough the top of the RBCs column (PCV = 44.9%)

 

When no reader is available:

Use a ruler to measure the length of the total column of blood (top of plasma to bottom of red cell column) in mm and the length of the red cell column (base to below buffy coat layer).

Calculate the PCV as follows:

 [length of the RBCs column (mm)] / [length of the total column (mm)] = … HIT

C) Macro-scale method

 1) Principle of the test 

The blood (mixed with anticoagulant) is placed in a graduated tube and centrifuged to pack the erythrocytes. The level of the column of erythrocytes is then read directly in the graduated tube.

Wintrobe tubes

Wintrobe tubes

 2) Materials and Reagents 

–  Centrifuge

–  Special graduated tubes (Wintrobe tubes), 9.5 cm long with a 0.6-cm bore, calibrated from 0 to 100

–  Long fine capillary Pasteur pipette (long enough to reach the bottom of the tube) with rubber teat

–  Anticoagulant EDTA dipotassium salt, 10% solution  or Wintrobe solution or EDTA tubes.

 

Centrifuge

Centrifuge

sedi-rack_bloodzUsing a capillary pipette to fill a graduated tube with blood220px-Pasteur_Pipets 3) Specimens 

In the macro-scale method we can’t use the capillary blood by puncturing the finger by a lancet because we will not get the need amount of blood.

Collect a venous blood specimen and by Using the capillary pipette add it to a graduated tube containing anticoagulant and fill the graduated tube with blood up to the 100 mark (see above).

–         making sure that no air bubbles form.

 4) Measurement technique 

measuring PCV by macro scale method1) Place the graduated tubes in the centrifuge and centrifuge for 30 minutes at 2300 g.

==> If the rotor arm of the centrifuge (measured from the axis of rotation to the base of the bucket holding the tube) is 15 cm long, 3600rpm will be needed to attain this force; with a 20-cm arm 3100rpm will be needed.

==> Important: a force of less than about 2300g will give a false result.

2) Read the level at which the erythrocytes meet the layer of leukocytes. Make sure that the correct set of graduations is being used, upwards towards the 100 mark.

The figure obtained is a percentage (the “packed cell volume”); divide by 100 to obtain the erythrocyte volume fraction.

D) Important caution

1)    Immediately after reading a PCV, discard the capillary into a puncture resistant container for incineration or burial in a deep covered pit. NEVER leave used capillary tubes on the bench from where they can easily roll to the floor, causing injury from broken glass and a serious biohazard risk.

2)    Report any abnormal appearance of the plasma (see previous text).

3) Examine the layer of leukocytes just above the column of erythrocytes. It is normally very thin; if it seems thick, determine the leukocyte number concentration (see section 9.6). The layer will seem abnormally thick if the leukocyte number concentration is greater than 20 ¥ 109/l. In cases of leukaemia, when the leukocyte number concentration may be 100–200 ¥ 109/l, the layer may be several millimetres thick.

E) Quality control of PCV

Tests should be performed in duplicate to check for imprecision (duplicate tests should not differ by more than 5%).

F) Sources of error in measuring PCV

1) Centrifuging at too low an RCF or for an insufficient length of time resulting in a PCV value being higher than it should be.

2) Delay in reading the PCV after centrifugation, allowing plasma to evaporate.

3) Using an anticoagulated blood sample containing excess EDTA (e.g. too little blood added to anticoagulant). This will cause the red cells to shrink, resulting in a PCV value lower than it should be. The opposite occurs if anticoagulated blood is left for more than 6 hours before being tested (the red cells swell, causing a falsely raised value).

4) Clots in an anticoagulated blood sample can result in a falsely low PCV value. False values will also be obtained when venous blood samples are not mixed adequately.

5) Rises in PCV (up to 6% error) can occur when there is an increase in trapped plasma due to changes in red cell size or shape, e.g. in spherocytosis, microcytosis and macrocytosis. Increases up to 20% of the PCV value can occur in sickle cell disease due mainly to the abnormal shape and rigidity of sickle cells.

6) Using capillary tubes that are not designed for measuring PCV.

7) Not cleaning and maintaining the microhaematocrit centrifuge as recommended by the manufacturer.

 

References:

Manual of basic techniques for a health laboratory Second edition; World Health Organization Geneva  2003

biology.clc.uc.edu

District Laboratory Practice in Tropical Countries, Part 2, Second Edition, Monica Cheesbrough

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