Packed cell volume (PCV)

Objectives
At the end of this chapter students will be able to:
Define packed cell volume
Identify the methods used in hematocrit determination
List the materials required in PCV determination using
the micro- and macro- hematocrit methods
Discuss the advantages of the microhematocrit method of
PCV determination
Objectives cont’d
Discuss the clinical significance of PCV determination
Indicate the normal hematocrit values in health
Perform PCV determination on a sample of blood using
the microhematocrit and Wintrobe method
List the sources of error in PCV determination
Apply QC measures for PCV determination
Packed cell volume (PCV)
I. Definition
Commonly referred to as hematocrit
is a measure of the ratio of the volume occupied by the red
cells to the volume of whole blood in a sample of capillary
or venous blood
The ratio is measured after appropriate centrifugation in
the manual methods
Expressed as a decimal fraction (in l/l) or as a percentage
(%) to the nearest 0.5%
 PCV cont’d

Buffy coat is
composed of
WBC and
platelets
Hematocrit Determination cont’d
Significance of the test
enables the calculation of the red cell indices that are
widely used in the classification of anemias. These are:
Mean cell volume (MCV),
Mean cell Hb concentration (MCHC) and
to screen for anemia when it is not possible to measure
hemoglobin, and
to diagnose polycythemia vera and to monitor its
treatment.
Hematocrit Determination
Methods
 Macro method
Wintrobe
 Micro methods
Adams microhematocrit method

 Electronic method
based on the principle that the average red cell volume is
determined, the red cell count made , and the hematocrit found by
calculation e.g. coulter counter
Microhematocrit Method
III. Principle of test
• Anticoagulated blood in a glass capillary of specified
length, bore size, and wall-thickness is centrifuged in a
microhematocrit centrifuge at 10000-15000 RPM for 5
minutes to obtain complete packing of the red cells
• The PCV value is read from the scale of a microhematocrit
reader or calculated by dividing the height of the red cell
column by the height of the total column of blood.
• A small amount of plasma remains trapped between the
packed red cells.
Microhematocrit Method cont’d
• Note: Due to trapped plasma, PCV values using a
centrifugation technique are 1-3% higher than those
obtained from an electronic cell analyzer which
computes the value from the MCV and red cell count
(PCV = MCV x RBC).

iv. Specimen:
• either well mixed EDTA anticoagulated blood or
• capillary blood collected into a heparinized capillary
tube
 Microhematocrit Method cont’d
v. Equipment
Microhematocrit centrifuge
fixed speed (10000-15000 rpm)
microhematocrit centrifuge with
essential safety features which include
a lid interlock,
metallic casing and
counterbalanced lid and fitted with a
digital timer is required
HCT reader
 Microhematocrit Method cont’d
• Capillary tubes
• plain (blue band) or heparinized (red band) capillaries
• measuring 75 mm in length
• internal diameter of 1 mm and
• wall thickness of 0.2 – 0.25 mm

• Sealant
• plastic sealant, modeling clay, or plasticine.
Note: heat sealing should be avoided since it distorts the end of
the tube resulting in breakage, or the heat damages the red cells
resulting in an incorrect PCV. Do NOT also use soap or any
other sealing substances for sealing Hct tubes since detergents
lyse RBCs
vii. Method
• If capillary blood from finger puncture is to be used, follow
SOP for capillary blood collection
• If anticoagulated venous blood, mix specimen well
• Allow the blood to enter the tube by capillarity
• Fill 3/4th of the capillary tube ; do in duplicate
• Seal the capillary tubes by vertically placing the dry end into a
tray of sealing compound (wax or plasticin)
• Rotate the capillary tube slightly and remove it from the tray.
The sealant plug should be 4-6mm long. Inspect the seal for a
flat bottom.
• Place the filled, sealed capillary tube in the groove (slots) of the
centrifuge with the sealed end toward the periphery.
Microhematocrit determination cont’d
Set the timer of the centrifuge at 5 minute and spin at
10,000-15,000g.
Read the PCV using a reading device that is either part of
the centrifuge or separate from it

Alternatively, the ratio of the red cell column to whole

column (i.e., plasma and red cells) can be calculated from
measurements obtained by placing the tube against
arithmetic graph paper
If no reader at all, use a ruler
Example: height of red cell column = 19mm
height of total blood column = 49mm
PCV = 19mm/49mm = 0.388 (l/l) or 38.8%
 vii. Quality control
For precision check
Perform in duplicate
duplicates must agree within 1.5 %

For accuracy check

Run a whole blood control with known results

Note: Only for QC purpose check “Rule of 3”

Hb x 3= Hct  3%
viii. Sources of Error in Hct Procedures
Specimen collection errors
Inadequate mixing of specimen (for EDTA blood)
Improper use of the hematocrit reader or including the
buffy coat layer
Improper centrifugation
Wrong RPMs (speed) of the centrifuge
Incorrect centrifuge time
Manually stopping the centrifuge
wrong sealing method used (e.g use of soap, heating)
Incorrect reading due to uneven clay plug
Excess anticoagulant
Sources of Error cont’d
Incomplete packing due to insufficient centrifugation.
Centrifuges should be regularly checked for proper
operation (use tachometer to check rpm & document)
Incorrect reading of results.
Hemolysis or clotting of samples:
Factors that cause hemolysis and clotting of samples should
be controlled.
Occasionally, the red cell – plasma interface is not clear-
cut and the hematocrit is difficult to read. In such cases
repeat the test ensuring proper filling and centrifugation.
Variation of the bore of the tubes cause serious errors if
they are not manufactured within the narrow limits of
precision that conform to defined standards
ix. Interpretation
• Reference range varies between age, gender and altitude
Children at birth 44-54%
Children 2-5 years 34-40%
Children 6-12 years 35-45%
Adult men 40-54%
Adult women 36-46

Ethiopia: Adult Males 41.6 – 55.1%

Adult Females 35.3 – 48.8%
Interpretation cont’d
Decreased in anemia
Increased in polycythemia

Limitation
Plasma trapping results in higher values (1-3% higher than
those obtained from an electronic cell analyzer)
Additional Note
The technician should cultivate the habit of inspecting both
the buffy coat and the supernatant plasma when reading the
hematocrit value.
A note should be made on the patient’s report if an
abnormal plasma or buffy coat is seen as this is often an
important clue for the clinician.
Example:
very thick buffy coat: may mean cases of leukemia
very thin buffy coat: may show marked leucopenia
yellowish plasma: may show jaundice (if pre-analytic error
can be ruled out)
When a thick buffy coat is seen, perform total and
differential WBC count
Macrohematocrit method
Procedure is no longer in routine use
Wintrobe tube is filled with well mixed EDTA
anticoagulated venous blood to the mark "0" on top using a
long stem Pasteur pipette making sure that no air bubbles
are trapped.
The ratio of EDTA to volume of blood should be 1.5mg/ml or
0.1ml 10%w/v K3EDTA/ml.
EDTA in excess of this proportion may cause a falsely low
PCV as a consequence of cell shrinkage.
The preparation is then spun at 2300g for 30 minutes.
The hematocrit is read from the scale on the right hand side
of the tube taking the top of the black band of reduced
erythrocytes immediately beneath the reddish gray
leukocyte layer.
Advantages of the Microhematocrit
Method
It enables higher centrifugation speeds with consequent
shorter centrifugation times and superior packing.
The amount of trapped plasma is less than that in the
Wintrobe method by virtue of the higher centrifugation
speed employed.
 Tips

RBC count – total # of red

cells in millions/uL
Hemoglobin –
concentration of Hb in red X3=15.1
cells reported in g/dL X3=45.0
Hematocrit – percentage
(%) of red cells in a known
volume of whole blood
RBC count, HGB and HCT
values parallel each other

22
 RBC Count, HGB, HCT

Each health institution should establish its own reference

ranges
Significance
Decreased RBC, HGB and/or HCT values….Anemia
Decreased production, increased loss/destruction
Increased RBC, HGB and/or HCT values….Polycythemia
Increased production
Critical values: HGB <7.0 or >18.5 g/dL
23
 Exercise: Result Evaluation
3 adults

 Patient # 1: All results are normal

 Patient #2: Anemia (and leukocytosis)
 Patient # 3: Polycythemia/erythrocytosis
24
 RBC Count, HGB, HCT
Correlation
Relationship: Hb x 3 = HCT + 3%
RBC x 3 = Hb or RBC x 9 = HCT
Used to estimate values or check data correlation
‘Rules’ only apply if red cells are normal in size and hgb
content
Which parameter does not correlate?
RBC = 4.00 million/cmm
Hb = 14.0 g/dl
HCT = 36.0%

25
 Hb error; RBC and HCT correlate
RED CELL INDICES
Objectives
At the end of this chapter, the student will be able to:
Define MCV, MCH and MCHC.
Explain the purpose of calculating the red cell indices
Calculate MCV, MCH and MCHC values from given
values
Define RDW
Discuss the clinical significance of RDW
Introduction
The red cell indices:
are absolute values calculated from:
 the measured hemoglobin,
PCV
RBC count
are of considerable clinical importance in the diagnosis and
classification of anemias
are dependent upon the accuracy of the various red cell
parameter estimations
Introduction cont’d
The red cell indices include
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Red cell distribution width (RDW) is another important
red cell parameter obtained by electronic methods
RDW measures the variation in size of the red blood
cells (degree of anisocytosis)
It must be remembered that the red cell count has the
greatest potential error and must be performed with
extreme care preferably using an electronic counter
The Mean Cell Volume (MCV)

 Is the average volume of a red cell expressed in femto litres

(fL)
Femtoliter is 10-15 of a liter
MCV is obtained by dividing the PCV by red cell number

 MCV (fl) = PCV (l/l)

No. of RBC/L
Example: PCV = 0.45(l/l)
RBC = 5  1012/l
MCV = 0.45 (l/l) = 90  10-15 = 90fl
5  1012
 Interpretation

Normal Values
Men and Women: 80-100 fl
MCV
 increased in
 macrocytic anemias
decreased in
iron deficiency anemia
Thalassemia
microcytic anemia
The Mean Cell Hemoglobin (MCH)

is the average amount of hemoglobin per individual red

cell expressed in picograms (pg).

It is given by:

MCH (Pg) = Hb (g/L)
RBC/L
Example: Hb conc. = 150g/L
RBC = 5  1012/L

MCH (pg) = 150 = 30  10-12 = 30pg

5  1012
Interpretation
Normal Value: Men and women: 27-31 pg

MCH is increased in
macrocytic anemia
MCH is decreased in - microcytic anemia
- iron deficiency anemia
The Mean Cell Hemoglobin Concentration (MCHC)

-Is the average hemoglobin per unit volume of red cells.

MCHC (g/l) = Hb (g/L)

PCV (L/L)

Example: Hb conc. = 148g/L

PCV = 0.45 (L/L)

MCHC = 148 = 328g/L

0.45
Interpretation
Normal Values: Men and women: 32-36 % (320-360
g/L)
MCHC is increased in some cases of hereditary
spherocytosis
MCHC is decreased in iron deficiency anemia
Red Cell Distribution Width (RDW)

Another index, the red cell distribution width (RDW), is

specifically designed to reflect the variability of red cell
size.
It is based on the width of the red blood cell volume
distribution curve
 larger values of RDW indicate greater variability.

 An elevated RDW may be an early sign of iron-deficiency

anemia
RDW cont’d
proposed as an aid in distinguishing iron deficiency
from other causes of microcytic anemia, such as
thalassemia,
 the RDW is not sufficiently specific to obviate the
need for more specific tests.
 The RDW can be used in the laboratory as a flag to
select those samples submitted for automated blood
count that should have manual review of the blood
film for red cell morphology
RDW cont’d

In the Coulter Model S plus, for example, a red cell

histogram is plotted and the RDW(%) is defined as the
coefficient of variation of the MCV:
RDW (%) = SD of MCV x 100
Mean MCV
The reference range for RDW is from 11% to 15%, but
varies with the instrument used.
Review Questions

1. Define: MCV, MCH, MCHC, and RDW.

2. What is the purpose of calculating the red cell indices?
3. A complete blood count was performed for a patient and
the following profiles were recorded:
WBC= 8,000/mm3
PCV = 50%
Hb = 15g/dl
RBC count = 5 x 106/mm3
• Calculate the MCV, MCH and MCHC values for the
patient. Interpret your results in the light of the normal
values for these indices.