2. In investigating physiological function and
malfunction of blood, accurate and precise
methodology is essential to ensure, as far as
possible, that tests do not give misleading
information because of technical errors
Obtaining the specimen is the first step
toward analytic procedures. It is important to
use appropriate blood containers and to
avoid faults in specimen collection, storage,
and transport to the laboratory.
3. Special care must be taken to avoid risk of
infection from various pathogens during all
aspects of laboratory practice, and the safety
procedures must be observer durring blood
The phlebotomist should wear disposable
plastic or thin rubber gloves. It is also desirable
to wear a protective apron or gown and, if
necessary, glasses or goggles.
Care must be taken to prevent injuries,
especially when handling syringes, needles,
4. Disposable sterilized syringes, needles, and
lancets should be used if at all possible, and
they should never be reused.
6. During Collection
Different times (diurnal variance)
Posture: lying, standing, or sitting
Haemoconcentration from prolonged
Excessive negative pressure when drawing
blood into syringe
Incorrect type of tube Capillary vs. venous
7. Handling of Specimen
Insufficient or excess anticoagulant
Inadequate mixing of blood with
Error in patient and/or specimen
Inadequate specimen storage conditions
Delay in transit to laboratory
8. It is collected by phlebotomist
The components of phlebotomy trays includes Syringes
and needles, Tourniquet, Specimen containers (or
evacuated tube system)—plain and with various
70% isopropyl alcohol swabs or 0.5% chlorhexidine
Sterile gauze swabs or cellulose pads
Self-sealing plastic bags
Rack to hold specimens upright during process of filling
(A puncture-resistant disposal container should also be
9. The common containers for haematology tests
are available commercially with dipotassium,
tripotassium, or disodium ethylenediaminetetra-
acetic acid (EDTA) as anticoagulant, and they are
marked at a level to indicate the correct amount
of blood to be added.
Containers are also available containing
trisodium citrate, heparin, or acid citrate
And containers with no additive, which are used
when serum is required
10. Evacuated tube systems, commonly used,
consist of a glass or plastic tube/container
(with or without anticoagulant)
An evacuated system is useful when multiple
samples in different anticoagulants are
The vacuum controls the amount of blood
that enters the tube, ensuring an adequate
specimen for the subsequent tests and the
correct proportion of anticoagulant when this
11. Firstly check the patients identity if it
corresponds to the details on the request
Make sure all required equipments are in
Clean the skin with 70% alcohol (e.g.,
Collect blood from an antecubital vein or
other visible veins in the forearm by means of
either an evacuated tube or a syringe
12. Care must also be taken when using a
tourniquet to avoid contaminating it with
blood, because infection risks have been
reported during blood collection.
The tourniquet should be applied just above
the venepuncture site and released as soon as
the blood begins to flow into the syringe or
delay in releasing it leads to fluid shift and
haemoconcentration as a result of venous
13. 1) Disposable syringes or vacutainer systems
2) Disposable lancets
3) Gauze pads or adsorbent cotton
5) Alcohol swab
6) Waste container
15. Check again if the patients identity if it
corresponds to the details on the request form
Label the specimen collected with adequate
patient identification immediately after the
samples have been obtained.
On the labels this should include at least
surname and forename or initials, hospital
number, date of birth, and date and time of
The same information must be given on the
16. Specimens should be sent in individual plastic
bags separated from the request forms to
prevent contamination of the forms in the
event of leakage.
Alternatively, the specimen tubes must be set
upright in a holder or rack and placed in a
carrier together with the request forms for
transport to the laboratory.
17. Dispose the used syringe without separating
the needle from the syringe in a puncture-
resistant container for disposal.
18. Used for obtaining a small amount of blood
either for direct use in an analytic process or
for collecting into capillary tubes coated with
heparin for packed cell volume
These methods are mostly used when it is
not possible to obtain venous blood (e.g., in
infants younger than 1 year, in cases of gross
obesity, or for point-of-care blood tests).
puncture of skin
with sharp point
to draw small
amount of blood.
Collected in small,
tubes, slides, or
20. In adults and older children blood can be
obtained from a finger; the recommended site
is the distal digit of the third or fourth finger
on its palmar surface
In infants, satisfactory samples can be obtained
by a deep puncture of the plantar surface of
Do not puncture the central plantar area for
infants to avoid the risk of injury and possible
infection to the underlying tarsal bones,
especially in newborns.
21. The capillary blood has slightly raised PCV,RBC
and hemoglobin concentration compared to
The total leucocyte and neutrophil counts are
higher by about 8%; the monocyte count is
higher by about 12%, and in some cases by as
much as 100%, especially in children.
Conversely, the platelet count appears to be
higher in venous than in capillary blood; this is
on average by about 9% and in some cases by
as much as 32%.
22. The difference between plasma and serum is
that the latter lacks fibrinogen and some of
the coagulation factors
Blood collected to obtain serum should be
delivered into sterile tubes with caps or
commercially available, plain
(nonanticoagulant), evacuated collection
tubes and allowed to clot undisturbed for
about 1 hour at room temperature
23. Ideally, blood films should be made immediately
after the blood has been collected.
Because blood samples are usually sent to the
laboratory after a variable delay, there are
advantages in preparing blood films when the
phlebotomy is carried out.
The phlebotomy tray might include some clean
glass slides and spreaders, and phlebotomists
should be given appropriate training for film
When films are not made on site, they should be
made in the laboratory without delay as soon as
the specimens have been received.
24. The tubes, with or without a serum separator,
are centrifuged for 10 min at about 1200 g.
The supernatant serum then is pipetted into
another tube and centrifuged again for 10 min
at about 1200 g.
The supernatant serum is transferred to tubes
for tests or for storage.
For most tests, serum should be kept at 4°C
until used, but if testing is delayed, serum can
be stored at -20°C for up to 3 months and at -
40°C or less for long-term storage.
25. EDTA and Citrate works by chelating Calcium
Heparin binds to antithrombin, thus inhibiting
the interaction of several clotting factors.
EDTA is used for blood counts; sodium citrate
is used for coagulation testing and erythrocyte
sedimentation rate (ESR).
For better long-term preservation of red cells
for certain tests and for transfusion purposes,
citrate is used in combination with dextrose in
the form of acid–citrate–dextrose (ACD),
26. The excess of 2g/ml of blood may cause
lower PCV, raised MCHC and higher platelets
27. It is thus the best anticoagulant for osmotic
fragility tests and is suitable for
However, heparin is not suitable for blood
counts because it often induces platelet and
It also should not be used for making blood
films because it gives a faint blue colouration
to the background when the films are stained
by Romanowsky dyes, especially in the
presence of abnormal proteins.
28. The RBC, white blood cell count (WBC),
platelet count, and red cell indices are usually
stable for 8 hours after blood collection,
although as the red cells start to swell the
PCV and MCV start to increase, osmotic
fragility increases, and the ESR decreases.
When the blood is kept at 4°C, the effects on
the blood count are usually insignificant for
up to 24 hours.
29. Storage beyond 24 hours at 4°C results in
erroneous data for automated white cell
differential counts, although the extent
depends on instrument performance and the
manufacturer's recommendation, which
should be followed when an automated
counting method is used
30. Reticulocyte counts are unchanged when the
blood is kept in either EDTA or ACD
anticoagulant for 24 hours at 4°C, but at
room temperature the count begins to
decrease within 6 hours.
Nucleated red cells disappear in the blood
specimen within 1-2 days at room
31. Haemoglobin concentration remains
unchanged for days, provided that the blood
does not become infected, as shown by
turbidity or discoloration of the specimen.
However, within 2-3 days, and especially at
high ambient temperatures, the blood begins
to lyse, resulting in a decrease in the RBC and
PCV, with an increase in the calculated MCH
32. Coagulation test stability is critical for
diagnosis and treatment of coagulopathies.
It is recommended that tests be carried out
within 2 hours when the blood or plasma is
stored at 22-24°C, 4 hours at 4°C, 2 weeks at
-20°C, and 6 months at -70°C.
33. Irrespective of anticoagulant, films made
from blood that has been standing for not
more than 1 hour at room temperature are
not easily distinguished from films made
immediately after collection of the blood.
By 3 hours, changes may be discernible, and
by 12-18 hours these become striking.
34. A number of factors affect hematological
values in apparently healthy individuals.
These include the technique and timing of
blood collection, transport and storage of
specimens, differences in the subject's posture
when the sample is taken, prior physical
activity, or whether the subject is confined to
Variation in the analytic methods used may
also affect the measurements. These all needs
to be standardized.
35. sex, age, occupation, body build, genetic
background, and adaptation to diet and to
environment (especially altitude). May also have
effects in hematological values.
Haematological values for the normal and
abnormal will overlap, and a value within the
recognized normal range may be definitely
pathological in a particular subject.
For these reasons the concept of “normal values”
and “normal ranges” has been replaced by
reference values and the reference range, which is
defined by reference limits and obtained from
measurements on the reference population for a
36. Ideally, each laboratory should establish a
databank of reference values that take
account of the variables mentioned earlier, so
that an individual's result can be expressed
and interpreted relative to a comparable
apparently normal population, insofar as
normal can be defined.
37. Find out the hematological reference ranges
used at KCMC CLINICAL LABORATORY
There is considerable variation in the red
blood cell count (RBC) and haemoglobin
concentration (Hb) at different periods of life
At birth the haemoglobin is higher than at
any period subsequently
After the immediate postnatal period, the Hb
,PCV and RBC falls fairly steeply to a
minimum by about the second month
39. In normal pregnancy, there is an increase in
However, at the same time, an increase in
plasma volume occurs, and this results in a
progressive decrease in haemoglobin, PCV,
The level returns to normal about a week
40. In healthy men and women, haemoglobin,
RBC, PCV and related parameters remain
remarkably constant until the sixth decade.
Aging is, however, a gradual process, the
start of which is arbitrary. In many studies it
is assumed to be 65 years, but anaemia
becomes more common in those older than
This is less marked in women than in men, so
that a difference of 20 g/l in younger age
groups is reduced to 10 g/l or less in old age
It is not clear whether light exercise increases
the RBC or haemoglobin significantly above
the baseline observed with the subject at rest;
the effects may be small enough to be
submerged in the technical errors of
However long-distance runners may develop
so-called “sports anaemia” with a slightly
lower haemoglobin and RBC, thought to be
the result of increased plasma volume.
42. Conversely, in sprinters who require a short
burst of very strenuous muscular activity, the
RBC increases by 0.5 × 1012/l and haemoglobin
by 15 g/l, largely because of reduction in
plasma volume and to a lesser extent to the re-
entry into the circulation of cells previously
sequestered in the spleen.
This is a transient event that occurs in athletes
immediately after a race, whereas at all other
times there are no significant differences in
haemoglobin and PCV between these athletes
43. There is a small but significant alteration in the
plasma volume with an increase in
haemoglobin and PCV as the posture changes
from lying to sitting, especially in women;
Conversely, change from walking about to
lying down results in a 5–10% decrease in the
Hb and PCV.
Thus, subjects should rest for 5–10 min before
their blood is collected. The difference in
position of the arm during venous sampling,
whether dependent or held at atrial level, can
also affect the PCV.
44. Changes in Hb and RBC during the course of
the day are usually slight, about 3%, with
negligible changes in the MCV and MCH.
However, variation of 20% occurs with
Serum erythropoietin has a marked diurnal
variation, being lowest at 8 a.m., with
increases by 40% at 4 p.m. and 60% at 8 p.
45. The effect of altitude is to increase the Hb and PCV
and increase the number of circulating red cells
with a lower MCV.
The magnitude of the polycythaemia depends on
the degree of hypoxaemia.
At an altitude of 2000 metres (c 6500 ft),
haemoglobin is 8–10 g/dl and PCV is 0.025 higher
than at sea level; at 3000 metres (c 10,000 ft),
haemoglobin is 20 g/dl and PCV is 0.060 higher,
and at 4000 metres (c 13,000 feet) haemoglobin is
35 g/dl and PCV is 0.110 higher. Corresponding
increases occur at intermediate and at higher
47. At birth, the total leucocyte count is high;
then falls over the next few weeks, and then to
a level at which the count remains steady.
The lymphocytes decrease during the first 3
days of life often to a low level and then rise up
to the 10th day.
after this time, they are the predominant cell
(up to about 60%) until the 5th to 7th year
when they give way to the neutrophils.
48. A moderate leucocytosis of up to 15 × 109/l
is common during pregnancy, owing to a
neutrophilia, with the peak in the second
49. There is a slight diurnal variation of about 5%;
this occurs during the course of a day as well
as from day to day.
Within the wide normal reference range, there
are some ethnic differences, and in healthy
West Indians and Africans platelet counts may
on average be 10–20% lower than those in
Europeans living in the same environment
50. A decrease in the platelet count may occur in
women at about the time of menstruation
There are no obvious age differences;
however, in the first year after birth the
platelet count tends to be at the higher level
of the adult normal reference range
Strenuous exercise causes a 30–40% increase
in platelet count....