Biochemistry lab 1

Specimen Collection
& Processing
By Hussein Yusuf

Physicians use lab. tests to:

1- Diagnose disease.
2- Monitor its progress or its response to treatment.
3- Screen for the disease in seemingly healthy individuals.
Many factors besides disease affect the composition of body fluids; these
factors may be either pre-analytical or analytical. The variability of test
results due to biological factors (pre-analytical) is often greater than the

.
variability due to analytical factors
Specimen Collection: Specimen containers must be labelled with the
patient’s name, hospital or identification number, location in the hospital,
date & time of collection. Before collecting any blood specimen, a
phlebotomist should put on disposable latex rubber gloves. If a patient is
known to have an infectious disease, the phlebotomist should wear a face
mask, glasses or goggles, & gown in addition to gloves
-Blood: Blood for analysis may be obtained from:
[Link] by venipuncture.
[Link] (arterial puncture).
[Link] (skin puncture). 1) Veins by venipuncture → specimen of
choice. * The patient should be fasting if fasting is necessary to ensure
medically useful results. * The patient should be comfortably seated or
supine for 20 min. before the specimen is drawn. * An arm with an inserted
I.V. line should be avoided, as should an arm with extensive scarring or
hematoma of the intended collection site. If a women has had a
mastectomy, arm veins on that side of the body should not be used
because the surgery may have caused lymphostasis, affecting the blood
composition .The median cubital vein is the preferred site for collecting v.
blood in adults because the v. is both large & close to the surface of the
skin.
* An arm containing a cannula or AV fistula should not be used without
consent of the pt’s physician.
* Specimens obtained from the opposite are or below the infusion site in
the same arm may be satisfactory for most tests except for those analytes
that are contained in the infused solutions ( e.g. glucose or electrolytes ).
* Cleaning of the puncture site should be done with a circular motion & from
the site outward with 70% isopropanol. The skin should be allowed to dry in
the air, traces of alcohol may cause hemolysis & invalidate test results.
* A tourniquet is applied ( 10 – 15 ) cm above the intended puncture site &
should not be left in position for more than 1 min. , the pt should not be
allowed to pump her or his fist while the tourniquet is in place because it
causes an increase in the plasma potassium, phosphate & lactate.
Alternatively, a blood pressure cuff may be used (inflate to ~ 60 mmHg).
* The most commonly used needle sizes are gauges 19 – 22 . the larger the gauge
size , the smaller the bore.

2) Arteries (arterial puncture) → used mainly for blood gas analysis.

Preferred sites are, in order:
[Link] radial Arteries at the wrist.
[Link] brachial Arteries in the elbow.
[Link] femoral Arteries in the groin.
In the neonate, an indwelling catheter in the umbilical Artery is best to obtain
specimens .
In the older child or adult in whom it is impossible to perform an arterial puncture,
a capillary puncture may be performed to obtain arterialized capillary blood (
acceptable values for pH , & pCO2 , but not always for pO2 ). Site: earlobe. In the
young child or infant: the heel.
No tourniquet is required for a puncture. The needle & syringe should be flushed
out with a heparin solution both to ensure adequate anti-coagulation & to eliminate
trapping of air in the needle & in the dead space of the nozzle

3) Capillaries (skin puncture)→ in young children.

If only a small volume of blood is required for the blood test ( e.g. a blood glucose
test ) , venipuncture is unnecessary .However , a skin puncture is time consuming ,
& there is a greater risk of infection than from a venipuncture .
1- In the adult or grown child blood may be obtained by puncturing the tip of a
finger or by piercing an earlobe.
The 3rd or the 4th finger of the non writing hand ( the best is the ulnar side of the
ring finger) is quickly punctured by a sharp stab with a lancet & the depth of the
incision should be ‹ 2.5 mm to avoid contact with bone.
Massage of the finger to stimulate blood flow should be avoided, because it causes
the outflow of debris & of tissue fluid, which does not have the same composition
as plasma.

To improve circulation of the blood, the finger may be warmed by application of a

warm , wet washcloth for 3 min. before pricking .The
1ST drop of blood is wiped off & subsequent drops are transferred to the
appropriate collection tube by gentle contact.
2-In an infant younger than 1 year of age : the lateral or medial planter surface of
the foot should be used for skin puncture.

3-In older children: the planter surface of the big toe may also be used .
Anticoagulants & Preservatives for Blood
If whole blood or plasma is desired for testing, an anticoagulant must be added to
the specimen during the collection procedure. Whole blood is rarely required for
clinical tests; indeed , only for blood gas, ammonia, & some trace elements
determinations is it the preferred specimen, although it may also be used for
glucose, urea nitrogen, lactate determination & for all tests in certain bedside or
physician office analyzers. Serum from coagulated blood is the specimen of choice
for many assay systems , but plasma obtained with an appropriate anticoagulant
may be an equally valid specimen, & in certain circumstances preferred to serum

Plasma use expedites analysis in medical emergencies because harvest of serum

requires 15 – 30 min. wait for completion of coagulation before centrifugation.
Furthermore, plasma yield from a given volume of whole blood is always greater
than the yield of serum. A disadvantage is the formation of fibrin clots or fragments
when plasma is stored & the subsequent risk of clogging sample probes of
automated analytical instruments
Anti-coagulant
1. Heparin:
Is the most widely used anticoagulant for clinical chemical analysis. It causes the
least interference with tests. It is a mucoitin polysulfuric acid & is available as
sodium, potassium, lithium, & ammonium salts.
It accelerates the action of antithrombin III , which neutralizes

thrombin & thus prevents the formation of fibrin from fibrinogen .

2. Ethylenediaminetetraacetic acid (EDTA):

This chelating agent is particularly useful for haematology. exam . because it
preserves the cellular compounds of blood. It is used as the disodium, dipotassium,
& tripotassium salts , the last 2 being more soluble .
EDTA prevents coagulation by binding calcium, which is essential for the clotting
mechanism.
Its chelation of calcium makes it unsuitable for specimen for calcium & iron
analysis .

3. Sodium fluoride:
It is usually considered as a preservative for blood glucose, however, it also acts as
a weak anticoagulant ( binds calcium but after several hours ), therefore, it is used
together with another anticoagulant such as potassium oxalate. Fluoride ions
prevent glycolysis by inhibiting enolase, an enzyme that requires magnesium

4. Citrate:
Sodium citrate solution is widely used for coagulation studies because the effect is
easily reversible by addition of calcium. It has little applications in the clinical lab.
Because citrate chelates calcium, it is unsuitable for specimens for measurement of
this element.
5. Oxalates:
Sodium, potassium, ammonium, & lithium oxalates inhibit blood coagulation by
forming rather insoluble complexes with calcium ions.
Potassium oxalate is the most widely used . At high conc., hemolysis is likely to
occur (oxalates cause shrinkage by drawing water into the plasma ).

6. Iodoacetate:
Sodium iodoacetate is an antiglycolytic agent & a substitute for sodium fluoride.
Because it has no effect on urease, it can be used when glucose & urea tests are
performed on a single specimen