This chapter discusses specimen collection, processing, and preservation for biochemical analysis. It describes the importance of proper collection and labeling of specimens and lists common specimen types including blood, urine, and body fluids. The chapter outlines factors that affect blood composition and details methods for blood collection as well as the use of anticoagulants and tubes with different colored stoppers. It also discusses the preservation of specimens through refrigeration, freezing, and use of chemical preservatives to inhibit reactions prior to analysis. Finally, the chapter reviews preanalytical factors that can influence test results such as collection procedures, sample handling and processing, and the presence of hemolysis.

1. Chapter 4
Specimen collection, processing, &
preservation
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2. Objectives
At the end of the chapter, the student will be able to
• Explain about different kind of specimens
• Discuss about factors affect blood composition
• Discuss about preservation, storage, and
transportation of specimens.
• Discuss different preanalytical factors affecting test
results
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3. Specimen for Biochemical analysis
• Proper collection, preparation, processing and
preservation of the specimen is essential for obtaining
best results.
• A properly filled test requisition slip should
accompany each specimen.
• Specimen must be labeled at least with the patient’s
name, hospital or identification number , ward, and
date and time of collection.
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4. Specimen types
• Biological specimens include:
– Whole blood, serum, plasma, urine, saliva, body
fluids (spinal, synovial, amniotic, pleural,
pericardial, ascetic fluid), and various types of
solid tissue, including specific cell types
• Majority of clinical chemistry tests are done on
blood : serum , whole blood, or plasma.
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5. Specimen types
• Random specimen
– collected any time
• Fasting specimen
– collected after no food has been eaten for
more than 6 hours and usually in the morning
• timed specimens
– collected at a particular time after an event
such as eating or taking a medication.
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6. Blood
• Blood for analysis may be obtained from veins,
arteries or capillaries.
• Venous blood is usually the specimen of choice and
venipuncture is the method for obtaining this
specimen
• Skin puncture is frequently used to obtain what is
mostly capillary blood
• Arterial puncture is used mainly for blood gas
analysis
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7. Venipuncture
• Venous blood is usually collected with
evacuated blood tubes
– Evacuated blood tubes are less expensive, more
convenient
– Different types: vary by the type of additive
present
– The color of the stopper used identifies the
additive present
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8. Coding of stopper color
COLOR Use Additive
Gray plasma or whole oxalate(Na,K)
blood with glycolysis fluoride(Na),
inhibition Iodoacetate(Na)
Yellow Sterile interior of tube None .
Green plasma or whole blood Heparin (Na,
Li, NH4
+ )
Red Serum None .
Blue plasma or whole blood citrate (Na) .
Lavender plasma or whole blood EDTA (Na2, K2)
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9. Colors of Tubes
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10. Serum and plasma
• Serum
– Whole blood is allowed to clot and centrifuged to
obtain the serum
– The most commonly used specimen in clinical
chemistry
• Plasma
– Clotting is preserved by allowing the whole blood to
mix an anticoagulant and centrifuged to obtain the
plasma
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11. Urine
• Urine is frequently requested specimen for assessment of
renal function and metabolic conditions
• The required type of urine depends upon the analyte to
be measured
– Eg, a timed 24 hout urine is required for clearance test
• If analysis is to be delayed, the urine specimen can be
retained at 20c or 40c for up to a week or at -20 oC if
longer delay is needed
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12. Other body fluids
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13. Anticoagulants for blood
• Anticoagulant: chemical substances added to
fresh blood to prevent clotting
• Used when whole blood or plasma is desired
for testing
• The right type of anticoagulant in correct
amounts should be used
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14. Anticoagulants for blood cont’d
• Precautions in the selection and use of anticoagulants:
– It should neither remove nor add the substance to
be measured;
• Eg: sodium salts of anticoagulants for sodium
determination, EDTA for calcium determination.
– Use neither excess nor less amount of an
anticoagulant.
– It should not be an enzyme inhibitor if sample is to
be used for enzyme assays.
• Eg: Sodium fluoride and chelating anticoagulants are
enzyme poisons
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15. Common Anticoagulants
• Heparin
• Sodium fluoride
• EDTA
• Oxalates
• Iodoacetate
• Citrate, Na
• What color coding corresponds with each of these?
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16. Heparin
• Available as Na, K, Li, and NH4
+ salts.
• Acts as antithrombin, preventing the transformation
of prothrombin into thrombin and thus the formation
of fibrin into fibrinogen.
• Most widely used anticoagulant for chemical
analyses.
• It causes the least interference with tests.
• Disadvantage: high cost and temporary action.
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17. Sodium fluoride
• Is usually considered as a preservative for blood
glucose, it also acts as a weak anticoagulant.
• Exerts preservative action by inhibiting
glycolytic enzymes
• Potent inhibitor to urease as well.
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18. Oxalates, Na, K, Li, NH+
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• Inhibit blood coagulation by forming insoluble
complexes with calcium ions.
• Used for glucose assay along with fluoride- as
fluoride-oxalate.
• Oxalate inhibits most enzymes- ALP, AMS, LDH,
CK and may also cause precipitation of calcium.
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19. Iodoacetate
• Sodium iodoacetate at a concentration of 2g/L is an
effective antiglycolytic agent and a substitute for
NaF.
• Since it has no effect on urease, it can be used when
glucose and urea are done on a single specimen.
• It inhibits CK but appears to have no significant
effect on other clinical chemistry tests.
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20. EDTA- Ethylene Diamine Tetra acetic Acid
• Prevent clotting by binding calcium, essential for
clotting mechanism.
• Available in disodium or dipotassium salt
• useful for hematologic exam as it preserves the
cellular components
• Not used in most of clinical chemistry tests
– Unsuitable for Ca, Fe, as it chelates them as well
– Inhibits ALP, CK activities by chelating of metallic
cofactors.
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21. Citrate, Na
• Acts by chelating calcium.
• Widely used for coagulation studies
• Little application in clinical chemistry
• Unsuitable for Ca, aminotransferase, and ALP
assays.
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22. Preservative methods
• Some of the changes which may occur in blood specimens
within a few hours of being collected include:
– Diffusion of potassium and some enzymes through the cell
membrane into serum or plasma
– Decrease in the conc. of glucose by glycolysis (at a rate of
about 10% hour)
– Reduction or loss in activity of certain enzymes
– Decomposition of bilirubin in light
– Decrease in volume by evaporation
– Diffuse of carbon dioxide off the surface of the blood
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23. Preservation methods
• BEST OPTION : Test FRESH Specimen as soon as
possible after collection and processing
• If analysis is to be delayed or the specimen has to be
transported:
– Separate the plasma or serum from the red cells as
soon as possible
– Refrigerate whole blood after the serum or plasma is
separated.
– Use antiglycolytic agents to prevent glycolysis
• fluoride-oxalate, sodium fluoride, or lithium
iodoacetate
– Protect specimen from light.
– Stopper tubes
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24. Preservation methods
• Physical means of preservation
A. Lowering the temperature
– Refrigeration(4-80C)
• Glucose is stable for 30 min at 200C otherwise it may be
consumed by bacteria, taking up by cells, converted to
others compounds
– Freezing
• Suitable for serum or plasma but not for whole blood
because RBCs may ruptures
B. Light protection
– Protect the specimen from sun light & excessive heat e.g.
bilurubin is light sensitive
C. Use of stoppers for tubes
– Used to prevent evaporation
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25. Preservation methods cont’d
• Chemical means of preservation
– Role of chemical preservatives
• In inhibition of bacterial action
–E.g.. Glucose----- consumed by bacteria
–urea ------ bacteria convert urea to
ammonia
• Inhibition of enzyme activity
• Inhibition of unwanted chemical reaction
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26. Pre analytical factors affecting test results
• The preanalytical phase of the testing process is
complex
• Many processes are involved in this phase:
– Request, sampling, transportation of sample, centrifugation,
sample preparation and storage in the laboratory
• Pre-analytical variables fall under two categories;
– controllable and non controllable
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27. Factors affecting test results cont’d
• Controllable preanalytical variables: have short-lived
effects and include:
– variables that are related to specimen collection
– physiological variables:
• posture, prolonged bed rest, exercise, physical
training, circadian variation, Pregnancy and
menstrual cycle
– those associated with diet, lifestyle, drugs smoking
• Non-controllable preanalytical variables
– Biological variables: Age , sex , race
– Environmental variables :Altitude, temperature,
geographical location and seasonal influence
– those related to underlying medical conditions 27

28. Factors affecting test results cont’d
• Specimen related variables It include variables related
to:
– specimen type and collection
– use of anticoagulant
– specimen handling and processing: storage,
transport and preparation
– duration of tourniquet application
– Specimen contamination from Infusion
– Hemolysis
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29. Duration of Tourniquet Application
• cause short-term venous occlusion, allowing palpation
of the veins
• > 1 min-hemoconcentration occurs due to fluid
changes
– Low molecular weight compounds (K+) leave the
capillaries and pool in the interstitial fluid region
– increase in the conc of large molecules(proteins) in
the remaining plasma.
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30. Duration of…
• Venous stasis from excessive fist clenching
– promotes anaerobic glycolysis with the accumulation
of plasma lactate and a reduction in blood pH
• Reduction in blood pH
– alter drug-protein or hormone-protein binding→
increased free drug or free hormone conc
– Levels of Ca2+, Mg2+ increase since they are released
from albumin
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Duration of Tourniquet Application…

31. Specimen Contamination from Fluid Infusion
• Dextrose(sugar) IV infusion→ extremely high glucose
results
• Total parenteral nutrition infusion:
– creates gross turbidity along with elevated lipid and
glucose values
• saline IV infusion: Na+ and Cl- falsely elevated
• blood transfusion: the extent of hemolysis may increase
and result in increased values for potassium, LDH
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32. Effects of Hemolysis
• disruption of the RBC membrane and release of
hemoglobin and other cellular components
• It is graded as mild, moderate, or gross hemolysis
based on visible presence of hemoglobin
• Serum shows visual evidence of hemolysis when
hemoglobin conc exceeds 20 mg/dl.
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33. Effect of hemolysis
• Caused by a variety of conditions during the
collection and processing steps
– poor placement of the needle into the vein, pulling
back the plunger on a syringe too quickly, and
allowing air leakage due to a poorly fitted needle
– Prolonged tourniquet application with leak of
interstitial fluid into tissue, Tissue trauma during
collection,
– Using a small-bore needle when compared to the size
of the evacuated tube or forcing blood through a
stopper into the evacuated tube
– Exposure of cells to excessive heat during storage or
transportation
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34. Effect of hemolysis…
• Hemolysis can be avoided by:
– Checking that the syringe and needle are dry and
that the barrel and plunger of the syringe fit well.
– Not withdrawing the blood too rapidly or moving
the needle once it is in the vein.
– Removing the needle from the syringe before
dispensing the blood into the specimen container.
– Allow the blood to run gently down the inside wall
of the container.
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35. • Effect of menstruation
– the reference intervals for estradiol, FSH, LH and
progesterone are influenced by the stages of menstrual
cycle
• Pregnancy
– A dilutional effect is observed due to an increase in the
mean plasma volume, which causes hemodilution
– heat-stable ALP increased (placental origin)
– Increased levels of hormones, such as estrogen,
progesterone, and human placental lactogen
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36. Circadian variation
• Responsible for the diurnal changes seen in the
circulating levels of some analytes
• Example:
– Cortisol: peaks at around 6:00 AM, with levels becoming
lower toward the evening and midnight
– Thyrotropin: peak during the late evening hours, with
the lowest values observed around midday
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37. Summary
• Proper collection, preparation, processing and
preservation of the specimen is essential for
obtaining best results.
• Specimen preservation techniques in clinical
chemistry includes, adding antiglycolysis chemicals,
refrigeration and prevent the specimen from direct
sun light.
• The types of anticoagulants used during specimen
collection for clinical chemistry depends on the
specific analyte required. How ever serum sample is
recommended in most analytes measurements
• Pre-analytical variables fall under two categories:
controllable and non-controllable
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38. Thank you !
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