Clinical Biochemistry

2. CLINICAL BIOCHEMISTRY
Dr. Mohammad Shiblee Zaman
Lecturer (Biochemistry)
Dhaka Medical College

3. Laboratory Hazards

4. Laboratory hazards
Any object or material in a laboratory that can
cause injury to human or harm the environment is
called hazard

5. Types of laboratory hazards
1. Biohazard eg. by infectious agents
2. Chemical hazard eg. by hazardous
chemicals
3. Physical hazard eg. by breaking of glass
wares
4. Electrical hazard eg. by electrical apparatus
5. Fire hazard eg. by gas/volatile substances
6. Radiation hazard eg. by UVR

6. Biohazard
The infectious materials or agents that present
a potential risk to the human health in the
laboratory

7. Source of infection
 Blood or blood products like serum or
plasma(mainly)
 Urine
 Body fluids eg. pleural/ pericardial/ synovial
fluid/ CSF etc.

8. Major infectious agents
1. HIV
2. Hepatitis B
3. Gonococcus
4. Treponema pallidum

9. Mode of infection
1. Aspiration
2. Inhalation
3. Inoculation
4. Contamination

10. Route of transmission
 Percutaneous: through accidental needle
prick, transfusion of infected blood.
 Non-intact skin: through minute cut or scratch
by contaminated glass ware
 Mucous membrane: through mouth pipetting,
splashing etc.

11. Prevention: 7 rules of biosafety
1. Avoidance of mouth pipetting
2. Treating all fluid as infectious
3. Restricted use of needle syringe
4. Wearing of personal protective
device eg. apron, eye shield, face
mask, hand gloves
5. Frequent hand washing
6. Before & after work,
decontamination of working
surface
7. Prohibition of eating or drinking
in the laboratory

12. Chemical hazard

13. Types of hazardous chemicals
1. Flammable: acetone, ethanol, toluene
2. Corrosive: HCl, H2SO4, HNO3, NaOH, KOH
3. Toxic: KCN, CCl4, barbiturate, chloroform
4. Oxidizing: chlorate, perchlorate, peroxides
5. Explosives: picric acid
6. Carcinogens: benzidine, nitrosamine

14. Chemical hazard occurs by
a) Direct contact with skin
b) Accidental swallowing during
mouth pipetting
c) Inhalation of vapour
d) Toxic effects of substance by
absorption from alimentary
tract, lungs, skin etc.

15. Prevention
1. Proper & complete labeling of all
chemicals
2. Always keeping chemicals below eye
level
3. Chemicals & reagents should be
returned to their storage site after use
4. Bottles of volatile substances should
not be kept open for extended period

16. Preventioncontd.
5. Toxic chemicals should store in a locked
cup board
6. Avoidance of mouth pipetting
7. Wearing of PPE
8. Frequent hand washing
9. Proper disposal of chemical waste

17. Physical hazard

18. Physical hazard may arise from
Broken glass ware, test tube.....cut injury
Sharp equipments eg. needle, syringe, scalpel,
blade etc.....cut injury
Flammable material e.g. Bunsen burner, boiled
water.....burn

19. Prevention
1. Use appropriate plastic containers for soaking
and decontaminating used glassware
2. Before reuse, inspect glassware for cracks,
broken and chipped ends
3. Discard broken glass in a separate puncture
resistant waste bin marked ‘Sharps’ and
dispose of the contents safely. Do not allow the
bin to overflow

20. Prevention
4. Never centrifuge cracked tubes or bottles
5. Wear protective gloves when cleaning
glassware
6. Store glassware safely
7. To avoid spillages and breakages, use racks or
trays to hold specimen containers and other
bottles

21. Electrical hazard

22. Sources
 Electrical equipments
 Electric shock

23. Prevention
1. Safe positioning and installation of equipment;
do not place electrical equipment near to water,
in direct sunlight or close to where chemicals
and reagents are used or stored
2. Make sure ventilation is adequate when
charging acid rechargeable batteries
3. Grounding of electrical equipments is essential

24. Prevention
4. Circuit breakers and earth-fault interrupters
should be fitted to all laboratory circuits.
[Circuit-breakers protect wiring from being overloaded with electric
current. Earth-fault interrupters protect people from electric shock]
4. Wires & switches should be well insulated
5. Use the equipment correctly

25. Fire hazard

26. Fire hazard
Results from gas or volatile
substances, flammable liquids
etc.

27. Prevention
1. Every laboratory should have
fire fighting equipment:
buckets of water, sand
buckets, fire blanket, dry
powder chemical fire
extinguisher and their use
must be understood
2. Avoid smoking in the
laboratory

28. Prevention
3. Laboratory should be well-
ventilated
4. Flammable liquid should be
heated in H2O bath
5. Everyone should know the
correct use of fire alarm

29. Radiation hazard
Sources are –
 Ionizing radiation e.g. X-ray, CT scan
 nonionizing radiation e.g. by UVR, visible
light, infrared & microwave
 Radioactive isotopes

30. Prevention
1. Staff exposed to such hazards
should be specially trained & will
require regular monitoring of the
degree of radiation received
2. Direct rays from source should
be properly shielded
3. Radioactive waste must be
disposed properly

31. Sample and its related issues

32.  Specimen.....material available for analysis
eg. blood, urine etc.
 Sample.....part of the specimen used for analysis
eg. plasma, serum etc.
 Analyte.....substance to be measured in sample
eg. glucose, urea etc.

33. Types of specimen
 Blood, plasma or serum
 Urine
 Stool
 Aspirates eg. CSF,
pleural, pericardial,
ascitic, synovial fluid
 Sputum
 Saliva
 Tissue and cells
 Calculus (stone)
 Hair and nail

34. Collection of blood

35. Blood specimen
Site selection
Vein
Antecubital vein
Any prominent vein
Artery
Femoral
Radial
Brachial
Capillary
Finger tip (middle & index finger)
Ear lobe
Heel (in infant)

36. Venous blood
 Collection of blood from vein.....venepuncture
 Person who collects blood.....phlebotomist

37. Vein is the commonest site as
 Veins are superficial
 Pressure of blood is low in vein
 Wider lumen containing more blood
 Wall is thin

38. Procedure of venous blood collection
1. A clean, dry test tube is taken
2. ID number is given
3. Application of tourniquet 4-6″ above the
puncture site
4. Cleaning of site with proper antiseptic
5. Venepuncture at 15⁰ angle is done by
disposable syringe

39. Procedure of venous blood collection
6. When needle enters into vein tourniquet must
be loosen
7. Blood is drawn slowly into the syringe to avoid
hemolysis
8. Needle is withdrawn and pressing the puncture
site with cotton for few min followed by
application of first aid band

40. Procedure of venous blood collection
9. Now needle is removed from syringe and blood
is poured slowly along the side of test tube
10.Sealing the tube with cap properly
11.Gently mixing of blood with anticoagulant, by
inverting in either direction few times, if
necessary

41. Precautions to prevent hemolysis
1. Disposable syringe, needle and test tube must
be dry and clean
2. Needle should be wide bored
3. Tourniquet should not be applied tightly
4. Blood is drawn slowly and steadily into syringe

42. Precautions to prevent hemolysis
5. Needle is removed from syringe and blood is
poured slowly along the side of test tube
6. Gently mixing of blood with anticoagulant by
inverting few times on either side, if needed.
Avoid vigorous shaking
7. Separation of serum/plasma by centrifugation
at low to moderate speed (2000-3000 rpm) for
5min

43. Changes in blood that occurs in long
standing with measures to prevent
Changes Prevention
Loss of CO2 since pCO2 is higher
in blood than in air leading to
diffusion to air)
Blood is drawn in heparinized
syringe
Wrapped with ice bag
Needle is sealed till analysis
Test is done within 15min
↓ blood glucose due to glycolysis Using NaF
Formation of NH3
+ from urea by
bacterial urease if bacterial
contamination
Blood should be chilled immediately
after collecting with sterile precautions

44. Changes in blood that occurs in long
standing with measures to prevent
Changes Prevention
Conversion of pyruvate to lactate Blood is mixed with protein
precipitate
↑ plasma inorganic PO4
- due to
hydrolysis of organic PO4
- in RBC
Serum/Plasma separated shortly
after collection
Passage of substances through
RBC membrane e.g. K+, LDH, AST
serum or anticoagulant mixed
plasma should be separated
shortly after collection
If stored, at 4°C.

45. Changes in blood that occurs in long
standing with measures to prevent
Changes Prevention
UV radiation or daylight exposure
destroys bilirubin
Keeping sample in dark or by
wrapping the tube with foil paper
Many hormones particularly
peptides are affected by proteases
in blood and become unstable

46. So, Net result is
↓ blood glucose, urea, CO2, pyruvate,
HCO3
-
↑ K+, PO4
-, Cl-, LDH, AST.

47. Anticoagulant
 Chemical substances
 Prevents formation of clot
 Needed when whole blood or plasma is required
 Not used in serum as it has NO CLOTTING
FACTOR

48. Commonly used anticoagulants
 Heparin
 EDTA
 Sodium fluoride (NaF)
 3.8% sodium citrate
 Sodium or potassium oxalate
 Sodium iodoacetate
 Paul Haler’s Mixture (K-oxalate:NH4-oxalate = 2:3)
 ACD (Acid Citrate & Dextrose)

49. How they act?
• Mostly act by chelating Ca2
+, thus making it
unavailable for blood clotting
• Heparin forms complex with antithrombin III,
thus inhibits thrombin
• NaF inhibits enolase, thereby inhibits glycolysis

51. STOP
LIGHT
RED
GREEN
LIGHT
GO

52. Collection of urine

53. Type of urine specimen
 Random...anytime
eg. spot urinary ACR
 Timed...at particular time
eg. during OGTT
 24 hrs...whole urine of last 24 hour (8am – 8am
next morning)
eg. 24hrs UTP, 24hrs urinary electrolyte, CCR

54. 24hrs urine collection
 The bladder should be emptied at the beginning
of collection and urine discarded (If starting time
is 8 am; voided urine of 8 am has to be
discarded)
 Fecal contamination should be avoided. Urine
should be collected before the act of defecation

55. 24hrs urine collection contd.
 Voided urine should be collected in a
separate container and then added to the
main container containing acid
preservatives to avoid splashing
 Starting time has to be noted in the main
container and stored at ~4°C
 All subsequent voiding of 24hrs is
collected including that of 8 am next
morning. No portion should be discarded

56. Changes in urine on long time storage
 Bacterial contamination
 Chemical decomposition of analyte
Bacterial fermentation of glucose
Conversion of urea to NH4
+
 Precipitation of analytes –
PO4
- precipitates
 Oxidation of unstable components –
Urobilinogen is oxidized to urobillin

57. Preservatives used
 Toluene
 Formalin
 HCl
 Boric acid
 Acetic acid

58. Accuracy
It is the degree of closeness
of a measured value to the
actual (or, true) value

59. Precision
 It means reproducibility
 A method is said reproducible when it produces
same test result on same specimen when
repeated on different days by different
technicians using different reagents
 Or, agreement between the replicate
measurements on the same or identical sample

61. Specificity
Ability of an analytical
method to measure
only that analyte
which is targeted to
be measured

62. Sensitivity
Ability of an analytical
method to measure
the smallest amount
of an analyte in a
sample

64. Control
Materials of known concentration that is used for
quality control purpose

65. Calibration material/Calibrator
 Material of known concentration that is used to
standardize an analytical method
 Or, a solution of known qualitative and
quantitative characteristics (concentration,
intensity and reactivity) used to calibrate a test
specimen or sample or an analytical technique

66. Standard
Materials of known
concentration with which
sample is compared to
determine the result

67. Blank
Solution consisting of all
components of a reaction
except analyte
Use:
1. To compensate any non-
specific color
2. To set the instrument at
100% T and ‘0’ (zero) OD