Laboratory hazard

HAzard

1. 1

LABORATORY HAZARDS A ND PREACUTIONS TO TA KEN WHILE WORKING
BIOCHEMICAL LAB
Laboratory Hazards
Definition
Hazard is anything that may cause injury, harm or damage. Danger or risk while working in laboratory is
called laboratory hazards. Hazard is an agent which has the potential to cause harm to a vulnerable
target.
The laboratory environment can be a hazardous place to work. Laboratory workers are exposed to
numerous potential hazards including physical, chemical and biological or radioactive hazards.

Types of Laboratory hazard
1. Physical hazard - eg Fire, Injury, noise, radiation, high voltage apparatus, machinery with
moving parts.
2. Chemical hazard- eg corrosive, flammable, toxic, carcinogenic, poisonous, explosive
3. Biological hazard - eg pathogenic microorganisms, animals, biological tissues, blood and other
body fluids (human and animal).

Physical hazard:
Hazard occurred by physical agent like heat, electrical or sharp materials is called physical hazard.
Research facilities inherently have significant physical hazards present. Included here are electrical safety
hazards, ergonomic hazards associated with manual material handling and equipment use, handling
sharps, and basic housekeeping issues.
Sharps containers are ubiquitous in labs and following a few safety rules can help prevent getting stuck
with accident reports. Use only puncture-proof and leakproof containers that are clearly labeled. Train
employees never to remove the covers or attempt to transfer the contents. Make sure these containers are
only used for “sharps” and that they get replaced when three-fourths full to prevent overfilling.

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Many injuries stem from poor housekeeping. Slips, trips, and falls are very common but easily avoided.
Start with safe and organized storage areas. Material storage should not create hazards. Bags, containers,
bundles, etc., stored in tiers should be stacked, blocked, interlocked, and limited in height so that they are
stable and secure against sliding or collapse. Keep storage areas free from an accumulation of materials
that could cause tripping, fire, explosion, or pest harborage.
Precautions
1. Always wear gloves, apron, and mask while working in lab.
2. Do not use cracked or broken glasswares.
3. Use tongs and/or protective gloves to handle hot objects.
4. Never reach across an open flame or burner.
5. When heating a test tube, move it around slowly over the flame to distribute the heat evenly.
6. When lighting a burner, wait until the striker is in place before you turn on the gas.
7. Lay electrical cords where no one can trip on them or get caught in them.
8. Be sure your hands and your lab area are dry before using electrical equipment.
9. Unplug cords by pulling the plug and not the cord.
10. Unplug all electrical equipment at the end of the lab period.
11. Keep fire extinguisher in biochemical lab.

Biological hazard:
Hazard occurred by biological agents like blood, other body fluids, experimental animals etc is called
biological hazard.
Biological hazards include potential exposures to allergens, infectious zoonotics (animal diseases
transmissible to humans), and experimental agents such as viral vectors. Allergens, ubiquitous in animal
research facilities, are one of the most important health hazards, yet they are frequently overlooked.
Biological hazards encompass microbes, recombinant organisms, and viral vectors. They also include
biological agents introduced into experimental animals. Health and safety issues such as containment,
ability for replication, and potential biological effect are all important. When working with biological
hazards, ensure that procedures can be conducted safely.

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Precautions
1. Always wear gloves, mask and apron while working in lab.
2. Use sodium hypochlorite as a disinfectant.
3. Properly dispose blood sample.
4. Properly dispose blood collected syringe.
5. Handle properly experimental animals.

Chemical hazard:
Hazard occurred by different chemicals is called chemical hazard.
Cleaning agents and disinfectants, drugs, anesthetic gases, solvents, paints, and compressed gases are
examples of chemical hazards. Potential exposures to chemical hazards can occur both during use and
with poor storage.
The use of chemicals in research laboratories is inevitable, and the potential for harm or injury could be
significant if they are misused or mishandled.
Precautions
1. Always wear gloves, mask and apron while working in lab.
2. Wear protective goggles and a lab apron whenever heating or pouring hazardous chemicals.
3. Never taste any chemicals (you should never taste anything in the lab).
4. If you need to smell the odor of a chemical, waft the fumes toward your nose with one hand. Do
not put your nose over the container and inhale the fumes.
5. Never pour water into a concentrated acid. Acid should be poured slowly into water.
6. Follow the instructions of your teacher when disposing of all chemicals.
7. Wash your hands after handling hazardous chemicals.

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SAFETY (PRECAUTIONS) IN THE BIOCHEMICAL LABORATORY

Laboratory safety rules and safe work practices or standard operating procedures (SOPs) should be
established by lab to meet specific operational needs and to reduce the risks associated with laboratory
hazards.
As a condition of entry to a laboratory, all individuals must complete a laboratory safety induction and
receive specific training in local safety rules and laboratory procedures relating to their work (including
relevant SOPs).
General Safety Rules
1. Wear safety goggles to protect eyes from chemicals, heated materials, or things that might be able
to shatter.
2. Listen to or read instructions carefully before attempting to do anything
3. Notify your teacher if any spills or accidents occur.
4. Use apron or gown while entering in Lab
5. Wear shoes that cover your feet
6. Always wear gloves while working in lab.
7. Always wear mask while working in lab.
8. After handling chemicals, always wash your hands with soap and water.
9. During lab work, keep your hands away from your face.
10. Tie back long hair.
11. Work areas/surfaces must be disinfected before and after use.
12. Label all materials with name, date, and any other applicable information.
13. Dispose of wastes in their proper containers.
14. Do not pour chemicals down the sink.
15. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes containing
infectious materials.
16. Know the location of the fire extinguisher, fire blanket, eyewash station, and first aid kit.
17. Keep your work area uncluttered. Take to the lab station only what is necessary.
18. No food or drinks are permitted in the laboratory at any time.
19. Never put anything into your mouth during a biochemical lab experiment.
20. Clean up your lab area at the conclusion of the laboratory period.
21. Never “horse around” or play practical jokes in the laboratory.

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INBORN ERROR OF META BOLISM


Inborn error: An inherited (i.e. genetic) disorder.

Metabolism: Chemical or physical changes undergone by substances in a biological system.

Inborn errors of metabolism, which are due to impaired activity of enzymes, transporters or co-factors,
results in accumulation of abnormal metabolites (substrate) proximal to the metabolic block or lack of
necessary products.



In fig the substrate A is converted by a series of reaction into product C. if one of enzymes is defective
(metabolic block), the substrate of reaction will be accumulate (A in this case) and can enter alternative
pathways of metabolic leading to the formation of byproduct (D in this case). At the same time, the
concentration of the product of the reaction (C) will decreased.
In born error of metabolic disease are,

Abnormalities of metabolism of carbohydrates
1. Galactosemia
2. Fructosaemia
3. Pyruvatekinase (PK) deficiency:
4. Glycogen storage disease

Abnormalities of metabolism of Protein
1. Phenylketonuria
2. Alkaptonuria

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3. Tyrosinaemia type-I
4. Tyrosinaemia type-II
5. Neonatal tyrosinaemia
6. Hereditary tyrosinaemia
7. Maple syrup urine disease
8. Albinism
Abnormalities of metabolism of Lipid
1. Lysomal storage diseases


GALACTOSEMIA
It is an inborn error of metabolism. The incidence is 1 in 35,000 births. Galactosemia is inherited
disorders in which there is not able to convert galactose to glucose in normal manner.

There is deficiency of enzyme galactose-1- phosphate uridyl transferase. Due to the block in this
enzyme, galactose-1- phosphate will accumulate in liver.

There is enlargement of liver, jaundice and severe mental retardation. Free galactose accumulates, leading
to galactosemia. It is partly excreted in urine (galactosuria)

Galactose is reduced to dulcitol. The accumulation of dulcitol in the lens results in cataract due to its
osmotic effect. This is called congenital cataract and is a very characteristic feature of galactosemia.
Galactose-1-phosphate may get deposited in renal tubules, producing tubular damage leading to
generalized amino aciduria.

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Diagnosis:
Clinical manifestation including congenital cataract and presence of galactose in urine as well as
elevated blood galactose levels will help in the diagnosis. Collection of fetal cells by amniocentesis may
be useful in prenatal diagnosis. Heterozygous parents could be detected by elevated galactose level in
blood after a galactose load.



FRUCTOSAEMIA
It is an inherited disorders manifesting with serve clinical features.

Enzyme defect: Aldolase-B deficiency

Administration of fructose in these patients leads to:
1. Excessive and prolonged rise of fructose increased and fructose 1 phosphate increased in blood.
2. Blood glucose decreased (hypoglycemia)

Symptoms

Nausea and vomiting (may be haemorrhagic), Profuse sweating, slight icterus, albuminuria.


Phenylketonuria (PKU)

Phenulketonuria is the most common metabolic disorders in amino acid metabolism. It is due to the
deficiency of the hepatic enzyme phenylalanine hydroxylase (which converts phenylalanine into
tyrosine). As a result, phenylalanine accumulates in tissue and blood and then excreted in urine. The
characteristics findings of this clinical condition are mental retardation, failure of growth, seizures and
tremor etc.

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Alternate pathways in phenyl ketonuria


Laboratory Diagnosis
1. Blood phenylalanine: Normal level is 1 mg/dl. In PKU, the level is >20 mg/dl. This may be
demonstrated by chromatography.
2. Guthrie test.
3. Ferric chloride test:
4. DNA probe


ALKAPTONURIA (BLACK URINE DISEASE)

It is also inherited metabolic disorders which is cause by the deficiency of homogentisate oxidase in
tyrosine metabolism. As a result, homogentisate accumulates in tissues and blood and is excreted in urine.


Diagnosis of Alkaptonuria

1. Urine becomes black on standing when it becomes alkaline. Blackening is accelerated on exposure to
sunlight and oxygen. The urine when kept in a test tube will start to blacken from the top layer.
2. Ferric chloride test will be positive for urine.
3. Benedict's test is strongly positive.

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4.

Fig: Flow chart of inborn error metabolism of protein

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QUALITY CONTROL IN C LINICAL BIOCHEMISTRY

Quality control (QC):
Quality control is the study of errors which is the responsibility of the laboratory and the steps taken to
recognize and minimize them. Quality control must be practicable, achievable and affordable.

Components of quality control

1. Internal quality control
2. External quality assessment
3. Quality management
Quality Assurance (QA)
According to WHO, Quality assurance is defined as, the total process whereby the quality of laboratory
reports can be guaranteed. It has been summarized as
 Right result,
 Right time,
 Right specimen,
 Right patient,
 Correct reference data, and
 Right price.
Quality assurance includes all those activities both inside and outside of laboratory.

QA = IQC + EQC + QM

Quality control involves consideration of a reliable analytical method.
Reliability of the selected method is determined by its accuracy, precision, specificity and sensitivity;
with major emphasis of QC being laid on monitoring the precision and accuracy of the performance of
analytical methods.

Accuracy:
It is the degree of closeness between the estimated value and the true value.

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Precision:
It is the measurement of reproducibility of the values. It may or may not be accurate.

(Reproducibility: same result obtained when the test is repeated)



High accuracy,
but low precision High precision,
but low accuracy



High precision with high accuracy



Sensitivity:
Sensitivity is the ability of an analytical method to detect small quantities of the measured analyte.

Sensitivity is a measure of the incidence of positive results in patients known to have a condition that is
True Positive (TP).

(The ability of the test to give a true positive result when certain disease is present)

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% Sensitivity =
TP
TP+FN
×100

Specificity:
Specificity is the ability of an analytical method to determine solely the analyte it is required to measure.
The ability of the test which give true negative result in a patient with no specific disease.


% Specificity =
TN
TP+FP
×100






1. Internal Quality control (IQC):

Internal quality control includes the strict measures taken every day by all staff and for all tests to make
certain that the laboratory test results are accurate.

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It is needed to ensure that test results are reliable and reproducible. It is carried out as part of the daily
work routine.

Internal QC can be maintained by the following ways:

1. Use of standardized glassware, reagents and equipment.
2. Employment of conscientious and well-trained staff.
3. Maintenances of:
a) Proper analytical skill throughout the test.
b) Required quality of regent.
c) Desired performance of the instruments.
4. Routine use of various primary standards QC sera, od previously analyzed specimen to evaluate
the obtained results.
5. Recording the temperature of the incubator or water bath and refrigerator daily.
6. Following SOPs.
7. Proper work organization.
8. Prepare Levey jenning’s chart

There are two phases in Internal Quality control:

1. Preventive phase 2. Retrospective phase

1. Preventive phase:
In this phase preventive precaution are taken at the following various stages of specimen analysis:
a. Collection of specimen
b. Separation of serum (preferably within 30 minutes of blood collection)
c. Specimen analysis
d. Photometric readings and
e. Calculation of test values etc.
2. Retrospective phase:
This phase includes the comparison between

a. Optimum condition variance (OCV) and

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b. Routine conditions variance (RCV)
c. OCV refers to the test results obtained under optimum conditions i.e. by using,
i) Freshly prepared reagents and ii) By using standardized A grade glassware.

d. RCV refers to the results obtained by using routine requirements i.e. by using routinely stored
reagents and glassware in regular use.
Note: The difference between OCV and RCV should not be more than 3%.


Advantage of internal QC:

1. Problems are identified immediately.
2. Errors are corrected immediately.
3. The quality of test result can be raised.

2. External Quality control (EQC):

It is needed to detect hidden problems and to compare the performance of one laboratory with another to
improve the quality of the results produced. Help and support is received from NPHL.

These programs are organized by national reference laboratories such as National Public Health
Laboratory (NPHL) in Kathmandu. The programme scheme is depicted below.

NPHL send control samples to laboratory along with the test request form
↓
Laboratories perform the required tests, record the result and report to NPHL
↓
NPHL gives feedback on the performance of the laboratories

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Advantages of External quality control

1. Hidden problems are identified.
2. Help and support from the NPHL are received.
3. If the laboratories test results are out of the control range. NPHL will contact us to discuss the
problem and find solutions so that the same mistaken are not made with the patient specimen.
4. Participation in EQA allows inter-laboratory comparison which result to improve quality.

3. Quality management
This includes the following additional measures to improve the quality of the test result.
1. Organization and management of laboratory.
2. Training and retraining laboratory staff
3. Maintenance SOP.
4. Buying standard instruments, reagents and other requirements.
5. Managing instrument
6. Routine supervision from higher authority.
Advantages
All the elements of QM will add up to produce a highly quality laboratory result.


Stages of QA
QA measures are taken all the time before the test, during the test and after the test.

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Pre-analytical stage
• Patients identification and preparation.
• Proper filling of the request form.
• Patient preparation.
• Specimen collection, transportation and storage.
• Checking the request form and the specimen when they are received by the laboratory (Specimen
receiving and recording in the laboratory).
Analytical stage
• Routine work organization.
• Aseptic techniques and safe handling of infectious materials.
• State of equipment
• SOP followed
• Test method used and interpretation of results.
• Disposal of specimens
• Internal Quality control procedures.
Post-analytical stage

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• Organization of recording and reporting.
• Result interpretation and reporting.

Accreditation
Accreditation is a valuable component of quality assurance in clinical microbiology
(Batstone, 1992). The purpose is simple: the audit by an external agency of an applicant’s
organizational and quality assurance programme to see that certain defined standards are met.
An accreditation
scheme should be comprehensive, covering all
aspects of the laboratory, including organization
and administration, staff development and education,
facilities and equipment, and policies and procedures.
Participation in all relevant quality
assessment schemes is a requirement for accreditation.
There are several accreditation schemes available
and before a laboratory embarks on the
process consideration should be given to the appropriateness
of the various schemes.


STANDARD DEVIATION ( SD):

A statistical value which is used to measure precision is called standard deviation or standard deviation is
deviation of standard from the mean values.

In statistical terms the distribution or scatter of values around the mean can be expressed as standard
deviation.
A range of 2 standard deviations (± 2 SD) is generally considered as the limit for a control value to be
acceptable.

A statistical value which is used to measure precision is called standard deviation or standard deviation is
deviation of standard from the mean value. It requires commercially produced or stabilized quality control
sera.

SD = √
∑(??????−??????̅)
2
�−1

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Where, ∑(??????−??????̅)
2
is the sum of the difference of each individual test from the mean, and n is the number
of observation.

n = no. of results



Coefficient of variation (CV):

The CV express standard deviation as the percentage of the mean.

CV =
????????????
�????????????�
× 100

For example, SD of 0.4 with a mean value of 25 shows the same precisions as SD of 0.8 with a mean value
of 50. Therefore,

CV=
0.4
25
× 100 = 1.6%
Or,
CV=
0.8
50
× 100 = 1.6%

The CV for several levels must be obtained for normal and abnormal ranges, and when using any CV, the
mean value should be established.


Establishing Performance Standard

Mean
Describes the midpoint of a population or in other words gives the calculated average of a set of values.
i.e.: If 10 tests have been performed, the 10 results are added and this sum is divided by 10.

No.
1..............................................107 mg%
2..............................................118 mg%

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3..............................................102 mg%
4..............................................114 mg%
5..............................................110 mg%
6..............................................108 mg%
7..............................................117 mg%
8..............................................109 mg%
9..............................................112 mg%
10............................................113 mg%

Total 1110 mg%

1110 mg% ÷10 = 111 mg% (mean)




Levey-Jennings’s chart (Daily Control Chart)

Levey-Jennings’s chart is graph that daily control data is plotted on to give a visual indication whether a
laboratory test is working well.

One of the methods of presenting the internal QC data is plotting Levey-Jenning’s charts.

1. Single batch of control serum is analyzed for 30 consecutive days.
2. Calculate the mean and SD values.
3. Draw a horizontal line through the mean value.
4. Draw line at 1SD, 2SD and 3SD values above and below the mean line.
5. The value obtained on each day by analysis of control samples is plotted in this chart.
6. If the analysis is satisfactory, the points will be scattered evenly on either side of the mid line that
is within 1SD limits. The pattern shows that the accuracy is maintained.
7. The value filling with the 2SD limit is acceptable.
8. When six consecutive values fall above or below the mean line it shows that the assay is out of
control.

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The distance from the mean is measured in standard deviations (SD). Lines run across the graph at the
mean, as well as one, two and sometimes three standard deviations either side of the mean. This makes it
easy to see how far off the result was. Rules, such as the Westgard rules can be applied to see whether the
results from the samples when the control was done can be released, or if they need to be rerun.


Preparation of Levey Jennings chart

 Example: Glucose determination.
 Method: Glucose oxidase

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Nearest significant number = 2.0
1
st
S.D. = + 2.0
2
nd
S.D.= ± 4.0
3
rd
S.D. = ± 6.0







Accept value below +2SD or -2SD

Variance:
Variance of a distribution is defined as the square of the standard deviation.

Thus,

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Variance (V) = (standard deviation)
2




CONTROL SERA:

It is the sample that simulates the physical and chemical pattern of a clinical sample being analyzed and it
is treated just like the clinical sample. The control serum will detect error due to reagents and standards
only. Most laboratories make use of commercially prepared control sera which are fairly expensive.
Commercially prepared control sera are available in two forms, freeze dried (lyophilized) sera, and
synthetically produced liquid sera. It is very important to follow the manufactures’s instructions for the
preparation, use and calibration of control sera. It is equally important to have information on a wide
variety of control sera from different manufacturers.


Differentiate between control and standard

Control Standard or calibrator
Matrix is added (e.g. glucose, urea, creatinine,
uric acid etc.)
No matrix (e.g. only one test like glucose or
urea or creatinine)

Serum Water
QC is used to before each run to be sure that
our result is Ok or Not.
Calibrator/Standard is used to calibrate the
instrument and both of them has exact
concentration of the analyte.
Use for multiple test Single test
Expensive Cheap



Standard Operating Procedures
(SOPs)

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1. SOPs are an important part of the quality assurance programme.
2. SOPs are written instruction protocols that include all aspects of laboratory work practices.
3. SOPs help prevent mistakes rather than detecting them.

SOPs have the following features:
1. SOPs are written in accordance with a standard format
2. SOPs are written in simple language, readily understood by employees.
3. SOPs contain sufficient procedural details to enable trained staff to perform the task without
supervision
4. SOPs are written by qualified and experienced laboratory officers
5. SOPs must be followed exactly by all staff.
6. SOPs must be given a title, identification number and date.
7. SOPs are reviewed and updated on a regular basis.

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Example SOP

Blood Sugar Estimation
GOD/POD METHOD

A – 001
Page 1

Authorised signature: Issuing Date: April 12, 2014
Next Revision Date: April 12, 2016

Staff able to perform test (Responsibility): Laboratory Assistant and higher

Principle of the Test Method:

Clinical Significance of the Test:

Specimen:
Equipments Requirements:
Reagents Requirements
Test Procedure Instruction
Reporting and Interpretation of Results
Internal Quality control and Sources of Error:
- Do not use haemolysed sample.
- Follow SOP
- Include a known internal quality control serum in each batch of analysis.
- Highly lipemic serum may require sample blank to prevent errors from turbidity.

25. 25

- Pipette the reagents and samples accurately.
-
Reference
1. District Laboratory Practice in Tropical Countries. Part 1 Monica Cheesbrough. Tropical Health
Technology
2. Varley’s Practical Clinical Biochemistry 6
th
Edition. Alan H Gowenlock. Heinemann
END OF DOCUMENT


AIMS AND OBJECTIVE O F QUALITY ASSURANCE

1. To achieve accuracy and precision of laboratory test.
2. To design a system for monitoring the collection, labelled transportation, processing and storage of
specimens before the test are performed.
3. To select and evaluated the new technique and new instruments.
4. To facilitate comparison between different techniques.
5. To develop safety programmed for clinical laboratories that includes protection of lab. Staff against
health and hazards while handling and processing of specimen.

COMMON ANTICOAGULANT S AND PRESERVATIVES USED IN BIOCHEMISTRY LAB

Collection and preservation of biological fluids
The different body fluids that are used for biochemical investigations are given below:

Sample Collection
1. Mainly venous blood sample collection for biochemistry.
2. Serum is used for most biochemistry tests carried out at a hospital.
3. Do not leave tourniquet on the arm for more than 2 minutes, as this will affect the concentration
of cells and substances in the blood.
4. Do not collect the blood from an arm in which an intravenous infusion is being given.
5. Remove the needle from the syringe before transferring the blood into the collection tube.
Transferring the blood through the needle may cause haemolysis, breaking of the red cells.

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6. The collection tube must be clean and dry.
7. Allow the blood to clot at room temperature away from direct sunlight.
8. Centrifuge the blood and separate the serum as soon as possible and not more than 1 hour after
collection.
9. After centrifugation remove the serum carefully without sucking up red cells.
10. Blood for sugar estimation is normally requested on a fasting specimen.

Sample Storage and Transportation:

1. Carry out the biochemistry test as soon as possible after separation of the serum from the blood
clot.
2. Not all analyses are stable, which means their concentration will reduce over time.
3. Generally, stability is prolonged if the serum is kept in the refrigerator.
4. Some analysts such as bilirubin are also affected by light.

Anticoagulants
Chemical agents that prevent coagulation are routinely used when whole blood or plasma is required.
Some of the commonly used anticoagulants are:

1. Heparin
2. EDTA
3. Oxalates
4. Sodium Fluoride
5. Citrate
1. Heparin:
Heparin is a naturally occurring anticoagulant produced by basophils and mast cells. It may be considered
to be a natural anticoagulant because it is already present in the blood, but in concentrations less than that
required to prevent clotting in freshly drawn blood. Heparin prevents coagulation by increasing the activity
of antithrombin III, an inhibitor of thrombin. This anticoagulant is used in a concentration of 0.2 mg / ml
of blood and since its molecular weight is large, it produces no change in erythrocyte volume.

2. Ethylene diamine tetracetic acid (EDTA):

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It is an anticoagulant which acts by virtue of removing calcium ions by chelating. A concentration of
1.5±0.25 mg/ml of blood is sufficient. Concentrations even greater than this produce no detectable change
in erythrocyte volume.

3. Oxalates:
Lithium, sodium and potassium oxalates act as anticoagulants by removing calcium ions essential for
blood coagulation. Potassium oxalate is commonly used. 1-2 mg of salt / ml of blood is required.
The disadvantage of the use of oxalate is the alteration of concentrations of plasma components. Shrinkage
of erythrocytes results from a water shift from the erythrocytes to plasma.
4. Sodium Fluoride:
It is used when blood is collected for glucose estimations. In the erythrocytes (RBC), it specifically inhibits
the enzyme Enolase of the glycolytic pathway, preventing the consumption of glucose by the RBC’s if
blood is left standing at room temperature. Though it has a weak anticoagulant action, it is usually
combined with another anticoagulant such as potassium oxalate.

5. Citrate
Sodium citrate solution at concentration 3.8% in ratio or 1 part to 9 parts of blood is widely used for
coagulation studies because its effect is reversible by addition of calcium.

6. Iodoacetate
Sodium iodoacetate at concentration of 2 mg/ml is an effective antiglycolytic agent and a substitute for
sodium fluoride. It is potent inhibitors of glyceraldehyde 3-phosphate dehydrogenase.


PRESERVATION, STORAG E AND TRANSPORT OF B LOOD SAMPLES

Specimen should be delivered as soon as possible to the laboratory, usually within 2 hours but in some
cases, they should be sent immediately. Passage of time affects concentration of many blood constituents
once it has been shed from the body. For blood the common anticoagulants used are fluoride and oxalate.

The sample must be processed as early as possible. If delay is inevitable, the serum must be separated and
refrigerated as these delays chemical change. The concentration of most of the commonly determined

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constituents remains stable up to a week if kept at 4ºC and longer if frozen. Some enzymes are, however,
unstable at 4ºC and should be frozen as soon as possible.

Preservative method used in biochemistry lab
A. Refrigerator method
- 2-8ºC
- Freezing
B. Chemical method:
1. Sodium fluoride (NaF)
2. Iodoacetate
3. Boric Acid
4. Formaldehyde
5. Toluene
6. Chloroform
7. Dilute HCL


ENUMERATE THE CHANGE S OCCURRING IN THE B LOOD AFTER COLLECTIO N,
STORE BLOOD AND TAKE PRECAUTIONS DURING B LOOD STORAGE.


Common changes that occur in blood sample after collection are as follows
1. Glucose is converted to lactate by glycolysis.
2. Due to haemolysis or passage through erythrocyte membrane (before separation of red cells from
plasma) plasma concentration of K
+
, lactate dehydrogenase and aminotransferase can increase.
3. Plasma phosphates increase due to hydrolysis of organic ester phosphates because of action of
enzymes on organic forms.
4. Some labile enzymes may be lost activity.
5. UV irradiation or daylight destroys bilirubin remarkably quickly so it is necessary to keep the same in
the dark as much as possible.

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6. At room temperature calcium is absorbed rapidly onto polystyrene containers (analyzers cups)
through this is minimized if the samples are placed immediately in the refrigerator. So, serum for
calcium estimation is best kept in glass.
7. Samples for blood gases and blood pH are taken in heparinized syringe and is left on with the needle
bend over so that blood does not come in contact with air. This is to avoid loss of CO2 because PCO2
of blood is higher than air. Syringe containing the blood should be kept on ice to slow down red cell
metabolism and to reduce glycolysis with consequent production of lactic acid.
8. Formation of ammonia from urea occurs in longstanding blood.

Average composition of different substances in different in plasma or RBC.

Quantity Units Plasma RBC
Glucose mmol/L 5.0 4.1
Calcium mmol/L 2.5 0.25
Phosphate mmol/L 1.2 4.2
Sodium mmol/L 140.0 16.0
Potassium mmol/L 4.0 100.0
Chloride mmol/L 104.0 52.0
Bicarbonate mmol/L 25.0 10.0
Urea mmol/L 5.5 4.0
AST IU/l\L 25.0 500.0
LDH IU/L 180.00 30000.0



ERRORS
Errors

30. 30

There are a number of potential errors which may contribute to the success or failure of the laboratory to
provide the correct results. Most of these problems arise during sampling called sampling errors which
mostly occurs before analysis the specimens.

Types of error
1. Intrinsic errors:
a. Due to an imprecise and inaccurate method.
b. Due to high blank values (in terms of OD)

2. Systemic errors: (Instruments error)
These are due to calibration problem and are reflected by a shift in mean value of a test monitored on a
daily basis. For rectification, the test reagents and protocols need to be checked and put in place.

a) All high values.
b) All low values.

3. Random errors: Technical error, temp, reagent preparation.
These are due to poor precision i.e. an inefficiency of carrying out the test properly. There could be
errors in pipetting, temperature variations, timing etc. They all indicate reproducibility problem. They
are reflected byb large standard deviations values without much change in mean value.

31. 31

REFERENCE RANGES OR NORMAL RANGES
Test Reference value Unit Diagnosis
Blood glucose Fasting (F) 60-110 mg/dl Diabetes mellitus
Blood glucose Post prandial (PP) 80-140 mg/dl Diabetes mellitus
Blood glucose Random (R) 60-140 mg/dl Diabetes mellitus
Blood Urea 15-45 mg/dl Renal disease
Serum Creatinine 0.3-1.4 mg/dl Renal disease
BUN 9-20 mg/dl Renal disease
Uric acid Male: 2-7
Female: 2-6
mg/dl Gout, Renal disease
Total protein 6-8 gm/dl Liver function
Serum albumin 3.5-5.5 gm/dl Liver function
Serum globulin 1.8-3.6 gm/dl Liver function
Plasma fibrinogen 0.2-0.4 gm/dl Liver function
Plasma prothrombin 10-15 mg/dl Liver function
Bilirubin Total (T)
Bilirubin Direct (D)
Bilirubin Indirect
0.2-1.2
< 0.4
0.2-0.7
mg/dl
Liver function
SGPT (ALT) 5-45
IU/L
Liver function
SGOT (AST) 5-40
IU/L
Liver function
ALP (M) 64-306 IU/L Liver function

32. 32

(F)
Upto 15 yrs
Upto 17 yrs
84-306
<644
<433
Sodium (NA
+
) 135-145
mEq/L
or
mmol/L
Renal function
Potassium (K
+
) 3.5-5.5
mEq/L
or
mmol/L
Renal function
Chloride 95-105
mEq/L
or
mmol/L
Renal function
Bicarbonate 22-28
mEq/L
or
mmol/L
Renal function
Gamma GT < 55
IU/L
Liver function
Amylase 15-220
IU/L
Pancreatic function
Lipase
< 38 IU/L IU/L
Pancreatic function
Total Cholesterol < 220
mg/dl
Lipid profile,
Triglyceride (TG)
Male
Female

65-170
45-145
mg/dl
Lipid profile,
HDL > 40
mg/dl
Lipid profile,
LDL < 150
mg/dl
Lipid profile,
VLDL 13-34
mg/dl
Lipid profile,
LDL/HDL ratio ≤ 2.5

Lipid profile,

33. 33

LDH 225-450
IU/L
Liver and cardiac
function
Serum Calcium (Total) 9-11
mg/dl
Renal function
Phosphorous 2.5-5.0
mg/dl
Renal function
Serum magnesium 1.0-3.0
mg/dl
Nerve function
HbA1C 4.5-6.5
%
Diabetes
CPK (Nac) 24-190 IU/L Cardiac function,
Muscle dystrophy
CPK (MB) < 25 IU/L Cardiac function
FT3 1.4-4.2 pg/ml Thyroid function
FT4 0.8-2.0 ng/dl Thyroid function
TSH 0.30-5.5 µIU/ml Thyroid function
aTPO < 60 IU/L Thyroid function
CA 125 < 30 IU/mL Ovarian cancer
CA 19 9 < 30.9 IU/mL Pancreas, colon cancer
AFP < 8.0 ng/mL Liver, biliary system,
stomach, lung cancer
CA 15 3 < 32.4 IU/mL Breast cancer
CEA
Non-smokers: < 5.0
Smokers: < 10.0
ng/mL
GI cancer
PSA < 4.0 ng/mL Prostate cancer
Vitamin D
<10 Severe
Deficiency
ng/ml Bone disease

34. 34

10-19 Deficiency
20-29 Insufficiency
30-80 Normal
>80 Toxicity