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208
10
05
Molecular weight of hydrated form
Weight of hydrated form
285 5
159 5
.
.
x
x =
= 44.7
V (Volume) =
M (mass)
49
100
)
Medical Laboratory Technology: Volume 1
In another example, the instruction calls for mixing 0.5 mL of blood with 9.5 mL of diluent
for cell count. The dilution or the relationship between the aliquot (0.5 mL) and the total
volume (0.5 + 9.5 = 10 mL) is expressed in the ratio of
= 20 times or 1:20.
.
Weight relationships of hydrated and anhydrous salts
You will often face a dilemma when directions for preparing a reagent solution involve a
chemical which is hydrated while your laboratory has only the anhydrous form, or vice
versa. The following simple formula and example may help to nd the solution.
Molecular weight of anhydrrous form
=
Weight of anhydrous forrm
Example The directions ask you to use 25 g of anhydrous copper sulphate (CuSO4) whereas
your laboratory has hydrated copper sulphate (CuSO47H2O). How much of the hydrated
form would you weigh in order to meet the requirement of the directions?
Molecular weight of anhydrous form (CuSO4) = 159.5 g
Molecular weight of hydrated form (CuSO4 7H2O) = 285.5 g
Apply the above formula:
=
159 525
.
25
285 5
.
Thus you should weigh 44.7 g of the hydrated form which is equivalent to 25 g of the
anhydrous form.
Figuring out the weight of a liquid from its volume
Weighing of a solid is not a problem in a laboratory. But suppose you need 49 g of sulphuric
acid to prepare 1000 mL of 1 N solution; the weighing of sulphuric acid can be dangerous,
erroneous (the acid absorbs water when exposed to air) and might create a number of other
problems. You can resolve this problem without weighing the sulphuric acid and the method
is applicable to any liquid reagent.
The label on the bole provides the density and purity of the chemical. If not, consult the
appendix at the end of the third volume which gives normalities, purities and densities of
commonly used acids and alkalis. Let us take the following gures and nd out the volume
of sulphuric acid that we need in order to meet the requirement of 49 g.
Specic gravity = 1.84
Purity = 98%
Weight needed = 49 g
Volume of the acid needed = x mL
Thus, V = 27.2 mL
If 27.2 mL of the given concentrated sulphuric acid is mixed with water and made to 1 L,
it will be about 1 N. In order to get exactly 1 N, you have to titrate and then make the nal
dilution. Let us continue further in order to get exact 1.0 N sulphuric acid.
Note It is always easier to dilute than to add more concentrated acid to reach our goal.
D (Density)
= (ratio t
oo compensate for impurity
184
98.
Units of Measurement and Preparation of Reagent Solutions
Or,
)
1000 1110.
mEq/L =
(mg/100mL)10
mmol/L =
)10
209
Let us suppose the strength of the acid, determined by titration, came to 1.1 N. Then the
new problem can be dened as follows: How much water should be added to the 1 L of 1.1 N
sulphuric acid in order to get exactly 1.0 N acid solution?
Apply the basic formula:
V1 × C1 = V2 × C
2
Where, Vı and V2 are the initial and nal volumes, respectively, and C1 and C2 are
the initial and nal strengths of the acid. In other words, the above formula comes to:
1000 × 1.1 = x × 1.0 (x is the nal volume).
1100 mL(the final volume
x
.
Thus, if we add 100 mL ( 1100−1000 mL) of water to the 1.1N acid solution, it results in exactly
1.0 N acid solution.
Mixing of two solutions of different concentrations
What will be the concentration of the nal solution when two solutions of dierent strengths
are mixed?
Problems
1. I have mixed 80 mL of 70% alcohol with 40 mL of 95% alcohol. What will be the
concentration of the nal mixture?
2. How much of 80% alcohol should be mixed with 30 mL of 95% alcohol in order to
prepare 90% alcohol?
Solutions
Let us take the basic formula:
(C1 × V1) + (C2 × V2) = C3 × (V1 + V2)
Where, С and V respectively stand for concentrations and volumes of solutions 1 (C1) and 2
(C2). Solution 3 (C3) is the concentration of the nal solution (x).
Note It is not important for you to nd out how this formula was derived. You are perfectly
justied in applying the formula without any idea of mathematical derivations.
Additional information
1. (70 × 80) + (95 × 40) = x ×(80 + 40)
Or, x = (70 × 80) + (95 × 40)/(80 + 40)
Or, x = 78.3
Thus, the new solution will be of 78.3% concentration.
2. (80 × x) + (95 × 30) = 90 (30 + x)
Or, 80x + 2850 = 2700 + 90x
Or, 10x = 150
x = 15
Thus, if 30 mL of 95% alcohol is mixed with 15 mL of 80% alcohol, the resulting
mixture will be of 90% concentration.
Eq. Wt.
(mg/100 mL
MW
210
Medical Laboratory Technology: Volume 1
revIew QueStIonS
1. What are the basic metric units of length, weight, and volume?
2. Why is the metric system preferred over the English system in clinical laboratory?
3. Convert the following units: (a) 0.001 g into mg; (b) 300 mg into g; (c) 750 µg into mg;
(d) 4000 mg into g; (e) 280 mg into µg; (f) 10 mg into pg
Answers (a) 1 mg; (b) 0.3 g; (c) 0.75 mg; (d) 4 g; (e) 280000 µg or 2.8 × 105 μg;
(f) 1 × 1010 pg.
4. Convert the following units: (a) 3 dL into mL and µL; (b) 0.3 L into dL and mL; (c) 45 cc
into L and mL; (d) 4 dL into L; (e) 60 µL into L and mL; (f) 6700 mL into L
Answers (a) 300 mL and 3 × 105 µL; (b) 3 dL and 300 mL; (c) 0.045 L and 45 mL; (d) 0.4 L;
(e) 6 × 10–5 L and 0.06 mL; (f) 6.7 L
5. Convert 98.6°F (normal body temperature) to Celsius (C) degrees.
Answer 37°C
6. Prepare 250 mL of a 2% solution of acetic acid using 10% acetic acid solution.
Answer 50 mL 10% acetic acid mixed with 200 mL of distilled water. Apply the for-
mula: C1 × V1= C2 × V2, where С1 and V1 are, respectively, the concentration and
volume of rst solution (10% acetic acid) and C2 and V2 are, respectively, the concentration and volume of the second solution (2% acetic acid). In other words:
10 × V1 = 2 × 250 or V1 = (2 × 250)/10 = 50 mL and the amount of distilled water will be
250 – 50 = 200 mL.
7. How much salt will you weigh to prepare 500 mL of saline (0.85%)?
Answer 0.85 × 500/100 = 4.25 g
8. To prepare 100 mL of 10% formalin from the commercial grade of 37% strength, how
much of the formalin would you measure?
Answer 100 × 10 = x × 37. Or, x = 1000/37 = 27.03 mL
9. To prepare 1 L of 2% acetic acid from concentrated acetic acid (glacial or 100%), how
much of acetic acid do you need?
Answer 20 mL
10. For preparing 4% suspension of red cells (to be used in blood bank) you centrifuged the
whole blood and discarded the plasma. The volume of packed red cells was found to be
2.5 mL. How much saline would you add in order to make a 4% suspension of red cells
in saline?
Answer When 96 mL of saline is mixed with 4 mL of packed red cells it yields 4% sus-
pension.
11. You are provided with concentrated sulphuric acid (specic gravity 1.98, 96% pure). In
order to prepare 500 mL of 1 N H2SO4 (approximately), what quantity of concentrated
sulphuric acid would you need?
Answer Eq. Wt. of sulphuric acid = 49; hence, 24.5 g needed for 500 mL solution. In
order to get 24.5 g weight of sulphuric acid, the volume of sulphuric acid that has to
be measured = 24.5/1.98 (V = M/D or volume is equivalent to mass divided by density)
= 12.37 mL. Finally the purity is to be compensated: 12.37 × 100/96 = 12.88 mL.
Units of Measurement and Preparation of Reagent Solutions
211
12. In a certain acid-base titration 6 mL of 1.5 N acid neutralized 4 mL of the alkali. What is
the strength of the alkali?
Answer 6 × 1.5 = 4 × x or x = 2.25 N
13. You have 1.15 N HCl solution (‘too concentrated’). How much water would you mix in
order to obtain 1 Lof 1 N HCl solution?
Answer 130 mL
14. One litre of 70% alcohol is needed. How much 95% alcohol is required to make that?
Answer 736.8 mL
15. How much KCl would you require for preparing 1 L of 5 mEq K/L?
Answer 1 mEq KCl = 39.1 + 35.5 = 74.6 mg KCl = 1 mEq К. Hence, for preparing 1 L
solution of 5 mEq K/L you should weigh 74.6 × 5 = 373 mg or 0.373 g of KCl.
Good Laboratory Practices and Statistical Quality Control
Chapter Outline
• Sources of Common Errors in Laboratory
• Prociency Testing
▪ Quality control issues by laboratory type
▪ Quality assessment and quality assurance
• Statistical Quality Control of Quantitative Data
• Basic Statistics
▪ Use of standard deviation in laboratory
▪ Preparation of quality control chart
▪ Interpretation of quality control chart
• Summary
• Review Questions
7
Aloka Chakravarty
“Quality is everyone ‘s responsibility. It is not enough to do your best;
you must know what to do, and then do your best. “
—W. Edwards Deming
Statistician and Father of Total Quality Management (1900–1993)
Laboratory ndings must be dependable so that accurate diagnosis can be based on them.
Medical laboratory personnel must be able to identify sources of variation and control for
them. For example, if a patient has a baery of laboratory chemistry tests, there is a possibility
that some tests will be abnormal, the so-called false positive results, purely due to chance
alone. It is important to identify and control by stringent margins each source of variation so
that we can minimize, control and quantify such errors.
As the laboratory also depends on external agencies, an eective quality control pro-
gramme will not only monitor internal laboratory procedures but will also communicate
needs and requirements of the clinical laboratory to other departments of the hospital. Strict
enforcement of laboratory policies creates an environment conducive to producing reliable,
reproducible and high-quality results. A relaxed approach toward quality control leads to
an equally relaxed approach toward the handling and testing of specimens and should be
avoided at any cost.
Areas where common errors are likely to occur and can be prevented are discussed further.
Good Laboratory Practices and Statistical Quality Control
213
SourceS of common errorS in Laboratory
Laboratory test request
All laboratory test requests should be made in writing on the appropriate request form as
indicated in Chapter 5. It may be worthwhile to insist on three time slots:
• when request was issued,
• when specimen was obtained,
• when results were reported (with the name of the technician who reported the result).
Specimen collection and processing
The technologist, trained nurse or the physician collects the specimen. Proper patient
preparation is an important component of quality control. Laboratory results are only as
good as the specimen. Hence, quality control is a joint responsibility and the cooperation
of the aending nurse is critical. If the specimen collected is not promptly delivered to the
laboratory, the specimen becomes useless for analysis.
The person who collects the specimen must report to the nursing station for entering
the information about specimen collection and identication of the patient; and must collect the specimen according to standards laid down by the laboratory. A frequent source
of error has been identied to be the failure to observe basic precautions and laboratory
rules. Identication of the patient is extremely important—identify the patient before
taking the specimen, check the name on the requisition slip, check also the name on the
patient’s wristband (if it is there) or ask the nursing sta as a double check. Specimens
must be in appropriate containers which must be carefully labelled. It is a good practice
to label the container before taking the specimen and use only waterproof ink so that
identication does not get ‘washed away’.
Proper collection procedure and specimen processing after its collection are both important
and must be strictly adhered to if meaningful results are to be expected. Haemolysed blood is
of lile value in most laboratory tests. Also, if the serum is not promptly separated from the
cells, the results may not be accurate. When the specimens are directed to other laboratories
for tests and follow-ups, care must be taken to ensure proper identication and re-labelling
of each specimen. Mix-up is more likely to occur within the laboratory.
Laboratory records
Each specimen arriving in the laboratory must receive the laboratory accession number
recorded in the laboratory register or the log book. The technician’s record must refer to the
accession number and when reports are dispatched by the laboratory, the results must be
noted in the accession register, with the time of reporting and the name of the technician who
reported the results. The technician’s register must indicate the quality control procedure
followed. Many advanced laboratories maintain an alphabetized test report card system for
each patient; these cards are further classied under dierent specimens and laboratories.
This helps to trace back the previous records of the patient.
Error in procedure
Well established laboratory procedures must be adopted and documented for the
physician’s information. The laboratory must maintain a procedure manual for each test
performed. Failure to adhere to established rules and procedures may provide results
that are not reproducible or reliable. Technicians must be discouraged from inventing
individual methods without appropriate documentation. A good technician keeps
214
abreast of newer techniques, is aware of drug interactions and is alert within limitations.
A method that is fast, economical, reliable and reproducible and can be done by personnel
without specialized training is optimal.
Reagents must be stored properly and outdated reagents must be discarded. Instruments
must be properly maintained. A regular maintenance programme is essential for the proper
running of the laboratory. All instruments, at regular intervals, must be checked for their
performance—wavelength of colorimeter, eciency of the autoclave, water bath temperature,
oven temperature, refrigerator temperature, cold room temperature, etc.
Calculation and transcription errors can occur at any point and may lead to wrong diag-
nosis. The technician must record the actual instrument reading, indicate calculations and
tally the report at least twice before sending it out of the laboratory.
Medical Laboratory Technology: Volume 1
Proficiency teSting
Only properly trained personnel should be appointed by the laboratory. The training
must be provided by a recognized institution and should not be only On the job training’.
The employee should be required to submit evidence of continuing education every year.
The technician must maintain daily ploing of the quality control chart explained in the
following pages and must participate in the prociency testing programmes organized by
various agencies, including the World Health Organization (WHO).
Prociency testing is an important component of quality control. An external agency may
send blind samples to the laboratory that have been assayed multiple times. The laboratory
then performs the assay and sends results back to the agency for comparison and a detailed
report on each component.
Quality Control Issues by Laboratory Type
Each laboratory has its own characteristic features and hence the quality control approach
varies. There are, however, some common features such as specimen collection, identication
and processing, laboratory records, maintenance of instruments, procedure and reagents
and supplies. The manufacturer also plays an important part in providing reliable data by
supplying reliable instruments and reagents. There should be close cooperation between
the manufacturer and the laboratory so that the former can meet needs of the laboratory
eectively on a continuous basis.
Haematology laboratory
Specimens must be processed correctly and tests must run within the specied time. Some of
the tests must be done within 2 h (ESR), within 4 h (WBC count) or can be delayed for 8–10 h
(RBC count, PCV or HCt). Fixed smears can be examined after several days.
The maintenance of the equipment must include the checking of centrifuge speed,
volume dispensed by diluters, temperature of the water bath, etc. Keep a record of
the maintenance plan. Manual RBC count is highly erroneous. Automated counters
have their own seings; run the control every day and set controls according to the
manufacturer’s directions.
Correlation of results is a good way to check on the results. For example, a normal haema-
tocrit should have normal haemoglobin; a stained smear must be correlated with the count
(Table 7.1) and the same holds for the platelet count (Table 7.2); a slide with hypochromic red
cells must have a low haemoglobin value; presence of macrocytes and megaloblasts should
correspond with increased mean cell volume (MCV). It has been found that indices stay close
to the normal values, hence, for standardization use indices.
Good Laboratory Practices and Statistical Quality Control
215
Table 7.1 Correlation of number of leucocytes under high-power objective (400X) and WBC count
Number of WBC under high power Estimated WBC count per mm
2–4 4–7 × 10
4–6 7–10 × 10
6–10 10–13 × 10
10–20 13–18 × 10
3
3
3
3
3
Table 7.2 Correlation between average number of platelets under oil-immersion objective (1000X)
and actual platelet count
Number of platelets per oil-immersion eld Comparable platelet count
Less than 1 Less than 2 × 10
5
6–15 (several platelets with occasional clumps) 3 × 10
More than 15 >5 × 10
(normal)
5
4
(thrombocytopenia)
Coagulation laboratory
The reagent is the biggest source of error in coagulation. Temperature of the water bath must
be checked regularly. All tests run must be accompanied by a control run with normal plasma.
Due to lack of appropriate facilities, lyophilized normal plasma is not yet very commonly
used in laboratories of developing countries.
Blood bank laboratory
Technical error is still high and all possible precautions should be adopted in order to avoid
wrong transfusion. Properly functioning equipment is equally important and so also is
the reagent supply. All reagents must be stored and frequently checked according to the
manufacturer’s directions. Check the Coombs reagent with Coombs control (sensitized cells).
Keep a semi-quantitative report of the haemagglutination reaction.
Before the matched blood leaves the laboratory for a particular patient, it must be
re-checked and countersigned by the technician and receiving nurse in order to minimize
clerical mistakes.
A complete record of every unit of blood received by the blood bank must be kept in a log
book that indicates: date of receiving donor’s blood, source, code number, blood type, result
of compatibility testing, name of recipient, date of transfusion and result of transfusion. In
case the blood is supplied by any outside agency, the blood type must be rechecked.
Microbiology laboratory
Collection of the specimen by recommended procedures and its proper transportation to
the laboratory are crucial for geing reliable results from the microbiology laboratory.
Performance of the equipment and reliability of reagents are the rst steps in quality
control, followed by appropriate culture technique or direct microscopic examination.
In case of bacteriology and mycology, testing of media and stains against type-cultures
is extremely important. In selecting the type-organisms, both ‘positive’ and ‘negative’
must be chosen. Table 7.3 gives the type of organisms used in testing the common media.
Table 7.4 gives the procedure for maintaining important test organisms. Sensitivity testing
must also be checked with appropriate organisms and the zone formed must tally with
the suppliers’ specications.
216
Medical Laboratory Technology: Volume 1
Table 7.3 Tests of performance of common media and biochemical reactions
Medium Test organism Result
Blood agar Streptococcus pyogenes Beta-haemolysis
Chocolate agar Neisseria gonorrhoeae Growth in 5% CO
McConkey Escherichia coli Red colonies
S-S agar E. coli Pink colonies
TSI agar E. coli Acid bu, acid slant, gas, no H
Sab-dextrose Candida albicans Growth
Citrate agar E. coli Negative
Klebsiella
Urease agar E. coli Positive
Proleus
Bile solubility S. pneumoniae Loss of turbidity negative
Indole oxidase E. coli Positive
E. coli Negative
Pseudomonas aeruginosa Positive (purple)
2
Table 7.4 Maintenance of important test organisms
S
2
Organism Medium Incubation temperature Interval between subcultures
E. coli Nutrient agar 37°/18 h 6 months
Shigella
Klebsiella Nutrient agar 37°/18 h 3 months
Proteus
Staphylococcus
Pseudomonas Peptone water 37°/18 h 3 months
Salmonella Dose egg 37°/18 h 12 months
Streptococcus Cooked meat 37°/18 h 3 months
Note Keep all type cultures at 5°C in the refrigerator.
Parasitology laboratory
The stool specimen is one of the most frequently neglected materials submied to the
laboratory. Only fresh, properly collected and uncontaminated stool specimens can yield
a proper diagnosis. In following the technique of faecal preparation, you must follow the
established procedure without unscientic modications. A parasitological reference book
must be available in the laboratory for identifying unusual eggs and other parasite-related
objects. An experienced technician must be consulted in case of doubt. Every tenth negative
report must be checked by the supervisor and every positive specimen must be tallied by the
supervisor. A set of prepared slides should be kept in the laboratory for reference.
Serology laboratory
All serodiagnostic tests must have a positive and a negative control. Most manufacturers
supply them along with the kit. The technician should have sucient experience to recognize
the positive reaction and whenever possible should grade the reaction (1+ to 4+). The
borderline reactions specially need personal judgement.
Good Laboratory Practices and Statistical Quality Control
217
Clinical pathology laboratory
A urine specimen must be processed within two hours. Quick chemical screening tests done
with the urine sample must be checked with the positive and negative controls. If necessary,
add an appropriate reagent to normal urine in order to prepare the positive urine. Do not add
an excessive amount so that the borderline specimens can be recognized. Follow the same
criteria for other body uids as is done in case of urine.
Urine culture must be done with well-mixed specimens and not with the sediment. This
gives the correct picture. If the bacterial population is less than 1 × 10, the specimen is rejected. The counting procedure must be standardized.
Laboratory ndings of histology and cytology are highly subjective to the decision
made by the pathologist during the examination of the specimen. Hence, selection of a
representative specimen and procedures of xing, cuing, embedding and staining are
all variable factors that may result in false ndings and artefacts. Stains must be checked
with known sections. Permanent slides may guide the technician towards ideal staining.
In case of poor quality, re-staining may have to be done. In most cases, staining can be
partially repeated.
Clinical biochemistry laboratory
The biochemistry laboratory requires quantitative analysis. All factors mentioned earlier,
e.g., specimen collection, instrument handling and procedures adopted, aect the reliability
of the ndings. Specimen-related errors must be minimized. For example, if the blood
specimen arrives with haemolysis for potassium determination, it must be rejected; a
specimen submied for the determination of blood gases must be in ice and under anaerobic
conditions; a specimen for bilirubin assay must not be exposed to strong light; and so on. At
regular intervals prepare a new calibration curve of the test in order to nd out whether the
assay region is in the linear range. Each morning the analysis of the control serum should be
done and the result ploed on the Quality Control (QC) chart which will be explained in the
following section.
Quality Assessment and Quality Assurance
Quality assessments are designed so that reliable laboratory results can be obtained and
reported as early as possible, by minimizing systematic sources of error. It evaluates preanalytical, analytical and post-analytical factors that can aect the results before, during and
after the test.
Pre-analytic factors include proper patient identiers, specimen collection and handling,
specimen rejection criteria, maintaining proper laboratory setup including calibration. Selecting right test methods, personnel and maintaining updated procedure manuals also are
important components.
Post-analytic factors are primarily in reporting and charting of the results, including
transcription and clerical errors. Use of computers as well as proper patient identiers,
laboratory request forms, labels and specimen, containers can greatly aid in minimizing these
errors.
Analytic factors that can aect laboratory tests are:
• Laboratory preparation of samples
• Instrument calibration and maintenance
• Standards and procedural control
• Test procedure logistics (reagents, pipeing, timing, etc.)
• Interfering conditions or substances
• Statistical analysis of control results