CLINICAL BIOCHEMISTRY LABORATORY & DO’S AND DONT’S Presented By: Dr. (Mrs). Meera Ghadge OIC, Clinical Biochemistry, Tata Memorial Hospital

AGENDA Uses of Biochemistry tests Samples used in biochemical investigations. Types of test carried out in a biochemistry lab Procedures carried out in the lab Quality Control Issues Do’s and don'ts (from receiving samples to the dispatch of results) Tips for lab personals. Conclusion

Clinical biochemistry is: Branch of laboratory medicine which studies chemical and biochemical methods (on blood, urine and other body fluids) Clinical biochemistry tests comprise over one third of all hospital investigations. Recent past has seen major advances in diagnostic methodologies and instrumentation (automation).

Uses of biochemical tests: diagnosis, screening the disease, monitoring patients or assessing the prognosis once a diagnosis has been made. research studies and in clinical trials of new drugs.

Samples used for biochemical investigations Blood Serum - clear liquid that can be separated from clotted blood. ( It does not have fibrinogen or the other clotting factors) Plasma - liquid portion of the blood, obtained by centrifugation of blood treated with anticoagulant Whole blood Urine Body fluids Cerebrospinal fluid

Samples used Tests done using Serum: (red top/plain evacuated tubes) urea, uric acid, creatinine, electrolytes, calcium, phosphorus, magnesium, total proteins, albumin, immunoglobulin, electrophoresis, bilirubin, all the enzymes, tumor markers, iron, ferritin, TIBC, transferrin, and lipids Tests done using Plasma: (gray top Sodium fluoride, Potassium oxalate) glucose Tests done using Whole blood: (Lavender cap 7.5% K3 EDTA) blood gases, glycosylated hemoglobin

Samples used Tests done using urine : albumin, calcium, VMA, 5 HIAA, BJP. Tests done using body fluids: glucose, proteins, LDH, amylase. Tests done using CSF: glucose, LDH, Chlorides and micro-proteins.

Instructions to the patients For all the biochemical investigations patients should be fasting for at least 8-10 hrs as lipemic serum interferes with the analysis. For lipid profile 14 hrs fasting is essential. For tumor markers, fasting is not required. Avoid alcohol, smoking and strenuous exercise before blood collection.

Methods 1. Manual procedures: - few labs - good for small sized labs - tedious and time consuming 2. Semi automated procedures 3. Automated procedures: - large labs - save time and manpower - require stringent quality control - expensive

Past and Present - Laboratory Procedures Volume of work increased, need for simplification arose. Increasing work load led to automation. Monostep methods replaced multistep methods. No manual pipetting Efficiency increased by the use of dispensers and diluters. The automated instruments not only save the labor and time, but allow reliable quality control, reduce the subjective errors, and work economically by using smaller quantities of sample and reagents.

Automation The early form of automation were semi auto analyzers. Than came auto analyzer based on continuous flow system. Later discrete analyzers were introduced; Batch analyzer : semi automated and fully automated. Stat or random access analyzers Automation leads to reduction in variability of results and error of analysis.

Semi-Auto analyzers Semi-auto analyzer: Here, the samples and reagents are mixed and read manually.

Batch Auto analyzers Batch analyzer: The reagent mixture is mixed and fed automatically. One reagent is stored in the machine at a time enabling one batch of a specific test to be automatically conducted e.g. RA 100, ASCA

Random Access auto analyzers Random Access auto analyzers: These analyzers can store more than one reagent. Samples are placed in the machine and the computer is programmed to carry out any number of selected tests on each sample e.g. Hitachi series, RxL series, AU 400,640,2700 etc. and many more

Small and Large Size Laboratory Large labs Small labs Multistep manual methods are used Reagents prepared manually Mouth pippetting is done Work load is less Method procedures are lengthy and time consuming Man power required is more for different tasks Monostep methods replaced multistep methods Reagents are commercially available Dispensers and diluters are used Workload has increased tremendously Test procedures are short & very fast Due to automations multitasks can be done.

Big labs and hospital based labs may have laboratory information system Laboratories today are held together by a system of software programs and computers that exchange data about patients, test requests, and test results known as a Laboratory Information System or LIS. The LIS may be interfaced with the Hospital information system

Common Biochemical Tests

1. Blood Glucose (plasma) and Lipids (serum) Glucose (fasting, post lunch or random) CV% (acceptable) : 2.9% Lipid (14 hrs fasting is compulsory) -Cholesterol :CV% (acceptable) : 2.7% -Triglycerides :CV% (acceptable) : 10.5% -HDL- Cholesterol -LDL- Cholesterol

2. Electrolytes (serum): Sodium, Potassium, Chlorides & Bicarbonates Mostly done in ICUs Urgent reporting

3. Renal function tests (serum) Urea : CV% ( acceptable ): 6.2% Uric Acid : CV% ( acceptable ): 4.5% Creatinine : CV% ( acceptable ): 2.7% Creatinine is more specific test for renal dysfunction

4. Liver function tests (serum) Total Proteins (Albumin, Globulins), Bilirubin (total and direct), Aspartate transaminase (AST); Alanine transaminase (ALT); Gamma- glutamyl transpeptidase (GGT); Alkaline phosphatase (ALP) GGT is the indicator for Alcoholism. AST, ALT, GGT and ALP cannot be done if the sample is hemolysed, as the ruptured RBCs will lead to the increased enzyme activity. Bilirubin absorbs light resulting in decreased bilirubin values, hence should be estimated immediately without exposure to light.

5. Cardiac markers (serum) Creatine kinase-MB (CK-MB), myoglobin, homocysteine, AST, LDH, C-reactive protein (CRP), troponin T (cTnT), & troponin I (cTnI) are all used for assessment of the suspected acute myocardial infarction CV% (acceptable) for Ck MB is 9.0% CK- MB levels rises within 3-4 hours, and remains elevated till 18- 24 hrs and returns to normal levels after 72 hrs. Troponin T is a more specific test for heart muscle damage.

6. Blood disorders (serum) Iron Transferrin TIBC Vitamin B12 Folic acid Ferritin These tests help in diagnosing and sub-typing anemia.

7. Blood gases (arterial / venous): whole blood Arterial oxygen tension (PaO2), Carbon dioxide tension (PaCO2), Acidity (pH), Arterial oxyhemoglobin saturation (SaO2) Sample should be transported immediately, and analyze it within 30 minutes after collection. Should be reported immediately Blood gases are important for patients with critical illness or respiratory disease. The most common tests performed on patients in ICUs.

8. Common tumor markers (serum) CEA CA 19-9 Beta-HCG PSA CA 15-5 AFP Ca 125 Beta-2 microglobulin Serum electrophoresis – M band

Circuit diagram of clinical biochemistry process Biochemical answer Clinical question 1.Request form 8. Reporting 7.Interpretation 2. Patient sample 6.Analysis 3. Transit to lab 4.Reception 5.Quality control

Sample reaches the lab, Step By Step Procedures Accessing and Receiving the samples Centrifuge the samples to separate the serum Decapping the samples Loading the samples in the Automated Analyzer Unloading the samples Reporting

Rejection criteria for biochemistry samples Mislabeled / wrongly labeled sample. Sample is highly lipemic, or haemolysed. No requisition form.

DO’s and DONT’s

DO’s and DONT’s 1. During centrifuging.. Samples should be balanced properly before centrifugation. Speed for centrifugation - 4000 rpm for 10minutes. If there is any breakage of sample tube while centrifuging the spills should be cleaned with proper precaution using spill kit. After centrifugation samples should be decapped and sent to the analyzer for analysis.

2. while loading samples on the analyzer DO’s and DONT’s 2. while loading samples on the analyzer 1. Check whether the maintenance procedure is performed according to the maintenance protocol. 2. Quality control is performed and is accepted. 3. Serum is sufficient to perform all the required tests. 4. Serum is non-hemolysed and non-lipaemic.

3. while reporting results DO’s and DONT’s 3. while reporting results The transferred data should be matched with the printout of results. Delta check should be done for all the test results. Remarks such as tests rechecked, sample hemolysed or lipemic, second sample receiving time, reports informed on phone etc should be mentioned. Discuss with referring doctors as and when warranted Report should be committed and dispatched within departments turnaround time.

Quality Control

Must in any Biochemistry Lab QC includes Instrument, Reagents, Staff, Process Two types - Internal QC - External Quality Assurance Scheme (EQAS) / Proficiency testing (PT) program

Internal Quality Control (IQC) To mention few aspects of IQC Frequency of Internal quality control Preferably, 2 to 3 levels of QC (normal and abnormal) are done for all the parameters. However, at least one QC level is a must Frequency of Calibration With every new lot of reagent. QC fails to follow westgard rules. Calibration and controls have different roles

Corrective measures taken when control is out Check the reconstitution date of QC. Check the temperature of the refrigerator. Check the reagent (expiry, opening date etc.) Check the instrument.(priming, dispensing etc) Rerun the controls. If problem persists run the fresh controls. If problem still persists, calibrate the parameters. If problem still persists, call the engineer.

Control material are run and LJ Charts are plotted

Internal QC is used to monitor precisions and accuracy of the tests Good Precision Only Neither Good precision Nor Accuracy Good Accuracy Good Precision 38

EQAS/ Proficiency Testing Programs PT is a must for all tests done in the laboratory Frequency: monthly for biochemical tests, whereas three monthly for other disciplines PT provider are outside agencies (national / international programs). When there is no PT program available Inter-laboratory comparison program Also recommended, retained and replicate sample testing for comparability study between the systems. However these are not substitutes for PT

Turnaround time ( TAT) Definition: It is time taken from receiving of the sample to dispatch of the report. Each lab defines its own TAT (based on international recommendations and local factors).

Maintenance of Instruments Maintenance of all the analyzers is performed according to the maintenance protocol by the MANUAL provided by manufacturer. It includes: Daily (By technical / scientific staff): - External & Internal cleaning of analyzers - Quality of water (pH, conductivity), cleaning of probes (Sample & reagent) - Temperature, Humidity, Stability of electric power. Weekly (By technical / scientific staff): - Cleaning of Cuvettes, water tank, filters, reagent assembly. - ISE cleaning Monthly (By technical / scientific staff) - Quality of water (ions & growth). - Precision check Quarterly (By engineer) : Total maintenance - changing of tubes, sample & reagent probes ( if required) - changing of lamp ( if required), cleaning of cuvettes. Yearly (By engineer): calibration of analyzer

Training of staff In-house calendar for lab staff: Every lab has to have its own teaching/academic program. Lab Director must encourage technicians to participate in CMEs, training programs, and conferences. Guest speakers may be invited for presentations.

Tips for the lab personnel Do not assume! Ask, if you don't know or even if you are unsure. Never put back chemicals into the container when you have already taken it out. Remove only the required quantity. Do not drink / eat in the lab (where tests are being performed). Biosafety: always wear a lab coat, hand gloves when working in the lab. Lab is an infected area. No mouth pipetting.

Document every procedure and Implement the same. Follow SOPs. Read instructions! Read other manuals of the lab including accreditation related documents. Label all containers with date of preparation and expiry. Spill control mechanism in place. Fire exits: Know about the fire extinguisher, eyewash station, and MSDS (material safety datasheet).

Conclude

Automation has become part and parcel of all clinical biochemical labs. Quality control is mandatory as is proficiency testing. Continual improvement should be the aim.

Thank you