The Department of Biochemistry in Sir Ganga Ram Hospital started as a small section of a basic clinical pathology laboratory over 50 years ago. In 1998, the Biochemistry section fractioned out of the initial Pathology Laboratory and, since then, under the able leadership of Prof. L.M. Srivastava, has evolved into a full-fledged independent department. The department works 24x7 and boasts of some of the top of the range, highly sophisticated fully automated analyzers and a wide range of test menu of routine and specialized tests. The laboratory is accredited by the NABL (ISO 15189 : 2007, NABL 112) since 2005 and has a highly experienced and accomplished faculty. This has translated into a large number of papers published in journals with high impact factor, oral and poster presentations in international and national conferences as well as a number of research projects, of which, some are funded by Indian Council Medical Research, New Delhi. In addition, it is the first department of the hospital to start a PhD programme.

Introduction:

"Clinical Chemistry is that branch of medical science that involves the analysis of biological materials, usually body fluids to provide diagnostic information on the state of the human body". Over one-third of all hospital investigations comprise of clinical chemistry tests. Biochemistry investigations are involved in every branch of medicine and are of use in diagnosis, prognosis and screening for disease, in monitoring of treatment and in research.

Figure 1: Uses of tests done in Clinical Biochemistry

The vision of our department is to provide leadership in clinical biochemistry and its application to patient care. We aim to achieve high reliability and efficiency in all the procedures including patient identification, specimen collection, assaying and reporting as well as follow up. The department has a faculty consisting of 6 Consultants, one Biochemist, 1 PhD student and 23 technical staff. It is headed by Prof.L.M.Srivastava, who has been the Head of Department of Biochemistry, All India Institute of Medical Sciences. He is the recipient of several national and international awards/recognitions which include Dr.B.C.Roy National Award, Dr.Vishwanath Gold Medal for outstanding work in the area of Biomedical Sciences, National Institute of Immunology Senior Scientist Oration Award of the Indian Immunology Society, fellowship of the National Academy of Medical Sciences, Dr.Shurveer Singh Trust Oration Award for meritorious services to Medical Education and Research, KEM Hospital and GS Medical College Oration Award of the Association of Clinical Biochemists of India, Oration Award of the Association of Biochemistry Alumni of Lucknow University, Fellowship of the Alexander von Humboldt Foundation of Germany, and the A.J.Thakur award of the Association of Clinical Biochemists of India. He is one of the founder members of the ACBI which was established in 1975 and a Trustee member of the Immunology Foundation. He is eminent in the field of Molecular and Clinical Biochemistry and Immunology, and has over 250 publications to his credit. He has been earlier Secretary, Vice President and President of the Immunology Society. At present, he is the President of the Society of Stress Sciences, India. He is also member of the Defence Research and Development Organisation and the Armed Forces Medical Research Committee (AFMRC).

The faculty of the department is as follows:

1. Dr. Seema Bhargava - Chairperson and Sr. Consultant
2. Dr. Anjali Manocha - Vice Chairperson and Sr. Consultant
3. Dr. Mamta Kankra - Sr. Consultant and Quality Manager, CLS
4. Dr. Parul Singla - Consultant
5. Dr. Anisha Sharma - Consultant
6. Dr. Reetika Saini - Associate Consultant

Figure 2: Biochemistry work - flow process

FIRSTS of the department:

1. First laboratory in South-East Asia to have installed the state-of the-art clinical chemistry auto- analyzer – Beckman
   Synchron LX-20 Pro (2003).
2. First department of the hospital to start a PhD programme (2005)
3. First NABL ISO:15189 accredited laboratory in India (2005)
4. First department in SGRH to be sanctioned an ICMR project (2008)

Work load and technology: In the last decade, the number of investigations performed has increased four-fold (figure 3) so that in 2011 we have performed over 14 lakh tests. The methods and the instrumentation used are of the latest advanced technology(figures 4-9), e.g. HPLC, chemiluminescence, TRACE (time-resolved-amplified-cryptate-emission).

Figure 3: The total number of investigations done annually has increased almost three-fold in the last decade.

Routine parameters done in SGRH Biochemistry laboratory: Test pertaining to all organ systems are included in our menu, separately as well as part of various panels, so as to best assess the patient's status. · Diabetes Assessment Tests: Glucose, glucose tolerance test(GTT), glucose spot test, glycosylated haemoglobin, BUN, creatinine, uric acid, calcium, phosphorus, sodium, potassium, chloride cholesterol, microalbumin (urine).

• Liver Function Tests: Bilirubin (direct and indirect), total protein, albumin, globulin, aspartate transaminase(SGOT), alanine transaminase (SGPT), alkaline phosphatase, gamma GT.
• Kidney Function Tests : BUN, creatinine, urinary protein, urinary creatinine clearance and random urine sodium, potassium, chloride and microalbumin.
• Extended Lipid Profile : Total cholesterol, HDL cholesterol, LDL cholesterol, VLDL cholesterol, triglycerides, apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), lipoprotein (a) {Lp(a)}.
• Cardiac Enzymes : Creatinine phosphokinase (Total CPK) CKMB (mass and activity), lactate dehydrogenase.
• Anemia Markers : Iron, TIBC, Transferrin and Ferritin
• Immunological Parameters : Immunoglobulins (IgA, IgG, IgM, IgE)
• Pancreatic & Intestinal Function Tests : Amylase, lipase, D-xylose excretion.
• Fluids : Urine, CSF, pleural, peritoneal, synovial aspirate, etc. These can be analysed for the routine glucose, protein, albumin and chloride and also specialized tests like LDH, Amylase and Immunoglobulins.

Specialized Parameters

1. Cardiac bio-markers: Tropinin I, High sensitivity CRP (hs-CRP), Brain Natriutretic Peptide (NT Pro BNP), Homocysteine
2. Inflammatory markers: Procalcitonin, Interleukin-6 (IL-6), Tumour Necrosis Factor alpha (TNF alpha)

Figure 4: The Beckman Coulter Unicel DXC-800

Figure 5: Beckman Coulter Access 2

(Chemiluminescent immunoassay)

Figure 6: BioRad D-10 for HbA1c  (Automated HPLC)

Figure 7: BioMerieux for cardiac markers

(Immunoflorescence)

Figure 8: Brahms Kryptor for PCT (TRACE)

 Figure 9: Abbott Architect  (CMIA)

1. Tumour markers: CEA, PSA, Free-PSA, PAP, CA125, CA19-9, CA 15-3, HE-4
2. Anaemia markers and vitamins: Folic acid, Vitamin B12, soluble transferrin receptor.
3. Total Antioxidant Status (TAS) : To assess preventive capabilities against oxidative damage to various tissues
4. Antibiotics : Gentamicin, Vancomycin and Amikacin.
5. Drugs : Carbamezapine, Phenytoin, Digoxin, Theophylline, Methotrexate
6. Hormones: Cortisol
7. Immunosuppressant drug: Tacrolimus
8. Miscellaneous:Magnesium, β2 Microglobulin, Ammonia, Lactate, prealbumin, fructosamine, ACE, ADA, DHEAS, Bicarbonate, VMA, Stone analysis, Food intolerance test.

Evidence based laboratory medicine is widely used in clinical practice, and not only are the number of tests done in our department on the increase but also we are introducing new parameters whenever clinically applicable. Laboratory results influence 70 % of medical diagnoses. It becomes imperative, then, for the entire health care system including clinical chemistry laboratory to become error free and to improve performance continually. For the appropriate laboratory results the entire testing process in laboratory is divided into three phases preanalytical, analytical and post analytical(figure 10): I. Preanalytical phase of testing cycle Preanalytical phase is an important component of total laboratory quality. A large proportion of errors (70%) in laboratory medicine occurs in the pre analytical phase of testing cycle. This phase includes understanding the principles for selecting, ordering the most rational laboratory test, careful sample withdrawal and transport of sample to the laboratory. Preanalytical variables are grouped broadly under three categories-

• Physiological factors
• Specimen collection and handling
• Endogenous variables like drugs and antibodies.

Physiological Factors The effects of age, sex, time, season, altitude, conditions such as menstruation and pregnancy, and lifestyle are some of the physiologic variables that affect laboratory results. So before withdrawing a sample the test requisition form needs to be filled up properly.

Specimen Collection and Handling Multiple variables can effect the molecular integrity of the biospecimen – patient preparation, use of open or closed evacuation system for blood collection, duration of tourniquet application, anticoagulants and stabilizing additives used for blood collection, and specimen transport to the laboratory. The Department of Biochemistry has laid down policies to monitor preanalytical variables

- Compartmentlization of Duties: samples are collected & received separately from general OPD, private OPD and IPD.

- Hospital Information System (HIS) with inbuilt delta check facility helps in monitoring and checking not only preanalytical error but also help in patient result authorization. This also displays the receiving time, collection and authorization time which further helps in monitoring turn around time of laboratory results.

- Commercially available closed evacuation system from BD Vacutainer system for sample collection help in checking and reducing error related to specimen acquisition, handling and collection like incorrect tube, anticoagulant and stabilizing additives used for blood collection.

Figure 10: Procedure And Quality Management At Preanalytical Phase

- Generation of specific 2D Bar Code numbers specific for each patient decreases problems like mislabelling, missing labels from vial/vacutainer.

- Implementation of Pneumatic Tube system for ward collection helps early delivery of samples to the laboratory and also helps in reporting urgent samples for better patient care.

- As part of continuous improvement plan, the department observed the type and percentage of preanalytical errors occurring over the last three months. It has been observed that the major preanalytical error is in the form of receipt of hemolyzed samples followed by receipt of inadequate samples (figure 11). To minimize these errors, the department has made a policy for notifying inadequate samples and haemolyzed specimen immediately to the concerned hospital staff or nursing station and keep a record of the same. This helps to better patient care and also to maintain quality of the preanalytical phase.

Figure 11: Type and percentage of preanalytical error in dept of biochemistry SGRH

Last but not the least, it is difficult to establish effective methods for monitoring and controlling preanalytical variables because many of the errors are outside the traditional laboratory area. Monitoring of preanalytical variables requires coordinated effort of laboratory staff and clinical department staff to maintain a high quality of services. II. Analytical Phase in the analytical phase, it is very important to chose the right methods and reagent vendors – the process of quality assurance.

Quality assurance (QA) is an overall term that may be used to describe all measures taken to ensure the reliability of laboratory testing and reporting. Internal quality control (IQC) and external quality assessment (EQA), sometimes referred to as proficiency testing, are two distinct, yet complementary, components of a laboratory quality assurance program.

Quality Control Materials

To assess the precision of a particular method, it is necessary to perform repeated analyses of aliquots of the same sample. It is important to include quality control (QC) samples with normal and abnormal values to ensure that a method is under control at different levels of a particular analyte, since relatively minor changes in an analytical process may be more apparent when testing an abnormal control.

The control material should be similar in properties to test samples and be analysed concurrently. All vials or aliquots of the control material should be practically identical, so that any variation in test results is not a consequence of vial-to-vial variation. Also, the QC material should also be stable for its intended period of use.

1. Internal quality control (IQC) is useful to detect, reduce and correct deficiencies in the laboratory's analytical process prior to the release of patient results. Stringent practice of quality control helps laboratories to achieve better performance goals. In addition to the daily routine, IQC material should be run after an instrument is serviced, after reagents are changed, after calibration and when patient results seem inappropriate. Quality control data is interpreted by both graphical and statistical methods using the Levey Jennings(LJ) chart. The control values are plotted against their respective dates. The mean value and one, two and three standard deviation limits are also marked. The pattern of plotted points provides information of random error, shifts or trends in the control runs. In 1981, Westgard published his first concept on the multirule chart for internal quality control in clinical chemistry.4 Multirule QC uses a combination of control rules, to decide whether an analytical run is within-control or out-of-control and also detects both random and systematic errors.

In our department, the following rules are followed (figures 12-16):

Figure 12: 13s refers to a control rule when the control limits are set as mean plus 3s and mean minus 3s. A run is rejected and troubleshooting is carried out when a single control measurement exceeds the mean plus 3s or the mean minus 3s control limit.

Figure 14: 22s refers to the control rule when 2 consecutive control measurements exceed the same mean plus 2s or the same mean minus 2s control limit. This indicates systematic error and the control run for the particular analyte is rejected.

Figure 15: R4s refers to the control rule when 1 control measurement exceeds the mean plus 2s and another exceeds the mean minus 2s. This can be a random error; the control value is rejected and is subjected to troubleshooting.

Figure 16: 10x refers to the control rule when 10 consecutive control measurements fall on one side of the mean. These control reasurements are rejected.

The internal quality control system helps in immediate intervention during the release of patients' results. The laboratory personnel performing this exercise determines the appropriate corrective action to be taken for QC data that fall outside the tolerance limits. In the event of a non conformance, the following steps are taken: - decision is taken whether action is necessary

- if action is required, type of error is determined

- once error is identified, specific steps are taken to eliminate it.

IQC runs: The following is the schedule of control runs in our department, which are in accordance with requirements for accreditation: If the number of patient samples analyzed for any parameter is > 75/day, two levels of control are run twice a day at appropriate intervals.

If the number of patient samples analyzed for any parameter is > 25 but <75/day, two levels of control are run once a day for these parameters.

If the number of patient samples analyzed for any parameter is < 25/day, one levels of control is run once a day.

For low volume parameters, i.e., parameters which are analyzed once in a few days/months, one level of control is run before analyzing patient samples.

The daily control values are documented along with calculation of % CV from the monthly quality control data and the control charts are maintained.

2. External Quality Assessment is used to identify the degree of agreement between one laboratory's results and those obtained by others. In large EQA schemes, retrospective analysis of results obtained by participating laboratories permits the identification, not only of poor individual laboratory performance, but also of reagents and methods that produce unreliable or misleading results. EQAS can provide information concerning the relative performance of analytical procedures, including the method principle, reagents, and instruments (Figure 17). Continued participation in EQA schemes has been linked to improved laboratory performance. This has been seen not only in the overall performance, evidenced by a reduction of the variability of results between laboratories, but also in respect of individual laboratories.

Figure 17: EQAS report showing the CV, SD, LJ chart, histogram and performance score of one analyte

The assessment of individual laboratory performance is an essential component of EQA schemes. The EQA scheme compares participants' results with the results on the same samples when analysed in large number of labs from around the world who participate in the External Quality Assessment Scheme. There are many reasons why a laboratory might produce results that are considered unsatisfactory. While the cause for this might be immediately apparent, the identification of the underlying problem is not always simple.

Total confidentiality is an important feature of all EQA schemes.

The process of evaluation of proficiency testing is stringent and an example of the report format is given below. Since the assessing is done by a third party (i.e. a party that neither provides the analysers nor the reagents for testing and IQC), the process is not biased. This too is a requirement for accreditation.

The performance of each analyte is evaluated in terms of the result as compared to other laboratories using the same equipment/method, the variance from the true value, the methodology and the overall score in comparison with worldwide laboratories as well as the gold standard of each analyte. Our laboratory has been enrolled in an external quality assessment scheme for over a decade now. There are separate schemes for routine analytes, immunological parameters, cardiac parameters, drugs and vitamins. A bar chart of our overall score for all parameters in the latest cycle of EQAS is shown in figure 18.

Figure 18: An Accuracy Score upto 2 is rated as excellent and upto 5 is rated as very good.

Thus, IQC and EQA act as guardians of the quality of analytical phase of testing.

3. Postanalytical phase This phase is comprised of the relay of the reports from the analyzing systems to the patients or the wards. Once the faculty are assured of the quality of the assays, they release the reports through the computerized hospital information system (HIS). The turnaround time (TAT) of reporting is monitored through the HIS as well as manually. Attempt is made to minimize this TAT so as to enable better management of the patients. As is evident, quality management is a continuous process, especially for a laboratory that runs 24 x 7. Quality management and accreditation of medical laboratories recognizes the need for providing and improving quality and competence in laboratory services. The accrediting process helps medical laboratories to provide high quality service by producing accurate, precise, relevant and comprehensive data that can be applied to the medical management of patients. Academic pursuits Our hospital is a seat of extensive academics in terms of postgraduate medical courses and PhD, and so is our department. Our faculty are actively involved in several academic activities including co-supervisorship of DNB theses, resulting in frequent publications in international and national indexed journals.5,6 In the last five years, we have had 13 publications and 21 presentations.

Our department has been the first to start a PhD programme and the first PhD of Sir Ganga Ram Hospital has been awarded to one of our senior consultants, Dr.Seema Bhargava with Prof. L.M.Srivastava (Head, Department of Biochemistry, SGRH) and Prof. Arif Ali (Head, Department of Biotechnology, Jamia Millia Islamia, New Delhi) as her supervisors.

Figure 19: Ongoing research projects of our Department

Research projects

In accordance with our motto "Perfection in Patient Care", our faculty are involved in several research projects (Figure 10) which result in better patient care. Our department was again the first in the hospital to have an ICMR-funded project with Dr.Seema Bhargava as principal investigator. Since then we have had several on-going ICMR-funded and hospital-funded projects (figure 19).

Since the global burden of vascular disease, diabetes, septicemia, etc is increasing at an alarming rate7, it is imperative to focus research in these areas and, hence, our research projects are aimed at these health issues.

Publications:

Text books (chapters and editorships)

1. Textbook of Biochemistry and Human Biology. 3rd edition 2002; Ed GP Talwar and LM Srivastava. Prentice Hall of India Pvt Ltd
2. Essentials of Practical Biohemistry, 1st edition, 2002, Ed. Lalit M Srivastava, Nibhriti Das and Subrata Sinha, CBS Publishers, New Delhi.
3. Conept o Biochemistry, 1st edition 2004, Ed. L M Srivastava, CBS Pubishers, New Delhi.
4. Essentials of Inborn Errors of Metabolic and Genetic Disorders. Ed PD Gupta, K Pushkala, K Tatsumi, LM Srivastava, Anjali Manocha, Mamta kankra. Capricorn Publishing House, Jaipur. 2010
5. Srivastava L M. Advances in Clinical Proteomics towards the Discovery of Biomarkers for Diagnostics. Ind J of Clin Biochem. 2011 Dec;26:25.
6. Vandana Bhatnagar, Seema Bhargava; Principles of Diagnostic Enzymology, Concepts in Biochemistry for Medical Students, Ed L.M.Srivastava, 1st ed 2004; : 115-126.
7. Seema Bhargava Biochemistry of blood and the Regulation of pH in Molecular Physiology, e-book of National Science Digital Library, National Institute of Science Communication And Information Resources (NISCAIR), New Delhi, 2008.

Papers

1. Bhargava S, Srivastava LM. Homocysteine – Marker of the millenium – A review of its evolution and clinical implication. Journal of Medical Sciences, 2001; 4(2):104-116.
2. Bhargava S, Jain A, Gupta V. Fractional excretion of sodium – a simple test for the differential diagnosis of acute renal failure. Clinical Nephrology 2002; 58 (1):79-80.
3. Bhargava S, Parakh R, Srivastava LM. Prevalence and treatment of hyperhomocysteinemia in Occlusive Vascular Disease. Trends in Clinical Biochemistry and Laboratory Medicine, Association of Clinical Biochemists of India 2003 : 150-156.
4. Srivastava LM, Kankra M, Manocha A, Bhargava S. Evaluation of antioxidant status in the critically ill with special reference to patients of cardiovascular disorders. Trends in Clinical Biochemistry and Laboratory Medicine, Association of Clinical Biochemists of India 2003 : 600-604.
5. Bhargava S, Parakh R, Srivastava LM. Studies on homocysteine demonstrating its significance as a possible tool for differential diagnosis in occlusive vascular disease. Indian Journal of Clinical Biochemistry 2004; 19 (1) :76-78.
6. Bhargava S, Parakh R, Manocha A, Kankra M, Aggarwal CS, Menon G, Srivastava LM. High Prevalence of hyperhomocysteinemia among Indian patients of vascular disease. Clinica Chimica Acta 2006; 374 (1-2): 160-162.
7. Bhargava S, Parakh R, Manocha A, Arif A, Srivastava LM. Prevalence of hyperhomocysteinemia in vascular disease – a comparative study of thrombotic venous disease vis-à-vis occlusive arterial disease. Vascular 2007; 15(3): 149-153.
8. Das S, Bhargava S, Manocha A, Kankra M, Srivastava LM. Procalcitonin – A specific and reliable marker of sepsis. CCLM 2007; W366 June, 2007.
9. Manocha A, Bhargava S, Kankra M, Das S, Srivastava LM. Hypertriglyceridemia in a 3-month infant – possibly LPL deficiency. Adv in Med Dent Sci, 2008; 2(2):50-52.
10. Manocha A, Bhargava S, Kankra M, Das S, Srivastava LM. Quality control evaluation using quality indicators – Experience of a Clinical Laboratory in Delhi. IJCB 2009; 24:203.
11. Das S, Bhargava S, Manocha A, Kankra M, Srivastava LM. The potential significance of procalcitonin and interleukin-6 in prediction of survival in patients with sepsis. IJCB 2009; 24:191.
12. Srivastava LM. Immunology. Foreword. First foreword 2011.
13. Singla P, Parkash A A, Bhatacharjee J. Preanalytical error occurrence rate in clinical chemistry laboratory of a public hospital in India. Clin. Lab. 2011; 57: 749-752.
14. Srivastava L M. Advances in Clinical Proteomics towards the Discovery of Biomarkers for Diagnostics. Ind J of Clin Biochem. 2011 Dec;26:25.
15. Das S, Bhargava S, Manocha A, Kankra M, Ray S, Srivastava L M. The prognostic value of hypocholesterolemia in sepsis. Asian J Pharm Biol Res 2011, 1(1), 41-46.
16. Anand D, Das S, Srivastava LM. Procalcitonin – a novel sepsis marker. Accepted for publication in Asian Journal of Pharmaceutical and Biological Research, 2011.
17. Bhargava S, Ali A, Parakh R, Saxena R, Srivastava LM. Higher Incidence Of C677T Polymorphism Of The MTHFR Gene In North Indian Patients Of Vascular Disease. Vascular 2012; 20(2): 88-95.
18. Bhargava S, Ali A, Bhargava EK, Manocha A, Kankra M, Das S, Srivastava LM. Lowering homocysteine and modifying nutritional status with folic acid and vitamin B12 in Indian patients of vascular disease. Journal of Clinical Biochemistry and Nutrition 2012; 50(3): 222-226.
19. Singla P, Parkash AA, Lal H, Nanda S. Benefits of vitamin d supplementation in pregnancy for prevention of preeclampsia. Int J Pharm Bio Sci 2012; 2 (3): 144-150.

Key Words: Cardiac risk factors – lipid profile, apolipoproteins, lipoprotein(a), homocysteine Diabetic profile – glucose tolerance test, glycosylated hemoglobin, fructosamine. 

Anemia profile – vitamin B12, folate, ferritin, soluble transferrin receptors

Tumour markers – HE4, PSA, CA 125, CEA, CA 19-9, CA 15-3 Steriods – DHEAS, cortisol

ADA, ACE, bile acids, prealbumin, methotrexate, Total Antioxidant Status (TAS), PCT, NT

Pro-BNP, CKMB-Mass, Troponin I, Tacrolimus.