Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Prepared By
Dr. Showkat Ahmad Bhat,
Assistant Professor,
Department of Biochemistry, Govt. Medical College Doda
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Laboratory Techniques and Equipment in Medical Biochemistry: A Comprehensive Guide, Study notes of Laboratory Practices and Management
A comprehensive overview of laboratory techniques and equipment commonly used in medical biochemistry. It covers essential topics such as sterilization methods, chemical bonding, solution preparation, and safety protocols. Particularly valuable for students and professionals in the field of medical biochemistry, offering detailed explanations and practical insights into laboratory practices.
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Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Prepared By
Dr. Showkat Ahmad Bhat,
Assistant Professor,
Department of Biochemistry, Govt. Medical College Doda
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Pre-Analytical Variables: A large number of variables can potentially affect the results
of a laboratory test and these can be divided into:
- Pre-analytical variables
- Analytical variables
- Post-analytical variables
There is increasing evidence that an awareness of the significance of the pre-analytical
variables that can potentially affect a lab test, will ensure the reliability of the final
result. Pre-Analytical Variables
These may be divided into three broad categories: Category Variables Sample Collection (^) Correct patient identification and sample labeling Correct phlebotomy technique Correct sample volume which may involve taking into account the patient's testing Correct s ample collection tube Sample Handling Storage Mixing & centrifugation Transport conditions and delays in transport Patient-specific Factors
Physiological variables Pathological states
Unit – I
Specimen Collection
Chapter at a Glance
The Learner will be able to answer questions on following topics Pre-Analytical Variables Collection of Blood Collection of CSF & other Fluid Urine Collection Use of preservatives Anticoagulants
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Arterial blood is rarely required and may be collected from radial, brachial, or femoral artery. Capillary blood may be collected from the tip of the thumb or finger or from ear lobe. Samples can even be taken for analysis during a surgical procedure.
Potential effects of unsafe phlebotomy: Unsafe phlebotomy can cause adverse effects for patients; such effects are rare, but range from pain or bruising at the site of puncture, to fainting, nerve damage and haematoma. Precautions Blood should be collected from venipuncture after cleaning the skin area with a cleaning agent. Prolonged venous stasis should be avoided to avoid prolonged rise in the concentration of large molecules like proteins. Hemolysis should also be avoided by avoiding dirty and wet tubes, repeated freezing/thawing and suction of syringe.
Collection of blood: - for the collection of the blood sample, following things are required:
1. The technician should be very well trained.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
2. Preparation of specimen collection materials. Disposable syringes and needles. Disposable lancets. Gauze pads or adsorbent cotton. Tourniquet. 70% (v/v) ethanol (or isopropanol). clean and dry wide mouth bottles 50 and 100 ml(called wiles) 3. Patients should be prepared. 4. Laboratory forms: - it should provide the following information: Patient‘s full name. Age and sex. Identification number. Test of specific tests. Urgent tests which are required for the immediate cases. Name of the physician ordering the test.
Collection: Check the paper works and tubes. The tubes and bulbs should be checked for appropriate kinds and for paper labeling. Select vein site. For most venipuncture procedures. The medium cubital vein is the one used for the patient. If the vein puncture of this vein is unsuccessful then one of the cephalic or basilica veins may be used. Cleaning the area by the sprit or 70% ethanol to prevent any contamination. Inspect the needles and syringes.
Storage and transport of samples The samples must be processed as early as possible. If delay is inevitable then sample must be stored properly. Concentration of most of the blood constituents remains stable up to one week at 4ºC. For longer periods it should be stored at - 20ºC or -80ºC. Specimen should be transported as soon as possible in the sterile conditions, usually within 2 hours. Passage of time affects concentration of many blood constituents once it is shed from the body.
Collection of CSF & other Fluid
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
(j) Proteins: 20 mg% whereas plasma has 6-8 gm%
CSF formation
In brain, there are four ventricles. They are two lateral ventricles (one each in cerebral hemispheres) and third and fourth ventricles. Choroid plexuses present more abundantly in lateral ventricles than in 3rd or 4th ventricles. These choroid plexuses (capillary network) are main source of formation of CSF. CSF is formed by two different processes, namely active secretion and ultrafiltration: Active secretion is brought about by choroid plexus. Ultrafiltration or dialysis is brought about by blood vessels that are present in ventricular regions
Circulation of CSF:
1. From lateral ventricles, CSF reaches 3rd ventricle through foramina of Monroe. 2. From 3rd ventricle to 4th ventricle, it reaches through cerebral aqueduct or aqueduct of Sylvius. 3. From 4th^ ventricle, it reaches spinal canal through foramen of Magendie and into subarachnoid spaces throughout CNS through foramina of Luschka. 4. CSF formed gets absorbed after its function is over. Most of CSF gets absorbed into arachnoid villi and granulations which dip into subdural venous sinuses.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Figure BI 11.15.2: The extension of spinal card and subarachnoid space
Subarachnoid spaces can be reached either by lumbar or cisternal puncture. Lumbar puncture is preferred because cisternal puncture requires insertion of needle into subarachnoid spaces near brainstem region. In the process, there are chances of brainstem neurons getting damaged. It is much easier to perform lumbar puncture to get a sample of CSF for diagnostic purposes (Figure BI 11.15.2).
Contents and Pressure of CSF CSF contains more sodium (Na+) and chlorine (Cl-) than blood, but less potassium, (K+), calcium (Ca++), proteins, and glucose. Normal CSF glucose is usually 2/3 of blood glucose. Normal CSF protein is 15-60 mg/100 ml and contains no red blood cells (RBC) or clotting factors and only 0-3 white blood cells (or WBC). CSF is in a closed system, so pressure is an important component of analysis. When lying down, the pressure is 10-18 cm H2O, or 8-15 mm Hg. In the sitting position, it is 20-30 cm H2O, or 16-24 mm Hg.
Blood-Brain Barrier: When trephan blue (an acidic dye) is injected into blood all tissues of body get stained except for certain regions in brain tissue. There is a barrier preventing diffusion of dye from blood into brain tissues and this barrier is known as blood-brain barrier. i) Present in CNS. ii) Provides protection to maintain constant internal environment in CNS.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
3. Interstitium of brain (microenvironment in brain) is maintained so that excitability of neuronal tissue is achieved and properly maintained. 4. Acts as shock absorber: Prevent damage to neuron in CNS because any damage to neurons in CNS is almost irreparable. When there is any impact on cranium, since brain is suspended in fluid environment of CSF, CSF acts as water cushion. The impact on head is dissipated to wider area and thereby direct impact on underlying brain tissue is minimized. 5. Tries to maintain contents of intracranial cavity as constant (relative volume of blood, neuronal tissue and CSF). Cranial vault is made of bony structures and hence cranial cavity volume is fixed. In this fixed volume, neuronal tissue, blood and CSF are present. If there is an increase or decrease in volume of either neuronal tissue or blood, corresponding changes in volume occupied by CSF is brought about.
CSF
CSF pressure is measured by lumbar puncture (LP) or spinal tap. The skin is anesthetized and a thin needle is introduced below the end of the spinal cord, allowing removal of CSF without damaging the spinal cord. Elevated pressure usually indicates swelling, infection, or mass (tumor or clot). Elevated CSF protein may indicate meningitis, other infections, tumors, or neurologic diseases. Analysis of protein types is needed for more definitive diagnosis. In infection and inflammation, there are increased WBCs as the body tries to fight the disease. In bacterial meningitis, the CSF glucose is decreased, as the bacteria use up the glucose for nutrition during growth. Composition of CSF in normal state and various disease conditions is present as shown in Table BI 11.15.A.
Table BI 11.15.A: Composition of CSF in normal state and various disease conditions Disease Colour &appearance
Cell count Protein Sugar Coagulation
Normal Clear and colourless
0 – 4 × 10^6 /l 10 – 30 mg/dl
50 – 70 mg/dl
Not seen
Bacterial meningitis (purulent meningitis)
Opalescent or turbiddue to high cell content.
Markedly increased Polymorphs
Marked increase
Marked decrease
May clot on standing
Tuberculosis meningitis May be opalescent Lymphocytes and Mononuclear
Lymphocyte s andmononuc lear cells
Increased Low but not very much decreased
Cobweb type Coagulation
Viral infection Clear and colourless
Increased Increased Normal Nil
Brain tumour Clear and Within Increased Low Solidifies
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
colourless normal range Subarachnoid Haemorrhage
Blood stained in freshHaemorrhage
RBCs and WBCs
Increased Not significant
Nil
CSF Collecting Methods Lumbar puncture (LP) It is the method for obtaining CSF sample for laboratory analysis. A special needle is introduced from posterior aspect into subarachnoid spaces of spinal cord between L3 and L vertebrae. Between L1 and L2 vertebrae, spinal cord ends and, therefore, when needle is introduced between L3 and L4 vertebrae injury to spinal nerves is prevented (Figure BI 11.15.3). Suspected CNS infections e.g. meningitis. Suspected Sub arachnoid haemorrhage with a negative CT scan. Suspicion of GBS or other CNS diseases. Non urgent indications Idiopathic intracranial hypertension (pseudo tumour cerebri) Carcinomatous meningitis. Tuberculosis meningitis Normal pressure hydrocephalus CNS syphilis CNS vasculitis Conditions in which LP is rarely diagnostic but still useful Multiple sclerosis Gullain Barre syndrome Para neoplastic syndromes LP is required as a therapeutic maneuver in the following situations Spinal anaesthesia Intrathecal administration of chemotherapy Injection of contrast for myelography or for cisternography. Contraindications for LP Although there are no absolute contraindications for LP however precautions should be taken in case of: Possible risk of increased intracranial pressure with risk of cerebral herniation. Thrombocytopenia or other bleeding disorders including ongoing anticoagulant therapy. Suspected spinal epidural abscess.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
iv) To induce spinal anesthesia.
Urine Collection
Urine specimens may be collected in a variety of ways according to the type of specimen required the collection site and patient type.
1. Randomly Collected Specimens : Randomly collected specimens are suitable for urinalysis in the clinical chemistry laboratory and for microscopic analysis. However, they are not regarded as specimens of choice because of the potential for dilution of the specimen when collection occurs soon after the patient has consumed fluids. In this situation, analyte values may be artificially low. Of necessity, pediatric urine specimens for urinalysis and microscopy are often of this type. 2. First Morning Specimen is the Specimen : First morning specimens are the specimen of choice for urinalysis and microscopic analysis since the urine is generally more concentrated (due to the length of time the urine is allowed to remain in the bladder) and, therefore, contains relatively higher levels of cellular elements and analytes. Abnormal constituents are also likely to be present in higher concentration and, thus, more likely to be detected. 3. Midstream Clean Catch Specimens : Midstream specimens are strongly recommended for microbiological culture and antibiotic susceptibility testing because of the reduced incidence of cellular and microbial contamination. Following instruction from a healthcare professional, patients are required to follow a prescribed procedure commencing with cleansing the urethral area. The patient should then void the first portion of the urine stream into the toilet. These first steps significantly reduce the opportunities for contaminants to enter the urine stream during collection of the clinical specimen. The urine midstream is then collected into a clean container after which the remaining urine is voided into the toilet. This method of collection can be conducted at any time of day or night. 4. Timed Collection Specimens : Timed specimens may be required for quantitative measurement of certain analytes, including those subject to diurnal variation. Analytes commonly tested using timed collection include creatinine, urea, potassium, sodium, uric acid, cortisol, calcium, citrate, amino acids, catecholamines, metanephrines, vanillylmandelic acid (VMA), 5 - hydroxyindoleacetic acid, protein, oxalate, copper, 17 - ketosteroids, and 17 - hydroxysteroids. A timed collection allows measurement of the excretion of these substances in urine over a specified length of time, usually, but not always, 8 or 24
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
hours. In this collection method, the bladder is emptied prior to beginning the timed collection. Then, for the duration of the designated time period, all urine is collected and pooled into a collection container, with the final collection taking place at the very end of that period. Half an hour before the end of the collection period, it is helpful to ask the patient to drink a glass of water, so that the last urine specimen can be obtained. If no specimen is produced, then the total volume and time of collection cannot be determined. It is also important to caution the patient not to lose urine specimens to the toilet during defecation. When a 24-hour urine specimen is required for the assay of catecholamines, metanephrines and/or VMA, for the diagnosis of pheochromocytoma, which causes persistent or episodic hypertension, it is advisable to monitor the blood pressure of the patient and collect the urine specimen when the blood pressure is high, in order to improve the chance of a positive finding. Timed specimens should be refrigerated during the collection period, unless otherwise directed by the laboratory. Accurate timing is very important as this information forms a critical part of the calculations performed to determine urine clearance values (e.g. creatinine clearance). Interpretations based on faulty calculations can result in improper diagnoses or medical treatment.
5. Collection from Catheters : Urine specimens can be collected from catheters (e.g. Foley catheter) using a syringe, followed by transfer to a specimen tube or cup. Alternatively, urine can be drawn directly from the catheter to an evacuated tube using an appropriate adaptor. Direct draw adaptor for urine specimen collection from Foley catheter 6. Supra-pubic Aspiration : Supra-pubic aspiration may be necessary when a non- ambulatory patient cannot be catheterized or where there are concerns about obtaining a sterile specimen by conventional means. This procedure involves collection of the specimen by needle aspiration through the abdominal wall into the bladder. 7. Pediatric Specimens : Urine collection from pediatric patients presents many challenges. For infants and small children, a special urine collection bag can be adhered to the skin surrounding the urethral area. Once the collection is completed, the urine is poured into a collection cup or transferred directly into an evacuated tube with a transfer straw. Urine collected from a diaper is not
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Use of preservatives
Anticoagulants
1. Ethylenediamine Tetra Acetic acid (EDTA) This anticoagulant preserves the cellular components well; it is used for hematological examinations. It is used as sodium or dipotassium salt. The dipotassium salt is preferred because it is more soluble compared to disodium salt. EDTA acts by its chelating effect on the calcium molecules in the blood.1. mg (app. 4 M mol) of anhydrous salt per ml of blood. Excess EDTA affects both red cells and leucocytes, causing shrinkage and degenerative changes. EDTA is not suitable for use in the investigation of coagulation problems and should not be used in the estimation of prothrombin time. 2. Oxalates Sodium, potassium, ammonium and lithium oxalates inhibit blood coagulation by forming insoluble complexes with calcium ions. Potassium oxalate is used in combination with sodium fluoride for blood used for glucose determination because sodium floride prevents glycolysis by inhibiting the enzyme systems involved in glycolysis. 3. Trisodium citrate Sodium citrate solution, at a concentration of 3.4 or 3.8 g/dl in a ratio of 1 part to 9 parts of blood is widely used for coagulation studies i.e. Study in the coagulogram. Since the effect is easily reversible by addition of Ca. Trisodium citrate prevents blood coagulation by chelating with calcium. (But it inhibits enzyme activities like SGPT, SGOT and anabolic phosphate. 4. Heparin It is available as sodium, potassium, lithium and ammonium salt, it is used at a concentration of 15 ± 2.5 in per ml of blood. It is dry anticoagulant. However heparinized blood is not used for making blood films as it gives a faint blue coloration to the background when the films are stained. Heparin prevents consolation of blood by acting as antithrombin to prevent the transformation of prothrombin to thrombin and thus the formation of fibrin from fibrinogen. 5. Acid – citrate Dextrose solution:- This is preferred for blood transfusion for preserving red cells, for enzyme studies. Standard preparation:-
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Sodium citrate 1.32 g Citric acid 0.40 g Dextrose 1.40 g and Distilled water 100 ml. And of ACD is sufficient to prevent coagulation of 4 ml of blood.
Vacutainers used in biochemical analysis Blood sampling vacuum-containing tubes /Vacutainer tubes arebeing widely used for venous blood sampling. Blood is collected by creating a negative pressure at the sight of collection_._ These evacuated blood collection systems or vacutainers are used to ensure leak proof, sterile and closed blood collection pathway. The evacuated tube system for blood collection in use for various laboratory tests consists of tubes of various sizes, with color coded tops indicating tube contents. Most blood collection tubes contain an additive that either accelerates clotting of the blood (clot activator) or prevents the blood from clotting (anticoagulant). Color coded Vacutainer tubes are used according to the specimen type required namely serum, plasma or whole blood_._
1. Red The red bottle is less common – it is used for biochemistry tests requiring serum which might be adversely affected by the separator gel used in the yellow bottle. Additive : None or contains silica particles which act as clot activators. What additive does : Clot activator promotes blood clotting with glass or silica particles. Laboratory Uses : Serum testing (glucose, cholesterol, triglycerides, HDL, potassium, amylase, alkaline phosphatase, BUN, CK, liver enzymes), blood bank, serology (RH Typing, Antibody screening, Red Cell Phototyping, DAT, RPR, monospot, rheumatoid factor, ANA) 2. Yellow Additive : anticoagulant SPS (Sodium Polyanetholsulfonate) & ACD (acid citrate dextrose) What additive does : Prevents the blood from clotting and stabilizes bacterial growth. Laboratory Uses : Blood and bodily fluid cultures (HLA, DNA, Paternity) Tubes with SPS – For Blood and bodily fluid cultures (HLA, DNA, Paternity). The SPS aids in the recovery of microorganisms by slowing down/ stopping the
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
What additive does : Forms calcium salts to remove calcium Laboratory uses : paediatric ESR
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Glass ware in laboratory
Commonly used glass ware in laboratory Name Use Glass ware/Apparatus/ equipments Beaker (^) Beaker are used to hold, mix, and heat liquids
Buret Burets are used for dispensing an accurate volume of a liquid.
Unit – II
Laboratory apparatus: Different types, use and maintenance
Chapter at a Glance
The Learner will be able to answer questions on following topics Glass ware in laboratory – significance of boro-silicate glass. Plastic ware in laboratory, cleaning of glass and plastic ware. Pipettes – glass and automated Burettes, beakers, Petri dishes, porocelain dish Flasks – different types (volumetric, round bottomed, Erlenmeyer, conical etc. Funnels – different types (conical, Buchner etc) Bottles – reagent, wash bottles Measuring cylinders, reagent dispensers Tubes – test tube, centrifuge tube, folin-wu tube Cuvettes and its use in measurements, Cuvettes for visible and UV range Racks – bottle, testube, pipette and draining racks Tripod stand, wire gauze, Bunsen burner, desiccators, stop watch, timers