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Biochemistry

TOTAL PROTEIN

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TOTAL PROTEIN (Biuret Method) INTENDED USE:The reagent kit is intended for in vitro quantitative determination of Total Protein in serum/plasma. CLINICAL SIGNIFICANCE:Proteins are conjugations of amino acids, enzymes, hormones and several other kinds of molecules, structural entities in the body. They are involved in the maintenance of the normal distribution of water between blood and the tissues. Considering many of albumin and globulin in the blood has many diagnostic and analytical values as several fractions are involved in many (diseases). Decreased levels are found mainly in malnutrition, hepatic synthesis, protein losses as in hemorrhage or excessive protein catabolism. PRINCIPLE:Proteins, in an alkaline medium, bind with the cupric ions present in the biuret reagent to form a blue-violet colored complex. The intensity of the color formed is directly proportional to the amount of proteins present in the sample. REACTION:Total Protein + Cu²⁺ → Violet complex. CONTENTS:Reagent 1: Biuret ReagentReagent 2: Protein Standard 6 g/dl MATERIALS REQUIRED BUT NOT PROVIDED: Clean Dry Glassware Laboratory Glass Pipettes or Micropipettes & Tips Colorimeter or Bio-Chemistry Analyzer SAMPLES:Serum, Heparinized/EDTA Plasma. Proteins are reported to be stable in the sample for 6 days at 2–8°C. PREPARATION OF REAGENT & STABILITY:All reagents are stable till the expiry date mentioned on the label at standard room temperature.If stored at 2–8°C, this is stable till expiry when not in use.All reagents are ready to use form. GENERAL SYSTEM PARAMETERS: Wavelength: 546 nm (530–570 nm) Temperature: 25°C Light Path: 1 cm Assay Type: End Point Reaction Time: 5 min Standard Concentration: 6 g/dl Zero setting: Reagent Blank Sample Volume: 10 µl Reagent Volume: 1.0 ml PROCEDURE:Pipette into clean dry test tube labeled as Blank (B), Standard (S) and Test (T): Addition sequence Blank (B) Standard (S) Test (T) Biuret Reagent 1.0 ml 1.0 ml 1.0 ml Distilled Water 10 µl — — Standard — 10 µl — Sample — — 10 µl Mix and incubate for 5 minutes at Room Temperature. Measure the absorbance of Standard and Sample (Aₛ) against the Blank (Aᵦ) at 546 nm. CALCULATION:Total Protein Conc. (g/dl) =(Aₛ / Aₛₜ) × 6 NORMAL VALUE:Serum: 6.0–8.0 g/dlIt is recommended that each laboratory establish its own normal range. LINEARITY:This procedure is linear up to 10 g/dl. Samples above this concentration should be suitably diluted and results should be multiplied by dilution factor. QUALITY CONTROL:For accuracy it is necessary to run known controls with every assay. LIMITATION & PRECAUTIONS: Storage condition mentioned on the kit must be adhered to. Do not mix reagents from different lot numbers. Maintain clean dry glassware for accurate results. Samples showing hemolysis are to be avoided. Avoid contamination of reagents with even traces of saliva (as causes false high absorbance due to dirt). BIBLIOGRAPHY: Henry, T.R., Clin. Chem., Am. J. Clin. Path., 16:40 Henry, T.R., Cannon, D.C., Winkleman, J.W., Clinical Chemistry, Principles and Techniques, Harper & Row, 2nd Edition, 1974. CODE NO / PACK SIZE / REAGENT 1 / REAGENT 2217 / 1 × 100 ml / 1 × 5 ml217A / 1 × 50 ml / 1 × 3.0 ml

Biochemistry, Uncategorized

SERUM ALBUMIN

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SUMMARY: Albumin a major plasma protein is synthesized in the liver from amino acids, which are absorbed from the liver. Its function includes regulation of distribution of extracellular fluid, transportation of various hormones, steroids, and amino acids. Aim: Estimation of Serum Albumin by BCG (Bromocresol Green) method. PRINCIPLE: Albumin binds with bromocresol green at pH 4.2 causing a shift in absorbance maximum of the Yellow BCG dye. The resulting bluish-green color is measured photometrically. The intensity of the color is directly proportional to the albumin concentration. The absorbance of the test and standard are measured against blank at 630 nm wavelength. Albumin + BCG → Albumin-BCG complex (blue-green color) REQUIREMENTS: Three test tubes Colorimeter with Automatic Pipetter Total Protein working reagent Distilled water Cuvette Pipette Serum Sample PROCEDURE: Contents Blank (ml) Standard (ml) Test (ml) Working reagent 1.0 1.0 1.0 Distilled water 1.0 – – Standard – 0.1 – Serum – – 0.1 For further help WhatsApp us: +91-9891068072 INSTRUCTIONS: Measure all content according to the chart in the test tubes. Mix well and incubate for 5 minutes at 37°C temperature in incubator. Read the optical density of the test and standard at 630 nm wavelength. Calculation: Serum Albumin (g/dL)= (Optical Density of Test × Concentration of Standard) / (Optical Density of Standard) Example Calculation: After testing:Optical Density of Test = 0.318Optical Density of Standard = 0.24 Therefore:= (0.318 × 4.0) / 0.24= 5.3 g/dL Normal Values: Parameter Normal Range Total Protein 6.0 – 8.0 g/dL Albumin 3.5 – 5.0 g/dL Globulin 2.5 – 3.5 g/dL A/G Ratio 1.0 – 2.0 Clinical Significance: Increased levels of albumin are present in cases of dehydration, especially noted for newborns.Decreased levels of albumin are present in conditions like malnutrition, nephrotic syndrome, hepatic

Biochemistry, Uncategorized

UREA

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UREA (NED METHOD):- The reagent set is intended for in vitro Quantitative determination of Urea in Serum and plasma. CLINICAL SIGNIFICANCE:Urea is the end product of the protein metabolism. It is synthesized in the liver from ammonia produced by the deamination of amino acids. It is transported by blood to the kidneys where it is excreted. Increased values are found in renal failure, urinary tract obstruction, shock, congestive heart failure and burns. Decreased levels are found in liver failure and pregnancy. PRINCIPLE:Urea forms with ortho–phthalaldehyde and Naphthylethylenediamine in the acidic medium a coloured complex. The value of colour formed is directly proportional to the urea concentration in the test sample and is measured by a fixed time method at 550 nm. REACTION:Urea + ODA → NH₃ + H₂ONH₃ + NED → Orange Color Complex. CONTENTS:Reagent 1 : ODA ReagentReagent 2 : NED ReagentReagent 3 : Urea Standard, 50 mg/dl MATERIALS REQUIRED BUT NOT PROVIDED: Clean Dry Glassware Laboratory Glass Pipettes or Micropipettes & Tips Bio–Chemistry Analyse SAMPLES: Serum, Heparinized/EDTA Plasma. Urea is reported to be stable in the serum for 5 days when stored at 2–8°C. PREPARATION OF REAGENT & STABILITY:All the reagents are ready for use and stable till the expiry date mentioned on the label when stored at 2–8°C. GENERAL SYSTEM PARAMETERS:Reaction type: End pointWavelength: 550 nm (520–550 nm) (Increasing)Temperature: 37°CReagent Volume: 1.0 mlSample Volume: 10 µlZero setting: Against Reagent BlankLight path: 1 cm. PROCEDURE:Pipette into clean dry test tubes labeled as Standard (S) and Test (T) Addition sequence (S) (T) ODA Reagent 1.0 ml 1.0 ml Sample — 10 µl Standard 10 µl — Mix well and incubate at 37°C for 5 minutes     NED Reagent 0.05 ml 0.05 ml Mix well and read the absorbance A₁ of the standard and test after exactly 5 minutes. Read A₂ after exactly 10 minutes. The absorbance reading to be recorded at 550 nm.Finally, take the difference A₂–A₁ for both the standard and test. For Standard: ΔA = A₂S – A₁SFor Test: ΔA = A₂T – A₁T CALCULATION:Urea Concentration (mg/dl) =(ΔAT / ΔAS) × 50 NORMAL VALUE:Serum/plasma: 15–40 mg/dlEach laboratory should establish its own normal range depending on the population. LINEARITY:The method is linear upto 200 mg/dl. The value exceeding 200 mg/dl should be diluted appropriately with distilled water and the values obtained multiplied by dilution factor. QUALITY CONTROL:For accuracy it is necessary to run known controls with every assay. LIMITATION & PRECAUTIONS: Storage condition of the reagent and kit should be strictly followed. Avoid contamination of reagents. All glassware must be dry and free from detergent or debris. BIBLIOGRAPHY: Goodwin, J., Hart, T., Am. J. Chem., 26 (1977) 707 CODE NO. PACK SIZE Reagent 1 Reagent 2 Reagent 3 Z18 100 ml 1 x 100 ml 1 x 50 ml 1 x 3.0 ml   BEACON DIAGNOSTICS PVT. LTD.424, NEW GIDC, KABILPORE, NAVSARI – 396 424, INDIA    

Histopathology

Fixation in Histopathology: Types, Techniques, and Fixatives Explained

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Primary aim: preserve the morphological and chemical integrity of the cell in as life-like manner. – Shape, structure, intercellular relationship and chemical constituents of tissues are preserved. – Prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body. Secondary goal: harden and protect the tissue from the trauma of further handling MAIN FACTORS INVOLVED IN FIXATION: Hydrogen Ion Concentration – pH 6 and 8 . Temperature – Formalin heated at 60C Thickness of section – 2cm queb for light microscopy Osmolality – slightly hypertonic Concentration – low conc. of glutaraldehyde Duration of fixation – 2-6 h in buffered formalin EFFECT OF FIXATIVES harden soft and friable tissues make the cells resistant to damage and distortion inhibit bacterial decomposition increase optical differentiation of cells and tissues act as mordants or accentuators reduce the risk of infection CHARACTERISTICS OF A GOOD FIXATIVE Cheap Stable Safe to handle Kills the cell quickly producing minimum distortion of cell constituents. Inhibit bacterial decomposition Produce minimum shrinkage of tissues Harden tissues making cutting sections easier Isotonic, causing minimal physical and chemical alteration of the cells and their constituents. Make cellular components insoluble to hypotonic solutions TYPES OF FIXATIVES :- 1. According to composition : A. Simple Fixative – made up of only one component substance such as- Formaldehyde ( Most used fixative), Glutaraldehyde, Mercuric Chloride, Potassium dichromate, Chromic acid , Picric Acid, Acetic Acid, Acetone ,Alcohol, Osmium tetra oxide etc. B. Compound Fixative – made up of two or more fixatives such as Zenker’s solution, Bouins Fluid etc. 2. According to Action A. Microanatomical Fixatives – permits the general microscopic study of tissue structures such as 10% Formol Saline 10% Neutral Bufered Formalin Heidenhain’s Susa Formol sublimate Zenker’s solution Zenker formol Ouin’s solution Brasil’s solution B. Nuclear Fixative – Preserve nuclear structures such as, Flemming’s fluid Carnoy’s fluid Bouin’s fluid Newcomer’s fluid Heidenhain’s Susa C. Cytological Fixatives – preserves cytoplasmic structures such as, Flemming’s fluid without acetic acid Kelly’s fluid Formalin with “post-chroming” Regaud’s fluid (Muller’s fluid) Orth’s fluid Histochemical Fixatives – preserve chemical contents of cells and tissues such as   D. LIPID FIXATIVE – Mercuric chloride and Potassium dichromate PHOSPHOLIPIDS FIXATIVE – Baker’s formal calcium CARBOHYDRATE FIXATIVE – Alcoholic formaldehyde PROTEIN FIXATIVE – Neutral buffered formal saline or formaldehyde GLYCOGEN FIXATIVE – Rossman’s fluid or absolute alcohol Composition, Advantage, Disadvantage & Use of Fixative Formaldehyde – A. 10% formaline widely used (10% formalin) Disadvantage – fumes are irritating to the nose and eyes prolonged storage may induce precipitation of white paraformaldehyde Notes –   Removal of precipitate is addition of 10% methano B. 10% formol – Saline – – 40% Formaldehyde + NaCl + Distilled water fixation of CNS Tissues and General post-mortem tissues preserves enzymes and proteins C. 10% Neutral Buffered Formalin/Phosphate-Buffered Formalin – Sodium dihydrogen phosphate + Disodium hydrogen phosphate + 40%Formaldehyde + Distilled water Preservation of surgical, post-mortem and research specimens Best fixative for iron-containing tissues D. Formol-Corrosive (Formol Sublimate) Aq. Mercuric Chloride + 40% Formaldehyde Routine post-mortem tissues Excellent in silver reticulum methods Fixes lipids, especially neutral fats and phospholipids E. Alcoholic Formalin (Gendre’s Fixative) 95% Ethyl Alcohol saturated with picric acid + Strong formaldehyde solution + glacial acetic acid. Immunoperoxidase studies on tissues Used for rapid diagnosis Good for preservation of glycogen and for micro-incineration Used to fix sputum, since it coagulate mucus F. Glutaraldehyde two formaldehyde residues linked by 3C chains used for enzyme histochemistry and electron microscopy preserves plasma proteins 2. METALLIC FIXATIVES   A. MERCURIC CHLORIDE Mercuric Chloride + Potassium Dichromate + Sodium Sulfate + Distilled Water most common metallic fixative Tissues fixed with mixtures containing mercuric chloride (except Susa) contain black precipitates of mercury. Routine fixative of choice for preservation of cell detail in tissue photography. Renal tissues, Fibrin, Connective tissues and muscles Black deposits may be removed by adding saturated iodine solution in 96% alcohol, the iodine being decolorized with absolute alcohol in the subsequent stages of dehydration. B. Zenker’s Fluid Mercuric Chloride + Glacial Acetic Acid fixing small pieces of liver, spleen, connective tissue and nuclei may act as mordant Mercuric deposits may be removed by immersing tissues in an alcoholic iodine solution. “de-zenkerization” C. Zenker-formol (Helly’s solution)   Mercuric chloride + Potassium dichromate + Sodium sulphate + Distilled water + Strong formaldehyde (40%) Fixative for pituitary gland, bone marrow and blood-containing organs such as spleen and liver. Preserves cytoplasmic granules Brown pigments are produced if tissues are allowed to stay for more than 24 hours. Pigments can be removed by immersing the tissue in saturated alcoholic picric acid or sodium hydroxide   D. Heidenhain’s Susa Solution   Mercuric chloride + Sodium chloride + Trichloroacetic acid + Glacial Acetic Acid + Formaldehyde (40%) + Distilled water tumor biopsies especially of the skin Excellent cytological fixative Mercuric chloride deposits may be removed by immersion on alcoholic iodine solution the tissue should be transferred directly to a high-grade alcohol, to avoid undue swelling of tissues caused by treatment with low-grade alcohol or water. E. B-5 Fixative Distilled water + Mercuric Chloride + Sodium acetate Commonly used for bone marrow biopsies

CCC

Fundamentals of Computer–Complete Guide for CCC, ADCA & DCA

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Introduction to Computer कंप्यूटर एक इलेक्ट्रॉनिक मशीन है जो हमारे दिए गए निर्देशों पर कार्य करती है। कंप्यूटर हार्डवेयर और सॉफ्टवेयर से मिलकर बना है। यह एक सार्वभौमिक मशीन होती है। कंप्यूटर शब्द अंग्रेजी के “Compute” शब्द से मिलकर बना होता है और कंप्यूटर को हिंदी में संगणक कहते हैं। कंप्यूटर के कार्य करने के तीन (Step) चरण होते हैं:- इनपुट – इसमें उपयोगकर्ता के द्वारा कंप्यूटर को निर्देश या कमांड दिया जाता है। प्रोसेस – इसमें कंप्यूटर निर्देश को संसाधित करता है और डाटा को सूचना में परिवर्तित करने का कार्य करता है। आउटपुट – इसमें कंप्यूटर उपयोगकर्ता को आउटपुट प्रदान करता है। कंप्यूटर सिस्टम के प्रमुख घटक केंद्रीय प्रक्रमन इकाई (CPU)  इनपुट डिवाइस आउटपुट डिवाइस सेंट्रल प्रोसेसिंग यूनिट (CPU) : CPU का पूरा नाम Central Processing Unit है। यह यूज़र के द्वारा दिए गए निर्देशों को प्रोसेस करता है और कंप्यूटर के सभी कार्यों को नियंत्रित (control) करता है और सीपीयू को कंप्यूटर का दिमाग कहा जाता है। सीपीयू निम्न तीन यूनिट से मिलकर बना होता है – CU (Control Unit) ALU (Arithmetic Logical Unit) Memory Unit (MU) 1. CU (Control Unit) – कंट्रोल यूनिट कंप्यूटर से जुड़े हुए सभी डिवाइस और उनके कार्यों को नियंत्रित (control) करती है ताकि कंप्यूटर के सभी कार्य सही ढंग से हो सकें। 2. ALU (Arithmetic Logical Unit) – ALU का पूरा नाम अर्थमैटिक लॉजिक यूनिट होता है। यह CPU का एक महत्वपूर्ण हिस्सा है। इसका इस्तेमाल अंकगणितीय और तर्किक कार्यों को करने के लिए किया जाता है। 3. Memory Unit (MU) – यह सीपीयू का एक हिस्सा होती है। मेमोरी यूनिट का इस्तेमाल कंप्यूटर में डेटा और निर्देशों को स्टोर करने के लिए किया जाता है। कंप्यूटर की विशेषताएं: 1. Speed (गति) – कंप्यूटर के काम करने की स्पीड काफी तेज़ होती है। कंप्यूटर इंसान की तुलना में बहुत तेजी से कार्य करता है। यह एक सेकंड में एक लाख से भी ज़्यादा कार्यों को पूरा कर सकता है। कंप्यूटर की गति को हर्ट्ज़ (Hz) में मापा जाता है। 2. Accuracy (शुद्धता) – कंप्यूटर बिना गलती के किसी भी काम को पूरा करता है। मनुष्य एक काम को करने में बहुत गलतियाँ करता है, जबकि कंप्यूटर बिना गलती के अपने कार्य को पूरा कर लेता है। हालांकि जो कार्य कंप्यूटर के द्वारा किया जाए वह बहुत ही तेज़ गति से शुद्ध गणना कर देगा। 3. Memory (मेमोरी) – कंप्यूटर की मेमोरी बहुत ही शक्तिशाली होती है। हम सभी चीजों को याद नहीं रख सकते लेकिन कंप्यूटर सभी चीजों को बिना भूले याद रखता है। 4. Diligence (परिश्रमी) – कंप्यूटर के बिना थके कार्य करने की क्षमता रखता है। यह मनुष्य के अंदर नहीं है, यह कार्य थकता नहीं है जबकि मनुष्य कुछ समय तक कार्य करने के बाद थक जाता है और उसे आराम की आवश्यकता होती है। 5. Automation (स्वचालित) – कंप्यूटर एक स्वचालित मशीन है। यह अपने कार्यों को खुद से पूरा करती है। जब एक बार यह अपने कार्य की शुरूआत कर देती है तो बिना किसी मनुष्य की सहायता के इसे पूरा कर देती है। कमियाँ (Weakness): No IQ – कंप्यूटर के अंदर मानव की तरह सोचने और समझने की शक्ति नहीं होती है, इसलिए कंप्यूटर भावनाओं का गुणात्मक विश्लेषण नहीं कर सकता। No Feeling – कंप्यूटर के पास मनुष्य के तरह कोई भी भावना (feeling) नहीं होती है। History of Computer (कंप्यूटर का इतिहास) कंप्यूटर का आविष्कार आज से लगभग 2000 हजार साल पहले हुआ था। लेकिन आधुनिक कंप्यूटर को अस्तित्व में आए हुए मुश्किल से 50 वर्ष ही हुए हैं और कंप्यूटर के विकास का इतिहास काफी पुराना है। कंप्यूटर का जब नाम लिया जाता है तब हम लोग इसे एक जटिल और वैज्ञानिक मशीन मानते हैं लेकिन यह हजारों वर्षों की वैज्ञानिक खोजों और विभिन्न प्रकार के आविष्कारों से संभव हुआ है। Abacus (अबेकस) अबेकस का आविष्कार चीन में 16वीं शताब्दी में ली काई चेन (Lee Kai-Chen) के द्वारा किया गया था। इसका प्रयोग जोड़-घटाने के लिए किया जाता था। अबेकस तारों का एक ढांचा होता है। इन तारों में बंधी हुई गोलियां (बीड्स) होती हैं, जिन्हें हिलाकर अंकगणितीय गणनाएं की जाती थीं। इसे दुनिया का पहला गणना यंत्र माना जाता है और आगे चलकर इसे आधुनिक कंप्यूटर का स्थान मिला। Napier’s Bones : Napier Bones एक कंप्यूटर डिवाइस है। Napier Bones का आविष्कार स्कॉटलैंड में 1617 में जॉन नेपियर के द्वारा किया गया था। इसका प्रयोग गुणा, भाग, जोड़ और घटाव के लिए किया जाता था। यह हड्डी के टुकड़ों से बनी होती थी जिन पर संख्याएं लिखी होती थीं। इसका उपयोग करके बड़ी-बड़ी संख्याओं का गणितीय हिसाब सरलता से कर लिया जाता था। इस मशीन की वजह से Napier Bones के नाम से जाना जाने लगा। ब्लेज़ पास्कल का कैलकुलेटर (Blaise Pascal’s Calculator) : Pascal’s Calculator पहला यांत्रिक कैलकुलेटर था। इसका आविष्कार फ्रांस में 1642 में ब्लेज़ पास्कल के द्वारा किया गया था। यह उस समय का पहला स्वचालित कैलकुलेटर (automatic calculator) था। यह लकड़ी का बॉक्स था जिसमें पहियों का प्रयोग किया गया था। इन पहियों का उपयोग संख्याओं को जोड़ने, घटाने, गुणा और भाग करने के लिए किया जाता था। ब्लेज़ पास्कल की इस मशीन को Adding Machine और Pascaline मशीन भी कहा जाता है जो सबसे पहले Mechanical Calculating Machine थी। डिफरेंस इंजन (Difference Engine)  : चार्ल्स बैबेज ने सन 1822 में एक मशीन का निर्माण किया जिसका नाम उन्होंने “डिफरेंस इंजन” रखा। इस इंजन की सहायता से Algebraic Expression एवं सांख्यिकीय तालिकाओं की गणना 20 अंकों तक शुद्धता से की जा सकती थी। इस मशीन का उपयोग उस समय बैंकों, बीमा तथा व्यावसायिक क्षेत्रों में व्यापार रूप से किया जाता था और यह मशीन भाप से चलती थी। एनालिटिकल इंजन (Analytical Engine) : चार्ल्स बैबेज डिफरेंस इंजन की सफलता से प्रेरित होकर Analytical Engine को बनाया। एनालिटिकल इंजन का आविष्कार वर्ष 1830 में किया गया था। यह कंप्यूटर जैसी मशीन थी जो गणितीय कार्य को बिना मनुष्य की सहायता किए कर सकती थी। कंप्यूटर सूचनाओं को स्थायी रूप से स्टोर कर सकता था। बैबेज का यह एनालिटिकल इंजन कंप्यूटर का प्रारंभिक रूप माना जाता है और इसी के कारण उन्हें कंप्यूटर के जनक कहा जाता है। Tabulating

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hemoglobin

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Hemoglobin is a protein found in red blood cells that plays a crucial role in transporting oxygen from your lungs to the rest of your body, and carbon dioxide from your body back to your lungs to be exhaled. Key facts about hemoglobin: Structure: It’s made up of four protein subunits, each containing an iron-containing molecule called heme. The iron in heme binds to oxygen. amet, consectetur adipiscing elit. Ut elit tellus, luctus nec ullamcorper mattis, pulvinar dapibus leo. Function: Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut elit tellus, luctus nec ullamcorper mattis, pulvinar dapibus leo. In the lungs: Hemoglobin binds to oxygen. In the lungs: Hemoglobin binds to oxygen. In the tissues: Hemoglobin releases oxygen and picks up carbon dioxide. Color: It gives red blood cells—and blood—their red color. Oxygenated hemoglobin is bright red; deoxygenated is darker.  

Hematology

Top 5 Common Anticoagulants in Laboratory Practice: EDTA, Heparin & More

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The anticoagulant prevents the clotting of blood. It is used in medical laboratories complete blood or plasma is required, depending upon the test to be done, the type of anticoagulant is decided. Most anticoagulants prevent clotting by removing calcium or iron, which are necessary for the clotting process. Every anticoagulant is added in fixed amount to blood. Anticoagulants are of following types: A – Chemical Anticoagulants These anticoagulants are prepared in the laboratory. These are as follows. Sodium Citrate CPD – Citrate phosphate dextrose EDTA – Ethylene diamine tetra acetic acid Oxalate Sodium Fluoride  (I)  Citrate:- Trisodium citrate –This anticoagulant is prepared by dissolving 3.8 g of trisodium citrate in 100 ml of distilled water. 0.4 ml anticoagulant is required for 2.0 ml of blood.Adding dextrose to anticoagulants provides nutrition to the red cells and helps in longer storage. Such anticoagulant is known as Acid Citrate Dextrose (ACD) because along with citric acid, trisodium citrate and dextrose are mixed in this anticoagulant. This anticoagulant is used in the solution form, as it is in the blood bank. The storage of blood with this anticoagulant is a maximum up to 21 days. (ii) CPD (Citrate Dextrose) In this anticoagulant, the citrate is dissolved in phosphate buffer, which maintains pH more accurately than ACD solution. The dextrose present in this provides nutrition to other cells. The storage period of CPD is 21 days. The disadvantage is that it is costly and difficult to prepare and adjust ph. (iii) EDTA (Ethylene Diamine Tetra-acetic Acid) It can be prepared by dissolving 10 gm of EDTA in 1000 ml of distilled water. 0.4 ml of anticoagulant is required for 2 ml of blood. This anticoagulant does not disturb the cellular structure. Therefore, it can be used for blood cell count, ESR, estimation, etc. EDTA however, cannot be used for biochemical tests. This anticoagulant is dried in the container by keeping in an incubator or hot air oven at 80 OC for an overnight period, i.e. for 12 hrs. The storage period with EDTA is 2 to 3 days. EDTA Advantage It is a powerful calcium-chelating agent. Used in concentrations of 1.5 to 2.0 mg (anhydrous) per ml of blood Dipotassium salt is preferred over disodium salt. Blood collected in EDTA can be used for TLC, PS preparation, Hb and DC. Disadvantage Excess of EDTA causes shrinkage of WBCs and RBCs and induces degenerative changes. It is unsuitable for coagulation studies. EDTA blood fails to demonstrate basophilic stippling of RBCs in lead poisoning. Activates naturally occurring antiplatelet autoantibodies, which cause platelet adherence to neutrophils.   (iv) Oxalate It is prepared by dissolving 1.2 gm of ammonium oxalate and 0.8 gm of potassium oxalate in 100 ml of distilled water.  0.2 ml (4mg) of anticoagulant is required for 2 ml of blood. This anticoagulant may disturb cellular structure if kept for a longer period; however, if used immediately, it can be used for the estimation of bilirubin, and prothrombin time, and estimation of blood cells, PVC (Packed cell volume). This anticoagulant is dried in a container by keeping it for an overnight period in a hot air oven. It is also called double oxalate.   (v)Sodium Fluoride It complexes with calcium to form calcium fluoride. 6 mg is used for 6 ml of blood. It is useful in the estimation of blood glucose levels (Fluoride prevents glycolysis by blocking phosphorylase enzymes in RBCs).   Biological (Natural) Anticoagulant – Heparin Heparin is the only biological anticoagulant, which cannot be prepared in a laboratory. It is obtained from leech. It is a good anticoagulant and does not alter size of RBC. It is used in concentration of 10-15 units / ml blood. This anticoagulant act by destroying thrombin or thromboplastin required for clotting. Heparin is used to determine the blood gases. It can be used for ESR, PVC, osmotic fragility and other hematological tests. Note:- Anticoagulant should be sterilized before use, and then blood is added to the anticoagulant, it should be mixed gently by inverting it 10 to 15 times or shacking it gently

Hematology

Improved Neubauer’s Chamber: How It Works, Grid Design & Clinical Use

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A Neubauer chamber is an important tool in the field of hematology and cell biology. It is a specialized counting chamber designed for the manual counting of cells, such as blood cells or sperm cells, under a microscope. Here are some key aspects related to Neubauer chambers: Grids:The Neubauer chamber has a central counting area and four corner counting area divided into grids. These grids help in systematic counting of cells. Cover Slip:The counting chamber is covered with a glass coverslip, which is essential for creating a consistent depth for the cells to be examined. Chamber Depth:The chamber has a specific depth 0.1mm that allows for an accurate volume of the sample to be loaded. Uses:Neubauer chambers are commonly used for manual cell counting, such as red and white blood cells, sperm cells, or any other cells present in a liquid sample.Concentration Calculation: Cleanliness: Keep the Neubauer chamber and coverslip clean and free from dust or debris. Clean them with a mild detergent and rinse thoroughly with distilled water.  

Hematology

What Are RBC and WBC Pipettes?

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RBC Pipette The RBC pipette is generally known as the thoma pipette or diluting pipette used for the dilute of blood in ratio of ‘1:100’ or ‘1:200’ for the Hemocytometer. The main use of the RBC pipette is as a liquid dispenser. It has been used in hematology laboratory. There is a red color bead present in this pipette bulb and it has marking of 0.5, 1 and 101. WBC Pipette WBC pipette has white bead in the bulb and it has marking of 0.5, 1 and 11. Used for the dilute of blood in ratio of ‘1:20 for the Hemocytometer. Cleaning RBCs & WBCs Pipette Cleaned by filling the pipette with distilled water and blowing it out twice. Finally, it will be filled with acetone or alcohol and blown out. Acetone or alcohol removes the water from it and dry it completely.

Hematology

Micro-Pipette: Types, Uses, and Handling Techniques in the Laboratory

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Micro-Pipette A micropipette is an essential laboratory instrument used for accurately measuring and transferring small volumes 1ml or less than 1ml of liquid. The three important parts of a micropipette are the plunger button, the volume adjustment dial, and the disposable tip. Plunger Button: The plunger button is pressed to aspirate (draw in) and dispense (release) the liquid. It is important to press the plunger button smoothly and steadily to avoid introducing air bubbles into the liquid. Volume Adjustment Dial: This part allows users to set the desired volume for aspiration and dispensing. It is crucial to set the volume accurately according to the requirements of the experiment. Disposable Tip: The tip is a crucial part of the micropipette. It comes in various sizes and should be selected based on the volume to be measured. Tips are disposable and should be changed for each new sample to prevent cross-contamination. Uses of Micro Pipettes: Micropipettes are commonly used in various scientific and medical applications, including: Molecular Biology: Used for DNA, RNA, and protein analysis. Clinical Diagnostics: Used for handling small volumes of blood, serum, or other biological fluids. Microbiology: Used for culturing and transferring small volumes of microbial samples. Chemistry: Used for preparing solutions and measuring reagents. Caring for Micro Pipettes: Proper care is essential to maintain the accuracy and longevity of micropipettes: Calibration: Regularly calibrate the micropipette to ensure accurate volume measurements.  Tip Usage: Use high quality, compatible tips, and change them for each new sample. Cleaning: Clean the external surfaces regularly with a mild detergent or disinfectant. Avoid Overloading: Do not exceed the maximum volume limit specified for the micropipette. Handle with Care: Avoid dropping or banging the pipette, as it can affect its accuracy.

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