Sikkim Manipal University

Assignment BLOO43

Roll No-621132459(Vipin Pant)4rh semester

1) What are acid-base disorders? Explain its biochemical findings. Add a note on regulation of acid-base balance

Ans- Acid–base imbalance is an abnormality of the human body's normal balance of

acids and bases that causes the plasma pH to deviate out of the normal range (7.35 to

7.45). In the fetus, the normal range differs based on which umbilical vessel is

sampled (umbilical vein pH is normally 7.25 to 7.45; umbilical artery pH is normally

7.18 to 7.38).[1] It can exist in varying levels of severity, some life-threatening. Classification

A Davenport diagram illustrates acid–base imbalance graphically.

An excess of acid is called acidosis and an excess in bases is called alkalosis. The

process that causes the imbalance is classified based on theetiology of the disturbance

(respiratory or metabolic) and the direction of change in pH (acidosis or alkalosis).

This yields the following four basic processes:

process pH carbon dioxide compensation

metabolic acidosis down down respiratory

respiratory acidosis down up renal

metabolic alkalosis up up respiratory

respiratory alkalosis up down renal

]Mixed disorders

The presence of only one of the above derangements is called a simple acid–base

disorder. In a mixed disorder more than one is occurring at the same time.[2] Mixed

disorders may feature an acidosis and alkosis at the same time that partially counteract

each other, or there can be two different conditions affecting the pH in the same

direction. The phrase "mixed acidosis", for example, refers to metabolic acidosis in

conjunction withrespiratory acidosis. Any combination is possible, except concurrent

respiratory acidosis and respiratory alkalosis, since a person cannot breathe too fast

and too slow at the same time.

Explain its biochemical findings-: Biochemists usually discuss acids and bases in terms of their ability to donate and

accept protons; that is, they use the Brønsted definition of acids and bases. A few

concepts from general chemistry are important to help organize your thoughts about

biochemical acids and bases:

1. A compound has two components — a conjugate acid and a conjugate base.

Thus, you can think of HCl as being composed of the proton-donating acidic

part (H+) and the proton-accepting basic part (Cl−). Likewise, acetic acid is

composed of H+ and the conjugate base (H3CCOO−).

2. The stronger the acid, the weaker its conjugate base. Thus, HCl is a stronger acid

than acetic acid, and acetate ion is a stronger base than chloride ion. That is,

acetate is a better proton acceptor than is chloride ion.

3. The strongest acid that can exist in appreciable concentration in a solution is the

conjugate acid of the solvent. The strongest base that can exist in a solution is

the conjugate base of the solvent. In water, the strongest base that exists is OH−.

If a stronger base, such as NaOCH3, is added to water, the methoxide ion rapidly

removes protons from the solvent:

4.

leaving the base OH- as the strongest base in solution. (Don't try these reactions

at home; they are highly exergonic!) The strongest acid that can exist in water in

appreciable amounts is H3O+, the conjugate acid of H2O:

5. Weak acids and bases — those less strong than H+ or OH− — exist in equilibrium

with water:

Regulation of acid-base balance. – 1. Chemical Buffer system: – Responds within seconds

– Does not eliminate or add H+ from body

– Operates by binding or to tied up H+ till balance is reestablished.

a. In ECF: – Mainly HCO-3/CO2 Buffer system

– Plasma Proteins

– HPO–4/H2PO-4 Buffer system

b. In ICF: – Proteins Mainly e.g.: Hb in RBCs

– HPO–4/H2PO-4 Buffer system

Routes of excretion of acids; lungs & kidneys

2. Respiratory Mechanisms: – Responds within minutes

– Takes 6-12 hours to be fully effective

– Operates by excreting CO2 or (adding H2CO3/HCO-3)

3. Renal Mechanisms: • Responds slowly (effectively in 3-5 days)

• Eliminates excess Acids or Base from body

• The most powerful mechanism

e.g. i. HCO-3/CO2 Buffer system

ii. NH3/NH+4 Buffer system

iii. HPO–4/H2PO-4 Buffer system

Chemical Buffer System

• Consists of a ‗pair of substances‘ present in a mixture of a solution that ‗minimizes

pH changes‘ when an ‗acid or base‘ is ‗added or removed‘ from the solution.

• Consists of; 1. Carbonic Acid – Bicarbonate Buffer System

2. Phosphate Buffer system

3. Protein Buffer system

Chemical Buffer System of ECF

1. Bicarbonate Buffer System: H2CO3/NaHCO3 consists of H2CO3 (weak Acid) + NaHCO3 (Bicarbonate salt)

– CO2 + H2O ↔H2CO3 ↔ H+ + HCO-3

– NaHCO3 ↔ Na+ + HCO-3 → H2CO3 → CO2 + H2O

Bicarbonate buffer system is quantitatively the most powerful ECF buffer system

Its two components HCO-3 & CO2 are precisely regulated by kidneys & lungs.

2. Phosphate Buffer System: – Not of major importance in ECF

– Only 8% of the conc. of HCO-3 Buffer system

– Comprised of HPO–4/H2PO-4

– Plays major role in ICF & in Renal tubules

3. Proteins: (ICF proteins, Hb, Plasma proteins) – Excellent buffers as proteins contain both Acidic & Basic groups.

– More important in ICF H2CO3 ← H2O + CO2

HCO-3 + H+ + HbO2 ↔ H.Hb + O2

– In RBCs, Hb is important

– 60-70% of total chemical buffering of body fluids inside the

cells & in ICF is by proteins.

– Hb buffers H+ ions generated by H2CO3

– Proteins are the most abundant buffers in cells & in blood

– Histidine and Cysteine are the two A. Acids that contribute

most of the buffering capacity of proteins

Respiratory Mechanisms in Regulation of Acid-Base

• Second line of defense against acid base disturbances

• Operates through regulation of ECF CO2 concentration by lungs

• Effectiveness between 50-75% [feedback gain is 1-3 i.e. fall in pH

from 7.4 to 7.0 is returned by Resp System to 7.2 to 7.3 within 3-12

minutes]

2) Discuss the advantages of automation in clinical biochemistry

laboratory. Make a list of few (at least five) automated instruments available for biochemical analysis. Discuss the principle of each .

Ans- Automation in clinical biochemistry laboratory- Clinical pathology or

laboratory medicine has a great influence on clinical decisions and 60–70% of the

most important decisions on admission, discharge, and medication are based on

laboratory results.1 As we learn more about clinical laboratory results and incorporate

them in outcome optimization schemes, the laboratory will play a more pivotal role in

management of patients and the eventual outcomes.2 It has been stated that the

development of information technology and automation in laboratory medicine has

allowed laboratory professionals to keep in pace with the growth in workload.3 In a

paper on ―robotics into the millennium,‖ the various types of automation have been

outlined4 while other authors have classified laboratory automation into total

laboratory automation, modular laboratory automation, and workcell/workstation

automation.5,6

This article evaluates the relationship of scientific staff, automation, and expert

systems in clinical chemistry with particular reference to the core laboratory and

ascertains staff requirements. The changes in work practices due to the introduction of

automation and computers in other industries are discussed and similarities with

clinical chemistry elucidated as it has been noted that the original total laboratory

automation was based on the manufacturing/factory model of production.7 Others

have also written on automation in various industries over the last century and how

the patterns of its implementation and effects can be applied to pathology.8 The goal

of a successful automation must be to change the way in which work is done in the

laboratory and this involves changing not only the tools and processes, but also the

job structure and ultimately the way people think about their work.7 The progress in

automation and convergence of technologies are two key factors, which particularly

affect how we think about the future of clinical chemistry.9 The role of the scientific

staff, use of automation and expert systems shall be discussed for a core laboratory

focusing on the Monash Medical Centre, Melbourne, Victoria, Australia where one of

the authors is based. It is our belief that to consolidate changes that are advocated,7 it

is important to look at skill requirements and training of the operatives in clinical

chemistry.

Four, flow type automatic biochemical analyzer

Flow type automatic biochemical analyzer can be divided into air staging system and

non segmented system. The former is a most typical flow analyzer.

(a) air staging system

The analyzer is through proportional pump extrusion elastic sample tube, air pipe and

the reagent tube (commonly known as the "tube"), the sample sequentially inhalation

and transported along the sample tube, on the other hand, by the air pipe into the

bubble will be determined by the same principle of inhalation and reagent

continuously flowing in pipeline is divided into segments of the reagent uniform,

sample and reagent flow in the process of continuous flow forward encounter, mixing,

absorption through (when necessary), thermal insulation, reaction and measured. The

analysis process is the flow process of continuous flow in the pipeline.

(two) non segmented system

Non segmented system is the reaction liquid, depending on the reagent blank or buffer

to interval of each sample, the continuous flow in the pipeline liquid can not be

segmented. Non segmented system can be divided into system and space system for

flow.

1, flow injection system

The system composition and air segmentation system is similar, but some structure

and working principle of different, air staging system is the use of the bubble to

prevent cross contamination of the reaction liquid in the pipeline in the flow process

of segmentation, and flow injection system is through the sample are injected to

prevent cross contamination to continuous flow reagent flow in a pipe.

2, clearance system

The system structure, composition and working principle and flow injection system is

similar, but its characteristic is each sample must be in the analysis process after the

end of the previous samples (including pipeline cleaning) to start, but not

continuously in turn into the sample, a time gap between each sample, it is person is

not continuous flow analyzer.

Principle-: air staging system

With increasing use of biomass in combustion processes, the reduction of the

related NOx

emissions which originate mainly from the fuel nitrogen becomes more and

more important.

Efficient primary measures for NOx reduction are staged combustion

techniques. Air staging

has been investigated earlier and has found its way into practice. Since fuel

staging has not

been applied with nonpulverized biomass yet, the aim of the present work was

to investigate the

potential of fuel staging for NOx reduction in fixed bed systems. For this

purpose, a prototype

understoker furnace of 75 kW thermal input with two fuel beds in series was

developed.

Experiments were performed with wood chips (low nitrogen content) and UF-

chipboards (high

nitrogen content) to investigate the influences of the main process parameters,

i.e., stoichiometric

ratio, temperatures, residence time, and fuel properties on the conversion of

fuel nitrogen to

N-species. The most important parameters were found to be the temperature

and the stoichiometric ratio in the reburn zone. The potential of fuel staging was

measured and compared with

air staging and unstaged combustion. The experiments show that low NOx

emissions are already

achievable with fuel staging at lower temperatures than with air staging, i.e.,

900-1000 °C, and

at a stoichiometric ratio of 0.85 in the reduction zone. The NOx reduction

achieved under optimum

conditions for UF-chipboard as main fuel was 78% which is higher than with

air staging, where

72% NOx reduction was measured. For wood chips both measures attained

about 66%. The

nitrogen conversion during air and fuel staging has also been simulated using

a furnace model

based on ideal flow patterns as perfectly stirred reactors and plug flow

reactors. A detailed reaction

mechanism including the nitrogen chemistry (GRI-Mech 2.11) was

implemented. The trends found

with this model are in good agreement with the experiments and they indicate

that even higher

NOx reduction may be reached with improved process design. The

investigations show that fuel

staging is a promising technology for NOx reduction also for fixed bed

biomass furnac

3) What is quality control? What are its components? Add a note on implementation of external quality control in various biochemical tests

Ans- quality control-:

The automated analyzers in clinical laboratories Nowadays, the overwhelming

majority of laboratory results in clinical laboratories is being generated by automated

analyzers. Modern automated analyzers are highly sophisticated instruments which

can produce a tremendous number of laboratory results in a very short time. This is

achieved thanks to the integration of technologies from three different scientific

fields: analytical chemistry, computer science and robotics. The combination of these

technologies substitutes a huge number of glassware equipment and tedious, repetitive

laboratory work. As a matter of fact, the laboratory routine work has diminished

significantly. Today laboratory personnel‘s duties have been shifted from manual

work to the maintenance of the equipment, internal and external quality control,

instrument calibration and data management of the generated results.

Components-:

The purpose of a control is to aid the operator in deciding whether an analytical

system is producing reliable results for a given assay, and ultimately whether to

release the results. This unit presents information on the techniques for determining

when results are in control or out of control.

External quality assessment

This is the evaluation by an outside agency of the performance by a number of

laboratories on specially supplied samples. Analysis of performance is retrospective.

The objective is to achieve between lab and between method compatibility, but this

doesn‘t guarantee accuracy unless the specimens have been assayed by a reference lab

alongside a reference preparation of known value. Schemes are usually organized on a

national or regional basis. Hence, EQA is mainly concerned with analytical part of the

test.

4) List various methods used for estimation of serum calcium. Explain the principle and procedure of any one method

Ans- estimation of serum calcium-: Calcium is the most abundant mineral1

and fifth

most common element in the body.2 Almost all blood calcium is present in

plasma and reference range

is 2.10 to 2.65 mmol/L. It is present as free or ionized (50%), protein bound

usually with albumin

(40%) and complexes with small anions (10%). Calcium is needed for bone

mineralisation, blood coagulation and influences the permeability and excitation

of plasma membranes. It is usually monitored

for hypoparathyroidism, hyperparathyroidism, vitamin D deficiency,

malnutrition, cancers, enhanced

renal retention, osteoporosis, etc.

There are many different methods for estimation of serum calcium like

spectrophotometeric, ion

selective electrode (ISE) and atomic absorption methods. The

spectrophotometric techniques use metallochromic indicators which change

color when

they bind to calcium. Arsenazo III and o-Cresolphthalein Complexone (CPC)

methods are the two spectrophotometric techniques frequently used.

Aim of our study was to compare serum calcium

estimation by CPC method using direct colorimetric

and volume / volume colorimeteric (v/v) methods.

The principle of CPC method is that calcium reacts

with CPC in an alkaline medium to form a red coloured complex. This complex

is measured at a wavelength at 570 nm. Sample is diluted with aci to release

protein bound and complexed calcium. Diethylamine, 2-amino-2-methyl-1-

propranolol or 2-

ethylaminoethanol is added to buffer the solution

and provide an alkaline medium.3 Effect of magnesium can be minimised either

by adding 8-hydroxyquinolone, buffering the solution to pH of around

12 or by measuring absorbance at 580 nm.

MATERIALS AND METHODS

The study was performed in a tertiary care laboratory in Rawalpindi from

March to June ‘2011. It was

a prospective comparative study. Seventy quality

control samples of Randox laboratories were used of

these thirty five were normal controls while thirty

five were abnormal controls. Controls were tested

simultaneously on both the kits provided by SS diagnostics using a fully

automated chemistry analyser

(Selectra E). Data was recorded using specially designed proformas and results

were analysed using

SPSS version 17.

RESULTS

Our results showed that in normal control samples

with a target of 2.33 mmol/L where the range of

calcium was 2.10 to 2.56 mmol/L. The v/v method

kit gave the mean result of 2.34 mmol/L ± 0.04

with a CV of 1.70%. With the direct calorimetric

Principle of GOD-POD-

Intended Use

The reagents are used for the quantitative determination of

Glucose in serum or plasma. For in-vitro diagnostic use only.

Introduction

Glucose is the reducing monosaccharide that serves as the

principal source of cellular energy in the body. It enters into the

cell under the influence of insulin and undergoes a series of

chemical reactions to produce energy. Lack of insulin or

resistance to its action at the cellular level causes diabetes.

Therefore, in diabetes mellitus the blood glucose level are

very high. However, high blood glucose level is also observed

in the pancreatitis, pituitary or thyroid dysfunction, renal

failure and liver disease whereas low glucose level is

associated with starvation, hyperinsulinaemia, neopalasms

or insulin induced hypoglycemia.

Method

GOD-POD method, End Point.

Principle

Glucose is oxidized by glucose oxidase(GOD) to produce

gluconate and hydrogen peroxide. The hydrogen peroxide is

then oxidatively coupled with 4 amino- antipyrene(4-AAP)

and phenol in the presence of peroxidase(POD) to yield a red

quinoeimine dye that is measured at 505nm. The absorbance

at 505 nm is proportional to concentration of glucose in the

sample.

Glucose +2H2O + O2

Gluconate + H2O2

2H O + 4-AAP + Phenol Quinoeimine Dye 2 2

Absorbance of the colored solution is directly proportional to

the glucose concentration, when measured at 505nm