Blood and Tests on Blood Cells

ByK.Sudarshan

M.Tech EI 1st yearR.No.314206522010

In larger animals including humans the nature has provided a special transport system for the exchange of chemical products between different organs-it is blood circulation.

The circulatory system of adult human consists of about 5 liters of blood.

Introduction

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Functions of Blood

Transport of: ◦ Gases, nutrients, waste products◦ Processed molecules◦ Regulatory molecules

Regulation of pH and osmosis Maintenance of body temperature Protection against foreign substances Clot formation

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Composition of Blood

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Plasma Liquid part of blood

◦ Pale yellow made up of 91% water, 9% other Colloid: Liquid containing suspended

substances that don’t settle out◦ Albumin: Important in regulation of water

movement between tissues and blood◦ Globulins: Immune system or transport

molecules◦ Fibrinogen: Responsible for formation of blood

clots

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Formed Elements Red blood cells (erythrocytes) White blood cells (leukocytes)

◦ Granulocytes Neutrophils Eosinophils Basophils

◦ Agranulocytes Lymphocytes Monocytes

Platelets (thrombocytes)

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Erythrocytes Structure

◦ Biconcave, anucleate

Components◦ Hemoglobin◦ Lipids, carbonic

anhydrase Function

◦ Transport oxygen from lungs to tissues and carbon dioxide from tissues to lungs

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Hemoglobin

Consists of: ◦ 4 globin molecules: Transport carbon dioxide

(carbonic anhydrase involved), nitric oxide◦ 4 heme molecules: Transport oxygen

Iron is required for oxygen transport

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LeukocytesProtect body against microorganisms and remove dead cells and debris

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Thrombocytes

These are masses of protoplasm 2-4 micrometer in diameter.

They are colourless and have no nucleus

Important in preventing blood loss

◦ Promoting formation and contraction of clots

Many diseases cause characteristic changes in composition of blood.

These variations can be ◦ Change in number◦ Size ◦ Shape of certain blood cells◦ Change in composition of blood serum

So chemical analysis of blood can provide information about the diseases.

Blood

Centrifugal sedimentation method:◦ In this method the blood sample is centrifuged,

making the blood cells sediment and form a packed column at the bottom of test tube.

◦ The volume of the packet red cells is called HEMATOCRIT.

It is expressed as a percentage of total blood Volume.

Tests on blood cells

By comparing the lengths of the column of capillary tube we can obtain % of cell volume i.e HEMATOCRIT

Centrifugal sedimentation method

Centrifugal sedimentation method

This method is also called conductivity method for blood cell count

The principal used here is blood cell have less electrical conductivity than the solution in which they are present.

It is an automated method for cell count

Coulter method

Coulter method

The count is completed in less than 20 seconds. With counts in the range of 100,000 the results

are statistically accurate.

The process is automated and the latest counters based on this principle are able to ◦ Determine count in 20 seconds◦ Performs dilution automatically◦ Determines not only RBC WBC count but also Hct, Hb,

MCV, MCH, MCHC.

Coulter method

It is another type of blood cell counter.

It uses the principle of Dark field microscope.

It consists of a thin cuvette in which diluted blood flows, a lamp , a ring aperture, lens , aperture and a phototube.

Dark Field Method

Dark field method

Dark Field Method

Blood serum is a complex fluid that contains numerous substances in its solution

The determination of these substances is performed by specialized chemical techniques

Most of the tests used are based on CHEMICAL Color reaction followed by a colorimetric determination of concentration

This principle makes use of the fact that many chemical compounds in solution appear colored, with saturation of color depending on the concentration.

Chemical Tests

Principle of Colorimeter analysis

The relation is expressed in terms of transmittance(T) and absorbance(optical density - A).

T = I1 / I0 *100 A = -log(I1 / I0) = log(1/T).

The amount of light absorbed depends only on the number of molecules in the solution that can interact with the light, which leads to the following equation:

A = aCL - Beer’s law

Colorimeter analysis

The absorbtivity can be obtained by measuring absorption of a solution with known concentration called standard.

Cu / Au = Cs / As

Colorimeter Analysis

In order to use the colorimeter to determine concentration of a substance, a suitable method for obtaining a colored derivative from the substance is necessary.

Thus a chemical reaction that is unique for the substance to be tested and that does not cause interference from the other substance must be found.

For this reactions may require several steps of adding reagents and incubating the sample at elevated temperatures.

CHEMICAL TESTS

Chemical tests

For most of the above said tests they can be performed by the color reaction.

In case of sodium and potassium a different property is utilized, one that causes a normally colorless flame to appear◦ Yellow for sodium◦ Violet for potassium

CHEMICAL TESTS

Flame Photometer

Spectrophotometer

Most chemical tests consists of steps like pipetting, diluting and incubating.

They are rather time consuming and require skilled technicians to prevent errors.

So attempts were made to automate these chemical tests.

The first automatic analyzer that found wide acceptance is AUTOANALYZER.

Automation of Chemical tests

Continuous Flow Analyzer

Continuous flow analyzer

The smallest model of autoanalyzer can perform a single test of upto 120 samples per hour.

Large later models perform upto 12 different type of tests on each of 90 samples per hour.

The major problem with continuous flow process is ‘carryover’

Autoanalyzer

Discrete type autoanalyzers use discrete samples to analyze.

Some of these perform the tests in test tubes mounted on a carrier or chain-belt with the test tubes being rinsed after the analysis.

These type of antoanalyzers use syringe-type pumps to dispense the sample and add the reagents.

After incubation the sample is aspirated into a colorimeter where its absorbance is measured

Autoanalyzers –Discrete type

Discrete type analyzers as well as continuous type analyzers require that all reagents be properly diluted.

One automatic analyzer uses a principle that always uses the correct dilution.

In this analyzer all reagents given for a test are sealed in premeasured quantities in dry form in packages.

Autoanalyzers

Autoanalyzers

Reagents Autoanalyzer

Autoanalyzer

Rotochem 2 parallel fast analyzer

The basic problem with automatic analyzers is the positive identification of samples.

In early devices the small sample cups were identified only by their position in the sample tray and the technician had to prepare a load-list for this purpose.

Now machine readable methods for sample identification are available and greatly reduce the mixups.

Autoanalyzers

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