SETTING UP HEMATOLOGY AND BIOCHEMISTRY LAB

Dr. Avinash Phadke

President

SRL & Piramal Labs

A VIEW TO HAEMATOLOGY

ErythrocyteLeukocyte

Thrombocytes

HAEMATOLOGY

Haematology - the scientific study of blood, its formation and disease

HEMATOLOGY Hematology, also spelled haematology (from the

Greek αἷμα haima "blood" and -λoγία), is the branch of internal medicine, physiology, pathology, clinical laboratory work, and pediatrics that is concerned with the study of blood, the blood-forming organs, and blood diseases.

Hematology includes the study of etiology, diagnosis, treatment, prognosis, and prevention of blood diseases.

The laboratory work that goes into the study of blood is frequently performed by a medical technologist.

Blood diseases affect the production of blood and its components, such as blood cells, hemoglobin, blood proteins, the mechanism of coagulation, etc

AUTOMATION IN HEMATOLOGY

Cell counting is reliable in automated method than manual method, since large number of cells a re counted rapidly.

Although better precision is obtained by automated cell counting, simultanoeus accuaracy can be obtained by microscopy.

ADVANTAGES -AUTOMATION

Automated cell counters determines 8-40 different parameters of a complete hemogram, which are not possible by manual methods.

Samle recognition by barcode, automatic mixing of ample & diluent.

Automated cell counters are calibrated and controls are available for precision & accuracy.

DIFFERENT PRINCIPLES …. VARIOUS TECHNOLOGIES FOR DIFFERENTIAL ENUMERATION

Impedance measurement Flow-cytometry Fluorescence Flow-cytometry

TYPES OF ANALYSERS-1.SEMI-AUTOANALYSERS

Semi-automatic analysers ( where the sample is diluted manually and only few parameters like Hb, PCV, WBC and or Platelets are given)

3-PART ANALYSERS

Sample is aspirated directly and diluted by the equipment. Parameters depending on the equipment are given with calculated indices …18-22 parameters.

Differential count graph is plotted approximately. Manual differential is indicated for WBC and morphology of RBC).Abnormal results are flagged . They indicate possibility of blasts, promyelocytes or myelocytes.

Flags or alerts indicate further examination of the specimen

IMPEDANCE MEASUREMENT

The principle takes advantage of the fact that blood cells are less conductive to electrical current than the cell diluting fluid.

Each change in the current is measured as pulse;indicating passage of a particle through the aperture. This allows the cells to be counted and the magnitude of pulse is proportional to the size of each cell.

IMPEDANCE METHOD

Vacuum

Resistance

(ca. 100 V)

Internal electrode External electrode

Aperture

Blood suspension

IMPEDANCE METHOD

Limitations

Only size not sufficient to differentiate five types of WBCs

Interference from NRBCs, Lyse Resistance RBCs etc.

Inability to identify abnormal cells Low linearity

CBC

ERRORS ….WHICH CAN OCCUR

Due to fibrin clots Sample deterioration Sample leakage(concentrated sample) Hemolysed sample

5-PART ANALYSERS

.Along with the parameter s of 3 part , WBC differentials are plotted on the graph.

Depending on the technology which is used either a laser or scattered light technology or flow cytometry based method.

Newer parametrs like RDW, MPV are also given by these equipments.

RDW:RDW in conjunction with RBC count and MCV is useful in interpretation of several hematological disorders.

MPV is derived from platelet histogram . There is an inverse elationship between number & size of platelets.Various hematological contions like ITP, aplastic states, B12 and folate deficiencies .

BASIC LASER FLOW CYTOMETRY

BASIC LASER FLOW CYTOMETRY

Limitations

Ability to differentiate only basic five types of WBCs

Limited ability to identify abnormal cells – hence unable to separate abnormal cells from normal cells

Interference from NRBCs & Lyse Resistance RBCs

LATEST ....BENEFITS OF 6-PART DIFF ANALYSIS

Reduces review rate significantly

User definable setting for diff review is still possible (IG > 2%, 3%, 4% or 5%)

Reduces operators‘ costs (FTEs)

Reduces TAT

Increases precision of the reported WBC differential (thousand of cells counted)

FLOURESCENCE FLOW CYTOMETRY

When the laser light beam strikes a flourescent stained cell, the light of various intensity is scattered with respect to the physical characteristics of the cell.

i.e the following characteristics are determined:

Cell size, Cell volume from forward scatter

Internal structure, e.g. granulation or vacuoles from side scatter

Content of nucleic acids, i.e. RNA and DNA from side flourescence

FLUORESCENCE FLOW CYTOMETRY

LASER FLUORESCENCE FLOW CYTOMETRY

Benefits

5 Part Differentiation of WBCs using patented Polymethine dye

Unmatched linearity of WBC from 0 - 4,40,000 cells / µL

Clear cut demarcation between normal and abnormal cells

Covers almost all the clinical conditions Dilution of samples is not required Interference from lyse resistance RBC’s

eliminated NRBC’s & PLT clumps are flagged and

quantified

DIFFERENTIAL CHANNEL FLUORESCENT STAIN EFFECT LASER (BY FLUORESCENCE FLOW CYTOMETRY )

A surfactant lyses and dissolves RBCs and PLTs and perforates WBCs.

Afterwards, Fluorescence stainer, polymethine dye enters perforated WBCs and stains their nucleic acids (RNA/DNA).

Reactive cells (ATL, IG ) absorb more stain and emit more flourescence

DIFFERENTIAL SCATTERGRAM(LASER FLUORESCENCE FLOW CYTOMETRY )

DIFFERENTIAL SCATTERGRAM (BY FLUORESCENCE FLOW CYTOMETRY )

SUMMARY Flourescence Flow Cytometry ascertain

Accurate 5 part Differentials Precise demarcation between normal &

abnormal cells Eliminates interferences from lyse resistance RBC’s,

NRBC’s, microcytic RBC’s & PLT clumps Identification of Morphological abnormalities such

as IG, ATL & Blasts

CLINICAL UTILITY OF IG COUNT

• Presence of IG in blood indicates a response to infection, inflammation or some other stimulus to the bone marrow.

• XE IG master provides accurate and valuable information for immediate action.

58 ICU PATIENTS

58 ICU PATIENTS

Through the use of the Sysmex XE-2100 analyser the IT Ratio could be used as

a FAST INEXPENSIVE RELIABLE indicator of sepsis

Reticulocyte Count• Ret # , Ret %

RET-He (Hemoglobinization of Reticulocytes)• Reflects the actual iron supply for hemoglobin synthesis in the bone marrow. • Early detection of iron depletion in erythropoiesis.• Distinguish Fe def (ID) and functional Fe Def (FID). • In FID the iron stores are replete, but the iron is not sufficiently

available for Hb systhesis

RET-Quantity

RET-Quality

Clinical Significance of RET-He

Clinical Untility of RET-He

Diagnostic• Help to distinguish classical iron deficiency and functional

iron deficiency in anemia of chronic disease• Detect early state of iron deficiency when biochemical

markers are influenced (acute-phase response, pregnancy)

Therapeutic• Monitoring of erythropoietin and / or iron therapy

Reference range : 28 - 35 pg

COAGULATION …

Equipments available are manual , semiautomatic and fully automatic equipments.

Parameters like PT, PTT, APTT , Fibrinogen ,FDP are routinely done in a medium size laboratory.

Blood coagulation factors like Factor V, VIII, Thrombin etc are availbale on fully automated equipments with good quality control data and calibrations.

AUTOMATION IN BIOCHEMISTRY

Why automation is required?

Since last few decades, in clinical biochemistry there has been increase in clinical demand for laboratory investigations.

When the volume of work is increased, there arose a need for work simplification.

Automation helps us to give correct results in short TAT

AUTOMATION IN BIOCHEMISTRY

What is automation?

Automation is a self – regulating process , where the specimen is accurately pipetted by a mechanical probe and mixed with the particular volume of the reagent and the results are displayed in digital forms and also printed by the printer.

DEVELOPMENT OF AUTOMATION IN BIOCHEMISTRY Initially Mono-step methods were introduced

to replace multistep cumbersome and inaccurate methods.

The efficiency of mono-step methods was further increased by the introduction of automatic dispensers and diluters.

In large labs for the common tests the above mentioned approach is still inadequate. Thus, instruments were developed which can handle the whole analytical process in a mechanized manner.

HISTORICAL ASPECTS OF AUTOMATIONFirst automated system was introduced in 1957

by L. T. Skeggs. 1.Initially Continuous flow analyzers– Two types Single channel continuous flow analyzers Multi channel continuous flow analyzers e.g.

SMA 2.Discrete Auto-analyzers- These analyzers co-

ordinate multiple operations into a smoothly functioning system.

Semi automated Fully automated

TYPES OF ANALYZERS

Semi automated analyzers- These analyzers are called semi automated,

because the initial stages of a specimen analysis are performed by Laboratory technician. These analyzers are suitable for medium complexity level laboratories.

SEMI AUTOMATED ANALYZERS

SEMI AUTOMATED ANALYZERS

SEMI AUTOMATED ANALYZERS

SEMI AUTOMATED ANALYZERS

TYPES OF ANALYZERS

Fully automated discrete analyzers- These auto analyzers perform all the functions

of semi automated analyzers and in addition to that , they also perform many functions like automatic dispensing of reagents & samples, mixing & incubation of reaction mixtures.

BATCH ANALYSERS & RANDOM ACCESS ANALYSERS

Batch analyzers perform only one type of test at a time.

Advantages…large no of sample batches can be tested accurately & precisely with appropriate quality control

Disadvantages…Not patient oriented , not equippped for stat samples, not equipped with facilities like “random access analysers”

RAA(RANDOM ACCESS ANALYSER)

RAA perform all the functions of batch analyser & additionaly they are equipped with random access mode .

Additional facilities like : cuvette disk with temperature control , on board refrigerator , level sensors for samples and reagents,sample rack system, bar code identification, continous loading of samples, faciltiy for autodilution, automatic plotting of daily & monthly QC charts.

DRY CHEMISTRY…

Analysers do not use wet chemicals, the chemicals are incorporated in to a series of thin films on a single use slide.

Patients samples permeated through the various layers on the slide and the end results are determined colorimetrically

Sample carry over is avoided because test recation takes place entirely within the slide.

FLEX TECHNOLOGY & DISPOSABLE CUVETTE TECHNOLOGY…

Analysers are discrete, random access clinical chemistry analysers, use test reagents as flex, have integrated multisensor technology to provide accurate and precise test results.

For each tests, a cuvette is manufactured on board and sealed and discarded seperately for each parameter

BIOCHIP ARRAY TESTS…

Biochip array consists of a single biochip which has many reagents coated.

Multiple tests can be done from a single chip. Convinient for panels of tests on a single

sample

AUTOMATED FLOOR MODELS

Continous sample loading facility Ion –selective electrode options On board laundry for cuvettes Facility for urgent samples Measures icteric, hemolytic or lipaemic index

ADVANTAGES OF AUTOMATION Large number of samples can be tested in a

short time. Variety of tests can be performed by using

various methods such as end point and rate of reactions.

Most of the methods performed on automation are accurate , precise, sensitive and specific.

Although the various types of auto-analyzers are expensive, in the long run , they prove to be cost effective because he amount of reagent and specimens required can be as low as 300 and 5 micro-liters respectively.

ADVANTAGES OF AUTOMATION Automation allows laboratories to process

much larger workload without a comparable increase in number of staff members.

Internal and external quality control programs can be implemented efficiently and effectively by using auto-analyzers.

In the case of fully-automated analyzers, the laboratory staff members do not come in contact with specimens and reagents and hence working on these analyzers is very safe.

THANK YOU