Setting up Hematology and

Biochemistry lab
Dr. Avinash Phadke
President
SRL & Piramal Labs
A vie to Haematology
Erythrocyte
Leukocyte
Thrombocytes
Haematology
Haematology ! the scienti"ic study o"
blood# its "ormation and disease
Hematology

Hematology# also spelled haematology $"rom the %reek

haima &blood& and !'o()*+# is the branch o"
internal medicine# physiology# pathology#
clinical laboratory ork# and pediatrics that is concerned
ith the study o" blood# the blood!"orming organs# and
blood diseases.

Hematology includes the study o" etiology# diagnosis#

treatment# prognosis# and prevention o" blood diseases.

,he laboratory ork that goes into the study o" blood is
"re-uently per"ormed by a medical technologist.

Blood diseases a""ect the production o" blood and its

components# such as blood cells# hemoglobin#
blood proteins# the mechanism o" coagulation# etc
Automation in hematology

.ell counting is reliable in automated method than

manual method# since large number o" 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 /!01 di""erent

parameters o" a complete hemogram# hich are not
possible by manual methods.

Samle recognition by barcode# automatic mi2ing o"

ample & diluent.

Automated cell counters are calibrated and controls are

available "or precision & accuracy.
Di""erent principles 3. Various
Technologies for Differential
Enumeration

4mpedance measurement

Flow-cytometry

Fluorescence Flow-cytometry
,ypes o" analysers!5.Semi!autoanalysers

Semi!automatic analysers $ here the sample is diluted

manually and only "e parameters like Hb# P.6# 7B.
and or Platelets are given+
8!part analysers

Sample is aspirated directly and diluted by the

e-uipment. Parameters depending on the e-uipment are
given ith calculated indices 35/!99 parameters.

Di""erential count graph is plotted appro2imately.

:anual di""erential is indicated "or 7B. and
morphology o" RB.+.Abnormal results are "lagged .
,hey indicate possibility o" blasts# promyelocytes or
myelocytes.

;lags or alerts indicate "urther e2amination o" the

specimen
4mpedance measurement

,he principle takes advantage o" the "act that blood cells
are less conductive to electrical current than the cell
diluting "luid.

<ach change in the current is measured as

pulse=indicating passage o" a particle through the
aperture. ,his allos the cells to be counted and the
magnitude o" pulse is proportional to the si>e o" each
cell.
Impedance Method
Vacuum
Resist ance
(ca. 100 V)
I nt er nal elect r ode Ext er nal elect r ode
Aper t ur e
Blood suspension
Impedance Method
Limit at ions

Only size not suf f icient to dif ferentiate f ive types

of WBCs

I nterf erence f rom NRBCs, Lyse Resistance RBCs

etc

I nabi!ity to identif y abnorma! ce!!s

Lo" !inearity
CBC
<rrors 3.hich can occur

Due to "ibrin clots

Sample deterioration

Sample leakage$concentrated sample+

Hemolysed sample
?!part analysers

.Along ith the parameter s o" 8 part # 7B. di""erentials

are plotted on the graph.

Depending on the technology hich is used either a laser

or scattered light technology or "lo cytometry based
method.

@eer parametrs like RD7# :P6 are also given by

these e-uipments.

RD7ARD7 in conBunction ith RB. count and :.6 is

use"ul in interpretation o" several hematological
disorders.

:P6 is derived "rom platelet histogram . ,here is an

inverse elationship beteen number & si>e o"
platelets.6arious hematological contions like 4,P#
aplastic states# B59 and "olate de"iciencies .
Basic Laser ;lo .ytometry
Basic Laser Flow Cytometry
Limit at ions

Ability to dif f erentiate on!y basic f ive types of

WBCs

Limited abi!ity to identif y abnorma! ce!!s # hence

unab!e to separate abnorma! ce!!s f rom norma! ce!!s

I nterf erence f rom NRBCs $ Lyse Resistance RBCs

Latest ....Bene"its o" C!part di"" analysis

Reduces revie rate signi"icantly

Dser de"inable setting "or di"" revie is still possible

$4% E 9F# 8F# 0F or ?F+

Reduces operatorsG costs $;,<s+

Reduces ,A,

4ncreases precision o" the reported 7B. di""erential

$thousand o" cells counted+
FLO!E"CE#CE FLO$ C%TOMET!%
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 sie, Cell !olume from forward scatter

"nternal structure, e.g. granulation or

!acuoles from side scatter

Content of nucleic acids, i.e. #$A and %$A

from side flourescence
Fluorescence Flow Cytometry
Laser Fluorescence Flow Cytometry
Bene"its

& 'art %ifferentiation of W(Cs using patented 'olymethine dye

)nmatched linearity of W(C from * - +,+*,*** cells , -.

Clear cut demarcation between normal and abnormal

cells

Co!ers almost all the clinical conditions

%ilution of samples is not re/uired

"nterference from lyse resistance #(C0s eliminated

$#(C0s 1 '.2 clumps are flagged and /uantified

Differential Channel
Fluorescent "tain 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 R!A"#!A$.
Reactive cells ATL, %& $ a'sor' more stain and emit more
flourescence
Differential "cattergram&Laser
Fluorescence Flow Cytometry '
Differential "cattergram &By
Fluorescence Flow Cytometry '
"MM(!%
Flourescence Flow Cytometry ascertain

Accurate ( part #ifferentials

Precise demarcation 'etween normal )

a'normal cells

*liminates interferences from lyse resistance RBC+s,

!RBC+s, microcytic RBC+s ) PLT clumps

%dentification of ,orpholo-ical a'normalities such

as %&, ATL ) Blasts
.linical Dtility o" 4% .ount

Presence o" 4% in blood indicates a response to in"ection# in"lammation or some

other stimulus to the bone marro.

H< 4% master provides accurate and valuable in"ormation "or immediate action.
?/ 4.D Patients
?/ 4.D Patients
,hrough the use o" the Sysmex XE-2100 analyser the 4,
Ratio could be used as

a FA32

"$45'4$3"64

#4."A(.4 indicator o" sepsis

Reticulocyte .ount

Ret I # Ret F
R<,!He $Hemoglobini>ation o" Reticulocytes+

Re"lects the actual iron supply "or hemoglobin synthesis

in the bone marro.

<arly detection o" iron depletion in erythropoiesis.

Distinguish ;e de" $4D+ and "unctional ;e De" $;4D+.

4n ;4D the iron stores are replete# but the iron is not su""iciently available "or Hb
systhesis
#42-7uantity
#42-
7uality
.linical Signi"icance o" R<,!He
.linical Dntility o" R<,!He
%iagnostic

Help to distinguish classical iron de"iciency and "unctional iron de"iciency

in anemia o" chronic disease

Detect early state o" iron de"iciency hen biochemical markers are
in"luenced $acute!phase response# pregnancy+
2herapeutic

:onitoring o" erythropoietin and J or iron therapy

#eference range : 9/ ! 8? pg
.oagulation 3

<-uipments available are manual # semiautomatic and

"ully automatic e-uipments.

Parameters like P,# P,,# AP,, # ;ibrinogen #;DP are

routinely done in a medium si>e laboratory.

Blood coagulation "actors like ;actor 6# 6444# ,hrombin

etc are availbale on "ully automated e-uipments ith
good -uality control data and calibrations.
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Why automation is required?
Since last "e decades# in clinical biochemistry there has
been increase in clinical demand "or laboratory
investigations.
7hen the volume o" ork is increased# there arose a need
"or ork simpli"ication.
Automation helps us to give correct results in short ,A,

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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.
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4nitially :ono!step methods ere introduced to replace

multistep cumbersome and inaccurate methods.

,he e""iciency o" mono!step methods as "urther

increased by the introduction o" automatic dispensers
and diluters.

4n large labs "or the common tests the above mentioned

approach is still inade-uate. ,hus# instruments ere
developed hich can handle the hole analytical process
in a mechani>ed manner.
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;irst automated system as introduced in 5M?N by
L. ,. Skeggs.
5.4nitially .ontinuous "lo analy>ersO ,o types

Single channel continuous "lo analy>ers

:ulti channel continuous "lo analy>ers e.g. S:A

9.Discrete Auto!analy>ers! ,hese analy>ers co!ordinate
multiple operations into a smoothly "unctioning system.

Semi automated

;ully automated
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emi automated analy!ers"
,hese analy>ers are called semi automated# because the
initial stages o" a specimen analysis are per"ormed by
Laboratory technician. ,hese analy>ers are suitable "or
medium comple2ity level laboratories.
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Fully automated discrete analy!ers"
,hese auto analy>ers per"orm all the "unctions o" semi
automated analy>ers and in addition to that # they also
per"orm many "unctions like automatic dispensing o"
reagents & samples# mi2ing & incubation o" reaction
mi2tures.
Batch Analysers & Random access Analysers

(atch analyers per"orm only one type o" test at a time.

Advantages3large no o" sample batches can be tested
accurately & precisely ith appropriate -uality control
Disadvantages3@ot patient oriented # not e-uippped "or
stat samples# not e-uipped ith "acilities like Qrandom
access analysersR
RAA$random access analyser+

RAA per"orm all the "unctions o" batch analyser &

additionaly they are e-uipped ith random access mode .

Additional "acilities like A cuvette disk ith temperature

control # on board re"rigerator # level sensors "or samples
and reagents#sample rack system# bar code identi"ication#
continous loading o" samples# "aciltiy "or autodilution#
automatic plotting o" daily & monthly S. charts.
Dry chemistry3

Analysers do not use et chemicals# the chemicals are

incorporated in to a series o" thin "ilms 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 ithin the slide.
;le2 technology & disposable cuvette
technology3

Analysers are discrete# random access clinical chemistry

analysers# use test reagents as "le2# have integrated
multisensor technology to provide accurate and precise
test results.

;or each tests# a cuvette is manu"actured on board and

sealed and discarded seperately "or each parameter
Biochip array tests3

Biochip array consists o" a single biochip hich has

many reagents coated.

:ultiple tests can be done "rom a single chip.

.onvinient "or panels o" tests on a single sample

Automated "loor models

.ontinous sample loading "acility

4on Oselective electrode options

Kn board laundry "or cuvettes

;acility "or urgent samples

:easures icteric# hemolytic or lipaemic inde2

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Large number o" samples can be tested in a short time.

6ariety o" tests can be per"ormed by using various

methods such as end point and rate o" reactions.

:ost o" the methods per"ormed on automation are

accurate # precise# sensitive and speci"ic.

Although the various types o" auto!analy>ers are

e2pensive# in the long run # they prove to be cost
e""ective because he amount o" reagent and specimens
re-uired can be as lo as 811 and ? micro!liters
respectively.
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Automation allos laboratories to process much larger

orkload ithout a comparable increase in number o"
sta"" members.

4nternal and e2ternal -uality control programs can be

implemented e""iciently and e""ectively by using auto!
analy>ers.

4n the case o" "ully!automated analy>ers# the laboratory

sta"" members do not come in contact ith specimens
and reagents and hence orking on these analy>ers is
very sa"e.
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