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Platelets
are the smallest blood cells in blood circulation , they participate in the bone
marrow . Enumaration of platelets are requested in the investigation of
bleeding disorder.
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Erythrocyte Sedimentation Rate (ESR)
Total WBC Count / T-Leukocyte Count
Reagent
:- WBC
Diluting fluid.
WBC Diluting fluid
hemolysis the RBC due to acidity. So that the counting of white cells become
easy. The following are the formula of turk solution -----
Calculation of Red Blood Cell indices
The
quantative measurements of the average size by volume (V) hemoglobin content by
weight (W) and hemoglobin concentration (W?V) of the Red Blood cells. Are of
substance aid in diagnosis of various types of anaemias. For the manual
procedure these various are calculated from the total number of Red cells. The
hemoglobin concentration and the hematocrit . these are called red cell indices
, three commonly used red cells indices are.
Total Erythrocyte count (TRBC)
Blood pipette
(Sahli’s) 50 micro liter and 20 micro liter
2) Hemocytometer
/Neubauer chamber
3) Microscope with
minimum 10x and 40x objective.
HEMATOCRIT ( Hct or PCV)
Hematocrit
or packed cell volume is the amount of packed red blood cell following
centrifuzation , expressed as a percentage of the total blood volume. Two
method are available for determining hematocrit.
a)
Microhematocrit Method
b)
Macro hematocrit Method
Specimen
:-
Only anti coagulated blood EDTA blood is used
in determination Hct value.
Equipment
:-
a) Wintrobe hematocrit
tube 110 mm long narrow test tube graduted from 0-10 cm(100mm). the scale with
the marking in ascending order from the to is used for ESR determination while
the scale with descending order is used for Hct determination
b) Transfer pipette a
long fine capillary pasteure pipette
c) Centri fuge
Procedure
:-
1)
The
blood specimen is mixed carefully
2)
The
wintrobe tube is marked labeled with the
identify number of the patient
3)
The
tube is filled with blood specimen by the help of pasteure pipette up to the 10
cm marked which blood or taken as blank to balance the centrifuge
4)
The
wintrobe tube are placed in the opposite cups of the centrifuge
5)
The
centrifuge is turned on to slow speed the increase gradually to bring up
finally the required speed.
6)
The
tube are centrifuge for 30 minutes at 3000 rpm ( Revolution per minute)
7)
After
30 minute centrifuge is switched off and left it self to stop spilling.
8)
The
wintrobe tube is taken out and observed that the blood sample has been lower portion of tube and plasma above. It
the amount of packed RBC are read directly from the graduation mark of the
tube.
Determination of Hemoglobin Concentration (HB%)
Hemoglobin
is a conjugated protein present in Red blood cell. It carries oxygen from the
lung to the
tissue cell and carbondy oxeyde the gaseous waste from the cells to lung. Hemoglobin contents to components Hem(Iron+Protophyrin+Globulin+Protein / amino acid chain)
tissue cell and carbondy oxeyde the gaseous waste from the cells to lung. Hemoglobin contents to components Hem(Iron+Protophyrin+Globulin+Protein / amino acid chain)
Specimen :- EDTA anti coagulated
vein us blood is commonly use but other anticoagulants heparin , Double oxalate
alasoyiclds the same result capillary blood can be used directly.
Method :- Cyanmethaemoglobin.
Equipments:-
1)
Photometer
with 540 nm filter
2)
Blood
pipette (Sahlys) of 20 micro l capacity
3)
Test
tube
4)
Surgical
Gauze/Cotton
5)
Other
Glassware
Reagent :-
1)
Drabkin’s
Solution (PIOSION)
2)
Cyanmethemoglobin
standard
Procedure :-
1)
Two
clean and dry test tube are taken and marked as “B” (blank) & “T” (Test).
2)
5
ml of Drabkin’s solution is pipette into the two tube
3)
The
blood specimen is well mixed by gently swinging.
4)
An
aliquot 20 micro l of well mixed EDTA anti coagulated blood specimen is drawn
into the pipette and out site of the
pipette is cleaned by use of gauze to avoid volume error.
5)
The
pipette blood sample is added to the “T” marked tube.
6)
The
contents of tube is well mixed and waited for 10 minutes.
7)
The
colorimeter is turned on and allowed to warm up for 10 minutes. The wavelength
selected filter is used in this range.
8)
The
instrument is set to “Zero -0” with out cuvette using the first control knob.
9)
The
both solution “B – Blank”, “T- Test” are transferred to two matched cuvettes.
10)
The
instrument “Zero -0” is rechecked , the cuvette with Blank solution “B” is
inserted in to the colorimeter cuvette socket.
11)
The
reading is adjusted to “Zero-0)” absorbance with the second control knob.
12)
The
cuvette with test solution “T” is inserted in to the socked of the colorimeter
and the absorbance reading is recorded . absorbance can be rechecked using
again the Blank solution.
13)
The
reference of the calibration curve is taken to find out the concentration of
the unknown. This can be simplied find by applying the flowing formula ……..
Abs of Test
Hb%.con (g/dl) =
--------------------- X 15*
Abs of stander
In
the above formula the value of stander and Absorbance of stander remain
constant Hence, it can be calculated as……
Con of std
-------------- = factor
Abs of std
There
fore the formula can be written as
hb.con (g/dl)=Abs of the test X factor.
Calculation – OD
of Test
Hb (g %) =
-------------------- X con of
std X 0.251
OD of std
OD of Test
= ------------------- X 60
X0.251
OD of std
OD of Test
= ----------------------- X
15.06
OD
of std
Routine Hematological Tests……..
The
flowing tests are done or considered to be routine Hematological tests…..
1)
Determination
of Hemoglobin Concentration (HB%)
2)
Determination
of hematocrit
3)
Routine
enumeration of formed elements- Total red Blood Cell and white blood cell
count. (TC)
4)
Calculating
of Red blood Cell indices – MCV,MCH & MCHC.
5)
Study
of stained blood smear differntical count (DC)
Study
of Red Cell Morphology
Study
of white Cell Morphology
6)
Reticulocyte
Count
7)
Erythrocyte
sedimentation rate (ESR)
8)
Eosinophils
Count
9)
Platelet
Count.
Capillary Puncture Method
A
capillary is a small blood vessel connecting the small arteries to the small
vein, the capillary blood is as tained by skin puncture of blood specimen for
making as blood smear (D.C) cell count or haematocrit determination . skin
puncture specimen is prefared over vein puncture specimen for the study of a
blood smear . the skin puncture is specially recommended for babies and where
vein puncture is different (old prolonged sickness three site chosen for skin
puncture are-------------
a)
Finger
puncture (Tip of the finger for adults are older children)
b)
Heel
puncture chosen for infants
c)
Ear
lob puncture chosen for all but not occasionally done.
Capillary Puncture Procedure : -
1)
The
necessary equipments are assembied lancet, alcohol pad, any surgical gauze ,
capillary tubes microscope slide and other supplies. It made sure that the
patient is seated comfortably.
2)
A stop on the middle finger or using finger of
the left hand is fond out. Toe puncture and ear puncture are done in case of
infants whenever possible previous puncture site should be avoided.
3)
The
site is clean with a sterial cotton swab deep 70 % alcohol then the site is
dried with a dry cotton swab. This will removed dirt and epithelial debris warm
up the part chosen for preking, increase the blood circulation and rive area reitivley ,sterile
clod skin may prevent a free flow of blood. If the puncture site is wet the
blood doesn’t form into well-roupeded
drops.
4)
The
finger is grasped firmly and made a quick firm puncture with a sterile lancet
that should be 2 – 3 mm deep. A deep puncture hun to know more than a superficial one and it gives much more
satisfactory flow of blood. If it doesn’t a gentake pressure is applied to form
a round drop blood.
5)
The
first drop of blood is wiped away eith cotton swab, the first drop of blood is
container with tissue fluid and will intentere laboratory result if used.
6)
The
specimen is collected by holding a capillary tube hot determination or by
seeking in to sahlis pipette for HB
determination and blood count or by touching the drop rapid collection is
necessary to prevent coagulation.
7)
After
the blood specimen is collected the patient
is asked gauze pice over the puncture site until the bleeding has stopped.
Collection of Blood
The
most common tests done for diagnostic purpose using Blood sample. Hence Blood
is an important specimen the Blood is collected by two method.
a)
Vein puncture method
b)
Capillary Puncture
Method.
Vein puncture method
The volume of Blood
obtained by vein puncture is sufficient to carry out either by the syringe
method or vacuum tube method.
Procedure : -
1)
All
the thing required during Blood collection are as sample.
2)
Carefully
the patient’s form identify of the patient id read and decided the total amount
of blood needed for all the test e.g . if a hemoglobin is requested 2 ml of
blood in E.D.T.A will be
sufficient, where as if L.F.T ( Liver Function Test) are to be done 10 ml of
Blood. In a plane test tube will be required.
3)
The
blood collection container are selected and labeled with patients proper
identify marked.
4)
The
patient is introduced with the phlebotomist the patient is asked to sit an the
collection chair used for blood drawing collection . his arm is Layer pain
upwards. The procedure of blood collection of blood collection should explaned
to the patient mini size apprehension
never draw Blood. Formastanding patient or patient sitting on a high chair or
tool. The collector should be prepared for the patient who may faint and should
be trained to first aid techniques.
5)
Selected
the puncture site carefully after inspecting both arms. Median Cephalic vein of
the forearm is most frequently used for vein puncture.
6)
The
tourniquet is applied on the appropiyet
place of the arm and puncture site vein where have to introduce the needle. The
thumb of left hand is placed over the vein just bellow the point of entrance to
fix the vein.
7)
The
syringe is removed from the protective wrap and assembled with out touching the
tip of the needle or wall of the piston the needle is fixed tightly.
8)
The
skin site to be punctured is disinfected using a swab dipped in 70 % alcohol by
rubbing.
9)
The
needle positioned with bevel upper most and pushed firmly and steadly with out
hesitation in to the needle and the vein should be at 30 – 40 degree angle.
The
needle is pushed along the line of the vein to a depth of 1 -1.5 cm.
10)
The
piston is pulled back firmly Blood should appear in the barel and if is
continued until the requisite amount of blood is with drawn.
11)
The
tourniquet is released by pulling on the keep and after the blood is drawn.
12)
A
swab of cotton well is placed over the hidden point of the needle. The needle
is removed in one rapid movement from under the swab.
13)
The patient is asked to press firmly on the
swab for 2-3 minutes . this steps the bleeding from the wound.
14)
The
needle is removed from the syringe an expelled gently the blood into a
appropriate container. Foaming or rupture of the planger the container is
stopper anti coagulant with the blood , if anti coagulant is used.
15)
The
blood is mixed immediately and through with the anti coagulant to prevent clotting.
The vials are labeled clearly with the patients proper indentify . it is good
practice to labeled first before the putting the blood specimen to the
container.
16)
In
the past year when a single syringe was used for blood collection. The syringe
was rinsed immedeatly with cold water . but now a days the disposable single
used syringe are used that dosn’t have to rinse but the disposed.
17)
Before
the patient leaves the vein puncture site is re-inspected that the bleeding as
stopped and adhesive tape. Is applied over the cotton swab on the wound, the patient
should not be relese until the bleeding stops .
Note : - Alaways removed the tourniquet before taking
out the needle of the vein to prevent the formation of hematoma.
Procedure of using microscope
Place
the microscope on a stable place are Laboratory bench . always sit straight
while working with the microscope . place the microscope near the window if
daylight is used for illumination
2)
Direct
the path of right to pass through the hole of the stage while setting the
mirror
3)
Put
the slide between the clips provided on the stage.
4)
Revolve
the nose piece and aligen the low power objective clox two examination the object on the slide. The
objective must into click into place
5)
Adjust
the illumination to improve contrast.
6)
Put
one hand on the focusing knob are fine one set time and the other on the screw to move the stage.
Use the adjustment knob for bringing the object closed two focus with the fine adjustment knob.
7)
Switch
to high power 40x and increase the illumination as needed. Bring the object of
interest in the centers as the use of fine adjustment knob may not be critical
and switched freely.
8)
If
high power Leans tends to touch a thick slide are slide with cover slip, always
focus by the anti clock wise movements. Of the coarse adjustment knob.
9)
After
screening under the low power and examination under the high power , oil
immersion objective is used for greeter details of the object. A tiny drop of immersion oil is applied on the place
where object and the reset will come over the path of light and the oil immersion objective.
Basic Needs of a clinical Laboratory.
The Basic needs
for a typical small size clinical Laboratory doing routine diagnosis tests is
listed bellow….
1)
Good
quality microscope along with oil immersion objective.
2)
Colorimeter
and accessories Cu vets filters queb etc.
3)
Bunsen
Burner and Glass.
4)
Centri
fuje
5)
Water
bath
6)
Balance
physical and chemical
7)
Test
tube rag
8)
Inoculating
needle or platineum were holder
9)
Burette
stand
10)
Tripod
stand
11)
Distillation
set
12)
Glass
ware plastic ware “Varity of volumetric and non volumetric glass wares
500. 100.
50 and 25 ml cylinders.
1000. 500.
250. 100. 50. And 25 ml flax
20 . 10 .
5 1 and 0.1 ml pipette and also serological (15.10.5 ml) tubes immersion oil
bottles. Dropping bottle (100ml)
Wash bottle
and cy lenders and petridiss.
13)
Filter
pump
14)
Hemocytometer
15)
ESR
tube & rag
16)
Microscope
slide cover slip and cavity slide or serological tests.
17)
Filter
paper, Gross pencil , note book, cotton gauze and swab
18)
Tharmometer
19)
Incubator
20)
Woven
21)
Refrigaretor
22)
Penicillin
vail for Blood collection
23)
Preser
cooker or auto clave
24)
Stain
& reagent