HEMATOLOGICAL

TESTS
INTRODUCTION :-

Gk. Hem(e) – Blood

Logy – Study of
• Hematology is defined as the study of normal and pathological aspects of blood and blood
cells.
• Hematology is the branch of medicine concerning the study of blood, the blood-forming
organs, and blood diseases. Hematology tests include laboratory assessments of blood
formation and blood and bone marrow disorders.
ROUTINE HEMATOLOGICAL TESTS

• Before the era of automaic count through automated machine routine hematology tests consists
of:-
° Hemoglobin level
° leukocyte count
° Differential count
° ESR
• Now Complete blood count(CBC) consists of :-
∆ RBC,MCV,MCH,MCHC,Ht,Hb,WBC,Differential count
∆ Platlet and ESR (Manual)
BLOOD CELL
COUNT
RBC COUNT
• Red blood cell count can be measured by manual method using an improved Neubauer counting chamber
or by automated analyzer.
At Birth- 6.0-7.0 millions/cumm
Adult Male- 4.4-6.0 millions/cumm
Adult Female- 3.8-7.0 millions/cumm
RBC Diluting Fluids:-
1.Hayem’s RBC Diluting fluid:: It contains mercuric chloride (prevent bacterial growth), Sodium
Chloride(provide isoosmolarity), sodium sulfate & Distilled water.
2.Dacie’s RBC Diluting Fluid:: It contains sodium citrate(anticoagulant) , formalin(fixation of cell &
retard bactrial & fungal growth) and Distilled water.
• Manual Method :-
Gently mix the blood in EDTA vial and draw the blood upto 0.5 mark of pipette. Wipe off if
excess blood drawn through the pipette tip.
Then draw RBC Diluting fluid through same pipetteupto mark 101. (Dilution 1:100)
Hold the pipette horrizontally in its long axis ,rotate it slowly to ensure thorough mix up of RBC
with dilutant.
Charge the improved Neubauer counting chamber by placing the tip of pipette just under the
cover slip and allow fluid to flow by capillary action till the counting chamber is just filled. Wait 2-5min.
For the cells to settle in the chamber.
Count RBCs using high power objective in the 5black small squares in the central big square and
total them together.
RBC Count= counted cells × 10,000 cells /cu mm

Causes of increased RBC:- Polycythemia (Polycythemia vera,Secondary Polycythemia)

Causes of decreased RBC:- Anemias, hemodilution
WBC COUNT

• TLC(Total Leukocyte Count) in the blood can be measured either by manual or by automated analyzer.
At Birth- 8000-22000cells/cumm
Childhood- 6000- 15000cells/cumm
Adult- 4000- 11000 cells/cumm
• WBC Diluting Fluid:-
1. Turk’s Fluid:: It contains glacial acetic acid(lyse the RBCs) and gentian violet(stain nuclei of
leucocyte,Distilled water).
2. Thomas diluting fluid
Manual Method::-
Gently mix the blood in EDTA vial and draw blood upto 0.5 mark of WBC pipette ,then fill
diluting fluid through pipette upto mark 11.
Hence dilution factor is 1/20 .Load the counting chamber, Cells in the 4corner squares each
1mm3 are counted.

WBC Count= Counted cells× 50 cells /cumm

Increased WBC Count::- Leukocytosis

Decreased WBC Count::- Leukopenia
PLATLET COUNT
• Platlet are small ,colourless and epithelial blood cells with a diameter of 2-4 micron .They Help in
coagulation ,integrity of vessels wall & for clot retraction.
• Normally RBC pipette used but if Platlet count us low a WBC pipette can be used instead of
RBC pipette.
Normal Range- 1,50,000 to 4,50,000/cumm
Platlet Diluting Fluid:-
Dacie’s Fluid:: It contains sodium citrate dihydrate,0.2% brilliant cresyl blue, formalin,
Distilled water
Rees-Ecker Fluid:: It contains Sodium citrate, Brilliant cresyl blue Formalin, Distilled water
Brecher- Cronkite Fluid:: It contains 1% ammonium oxalate solution
• Manual Mathod:-
Gently mix the blood in EDTA vial and draw the blood in RBC pipette upto 0.5 mark ,Filldilution fluid
upto 101 mark, mix it by holding pipette horrizontally Load counting chamber , wait for 5-10 min.
Calculate the cells in entire central 1×1 mm3 square.

Platlet Count= Total counted cells in central square× 2000 cells/cumm (if used RBC Pipette)
Platlet Count= Total Counted cells in the central square × 200cells/cumm(if used WBC Pipette)

Increased Platlet Count::- Thrmbocytosis

Decreased Platlet Count::- Thrmbocytopenia
“
Neubauer’s chamber cell calculation:-
• RBC Count- Central 5big squares
”
• WBC Count- 4 corner squares(1,3,7,9)
• Platlet Count- Central 25 big squares
OTHER BLOOD
COMPONENTS
COUNT
RBC INDICES::-

• Red blood cell (RBC) indices are part of the complete blood count (CBC) test. They are used to help
diagnose the cause of anemia, a condition in which there are too few red blood cells.
• Red blood cell indices are blood tests that provide information about the hemoglobin content and
size of red blood cells. Abnormal values indicate the presence of anemia and type of Anemia.
• The indices include::-
• ∆ (MCV) Average red blood cell size
• ∆ (MCH) Hemoglobin amount per red blood cell (MCH)
∆ (MCHC) The amount of hemoglobin relative to the size of the cell (hemoglobin concentration)
per red blood cell (MCHC)
• The blood Indices are Calculated from::- PCV
RBC Count
Hb value
• PCV(Packed Cell Volume)/ Haematocrit
• RBC Count(Red Blood Cell count)
• Hb Value(Hemoglobin conc.)
• PCV/HCT. ::-
Hematocrit is the volume of red cells expressed as a percentage of volume of whole blood in the
sample.
Manual Methods:-
1. Using Wintrobe’s tube
2. Using Capillary tube(Micro Hematocrit method)
• Wintrobe’s Method:-
Fill the Wintrobe’s tube with anticoagulated blood sample till mark 100 on top with help of
Pasteur’s pipette or syringe with long needle.
Centrifuge for ½ an hour at 3000 rpm.
Blood is now separated into 3layers a column of – RBC at bottom ;WBC & platlet in middle layer
(Platlet layer is known as Buffy coat; the top most layer formed by plasma.
The height of column of blood occupied by RBC gives PCV in percentage.
Roughly, PCV= 3× Hb value
RBC count= 1/3 rd of Hb value
• Micro Haematocrit /Capillary Tube Method:-
Commonly used in well equipped labs. The capillary tube is coated with anticoagulants are filled
with blood ; one end of tube seal with wax & kept for centrifuge for 3min.at high speed.
By reading packed cell height and height of entire sampl ,finally Hematocrit value comes out.
Normal Values:- Adult Male-40-50%
Female—37-47%
Children- 35-37%
HAEMOGLOBIN ESTIMATION:-
• Haemoglobin Estimation can be done through different omplex substance present in the RBC which
gives red color to the blood as is composed of iron containing pigment heme and globin. (Anaemia
causes mostly due to reduction in Hb%)
• Haemoglobin estimation can be done through different method :-
1. Color
2.Specific Gravity
3. Iron Conten
4. O2 combining capacity of blood
• The most commonly used methods include Calorimetric Method (namely Sahil’s Method &
Cyanmethemoglobin Method) and automated analyzer.
SAHLI’S METHOD :- (Estimate Hb by Acid Haematon Method)
• Apparatus Needed:- Sahli’s haemoglobinometer tube ,
pipette, glass rod, N/10 HCl, pricking needle, spirit, sterile
cotton, blood.
• Method:- Take N/10 Hcl through pipette upto lowest mark
of hemoglobinometer tube ; Prick finger under sterile
technique and draw blood by pipette upto 0.02ml(20cumm). ;
Pour the blood into Graduated hemoglobinometer tube
immediately; mix it by using stirrer/ Glass rodwait for 10
min. ; Now Acid hematin formed and appear brown in color ;
Add distilled water till the color approach the standard color
by holding against natural light or fluorescent tube light.
Hemoglobin is record as gm/dl.
• Increased Hb Conc. [( Physiological- Newborns,
diurnal(higher in afternoon ), after muscular exercise,
emotional state , higher altitude)(Pathological- Polycythemia
vera, cyanotic heart disease )]
• Decreased Hb. Conc. ( Anaemia,hemodilution,leukemia)
MCV:-

• MCV is stands for Mean Corpuscular Volume.

• MCV indicates average volume of single red cell in cubic microns (Average red blood cell size (MCV)).
• MCV classify anaemias depending on the size of the cells.
• Normal Value- (92 +/- 9) cumm
MCV=[{ PCV/RBC(in millions/cm)}× 10]
• Value greater than 100= Macrocytic Anemia
• Value lesser than 100= Microcytic anaemia (MCV less than 83)
MCH:-

• MCH indicates average amount of Hb present in one red cell(Hemoglobin amount per red blood cell ).
• It is expressed in unit picograms(pg).
• Normal Value- (29.5 +/- 2.5 pg)
MCH= {( Hb per lit. Blood)/ RBC(million/cm.)} ×10
. Less MCH = Hypochromic RBC’S
. More MCH = Macrocytic RBC’S
MCHC :-

• MCHC stands for Mean Corpuscular Hemoglobin Conc. ( average concentration of hemoglobin per
unit volume of red blood cells) .
• Calculated by dividing the hemoglobin by the hematocrit.

• Normal Value= 31-35 %

MCHC= [{Hb( lit. Of blood)/ PCV}×100]
• Increased MCHC in case of spherocytosis.
ESR(ERYTHROCYTE SEDIMENTATION
RATE):-
• ESR is the rate at which Erythrocyte sediment on their own weight when anticoagulated blood is left
undistributed in a vertical column and form 2layers. This process is called Sedimentation.
• Methods:-
1. Wintribe’s method ( most common methid).
2. Westergren Method
3. Didposable method(ESR Kit)
4. Capillary method(micro ESR method used in pediatric case)
5. Zeta Sedimentation Rate ( Automated ESR)
• ESR is a prognostic test rather than diagnostic. It is useful in monitoring disease activity not able to specify the
disease ( non-specific prognostic test).
• Normal Value(in 1st hr.) - 0-10 mm ( Adult Male)
0-12 mm ( Adult Female)
0-10 mm ( children)
COAGULATION TESTS (BT,CT & PT) :-

• BT:-
BT is used to test platlet vessel wall interaction it mean the BT measures the time required for bleeding to stop after a standardized
superficial cut of skin capillary bed. [BT= Time from start of bleeding to stop]
Normal= 3-5 min.
• Methods:-
1. Duke’s Method ( Clean finger with spirit – puncture deeply as it bleed drop 💧 by drop 💧 without squeezing- at ½ min.
Interval remove drops of blood on filter paper without touching skin surface– Note appearance of 1st drop and removal of last
represent BT ).
2. Ivy’s Method (blood pressure cuff applied to arm and inflate just above diastolic pressure - clean the forearm with
spirit & dry – make puncture on forearm except vein – wipe the blood in every 15 sec. Interval by filter paper without touching
skin surface – Note appearance of 1st drop and removal of last represent BT ).
* The blood oozing from the cut site is blotted with filter paper every time interval (30sec/½
min.) untill bleeding stops.
• CT :-
CT measures time taken to clot fresh blood.
Normal- (5-11)min.
• Methods:-
1. Lee-White Method:- ( Label 3testtube as 1,2 & 3 and keep in water bath at 37°C ( Saline) - Collect 3ml. Of venous
blood in aseptic technique – deliver 1ml. Of blood to each – 1 st,2nd & 3rd tube should gently tilt every 30sec. Untill blood clots –
record the coagulation time interval between appearance of blood in syringe and time at which blood no longerflow/clot)
2. Slide Method:- ( Place several drops of blood on aclean slide from a deep finger puncture- at 30sec. Interval draw a
needle through each drop- as soon as needle pick-up fibrin thread drag them along – leads to coagulation is complete- Note the
time interval between the drop place on slide upto the formation of fibrin thread.)
3. Capillary Tube Method:- ( clean finger with spirit &dry- puncture deeply to obtain spontaneous free flow of blood –
allow 2capillary tube to fill 4inches( upto 3inch should be continuos) – observe tube after 2min. And in every 30sec break 1cm length
of tubing – onset of coagulation indicate by development of fibrin bridge on to broken end. )