Bst unit-iii

UNIT -III
• Haematological investigation: Blood composition,
blood sample collection and smear preparation,
Differential cell counts – RBC, WBC, Platelets,
hemoglobin estimation, erythrocytic sedimentation
rate (ESR), Blood platelet count by hemocytometer,
and testing of blood glucose using glucometer.
Dr. I. Manjubala
SBST, VIT University
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Dr. I. Manjubala
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Laboratory work flow cycle:
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The phlebotomist
The phlebotomist :
Is the technician who collects blood, should be trained to:
1) Prepare specimen collection material
2) Instruct patient appropriately
3) Collect, preserve and transport specimen carefully
4) Separate serum or plasma properly
5) Maintain proper record of collection
6) Handle the specimen carefully
7) Analyze the specimen accurately
8) Maintain proper record of reports
9) Work with appropriate safety precautions
Dr. I. Manjubala
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Dr. I. Manjubala
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Phlebotomy or blood
collection:
The term phlebotomy refers to
blood draw from a vein, artery, or
the capillary bed for lab analysis
or blood transfusion.
The phlebotomy equipments:
For specimen collection, the
following materials will be
required:
Phlebotomy
Disposable syringes Vacationer systems Disposable lancets
Gauze pads absorbent cotton Tourniquet
Alcohol swap Plastic bandage Waste container

VENEPUNCTURE
•Venipuncture is performed to obtain laboratory results that
provide prevalence estimates of disease, risk factors for
exam components, and baseline information on health and
nutritional status of the population.
•Definition –venepuncture describes the procedure of
inserting a needle into a vein, usually for the purpose of
withdrawing blood for haematological, biochemical or
bacteriological analysis
•It is one of the most commonly performed procedures
which, carried out skilfully, carefully and accurately, will
provide high quality blood samples without causing
discomfort to the patient –

• The superficial veins of the arm
are usually chosen for
venepuncture, namely basilic,
cephalic and median cubital
veins in the antecubital fossa
• – These veins are recommended
as they as they are well supported
by muscle and connective tissue,
visible and easy to palpate

• The walls (outer structure) of veins
consist of three layers of tissues that
are thinner and less elastic than the
corresponding layers of arteries
• Veins include valves that aid the
return of blood to the heart by
preventing blood from flowing in the
reverse direction

Usually vein is used to collect blood by
veinpuncture procedure.
In adults: most venipuncture procedure use
arm vein.
On arm, one of three arm veins is used:
median cubital vein "located on the
middle", cephalic vein or basilic vein
"located on both sides".
Median cubital vein is the best choice
(why?) because it has good blood flow than
cephalic and basilica which has slower blood
flow.
However if veinpuncture procedure is
unsuccessful in median capital; cephalic or
basilica is used.
Artery blood is rarely used in special
cases as when blood gases, pH, PCO2,
PO2 and bicarbonate is requested. It is
usually performed by physicians.
Selecting vein site
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• There are two stages to locating a vein:
1. Visual inspection 2. Palpation
• Visual Inspection – The scrutiny of the veins in both arms is
essential prior to choosing a vein
• Veins to avoid:
– Veins close to an infection : Veins close to bruising and
phlebitis : Oedematous limbs as there is danger of stasis of
lymph, predisposing to such complications as phlebitis and
cellulites
• Areas of previous venepuncture should be avoided as a build
up of scar tissue can cause difficulty in accessing the vein and
can result in pain
• Avoid veins that are thrombosed
• Do not use the affected arm in CVA or mastectomy patients
• A vein sited in the region of a drip site should never be used
as it may result in the collection of a diluted samples

• Palpation is also an important assessment technique as it: –
determines the location and condition of the veins –
distinguishes veins from arteries and tendons – identifies the
presence of valves
• Healthy veins feel soft and bouncy and will refill when depressed

Improving venous access – There are a number of methods
to improve venous access, for example:
1. Application of a Tourniquet -Promotes venous distension –
The tourniquet should be tight enough to impede venous
return but not restrict arterial flow – The tourniquet should be
placed about 7 – 8 cm above the venepuncture site
•The tourniquet should not be left on for longer than 1 minute
as it may result in haemo-concentration or pooling of the
blood, leading to inaccurate blood results

2. Opening and closing of the fist – The muscles will force
blood into the veins and encourages distension. However this
action may affect certain blood results, e.g. potassium
3. Light tapping of the vein – May be useful but can be painful
and may result in the formation of a haematoma in patients
with fragile veins
4. The use of heat: In the form of warm pack to encourage
venodilatation and venous filling
5. Lowering the arm below the level of the heart

Skin preparation
– Asepsis is vital when performing
venepuncture as the skin is breached and
a foreign device is introduced into a sterile
circulatory system
– Skin cleaning is a controversial subject
and it is acknowledged that a cursory wipe
with an alcohol swab does more harm than
good as it disturbs the skin flora
- For blood culture sampling or if the
patient is at increased risk of infection the
skin should be cleaned with an alcohol
swab BUT you must allow at least 2
minutes for the area to dry thoroughly
before proceeding with venepuncture

• Hand hygiene – Is the single most important activity for
reducing the spread of disease, yet evidence suggests that
many health care professionals do not decontaminate their
hands as often as they need to, or use the correct technique
which means that areas of the hands can be missed

• Complications
– Inability to obtain specimen due to: – Inappropriate choice
of vein – Thrombosed vein (due to previous or repeated
attempts) – Inexperience of operator – Patient shocked,
cold or dehydrated causing vasoconstriction
• Formation of haematoma due to: – Poor technique – Pressure
not being applied to puncture site following removal of needle
• Puncture of an artery – If an artery is punctured: release the
tourniquet, remove the needle and apply firm pressure for a
minimum 5 minutes. Cover the site with a dressing once
bleeding has stopped. Re-check for signs of bleeding in 20
minutes.

Preparation of Blood Sample
One of three different specimens may be used:
•whole blood
•serum
•plasma
First: Whole-blood specimen:
It must be analyzed within limited time (why?)
– Over time, cells will lyse in whole-blood which will
change the conc. of some analytes as potassium,
phosphate and lactate dehydrogenase.
– Some cellular metabolic processes will continue which
will alter analytes conc. like glucose and lactate.
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Serum
Difference between Serum and plasma:
•Serum is the same as plasma except it doesn't contain
clotting factors (as fibrin).
•Plasma contains all clotting factors.
•So, serum and plasma all has the same contents of
electrolytes, enzymes proteins, hormones except clotting
factors
•Serum is mainly use in chemistry lab & serology.
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Procedure of Serum preparation
• Draw blood from patient. Select vacutainer with no
anticoagulant.
• Allow to stand for 20-30min for clot formation.
• Centrifuge the sample to speed separation and affect
a greater packing of cells. Clot and cells will separate
from clean serum and settle to the bottom of the
vessel.
• The supernatant is the serum which can be now
collected by Dropper or pipette for testing purposes or
stored (-20°C to -80°C) for subsequent analysis or
use.
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Plasma
• The tube will have anti-coagulation
• After centrifugation the blood sample
got separated into three layers
Procedure for Plasma Preperartion
•Draw blood from patient. Select vacutainer with an appropriate
anticoagulant.
•Mix well with anticoagulant.
•Allow to stand for 10min.
•Centrifuge the sample to speed separation and affect a greater
packing of cells.
•The supernatant is the plasma which can be now collected for
testing
•Purposes or stored (-20°C to -80°C) for subsequent analysis or
use.

Specimen rejection criteria
1- Specimen improperly labeled or unlabeled
2- Specimen improperly collected or preserved
3- Specimen submitted without properly completed request
form
4- blood hemolysis
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Hemolysis of blood
Hemolysis :
•It means liberation of hemoglobin due to rupture of RBCs.
•Due to hemolysis plasma or serum appears pink to red color.
•It causes elevation in: K+
, Ca2+
, phosphate, SGOT, SLDH and acid
phosphatase.
•Hemolysis is occurred due to sampling, transporting and storage (too hot
or too cold).
•According to the degree of hemolysis it is classified as H+, H++ and H++
+. H+ accepted for tests, H++ and H+++ not acceptable for test.
Changes in the serum color indicate one of the following:
•Hemolyzed: serum appears pink to red due to rupture of RBCs
•Icteric: serum appears yellow due to high bilirubin.
•Lipemic: serum appears milky or turbid due to high lipid.
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Blood collection tubes:
Two major types of blood collecting tubes:
•Serum separating tubes (SST)
•Plasma separating tubes (PST)

Top Color Additives Principle Uses
Lavender EDTA -The strongest anti-coagulant
-Ca+2
chelating agent
- To preserve blood cells
components
- Hematology
- Blood bank
(ABO)
- HbA1C
(Glycosylated
Hb)
Light Blue Sodium
Citrate
Ca+2
chelating agent - PT: Prothrombin
Time
- PTT: Partial
Thromboplastin
Time
( in case of
unexplained
bleeding and liver
disease)
Green Sodium
Heparin or
Lithium
Heparin
Heparin binds to Thrombin and
inhibits the second step in the
coagulation cascade
(Prothrombin Thrombin)
Fibrinogen Fibrin
Enzymes
Hormones
Electrolytes (Na+
,
K+
, Mg+
, Cl-
Heparin
Plasma Separating Tubes (PST)

Top
Color
Additives Principle Uses
Black Sodium Citrate Ca+2
chelating
agent
ESR ( Erythrocyte Sedimentation
Rate)
to test how much inflammation
in the patient, unexplained fever,
Arthritis, Autoimmune Disorder
Gray -Sodium Fluoride
-Potassium
Oxalate
Glycolysis
inhibitor
Anti-
Coagulant
Glucose tests
Royal
Blue
Heparin
Na-EDTA
Anti-
Coagulant
Tube should
not be
contaminated
with metals
Toxicology
Trace Elements and metals
Yellow ACD ( Acid-Citrate
Dextrose)
Anti-
Coagulant
DNA Studies
Paternity Test
HLA Tissue Typing
(Human Leukocyte Antigen)
The body used this protein to
differentiate the self-cells from
non-self cells

Top Tubes Additives Principle Uses
Red ------
Sometimes it
has gel or silicon
at the bottom of
tube to reduce
hemolysis
Enhancing the
formation of
blood clot
Serology
-Antibodies
-Hormones
-Drugs
Virology
Chemistry
Blood cross
matching before
blood
transfusion
Gold -------
It has gel at the
bottom of the
tube to separate
serum from the
blood
Serum
separating from
the blood
through the gel
in the tube
Serology
Chemistry
Serum Separating Tubes (SST)

Most commonly used:
a. Red-stopper tubes – are for tests requiring clotted blood
b. Lavender stopper tubes – contain EDTA in concentrated
liquid or desiccated powder form
c. Green stopper tubes – contain heparin and are used for
blood gases, PH, (CO2, O2)….
d. Gray stopper tubes – contain oxalates, fluorides, or
citrates
e. Yellow stopper tubes – available with Acid Citrate
Dextrose (ACD) solution or physiological saline solution
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Hemoglobin Determination
Significance of hemoglobin
a. It serves as an index of blood condition of the patient.
b. If the hemoglobin [Hb] content falls below the normal levels, it
indicates anemia, or pregnancy (physiological).
c. If it increases than the normal value, it indicates polycythemia,
decrease in O2 supply, heart disease, emphysema etc.
Method: Acid hematin method
Requirements: Sahlis instrument,
blood sample
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Procedure
• Take 0.1N HCl (1%) into central graduated tube up to mark 2.
• Suck the blood exactly up to mark 20 (20 μl) with the help of sahlis
pipette.
• Transfer the blood from pipette to central graduated tube of the
hemometer.
• Mix it well with the help of stirrer or rod and allow it to react for two
minute.
• Make up with distilled water by adding drop by drop until the color
matches with the Standard comparator tube and mix well.
• When the color matches take out and record the values on the
side as gm/100ml and or in percentage.
• Repeat 5 to 6 times and take the average value
Normal value:
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Hemoglobin Determination

Principle: Blood compartment is separated into three parts using
capillary tube in a hematocrit centrifuge.
Method: Wintrobe hematocrite method
Significance
• Packed Cell Volume (PCV) = erythrocyte mass; anemia when PCV
falls dawn.
• Buffy coat; white to gray layer above PCV. It will give number of
WBC (0.5mm to1.5mm).Leukopenia or leukocytosis.
• Plasma content: usually about 55%, Yellowish in color. Degree of
yellowness indicates icterus (jaundice).
Requirements: Hematocrit tube, hematocrite centrifuge, hematocrit
reader and sealer.
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Hematocrit Determination (PCV)

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Procedure
• The blood is filled in to a micro hematocrit tube (3/4th)
and seals it with sealer.
• Centrifuge the filled hematocrit tube in a hematocrite
centrifuge at 2000 rpm for 4-5 minutes.
• Read the value (the tube) with hematocrit reader and
record the result.
Normal value
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• Normal Range
• Reference Index : the concept of
'Universal RIs' or 'Global Ris’ ??
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• Smear Preparation
• Wet smear
• Thin smear
• Thick smear
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Wet blood smear/film preparation
A drop of blood is placed at the centre of a clean slide
• Cover with a clean, dry cover slip
• Examine the film under the microscope (40 × objective)
The method does not require staining. It is rapid and simple to
perform.
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Thin/Thick blood smears preparation
It can be made by spreading a drop of blood evenly across a clean
grease free slide using a smooth edged spreader.
Thin Smear
Make a drop of blood on one end
of glass slide
• Place the end of second glass slide
/spreader slide / against the surface
of the first slide, holding at an angle
of 30-45 degrees
• Draw the spreader slide gently into
the drop of blood and when the
blood has along 2/3 of width of the
spreader slide by capillary action,
push the spreader slide forward with
a steady even motion
• Dry by waving rapidly in the air
Thick bloods smear
preparation
• A large drop of blood is put at
the centre of a clean dry slide
• The drop is spread with an
applicator stick, needle or corner
of another slide to cover an area
of ½ an inch square
• The smear is thoroughly dried in
a horizontal position so that the
blood could not ooze to one edge
to the film and protected from
dust, insects and direct sunlight.
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Problems in Smear preparation
Entire smear is too blue
• Buffered water, wash water or stain too alkaline, Alkaline residue to the slide,
Insufficient washing
• Excessive thickness of the smear
• Prolonged staining before diluting with buffered water
Pale blue:
Buffered water, wash water or stain too acid, Acid residue on slide
Entire smear has a pale stain: Under staining, Weak stain, Excessive washing or
allowing water to stand on slide, using warm or hot water for washing slide
Variation in staining on different areas of the smear
Buffered water un evenly applied and not thoroughly mixed with Wright’s stain,
Acid or alkaline residue on the slide, Water not properly drained from slide after
washing
Precipitated stain
Lack of through washing, Precipitate in Wright’s stain not properly filtered,
Evaporation of alcoholic stain
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Analysis
1.Total Count of RBC
Objective: To enumerate the total count of RBC/cumm of a given
blood sample.
Method: Hemocytometry method
Significance
• It performs some functions such as transportation of O2 and CO2
• A decrease in RBC accounts for less hemoglobin i.e., anemia
• An increase in RBC is referred as Polycythemia
Requirements: Hemocytometer, cover slip, microscope, RBC
diluting fluid, Haeyem’s solution or Physiological saline 0.85%
Nacl.
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Procedure
• Take the blood in to RBC pipette up to 0.5 marks
• Immediately draw the RBC diluting fluid up to mark 101.
• Rotate the pipette between thumb and other fingers with finger eight
movements. This gives a dilution of 1:200.
• Clean the counting chamber of hemocytometer and cover slip
• Place the cover slip in position over counting chamber by
gentlepressure
• Expel a drop of blood on to the counting chamber by holding the pipette
at an angle of 45º.
• Allow the hemocytometer for 2-3 min to settle down the RBC in counting
chamber
• Counting: Counting rules
- Count less than 40 × microscope objective
- Count cells touching the left and top side lines.
- Don’t count cells touching the bottom right side lines.
- Count first left to right direction, then to vise verse.
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Calculation
Volume of one small square
= 1/20mm × 1/20mm × 1/10mm = 1/4000mm3
Volume of 80 small square
= 80 × 1/4000mm3 = 1/50mm3
Total number of RBC =
Cells counted (N)
Volume of all squares × dilution factor
Total RBC =
N (cell counted) = N × 10,000
1/50mm3 × 1/200
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Self study
Analysis
2. Total Count of WBC (White Blood Cells)
• Differential Leukocyte Count
• Neutrophils, Eosinophils, Basophils, Lymphocytes,
Monocytes,
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Principle:
The distance (mm), the erythrocyte fall, in a given period of time when blood
(anticoagulant added) in a tube, placed in a vertical position
Significance
• It is not a specific test, but reflects change in plasma protein accompanying
most of acute and chronic infection
• Some pathological condition causes rouleax formation
• The greater the ESR reading, the more the severity of pathological condition
• During TB and rheumatic disease ESR increases
Method: Westergrens method
Requirements: ESR stand, ESR tube, blood sample
Protocol
• Take the anticoagulant blood in to ESR tubes exactly up to ‘0’ mark.
• Place the tube vertically (upright position) in ESR stand.
• Take reading after 5min as ‘zero’ hour reading and again note the reading after
1 hour and 2 hours.
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3. Determination of Erythrocyte Sedimentation Rate (ESR)

4. Coagulation Time Determination
(Whole Blood Clotting Time)
Lee-white method
• Obtain at least 3ml of blood in a plastic syringe by careful vein
puncture (start a stop watch)
• Place 1ml of blood into each of the three tubes
• Place the test tube in a water bath at 37°C
• After 2 minutes one of the three test tubes is tipped gently at
one minute interval
• Test the third test tube in the same manner
• The time elapsed between the first appearance of the blood in
the syringe and clot formation in the third tube is clotting time
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Capillary tube method
Capillary tube, filter paper, clock watch should be used as requirements
• A skin puncture is made and wiping away the first drop, fill a special capillary
tube with blood noting the time when the blood first appeared
• Holding the tube between the thumb and index finger of both hands, gently
break the tube every second until a strand of thread fibrin is seen extending
across the gap between the ends of the tube
• The interval between the appearance of the blood and the appearance of the
fibrin stand is the coagulation time
Interpretation
Normal value
• Lee- white method in glass tube --- 3-12 minutes
• Capillary tube method---3-15
Prolonged
• Deficiency in coagulation factors, • Vitamin K deficiency
• Thrombocytopenia, • The presence of circulating anticoagulants
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5. Bleeding Time
Principle:
Determination of bleeding time is a simple and sometimes useful
tool for evaluating the efficiency of the capillary – platelet aspect
of homeostasis
Purpose: To determine the bleeding time
Method: Dukes method
Significance
• The study helps in diagnosis, treatment, and study of
hemorrhagic diseases
• The prolonged time indicates coagulation defect
Requirements: Blood lancet, filter paper, clock (stop clock)
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Protocol
• Make a moderately small, deep puncture in clean, sterile blood
lancet or sterile needle, and note the time when blood first appears
(nose is preferable)
• Remove the drops of blood with filter paper every 30 second
being careful not to touch the skin. The use of highly absorbent
paper such as cleaning tissue tends to prolong bleeding time due
to more effective removal of surface blood
• Note the end point, when blood no longer appears from the
puncture site
Interpretation
Normal values: 1- 5 min
Prolonged due to
• vascular lesions, • platelet defect, • severe liver disease, uremia
• anticoagulant drug administration
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Blood platelet count by
hemocytometer
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HEMOCYTOMETER
• The hemocytometer is a specimen slide which is used to determine the
concentration of cells in a liquid sample.
• It has a rectangular indentation that that creates a chamber
• The device is carefully crafted so that the area bounded by the lines is
known, and the depth of the chamber is also known.
• Given the known parameters it is possible to count the number of cells
or particles in a specific volume of fluid, and thereby calculate the
concentration of cells in the fluid overall
• The hemocytometer is frequently used to determine the concentration of
blood cells (hence the name “hemo-”)
• However, it can also be used for other samples, such as sperm cells.

HEMOCYTOMETER
• The cover glass, which is placed on the sample, does not simply float on
the liquid, but is held in place at a specified height (usually 0.1mm).
• Additionally, a grid is etched into the glass of the hemocytometer.
• This grid, an arrangement of squares of different sizes, allows for an
easy counting of cells.
• This way it is possible to determine the number of cells in a specified
volume.

HEMOCYTOMETER
NEUBAUER COUNTING CHAMBER

SAMPLE PREPARATION
• Proper mixing:
The fluid should be a homogenous suspension.
Cells that stick together in clumps are difficult to count and they are usually not
evenly distributed.
Appropriate concentration:
• The concentration of the cells should neither be too high or too low.
• concentration - too high, - the cells overlap and are difficult to count.
• Low concentration - a few cells per square results - then necessary to count
more squares (which takes time).
• Suspensions that have a too high concentration should be diluted 1:10, 1:100
and 1:1000. (1:10 dilution means 1 part sample and 9 parts normal saline)
• The dilution must later be considered when calculating the final concentration.

COUNTING CELLS
Counting cells that are on a line:
•Cells that are on the line of a grid require special attention.
•Cells that touch the top and right lines of a square should not be counted
•Cells on the bottom and left side should be counted.
Number of squares to count:
•The lower the concentration, the more squares should be counted.
•Otherwise one introduces statistical errors.
•Cells should be counted on both sides of the chamber.
•If the final result is very different, then this can be an indication of sampling
error.

NEUBAUER COUNTING
CHAMBER
• When a liquid sample containing immobilized cells is placed on the
chamber, it is covered with a cover glass, and capillary action
completely fills the chamber with the sample.
• Looking at the chamber through a microscope, the number of cells in the
chamber can be determined by counting.
• Different kinds of cells can be counted separately as long as they are
visually distinguishable.
• The concentration of the cells can be calculated from the cells counted
from the mixture using simple formulas

NEUBAUER COUNTING
CHAMBER
• Rulings cover 9 square millimeters.
• Boundary lines of the Neubauer ruling are the center lines of the groups of
three
• The central square millimeter is ruled into 25 groups of 16
small squares
• The ruled surface is 0.10mm below the cover glass
• One (1) Milliliter = 1000 cubic millimeters (cu mm)
• One (1) Microliter (ul) = One (1) cubic millimeter (cu
mm)
• The number of cells per cubic millimeter =
Number of cells counted per square millimeter X
dilution (eg. 100 for WBC count) X 10 (depth factor)

CALCULATE AREA AND VOLUME
• Depth: 0.1 mm
• Red square = 1 x 1 mm = 1 mm2
(AREA) = 0.1 cubic millimeter
• Green square = 0.25 x 0.25 mm = 0.0625 mm2
= 0.00625 mm3
• Yellow square = 0.2 x 0.2 mm = 0.04 mm2
= 0.004 mm3
• Blue square = 0.05 x 0.05 mm = 0.0025 mm2
= 0.00025 mm3

SOURCES OF ERRORS
• There are different types of counting chambers available, with different grid
sizes.
• Know the grid size and height (read the instruction manual) otherwise
you’ll make calculation errors.
• The provided cover-glasses are thicker than the standard 0.15mm cover
glasses.
• They are less flexible and the surface tension of the fluid will not deform
them. This way the height of the fluid is standardized.
• Moving cells (such as sperm cells) are difficult to count.
• These cells must first be immobilized.
• The hemocytometer is much thicker than a regular slide.
• Be careful that you do not crash the objective into the hemocytometer when
focusing

UNOPETTE MICROCOLLECTION SYSTEM
• The Unopette system is a system of prefilled blood dilution vials
containing solutions that will preserve certain cell types while
lysing others.
• It utilizes a premeasured volume of diluent in a chamber into
which a specified amount of blood is drawn
• The Unopette test system consists of a self-filling capillary
pipette
• It consists of a straight, thin-wall, uniform-bore plastic capillary
tube fitted into a plastic holder
• Also has a plastic reservoir containing a premeasured volume of
reagent for diluting
• The reservoir is punctured to open access to the reagent
• The dilution is determined by the type of capillary used since
each type have different volumes
• The diluted blood is added to a hemocytometer chamber and
cells are counted in a specified area.

BMP LEUKOCHEK SYSTEM
• Unopette System discontinued
• The BMP LeukoChek is used to measure and dilute
whole blood for manual counting of leukocytes
(WBC) and platelets
• It replaces the Unopette system
Tested to CLIA guidelines
Clinical Laboratory Improvement Amendments (CLIA) – establish quality
standards for all laboratory testing to ensure the accuracy, reliability and
timeliness of patient test results regardless of where the test was performed

MANUAL DETERMINATION OF
WBC AND PLATELETS
PRINCIPLE
• Whole blood is added to the diluent (ammonium oxalate) , which lyses red
cells but preserves platelets, leukocytes
• When erythrocytes are completely lysed, the solution will be clear red and
counting can proceed.
• The diluted blood is placed in a hemocytometer according to accepted
technique.
• Cells are allowed to settle for 10-15 minutes before leukocytes and platelets
are counted.
• Under 100X magnification (x10 objective) using bright-light microscopy,
leukocytes appear refractile (can be seen as dark dots)
• Under 400X magnification (x40 objective) using bright-light microscopy,
platelets appear oval or round and frequently have one or more dendritic
processes.

Reagents and Equipment
• BMP LeukoChek containing ammonium oxalate
Check expiration dates, do not use expired test kits. Protect from sunlight.
• BMP LeukoChek capillary pipette, 20 μL.
• Hemocytometer : improved Neubauer ruling
• Hemocytometer coverslips
• Petri dish lined with filter paper that has been moistened and two applicator
sticks to hold the hemocytometer
• Microscope, Hand counter, EDTA whole blood
DILUTION RATIO
• Sample to total volume.......................1:100
• That is 1.98 ml of diluent to 20μl of sample

PROCEDURE FOR DETERMINATION OF WBC AND
PLATELETS
• 1. Specimen should be well mixed and left on a rocker for at least
5 minutes before using.
• 2. Check BMP LeukoChek for clarity and contents. If the BMP
LeukoChek chambers appear cloudy or the amount of reagent
looks questionable, do not use.

3. With the reservoir on a flat surface, puncture the diaphragm of the reservoir
using the protective shield of the capillary pipette.
A. Using a twist action, remove protective shield from the pipette assembly.
B. Holding the pipette and the tube of blood almost horizontally, touch the tip
of the pipette to the blood (fill with 20μl of blood).
The pipette will fill by capillary action and will stop automatically when the
blood reaches the end of the capillary bore in the neck of the pipette
C. Wipe the excess blood from the outside of the capillary pipette.
Be careful not to touch the tip of the capillary when wiping off excess blood.
D. Before entering the reservoir, it is necessary to force some air out of the
reservoir by squeezing it. Do not expel any liquid and maintain pressure on
reservoir.

E. Place an index finger over opening of overflow chamber and position
pipette into reservoir neck.
F. Release pressure on reservoir and then remove finger. The negative
pressure will draw blood into pipette.
G. Rinse the capillary pipette with the diluents by squeezing the
reservoir gently two or three times.
This forces diluent up into, but not out of, the overflow chamber and
releases pressure each time to ensure the mixture returns to the reservoir.
H. Return protective shield over upper opening and gently invert several
times to mix blood adequately.
I. Allow the BMP LeukoChek to stand for 10 minutes to allow RBCs to
hemolyze. Leukocyte counts should be performed within 3 hours.

PROCEDURE FOR DETERMINATION OF WBC AND
PLATELETS
4. Charge hemocytometer
A. Mix the dilution by inversion and convert the BMP LeukoChek to the
dropper assembly.
B. Gently squeeze BMP LeukoChek and discard first 3 or 4 drops.
This allows proper mixing, with no excess diluent in the tip of the capillary.
C. Carefully charge hemocytometer with the diluted blood, gently
squeezing the reservoir to release contents until chamber is properly filled.
Be sure to charge both sides and not to overfill chambers.
5. Place the hemocytometer in the pre-moistened Petri dish and leave for 15
minutes.
This allows the sample to settle evenly.
6. Cell count can now be performed

CELL COUNT AND CALCULATION - WBC COUNT
• A WBC count is performed with a Neubauer
hemocytometer.
• Using the X10 microscope magnification, count WBC using
the four outer large squares on the outer sections of the
counting chamber
• Count both sides of the chamber and average the count.
• When counting, the cells that touch the extreme lower and
the extreme left lines are included in the count.
Those on top and right are not included.
Count both sides of the chamber and average the numbers
•

CELL COUNT AND CALCULATION - WBC COUNT
• Use the following formulas to calculate the WBC.
• Cells/mm3 = Average No. of cells + 10% X depth factor (10) X
dilution factor (100) divided by the Area (number of squares
counted)
• Depth factor is multiplied by 10 to convert area to volume in μl
• Area of each large square = 1mm, so for the 4 large squares =
4mm
Normal Value:
• Adult: 4,000 – 10,000
• Newborn: 10,000 – 30,000

CELL COUNT AND CALCULATION -
PLATELET COUNT
• Platelet counts are performed with
a Neubauer hemocytometer
• Counting is done using x40 dry
phase contrast objective.
Platelets will have a faint halo.
• The middle square of the
hemocytometer chamber is
counted.
• It contains 25 small squares.

CELL COUNT AND CALCULATION
- PLATELET COUNT
• Count the 25 squares in the middle of the counting chamber
No. of platelets/mm3 =
Multiply No. of platelets (+ 10%) X 1000
OR
• Count 5 of the 25 squares
• Take the average of both sides add 10%
• Multiply No. of platelets x 5000 = No. of platelets/mm3
• Normal Value: Platelets: 150,000 to 400,000 mm3

WBC AND PLATELET CELL COUNT - UNOPETTE
LIMITATIONS
1. Specimen should be properly mixed and have sufficient volume of blood so
there is no dilution of anticoagulant.
2. The capillary tube must be filled completely and be free of any air bubbles.
3. After the hemocytometer is charged, it should be placed in a pre-moistened
Petri dish to prevent evaporation while the cells are settling out.
4. The light adjustment is critical. Important for WBCs platelets. If the
condenser is not in the correct position, it will fade out platelets.
5. Debris and bacteria can be mistaken for platelets.
6. Clumped platelets cannot be counted properly The anticoagulant of choice is
EDTA for preventing platelet clumping.
7. Avoiding overloading of hemocytometer chamber.
8. A highly elevated leukocyte or platelet count - makes counting difficult. A
secondary dilution –needed. secondary dilution calculating the total count,
9. All WBC and platelet counts are done in duplicate.WBC counts should agree
+/- 15%., Platelet counts must agree +/- 25%.

Causes of elevated WBC (Leukocytosis)
• Infections – most common is bacterial infections
It also occur in viral (lymphocytosis)
• Allergy and drug hypersensitivity
• Parasitic infections
• Inflammation: eg. Inflammatory bowel disease, RA, and
vasculitis
• Extremely low birth weight
• Malignancy and myeloproliferative disorders: eg.
Leukemias, lymphomas
• Increased release of WBC from bone marrow:- This occurs
in infection, stress, and hypoxia
it also occurs due to endotoxin stimulation and steroid
administration

Causes of low WBC (Leukopenia)
• Viral infections – eg. HIV
• Medications – abx, diuretics
• Chemotherapy/Radiation therapy
• Hyperthyroidism
• Malignancy
• Leukemia
• Lupus
• Aplastic anemia

Causes of thrombocytosis
• Acute blood loss/Hemolytic Anemia
• Malignancy
• Splenomegaly
• Inflammatory conditions – RA, IBS, Celia disease, Connective
tissue disorder
• Pancreatitis
• Kidney disease

Causes of thrombocytopenia
• Idiopathic thrombocytopenic purpura (ITP)
• Thrombotic thrombocytopenic purpura (TTP)
• Hemolytic uremic syndrome – heparin, sulfa drugs, quinidine
• Bacteremia
• Autoimmune diseases
• Pregnancy
• Trapping of platelets in the spleen
• Reduced production of platelets
• Increased breakdown of platelets

Dr. I. Manjubala
78
• https://www.youtube.com/watch?v=WWS9sZbGj6A
https://www.youtube.com/watch?v=pP0xERLUhyc
https://www.youtube.com/watch?v=vEXMajaF6zo
https://www.youtube.com/watch?v=7AWu4Qb_Emk

Blood Glucose - Glucometer
Dr. I. Manjubala
79

Diabetes - Types
Diabetes: (too much glucose in the blood)
•a condition where the body is unable to regulate the amount of glucose
in the blood due to lack of insulin or the body’s inability to produce
insulin.
Type 1:
•also known as “juvenile diabetes” or “insulin-dependent diabetes”
•usually develops in children or young adults (must take insulin daily)
•the body does not produce enough insulin to control the amount of
glucose in the blood. (autoimmune disease - destroying the cells of the
pancreas)
Type 2
•known as “adult-onset diabetes” or “non-insulin dependent diabetes”
•Adult, in obese children: a genetic disorder due to deficiency
•Based on diet, exercise and medicine type 2 diabetics may not need to
take insulin daily

Glucose
Glucose:
• a simple sugar that serves as the chief source of energy for
the body.
• Hypoglycemia:
• Low blood sugar
• 60mg/dL or less
• Occurs mostly in Type 1 diabetes or in elderly
• Hyperglycemia:
• High blood sugar
• 240mg/dL or higher
• Can cause damage to eyes, kidneys, heart and nerves
•

Glucose Monitoring
Blood Glucose Monitoring is a way of checking the concentration of
glucose in the blood using a glucometer.
What is the purpose?
–Provides quick response to tell if the sugar is high or low indicating a
change in diet, exercise or insulin.
–Over time, it reveals individual of blood glucose changes.
Why ?
• Reduces risk of developing complications with diabetes.
• Allows diabetics to see if the insulin and other medications they are
taking are working.
• Gives an idea as to how exercise and food affect their blood sugar.
• May prevent hypoglycemia or hyperglycemia

When ?
•When you wake up
•Before meals
•1 to 2 hours after meals
•Before physical activity
• 15 minutes after physical activity
•Before bed
Glucose Test Person without
diabetes
Person with diabetes
Fasting Test 70-110mg/dL > 140mg/dL
2 hours after eating <110mg/dL > 200mg/dL

Glucometer
• A glucometer is an electronic device used to test the
amount of glucose in the blood.
• New models are able to read and calculate the blood
sugar within seconds.
• Some models not only display the glucose reading but
also say it.

Advances in Blood Glucose Monitoring
• Alternate site-testing
• Continuous glucose monitors
• Non-Invasive
• Semi-Invasive
• Surgical
• Laser

AtLast Blood Glucose Meter
• What is it?
– An alternate test site glucose monitor. Instead of pricking
their finger, diabetics are able to draw blood from their thigh
or forearm to test their blood sugar.
• How does it work?
– The AtLast Glucose Meter works just the same as any other
meter, except blood is taken from alternate sites to relieve
the pain of pricking fingertips. The blood drawn is then put
onto a strip which the meter reads and displays the blood
glucose level.
• Why is it advanced?
Alternate site testing is less painful then the
raditional finger testing because there are less
nerve endings in the alternate sites then the finger tips.
• Where can you test?
– Upper arm, forearm, thigh, hand,
palm & calf
•

GlucoWatch Biographer
• What is it?
– Warn like a wristwatch, the Biographer measures glucose every
ten minutes through the skin.
– Using an AutoSensor, a replaceable pad that sticks to the back of
the watch, that is adhesive to the skin
which allows it to come into contact with an electrical charge. This
electrical charge then brings the glucose to the skin surface where
an enzyme reaction generates electrons in the glucose, similar to
that of regular meters, allowing the glucose to be closely estimated.
– First noninvasive glucose monitor
– Provides glucose readings every ten minutes
– Very helpful at showing patterns of glucose levels

HypoMon®
• What is it?
– The HypoMon® System noninvasively detects low blood sugar in
diabetes throught skin contact. The HypoMon® includes a battery
power pack worn on the chest and a wireless receiver where the
readings are sent to and can be read.
– With the battery powered unit attached to the chest, the four skin
sensors measure skin moisture and heart activity which are two known
symptoms of hypoglycemia. The readings are then sent to the
wireless receiver where they can be read.
– Alerts sound when the blood glucose level falls
below 45mg/dL.
– Enables monitoring during the day and night.
– Alters allow the diabetic to treat hypoglycemia
at an earlier stage.

Silicon Micro Needle
• What is it?
– Silicon Micro Needle consists of a hand-held battery-powered
electronic monitor which holds a cartridge loaded with 10
disposable sampling devices. Each disposable consists of the
micro-needle and a receptacle into which the blood sample is
drawn.
– The cartridge is loaded into the monitor and pressed up to the
skin. This penetrates the skin, drawing a very small amount of
blood into the disposable needle. Chemicals in the microneedle
react with the glucose to produce a color. The monitor then
analyses this color using a laser light and displays the glucose
level.
– Pain free testing and the amount of blood
required is 1/100th
of a drop of blood.

REAL-Time Continuous
Glucose Monitoring System
• What is it?
– An insulin pump integrated with REAL-Time continuous glucose
monitoring that measures the glucose levels for up to 72 hours.
– Diabetic must use MiniMed Paradigm insulin pump, a device that
delivers insulin to the body though a small plastic catheter. They must
also wear a sensor that monitors glucose for up to 3 days that is
connected to the MiniLinkTM
Transmitter. This transmitter sends the data
from the sensor to the insulin pump through radio frequency wireless
technology. The insulin pump with REAL-Time alarms diabetics when
their glucose levels are high or low.
• Why is this advanced?
– Warns diabetics of glucose levels a finger stick
– Helps take action and gain control sooner.
– The REAL-Time trend graph shows how meals,
exercise, insulin and medication affect glucose.

Cell Robotics' Lasette
• What is it?
– A laser lancing device that uses a laser beam to draw a drop of
blood rather then using a steel lancet.
– The fingertip is placed over the disposable lens cover where
the laser beam comes out of. Water in the skin absorbs the
energy from the laser beam, instantly vaporizing tissue which
draws blood.
– Virtually painless
– No more finger pricking

Glucose Control Benefits
• Keeping blood glucose levels as close to normal as possible:
– Few or even no complications
– Normal life span
• Short term benefits of glucose control
– Feel better
– Stay healthy
– Have more energy
– Reduce risk of hyperglycemia and hypoglycemia
• Long term benefits of glucose control
– Lower chances of having eye, heart and kidney disease and nerve damage
– Enjoy a better quality of life

Glucose Tolerance Test- An overview
• The ability to utilize carbohydrates can be determined by
Glucose tolerance test.
• Initially fasting blood glucose is estimated
• A loading dose of glucose is given.
• The blood glucose levels are estimated at regular intervals
after the glucose load
• In conditions of insulin deficiency, blood glucose levels get
elevated due to impaired utilization of glucose.
07/30/17 93

Glucose tolerance
A) Decreased Glucose tolerance
Decreased carbohydrate tolerance (non-utilization of
carbohydrate load) is observed in conditions causing
hyperglycemia, for example:
• Diabetes mellitus
• Hyperactivity of anterior pituitary and adrenal cortex
• Hyperthyroidism
• Stress
B) Increased Glucose Tolerance
Increased carbohydrate tolerance is observed in all conditions that
cause hypoglycemia-
i) Hypopituitarism
ii) Hyperinsulinism
iii) Hypothyroidism
iv) Adrenal cortical hypofunction
v) Decreased gastro intestinal absorption like sprue, celiac disease.
07/30/17 94

When to do, for whom to do GTT
i)In asymptomatic persons with sustained or transient glycosuria
ii) In persons with symptoms of diabetes but no glycosuria or
hyperglycemia
iii) Persons with family history but no symptoms or positive blood findings
iv)In persons with or without symptoms of diabetes mellitus showing one
abnormal blood finding
v) In patients with neuropathies or retinopathies of unknown origin
vi) In women with H/o having delivered large babies.
NOT TO DO:
a) In proven cases of diabetes mellitus the test is not required.
b) GTT is required only in doubtful cases, it is not recommended for follow
up of patient.
c) The test should not be carried out in acutely ill patients
07/30/17 95

GTT - Precautions
a) The patient is instructed to have good carbohydrate diet for 3
days prior to the test. Further , diet containing about 30-50 G
of carbohydrate should be taken on the evening prior to the
test.
b) should avoid drugs likely to influence the blood glucose levels,
for at least, 2 days prior to the test
c) should abstain from smoking during the test.
d) Strenuous exercise on the previous day is to be avoided.
e) The exercise is also to be avoided on the same day prior to
the test
07/30/17 96

Types of glucose tolerance test
• Standard Oral glucose tolerance test
• I/V Glucose tolerance test
• Mini Glucose tolerance test
07/30/17 97

Procedure of standard oral glucose
tolerance test
a) At about 8 a.m. the fasting blood and urine samples are collected.
These are called zero samples.
b) A loading dose of 75 g. anhydrous glucose dissolved in 250-300 ml of
water is given to the patient.
07/30/17 98
Procedure
•In children 1.75 g of glucose /kg body weight is
given.
•In the classical procedures, the blood and urine
samples are collected at half hourly interval of the
next two and a half hour or three hours.
•Glucose is estimated in all the blood samples.
•Urine is analyzed for the presence of glucose.

Glucose tolerance curve
• A curve is plotted with the blood glucose levels on the vertical
axis against the time of collection on the horizontal axis.
• The curve so obtained is called glucose tolerance curve.
07/30/17 99
Fasting
(Zero
sample
)
30
minute
s
60
minute
s
90
minute
s
120
minute
s
150
minute
s
180
minute
s
Blood
Glucose
(mg/dl)
90 100 150 120 110 80 70
Urinary
Glucose
nil nil nil nil nil nil nil
Laboratory profile of a normal person after glucose load

Normal Glucose tolerance curve
07/30/17 100

Normal glucose tolerance curve
i) Fasting blood glucose (Zero hour sample)- is 90 mg /dl, which
is well within the normal range(Normal 60-100 mg/dl).
ii) There is rise of blood glucose after glucose load and the peak
value is observed at I hour. This is due to absorption of
glucose from the intestine.
iii) Insulin is released upon increase of blood glucose level.
There is fall in blood glucose with time due to glucose
utilization promoted by insulin.
iv) The normal blood glucose level is achieved after 150 minutes.
07/30/17 101

Diabetic curve
1) Fasting blood glucose is higher than normal
2) The highest value is attained at 1 hour to 1 hour 30 minutes.
3) The highest value exceeds the renal threshold
4) Glucose is found in almost all the urine samples.
5) The blood glucose level does not return to the fasting level even
within 2hour 30 minutes.
07/30/17 102
Fasting
(Zero
sample
)
30
minute
s
60
minute
s
90
minute
s
120
minute
s
150
minute
s
180
minute
s
Blood
Glucose
(mg/dl)
200 225 350 300 275 250 225
Urinary
Glucose
+ + + + + + +
Laboratory profile of a diabetic patient after glucose load

Diabetic curve
Time in minutes
Bloodglucose(mg/Dl)
07/30/17 103

Diabetic V/S Normal curve
Time in minutes
Bloodglucose(mg/Dl)
07/30/17 104

Renal Glycosuria
• Blood glucose levels are within the normal limits.
• Glucose tolerance curve is normal.
• There is lowering of renal threshold.
• Thus glucose is found in some of the samples depending upon
the renal threshold.
Causes of Renal Glycosuria
• Early diabetes mellitus,
• Pregnancy,
• Renal disease,
• Heavy metal poisoning
• Renal glycosuria can also be observed in children of
diabetic parents.
07/30/17 105

Laboratory profile with Renal glycosuria
Fasting
(Zero
sample
)
30
minute
s
60
minute
s
90
minute
s
120
minute
s
150
minute
s
180
minute
s
Blood
Glucose
(mg/dl)
90 130 150 140 120 100 90
Urinary
Glucose
nil + + + + ± nil
07/30/17 106

Lag Curve
• Fasting blood glucose is normal.
• Sharp rise within 30 minutes to one hour
• The blood glucose levels exceed the renal threshold.
• The decline is rapid and the normal levels are attained back.
• Some of the urine samples contain glucose, - where the blood glucose
is above the renal threshold.
Cause of Lag curve: Hyperthyroidism, Pregnancy, After gastro-
enterostomy, Early diabetes mellitus
07/30/17 107
Fasting
(Zero
sample)
30
minutes
60
minutes
90
minutes
120
minutes
150
minutes
180
minutes
Blood
Glucose
(mg/dl)
90 230 180 150 120 100 90
Urinary
Glucose
nil + + nil nil nil nil
Laboratory profile of a patient having lag curve

I/V Glucose tolerance test
•This test is undertaken for patients with malabsorption (Celiac
disease or enteropathies),
•Under these conditions oral glucose load is not well absorbed and
the results of oral glucose tolerance test become inconclusive.
07/30/17 108
I/V Glucose tolerance test- Procedure
•I/V glucose tolerance test is carried out by giving 25 g of glucose
dissolved in 100 ml distill water as intravenous injection within 5 min.
•Completion of infusion is taken as 0 time.
•Blood samples are taken at 10 minutes interval for the next hour.
•The peak value is reached within a few minutes and the value
touches to near normal in 45-60 minutes.
Interpretation
In normal individuals, blood glucose level returns to normal within 60 min
In diabetes mellitus, decline is slow
The initial values are attained in 120 minutes.

Mini or Modern GTT
• As per current WHO recommendations, in the mini or modern
glucose tolerance test, only two samples are collected,
• Fasting (zero hour) and 2 hour post glucose load.
• Urine samples are also collected during the same time.
• The diagnosis is made from the variations observed in these results.
07/30/17 109
Time of sample
collection
Normal person Criteria for
diagnosing
diabetes mellitus
Criteria for
diagnosing IGT
Fasting <110 mg/dl
<(6.1m.mol/L)
> 126 mg/dl
>(7.0m.mol/L)
110- 126 mg/dl
2 hour after
glucose load
<140 mg/dl
<(7.8mmol/L)
> 200 mg/dl 140-199 mg/dl
The Diabetes Expert Committee criteria

Factors affecting GTT
a) Acute infections- Cortisol is secreted, the curve is elevated
and prolonged
b) Liver diseases- The curve is elevated and prolonged.
c) Hyperthyroidism- There is steep rise in curve.
d) Hypothyroidism-A flat curve is obtained in hypothyroidism.
Thyroid hormone increases the absorption of glucose from
the gut.
e) Starvation- There is rise of counter regulatory hormones,
which show increased glucose tolerance
07/30/17 110

Some more precautions before GTT
• For proper evaluation of the test, the subjects should be
normally active and free from acute illness.
• Medications that may impair glucose tolerance include
diuretics, contraceptive drugs, glucocorticoids, niacin, and
phenytoin should be avoided on that day.
07/30/17 111
GTT Under special conditions
Cortisone stress test- used for detecting pre diabetes or
Latent diabetes
Extended GTT- To diagnose the cause of hypoglycemia
especially 2-3 hours after meals.

Criteria for diagnosis of Diabetes mellitus
• If the fasting plasma glucose level is 126 mg/dL or higher
on more than one occasion, further evaluation of the
patient with a glucose challenge is unnecessary.
• However, when fasting plasma glucose is less than 126
mg/dL in suspected cases, a standardized oral glucose
tolerance test may be done .
• A random plasma glucose concentration 200 mg/dL
accompanied by classic symptoms of DM (polyuria,
polydipsia, weight loss) is sufficient for the diagnosis of
DM.
07/30/17 112

Dr. I. Manjubala
113
Regulation of blood glucose level
https://www.youtube.com/watch?v=ae_jC4FDOUc
https://www.youtube.com/watch?v=OYH1deu7-4E

Bst unit-iii

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