INSTRUMENTS

-DR .M .Vijayalakshmi
Assistant professor
GSMC
• [Link] needle
• [Link] punctur needle
• [Link]’s PCV / ESR Tube
• [Link]’s ESR Tube
• [Link]’S HEMOGLOBINOMETER
• [Link] NEUBAUER CHAMBER
• 7. RBC Pipette
• [Link] Pipette
• 9. Urinometer
• 10.L Mould (Leuckhart’s Embedding Mould)
• [Link] bag
• [Link]’s
• 13. Coplin Jar
• 14. Paraffin Block
1.
Salah’s Bone Marrow Aspiration
Needle:

[Stout needle with a short bevelled tip

& a wide bore with a close fitting
stylet. There is an adjustable guard
which prevents penetration of the
needle further than desired]
Sites of aspiration:

Posterior iliac crest

Sternum

Tibial Head (In Infants)

Indications for the BMA procedure:

[Link] Leukemias

[Link]

[Link] cytopenia (Anemia/

Leucopenia/ Thrombocytopenia)

[Link] Plasma cell dyscrasias

[Link] storage disorders

 BMA PROCEDURE

1. For aspiration from posterior superior iliac spine, patient should lie on
one side with back towards the physician, knees and hips flexed, and the
knees drawn towards the chest.
2. Antiseptic solution is applied in a circular fashion at the site for
aspiration, moving from center towards periphery.
3. Skin and periosteum are infiltrated with a local anesthetic.
4. After waiting for 5 minutes for anesthesia to take effect, bone marrow
aspiration needle is inserted along with the fitted stylet. (Stylet prevents
blockage of lumen of needle by tissues through which needle passes).
5. When the bone is reached, the needle is rotated clockwise and
anticlockwise and slowly advanced into the bone, maintaining steady
and firm pressure. When the marrow is reached, a slight “give” (decrease
in resistance) will be noted.
[Link] needle is advanced for 1-2 mm into the marrow and the stylet
is removed. If the needle is placed correctly, it will be fixed by the
surrounding bone and will remain rigid and unmoving.
7.A 5 or 10 ml syringe is attached to the needle and a small amount
of marrow is aspirated (till the first drop of blood appears i.e.
0.25-0.50 ml) by quickly pulling the plunger of the syringe.
Aspiration of larger amount of blood causes dilution of marrow
sample by peripheral blood.
[Link] completion of aspiration, the stylet should be reinserted into
the needle and the needle is removed. Sterile gauze is placed
over the site and light pressure is applied till bleeding ceases. A
larger dressing is then applied
Bone marrow aspiration USES:

• Morphology of marrow cells.

• Assessment of hematopoiesis
• Cytogenetic analysis
• Immunophenotyping in leukemias.
• Cytochemistry for typing of leukemia
• Iron stain for assessing iron stores
• Microbial culture, e.g. for tuberculosis
Other BMA Needles:
 Klima Needle
 Jamshidi Needle (Also for bone marrow
biopsy)

Complications:
 Local Infection, Hemorrhage
2.
Lumbar Puncture Needle

[LP needle is about 10cm long fitted with a

long stylet used for doing lumbar puncture to

obtain CSF]
Common site for the procedure

 Between the L4 & L5 vertebra with patient

in lateral flexed position

Indications for the procedure:

(i) In the differential diagnosis of meningitis

(ii) For cytological study in diseases

infiltrating meninges. Ex: Leukemias,
Tumors.

(iii) For injecting anaesthetic agents

(iv) For injecting chemotherapeutic agents

Contraindications:
 Increased intracranial pressure as
evidenced by papilloedema on fundoscopy
 Bleeding diathesis
Complications of Procedure:
 Post puncture headache

 Subdural hematoma

 Infections

 Herniation of brain through foramen

magnum (When CSF pressure is high)
 PROCEDURE
 Patient is in a side-lying (lateral recumbent) position with his back absolutely
vertical and at the edge of the bed, with the knees drawn up and the head
flexed onto his chest. This position increases the space between the lumbar
vertebrae.
 The selected site is thoroughly disinfected, covered with sterile drapes, and
after injecting a local anesthetic, a sterile lumbar puncture needle,
preferably 22 gauge, is inserted slowly.
 There is an increased resistance as the needle passes through the spinal
ligaments and dura mater. As the needle enters the subarachnoid space, a
sudden 'give' (or loss of resistance) is felt.
 The stylet is withdrawn slowly. When CSF drops appear, a pre-assembled
manometer is attached to the needle and the opening pressure is recorded.
 With the three-way stopcock in appropriate position, CSF is collected in
sterile plain tubes as follows:
Tube 1: Chemistry (glucose and protein) and serology
Tube 2: Microbiology (Gram's staining, bacterial culture & sensitivity)
Tube 3: Hematology (Total cell count and differential count)
Tube 4: Cytology, special studies
Xanthochromia refers to yellowish
discoloration of CSF.
- Commonly seen in Subarachnoid
hemorrhage
- [Red blood cells in CSF are hemolyzed with
release of oxyhemoglobin. About a week
after bleeding, macrophages and other
cells of leptomeninges convert
oxyhemoglobin to bilirubin. This produces
yellowish discoloration of CSF]
- Other Causes of Xanthochromia are
Jaundice & Increased CSF Protein.
3.
WINTROBE’S PCV / ESR TUBE

 Thick walled glass tube with

length 11cm
 Uniform internal diameter
2.5mm
 Flat Base
 Calibrated at 1mm intervals
from 0-110 mm in ascending
order
 Holds 1ml of blood
USES OF PCV:
 Estimation of red cell indices
(mean cell volume and mean
corpuscular hemoglobin
concentration)
 Checking accuracy of hemoglobin
value (Hemoglobin in grams/dl × 3
= PCV).
 In anemia, PCV is below the lower
level of normal range.
 PCV is raised in dehydration,
shock, burns, and polycythemia
PACKED CELL VOLUME / HEMATOCRIT PROCEDURE:
 Packed cell volume (PCV) is the volume occupied by the red cells when a
sample of anticoagulated blood is centrifuged
 Mix the anticoagulated blood sample thoroughly. Draw the blood sample
in a Pasteur pipette and introduce the pipette up to the bottom of the
Wintrobe tube. Fill the tube from the bottom exactly up to the 100 mark.
 During filling, tip of the pipette is raised, but should remain under the
rising meniscus to avoid foaming.
 Centrifuge the sample at 3000RPM for 30 minutes.
 After centrifugation of anticoagulated whole blood, three zones can be
distinguished in the Wintrobe tube from above downwards - Plasma,
Buffy coat layer (a small greyish layer of white cells and platelets, about 1
mm thick), and Packed red cells
 Take the reading of the length of the column of red cells. PCV
(Hematocrit) can be expressed either as a percentage or as a fraction of
the total volume of blood sample.
 PCV = (RBC column / Total volume of blood taken) X 100
NORMAL VALUES:
Adult males: 40-50%
Adult females (Nonpregnant): 38-45%
Adult females (Pregnant): 36-42%
4.
Westergren’s ESR Tube.
[Pipette open at both ends; 30cm long; Bore Calibrated in millimeter from 0
– 200 above downwards]
*ESR – Erythrocyte Sedimentation Rate
ANTICOAGLUANT: 3.8% Trisodium Citrate
[1.6ml of venous blood is added to 0.4ml of 3.8% Trisodium Citrate in the proportion of 4:1.
Westergren tube is filled with the blood sample up to the “0” mark. The tube is placed in a
strictly vertical position in the ESR stand and left undisturbed for 1 hour. After exactly 1
hour, read the height of the column of plasma above the red cell column in mm]
STAGES OF ERYTHROCYTE SEDIMENTATION RATE:
Stage 1: Phase of Rouleaux Formation (10 minutes). RBCs stack together like a pack of coins.
Stage 2: Phase of Settling (40 minutes). Rapid and constant sedimentation
Stage 3: Phase of Packing (10 minutes). Sedimentation slows and packing of RBCs occur.
Factors increasing ESR:
– Old age
– Pregnancy
– Anemia
– Elevated fibrinogen
– Macrocytosis
– Technical factors: High temperature, tilting of ESR tube

Factors decreasing ESR:

– Microcytosis
– Low fibrinogen
– Polycythemia
– Marked leukocytosis
– Technical factors: Vibration of tube during test
NORMAL VALUES: Other Methods to Estimate ESR:
Males: 3-5 mm in 1 hour  Wintrobe method
Females: 4-7 mm in 1 hour  Zeta sedimentation ratio
 Micro-ESR
Causes of increased erythrocyte sedimentation rate:
 Infections: Acute rheumatic fever, Osteomyelitis, Bacterial
endocarditis, Tuberculosis, Acute hepatitis
 Inflammatory diseases: Rheumatoid arthritis, Systemic lupus
erythematosus
 Acute myocardial infarction

 Malignancy

 Multiple myeloma
[Link]’S HEMOGLOBINOMETER
SAHLI’S HEMOGLOBINOMETER: Consists of a standard brown glass
mounted on a comparator and a graduated tube. The tube shows
graduations in percent on one side and grams on the other side.
HEMOGLOBIN PIPETTE: A special pipette to measure 20 microliters of blood
INDICATIONS FOR Hb ESTIMATION

 To determine presence and severity of anemia

 Screening for polycythemia

 To assess response to specific therapy in anemia.

 Estimation of red cell indices

 Selection of blood donors.

(EDTA Anticoagulant)
SAHLI’S ACID HEMATIN METHOD
 Principle:
Blood is mixed with an acid solution so that
hemoglobin is converted to brown coloured acid
hematin.
This is then diluted with water till the brown colour
matches that of the brown glass standard.
The hemoglobin value is read directly from the scale.
EQUIPMENTS & REAGENTS

Reagents: N/10 HCL, Distilled water

 PROCEDURE
• After ensuring the hemoglobin pipette and tube are
dry, add N/10 HCl into the tube up to mark 2g%
• Mix the EDTA-Blood sample gently and fill the
pipette with 20 microlitre blood. Make sure that no
air bubbles enter into the pipette. If it enters,
discard and pipette again. Wipe the external
surface of the pipette to remove any excess blood.
 PROCEDURE
• Add the blood into the tube containing HCl. Wash
out the contents of the hemoglobin pipette by
drawing in and blowing out the acid few times so
that the blood is mixed with the acid thoroughly.
• Allow to stand undisturbed for 10min.( This is
because, maximum conversion of hemoglobin to
acid hematin, occurs in the first ten minutes )
 PROCEDURE
• Place the hemoglobinometer tube in the comparator and add
distilled water to the solution drop by drop. Stir with the glass
rod till its colour matches with that of the comparator glass.
• While matching the colour, glass rod must be removed from
solution and held vertically in the tube. (Note that the stirrer
should be above the level of the solution and not out of tube
)
• The reading of the lower meniscus of the solution should be
noted as the result. Express the hemoglobin content as g%.
 ADVANTAGES
• Easy to perform
• Quick
• Inexpensive
• Used as a bedside procedure
• Does not require technical expertise
 DISADVANTAGES
• For maximum colour, longer time is required

• Perfect matching with brown glass standard is not

possible
• Carboxyhemoglobin, methemoglobin & sulfhemoglobin
are not converted to acid hematin
• Development of colour is slow and acid hematin is not
stable
• Source of light will influence the development of colour.
 OTHER METHODS
• Tallqvist hemoglobin chart
• WHO hemoglobin colour scale
• Specific gravity method
• Alkaline hematin method
• Cyanmethemoglobin method
6.
IMPROVED NEUBAUER CHAMBER: There are two ruled areas on the
surface of the chamber. Each ruled area is 3 mm × 3 mm in size and
consists of 9 large squares with each large square measuring 1 mm ×
1 mm. When the special thick cover glass is placed over the ruled
area, the volume occupied by the diluted blood in each large square
is 0.1 ml
IMPROVED NEUBAUER CHAMBER: In the improved Neubauer chamber,
the central large square is divided into 25 squares, each of which is
further subdivided into 16 small squares. A group of 16 small squares
is separated by closely ruled triple lines. Metallized surface makes
background rulings and cells easily visible. The 4 large corner
squares are used for counting leukocytes, while the central large
square is used for counting platelets and red blood cells .
USES:
 RBC Count

 WBC Count

 Platelet Count

 Sperm Count
7.
RBC Pipette
 Also known as Thoma’s Red Cell
Counting Pipette
 Consists of

- Capillary Tube

- Bulb with a rubber Bead

- Short capillary tube

- Rubber Tube
USES:
 Counting red cells

 Counting platelets

 Counting WBCs when count is high

as in leukemia
 Capillary tube is divided into 10
parts; Marked 0.5 & 1
 Bulb has a red colored rubber bead;
In the dry state, the bead should be
free within the bulb
 Short capillary tube is marked 101

 Blood is drawn up to 0.5 mark and

RBC diluting fluid (Hayem’s or
Dacie’s fluid) is drawn up to 101
mark (Blood diluted 200 times) and
mixed well
 Discard the first 1-2 drops of blood

 Charge the Neubauer chamber by touching the tip of pipette and leave
it undisturbed for 3 minutes
 Count the RBCs in the 5 squares marked in the picture

 RBC Count = (No. of RBCs counted X Dilution Factor) / Volume of

chamber in which RBCs are counted  (Nx200) / (1/50)
8.
 Discard the first 1-2 drops of blood

 Charge the Neubauer chamber by touching the tip of pipette and leave
it undisturbed for 3 minutes
 Count the RBCs in the 5 squares marked in the picture

 RBC Count = (No. of RBCs counted X Dilution Factor) / Volume of

chamber in which RBCs are counted  (Nx200) / (1/50)
WBC Pipette
 Also known as Thoma’s White Cell
Counting Pipette
 Consists of

- Capillary Tube

- Bulb with a white rubber Bead

- Short capillary tube

- Rubber Tube
USES:
 Counting WBCs

 Counting sperms

 Cell count in fluids like pleural fluid,

ascitic fluid
  Capillary tube is divided into 10
parts; Marked 0.5 & 1
 Bulb has a white colored rubber
bead; In the dry state, the bead
should be free within the bulb
 Short capillary tube is marked 11

 Blood is drawn up to 0.5 mark and

WBC diluting fluid (Turk’s fluid) is
drawn up to 11 mark (Blood diluted
20 times) and mixed well

[Turk’s Fluid: Glacial acetic acid 2 ml lyses the RBCs + Gentian violet 1 ml stains the WBCs +
Distilled water to make 100 ml]
 Discard the first 1-2 drops of blood

 Charge the Neubauer chamber by touching the tip of pipette and leave
it undisturbed for 3 minutes
 Count the WBCs in the 4 large squares marked in the picture

 WBC Count = (No. of WBCs counted X Dilution Factor) / Volume of

chamber in which RBCs are counted  (Nx20) / (4/10)
9.
URINOMETER:

 Used to measure specific

gravity of urine
 Consists of a weighted
cylinder which is floated in
urine
 Has a scale with readings
from 1.000 – 1.060
 Normal SG of urine: 1.003 –
1.030
SPECIFIC GRAVITY OF URINE - PROCEDURE:
Based on the principle of buoyancy; (i.e. the ability of a fluid to
exert an upward thrust on a body placed in it).
Urinometer is placed in a container (3/4) filled with urine. Insert
urinometer into it so that it floats in urine without touching the
wall and bottom of container
When solute concentration is high, upthrust of solution increases
and urinometer is pushed up (high SG). If solute concentration is
low, urinometer sinks further into the urine (low SG)
Read the graduation on the arm of urinometer at
lower urinary meniscus.
SPECIFIC GRAVITY OF URINE – CORRECTION FOR TEMPERATURE:
Density of urine increases at low temperature and decreases at
higher temperature. This causes false reading of SG.
Therefore, SG is corrected for difference between urine
temperature and calibration temperature. Check the temperature
of calibration of the urinometer.
Distilled water  Urinometer shows SG of 1.000 at the
temperature of calibration
To get the corrected SG, add 0.001 to the reading for every 3°C
that the urine temperature is above the calibration temperature
Similarly subtract 0.001 from the reading for every 3°C below
the calibration temperature
URINOMETER – SPECIFIC GRAVITY
.
L MOULD (LEUCKHART’S EMBEDDING MOULD):

2 L shaped pieces of brass metal used for paraffin embedding in

histological tissue processing
L MOULD (LEUCKHART’S EMBEDDING MOULD):
 L moulds are kept on a glass; Tissue is placed in the bottom

 Space is filled with molten wax

 Wax is cooled and allowed to solidify; L moulds are removed

 Brick like wax with embedded tissue is called paraffin block

STEPS IN TISSUE PROCESSING:
 Fixation (Using 10% Neutral buffered formalin  Fixes tissue)

 Dehydration (Using alcohol  Removes water and formalin)

 Clearing (Using Xylene  Removes alcohol)

 Impregnation (With Paraffin wax)

 Embedding (With Paraffin wax)

 Section cutting (Using Microtome)

 Staining (Using H & E)

 Mounting of slide (Using DPX)

10. Blood bag
CC NNN
– Citrate –(major anticoagulant- chelates
Ca2+ )
– Phosphate (buffer to maintain pH)
– Dextrose (provides substrate for
metabolism of stored cells)
– Adenine (for viability of cells by
enhancing ATP production)

• 350ml bag contains 49ml CPDA

mixture
[Link]
• Antisera are essential for blood grouping, a
process used to determine a person's blood
type. They are used to identify antigens on the
surface of red blood cells.
• Specifically, the test that uses antisera and red
blood cells is known as the "front cell group"
or "forward typing,"
 Anti-D
• Rh typing antisera - to determine a person's
Rh blood type (positive or negative).
• It detects the presence or absence of the
Rh(D) antigen on red blood cells.
• If the Rh(D) antigen is present, the individual is
Rh positive
• if it's absent, they are Rh negative.
• Anti-A – blue in colour due to Methyelene
blue
• Anti-B –yellow in colour due to Tartrazine
• Anti AB- pink due to eosin ,now available as
colourless
• Anti-D -colourless
• STORAGE: 2-8 Celsius
[Link] JAR
• Coplin jars are primarily used in laboratories,
particularly in histology and microscopy, for
staining and storing microscope slides.
• They are designed to hold multiple slides
upright, ensuring uniform staining and easy
access.
• Additionally, they can be used for transporting
slides and storing specimens WITH FIXATIVES
14 .Paraffin blocks
PARAFFIN BLOCK (TISSUE BLOCK):
 Paraffin embedded tissue
block
 Method used to preserve and
prepare tissue for cutting using
microtome (Very thin sections
of 3-5 microns)
PARAFFIN BLOCK (TISSUE BLOCK):
 Embedding is the process by
which processed tissues are
surrounded by a support medium
such as wax, which on
solidification will provide
sufficient support during
sectioning.
STEPS IN TISSUE PROCESSING:
 Fixation (Using 10% Neutral buffered formalin  Fixes tissue)

 Dehydration (Using alcohol  Removes water and formalin)

 Clearing (Using Xylene  Removes alcohol)

 Impregnation (With Paraffin wax)

 Embedding (With Paraffin wax)

 Section cutting (Using Microtome)

 Staining (Using H & E)

 Mounting of slide (Using DPX)

Thank you