LAB - PLATELET COUNTING

Equipments:

1. Formal citrate dilution solution
2. Neubauer chamber
3. Red blood cell Thoma pipette
4. Microscope
5. Blood sample

Reagents:

Dilution solution: Formal citrate

1. 1% (l/l) Formalin (40% Formaldehyde) in 3% Trisodium Citrate.
2. 1-2 drops of Brilliant Cresyl blue (1% in isotonic saline) in each litre of formal citrate solution.

Procedure:

1. Using a red blood cell Thoma pipette draw well mixed venous blood or capillary blood and fill till the 0.5 mark. Clean the tip of the tube.
2. Draw diluting fluid to the 101 mark.
3. Shake and mixed well for 5 minutes.
4. Discard the first three to four drops of mixture and gently fill in the mixture into the Neubauer chamber.
5. Under 10X magnification, scan to ensure even distribution.
6. Count the red blood cells using 40X objective in the 80 smallest squares as indicated in the diagram of the chamber.

Normal range:

150-400 x 10⁹ / L

Questions:

1. List TWO conditions for thrombocytopenia
2. List TWO conditions for thrombocytosis
3. State the normal range for platelets.

LAB - RED BLOOD CELL COUNT

Introduction:

Red blood cell counting is an important diagnostic test towards observation of peripheral blood film. Changing in total number of red blood cells is related to physiological or pathological method. Increase in red blood cell is related to polycythaemia cases while decrease of total number of red blood cells is related to anaemia cases.

Equipments:

1. Formal citrate dilution solution
2. Neubauer chamber
3. Red blood cell Thoma pipette
4. Microscope
5. Blood sample

Reagents:

Dilution solution: Formal citrate

1. 1% (l/l) Formalin (40% Formaldehyde) in 3% Trisodium Citrate.
2. 1-2 drops of Brilliant Cresyl blue (1% in isotonic saline) in each litre of formal citrate solution.

Procedure:

1. Using a red blood cell Thoma pipette draw well mixed venous blood or capillary blood and fill till the 0.5 mark. Clean the tip of the tube.
2. Draw diluting fluid to the 101 mark.
3. Shake and mixed well for 5 minutes.
4. Discard the first four drops of mixture and gently fill in the mixture into the Neubauer chamber.
5. Under 10x magnification, scan to ensure even distribution.
6. Count the red blood cells using 40X objective in the 80 smallest squares as indicated in the diagram of the chamber.

Normal range:

Men                 :           4.5-6.5 x 10¹² / L

Women            :           3.8-5.8 x 10¹² / L

Babies             :           4.0-6.0 x 10¹² / L

Questions:

1. State FOUR (4) sources of error and indicate if the cell count would be falsely increased or decrease.
2. What is the purpose to dilute the blood with formal citrate?

LAB - PREPARATION OF CYANMETHEMOGLOBIN STANDARD GRAPH

REAGENTS AND EQUIPMENTS

1.         Drabkin’s solution

2.         Test tubes

3.         Micropipette

4.         Spectrophotometer

Specimens

EDTA / capillary blood

Preparation of cyanmethemoglobin standard graph

Procedure

1.         Prepare and label 5 test tubes.

2.         Dilution factor use is 1:300 if 6ml of Drabkin’s solution are used. The given standard 

            concentration (cuanmethemoglobin) is 57.2 g/ dL.

3.         The contents are mixed thoroughly.

4.         Read the absorbane of each solution at 540 nm.

5.         Record the results and prepare a standard graph.

Table for cyanmethemoglobin standard graph

Number	Drabkin’s solution	Standard Cyanmethemoglobin solution	Standard Cyanmethemoglobin concentration	Absorbance reading
1	6.0	0.0
2	5.0	1.0
3	3.0	3.0
4	1.0	5.0
5	0.0	6.0

Procedure

1.         Transfer 20µl of blood to 6.0 ml Drabkin’s solution

2.         Mix well and leave in a dark place for 10 minutes

3.        Read the absorbance of each solution at 540nm using a spectrophotometer and use Drabkin’s solution as ‘Blank’.

Tests tube	Blood A	Blood B
Drabkin’s solution	6.0 ml	6.0 ml
Blood sample	20µl	20µl
Standard cyanmethemoglobin concentration	X	Y
Absorbance reading	a	b

QUESTIONS

1.                List 3 errors that can interfere with haemoglobin determinations.

2.               State the normal range for haemoglobin for Male and Female.

3.              What is the wavelength used to measure haemoglobin manually on the spectrophotometer?

PREPARATION OF SECTION IN HISTOLOGICAL TECHNIQUES

Equipment 
  

i)          rotary microtome

         -for production of large number of sections

                     -serial section

ii)         floatation bath

                   -thermostically  controlled

                   -for paraffin wax 56^oC melting point water bath temperature is about                             

                     45^oC

                   -a variety of fluids are recommended from distilled water to quite high                      

                     concentration of alcohol

iii)         hot plate or drying oven

                   -recommended drying of section onto slides should take place at

                    temperature as low as 37^oC

                   -section dried at low temperature drying is prolonged - over night

                   -drying section on a hot plate is necessary if staining is to be carried out 

                     urgently

iv)        brush, seeker, forceps

                   -necessary  during sectioning for removal of folds

 v)        slides, glass marker

                   - size 76mm x 25mm, 1 to 2mm thick

                   -mark slides with a glass marker

vi)        section adhesives

             -most tissue section do not requires any adhesive  provided sections are                  

              initially  dried adequately on to slides

             -if using adhesive solution tend to remove section from slides so

               adhesive should be use

            

      -types of adhesive

                      -albumen

                      -gelatine

                      -starch

                      -cellulose

                      -sodium silicate

                      -resin

  

-tissues impregnated with ester wax or polyester wax will usually require the use of   

  section adhesive - to minimize loss

a)         Mayer`s glycerol albumen

                 Fresh egg white                             50cm³

                 Glycerol                                         50cm³

                 Sodium salicylate                           1cm³

             -mix and agitate ingredient

 -filter through coarse filter paper

 -thin smear is needed on the slide

Sectioning

i)          setting up the microtome

                         -angle of slope of knife at 90^o   (clearance 5-10 ^o  )

                                 -tighten knife clamp screws securely

   block clamp screw must firmly

                          -exposed ends of knife must be protected

ii)         trimming of tissue block

                         -to trim any surplus wax

                         -to expose suitable area of tissue for sectioning - thickness at 20um

                         -on exposing - section thickness set to 0.5-4um

                         -top and bottom of block parallel and horizontal to the edge of knife at the

                          moment of impact

iii)         cutting sections

                -all tissues desired on the slide should be exposed on the surface

                        -no scratch marks on the surface

                        -if scratch marks are visible the knife must be moved laterally

                         the face trimmed again

               -speed of block is important

                        -softer tissues - at a slower rate

                       -optimal speed is obtained through experience

                       -maintain  a regular cutting rhythm

             -a ribbon section is produced

                         -use fine forceps to hold the free end of the ribbon

                         -use soft paint brush to brush away the last section from the knife

                         -transfer ribbon to water bath

            -to obtain flat section its is necessary to spend time in the cutting and gentle               

                         stretching of the ribbon - before floating on the water surface

      

iv)        floating out sections

                -action in floating out must be smooth with trailing end of the ribbon    

                   making contact  with the water

                -slight  drag when ribbon touches the water will produce tension in ribbon 

                   remove folds from section

                -when ribbon has come to rest on the water - remaining wrinkles and folds

                 are removed by teasing a part using forceps or seeker

                -prolonged floating out of section on the water bath must be avoided as

                 tissues may expand and become distorted

               -advantage if water bath has a black base - section are more easily seen

               -some tissues eg. cartilage  are difficult to flatten on the water surface

   - to overcome this prolonged floating on the water bath - teasing with forceps

     or seekers                                                           

 -trimming away surplus wax from the block

v)         picking up sections

                    -is done by immersing the slide

                    -vertically in the water bath

                    -position the section into contact with the slide

                    -drain it in a vertical position for several minutes

vi)        drying sections

                        -if staining is required urgently drying section on a hot plate is necessary

                        -drying time is 10 minutes

            -dried in an oven - 45 minutes at 45^o  C    - section loss if less than 30

              minutes

                        -advantage of oven drying - dust is less likely to settle on sections

                        -after removal from hot plate or oven - cooled sections are stored in dust

                           free containers

            -if overnight the temperature is 37^o C   

Faults and remedies in paraffin wax sectioning

i)          Sections will not join to form a ribbon

-Cause            -wax too hard

                                                -dust on knife edge

                                                -knife angle too wide or narrow

                        -Remedy         -warm or re-embed

                                                -clean with xylene

                                                -adjust angle

ii)         Sections roll into a coil

                        -Cause            -knife blunt

                                                -knife angle too wide

                                                -section thickness too great

                        -Remedy         -replace knife

                                                -reduce knife tilt

                                                -reduce section thickness

iii)         When sections are curved

                        -Cause            -knife blunt in one area

                                                -excess wax in one side

                        -Remedy         -use different part of knife or replace with a new one

                                                -trim away excess wax

iv)        Splitting of sections

                        -Cause            -damage in knife edge

                                                -hard particles in tissue

                                                -hard particles in wax

                        -Remedy         -use different part of knife or replace with a new one

                                                -remove particles from tissue

                                                -re-embed in fresh wax

v)         Compression of sections

                        -Cause            -blunt knife

                                                -angle knife too wide

                                                -wax too soft

                        -Remedy         -replace with a new knife

                                                -reduce knife tilt

                                                -cool block in refrigerator

vi)        Areas of tissue blocks not present in sections

                        -Cause            -incomplete impregnation of tissue

                                                -wax block become detached

-Remedy         - return tissue to vacuum impregnation container for a few       

                          hours

                                                -re-attach block

vii)       Sections expand and disintegrate on water surface

                        -Cause            -poor impregnation of tissues

                                                -water temperature too high

                                                -floatation bath dirty

-Remedy         -return tissue to vacuum impregnation container for a few    

                         hours

                                                -cool water

                                                -clean floatation bath

PROCESSING OF TISSUES IN HISTOLOGICAL TECHNIQUES

·                     Definition of tissue processing    

-          any treatment of tissues necessary

-           to impregnate them with solid medium

-          to facilitate the production of sections for microscopy

·                     Aim

-          to embed tissues in solid medium

-          to support the tissues

-          to enable the knife to cut the section with little damage to knife or tissues

Dehydration

·                     A process by which water is removed from fixed tissues

·                     Also removal of aqueous and some of the lipid tissues fluids

·                     Tissue blocks are placed in containers or cassette with tag

                         - lid securely closed

                         - tiny fragments should be wrapped in a single layer of  lens paper

·                     Tissues are passed through a series of increasing concentrations of alcohol

                         - with one change in each concentration

                         - duration of one hour in each

                         - keep covered  to avoid evaporation

·                     Starting alcohol is usually 80%

  - for soft tissues 30%, 50%,70%

                          - it is important that the final bath of alcohol is pure and change 3 times

                          - free from water

                   

Dehydrating agents   

i)          Ethyl alcohol (ethanol) C₂H₅ OH

         -clear colourless inflammable liquid with pleasant odour

         -hydrophillic - miscible with water in all volumes

   - the most common

ii)         Methylated spirit (denatured  alcohol)

         -some physical characteristics as ethanol

         -stronger smell

         -consists of ethanol to which has been added a proportion of methanol

iii)         Methyl alcohol (methanol) CH₃OH    

         -clear ,colourless inflammable and poisonous fluid

         -unpleasant  odour ,miscible with water, ethanol

iv)        Isopropyl  alcohol (2 propanol) CH₃CHOHCH₃

         -miscible with water, ethanol

         -does not harden the tissue

   -99% is the best substitute for ethyl alcohol

v)         Acetone CH₃COCH₃

         -clear ,colourless ,inflammable fluid

         -pungent odours

         -miscible with water, ethanol

         -more volatile 

         -causes brittleness of tissue if treatment is prolonged

         -when speed is essential - acetone is preferred

Clearing

·                     The removal of dehydrating agents from tissue

·                     To make tissue transparent

·                     Selection of a suitable clearing agent must be based on

         -speed of removal of alcohol

         -ease of removal

         -gentleness towards tissue

         -flammability

         -toxicity

         -cost

Clearing agents

i)              xylene

         -for regular size

         -not thicker than 3 -4mm

         -immersion time must not be prolonged

         -tissues become brittle

ii)         Toulene

         -less damaging on prolonged immersion of tissues

         -suitable for automated tissue processing

iii)         Chloroform

        -slower in action causes brittleness

        -thicker tissue up to 1cm thickness can be processed

        -dangerous - releases toxic gas

iv)        Benzene

        -carcinogenic properties

        -similar to xylene

v)         Carbon tetrachloride

        -similar properties to chloroform

        -toxic

vi)        Paraffin

        -variable mixture of hydrocarbon

        -less flammable

        -less dangerous

vii)       Petrol

        -similar properties to xylene

        -various additives

        -not recommended

viii)       Carbon disulphide

        -unpleasant odour

        -danger of fire –not recommended

ix)        Amyl acetate

        -remove alcohol fairly rapidly

        -costly

x)         Methyl benzoate and methyl salicylate

        -fluids moderate in speed action

        -cause minimal distortion of tissue

xi)        Cedarwood oil

        -slow in action

        -causes little hardening or shrinkage of tissues

        -low volatile properties

xii)         Clove oil

              -similar to cedar wood oil

              -more expensive

             -use in humid climates

Infiltration and impregnation

·                     Tissues are transferred to a bath of molten paraffin wax

·                     During this process xylene is eliminated from tissues by diffusion

      -is called infiltration

·                     Wax diffuses into the tissue to replace the clearing agent

        -is called impregnation

·                     To completely remove the clearing agent

·                     Inadequate impregnation lead  to drying and shrinking of tissues

        -crack and crumble develop                 

·                                 High temperature will over - harden the tissues

Embedding

·                     Placing the infiltrated impregnated tissue in warm liquid paraffin wax that

            solidifies into a firm block when cool to room temperature

·                     Known as casting or blocking - use plastic mould

·                     Precaution should be taken

           -wax to be used must contain no trace of clearing agent

           -no dust particles must be present

           -after embedding the wax must be rapidly cooled to reduce the wax crystal

             size

·                     Orientation of tissues

           -tissues of tubular nature cut transversely

     -skin- cut in a plane at right angles

     - muscle biopsies are sectioned both transverse and longitudinal planes

·                     Wax is dispensed into the mould to a depth to cover the thickest tissue block

·                     When a thin film of semi-solid wax has formed on the base of the mould

      -tissue is introduced with warm forceps gently pressing the tissue into the

             semi-solid wax correctly orientated plane

·                     Then fill the mould with wax

           -make sure that there is no air bubbles

·                     Transfer blocks to refrigerator to complete hardening

·                     Identification label must accompany the specimen through all types of  tissues

      processing

Embedding media

i)          Paraffin wax

-most popular

-large number of tissue blocks may be processed in a short time

-sectioning and staining - fewer difficulties

-cheaper

-melting point in range 40-70^oC

-for satisfactory sections melting point 54-58^oC

-additives have been proposed

          -to increase hardness to cut thinner section and to support hard tissues

          -to increase stickiness to produce serial ribbon

          -to reduce crystal size

    eg. bee wax (stickiness) stearic acid (hardness)

ii)         Other waxes

         -ester wax

         -microcrystalline wax

iii)         Resins

          -acrylic

  -epoxy

  -urea - formaldehyde

iv)        Other media

         -agar

         -gelatin

          -celloidin

·                     Different medium is required if

          -medium not sufficiently hard and fail to provide adequate support

          -affected by heat

          -distort the tissue

    -tissue breaking away from the wax during sectioning

          -sections cannot be cut thin enough

Manual tissue processing

·                     Necessary in the following

          -large tissue slices

          -speed processing

          -electrical power failure / machine breakdown

          -small tissue slices - need optimum time

·                     Advantage

         -flexibility tissues are treated for the optimum duration in each fluid

         -use of fluids-not dangerous when using -flammable

                                                                          -volatile   eg. acetone

·                     Rapid technique for thin slices of tissue

i)          10% formalin 60^oC                             20min

      ii)         fresh acetone                                       20min

      iii)        fresh acetone                                       20min

     iv)         fresh acetone                                       20min

     v)          xylene                                                 10min

     vi)         xylene                                                 15min

     vii)        wax                                                     30min

     viii)       wax                                                     60min (185 min)

 -agitate frequently to assist in the transfer of fluids

Automated tissue processing

·                     Most automated tissues processor have 12 containers processing cycle

·                     Some machines apply - heat and vacuum to increase the rate of processing

·                     Care must be taken for type of tissue eg. spleen, muscle, skin, decalcified

       tissue become hardened if heat and vacuum is applied

·                     Vacuum

 -the degree of vacuum should not exceed 40-50mmHg

-normally in a sealed container of molten paraffin wax-applying suction to the 

  container

                  aim   -to remove air bubbles in the tissue

                     -to remove clearing agents

        

           -tissues particularly - lung ,muscle ,spleen, decalcified bone, skin

·                     For increasing speed of processing

                    -use warm (40-50^oC) fixative to ensure fixation is complete

                    -95% alcohol

                    -use a fast - acting clearing agent (eg. xylene)

                    -use of vacuum infiltration at all wax stages

                    -agitation at all stages ,even during fixation

                    -minimum of time at all stages

i.              Routine overnight processing

1                      4% formol saline (1.5 lit.)                                                        2 hrs

                        (40 ml formaldehyde 960 ml water add 9 g. NaCl)

2                      4 % formol saline (1.5 lit.)                                                       2 hrs

3                      70 % alcohol (1.5 lit.)                                                              1 hr     

                        (700 ml alcohol 300 ml water)

4                      90 % alcohol (1.5 lit.)                                                              1 hr

                        (900 ml alcohol 100 ml water)

5                      absolute alcohol (100% alcohol) (1.5 lit.)                                1 hr

6                      absolute alcohol (1.5 lit.)                                                         2 hrs

7                      absolute alcohol (1.5 lit.)                                                         2 hrs

8                      xylene (1.5 lit.)                                                                        1 hr

9                      xylene (1.5 lit.)                                                                        11/2 hrs

10                    xylene (1.5 lit.)                                                                        11/2 hrs

11                    wax                                                                                         2 hrs

12                    wax                                                                                         3 hrs

                                                                                                Total                20 hrs

ii.             Day time processing -for small specimen 

 

1.    formal saline                   15min

2.    formal saline                   15min

3.    70% alcohol                    15min

4.    90%alcohol                     15min

5.    100%alcohol                   15min

6.    100%alcohol                   15min

7.    100%alcohol                   15 min

8.    toluene                             15min

9.    toluene                             15min

10.  toluene                            15min

11.  wax                                 30min

12.  wax                                 30min                                 

                                                 3hrs 30min

Factors influencing the rate of impregnation  

i)    Agitation

    -allow the tissue to sink to the base of container - to make sure interchange of fluids   

      occur

    -mechanical device - vertical agitation

    -average speed of tissue movement is 10-12 inches per minute

    -improved impregnation  by 25-35%

ii)    Heat

      -increases the rate of penetration

      -care must be taken not to overheat tissues and cause shrinkage, brittleness and                                                 

        difficulties  in sectioning

iii)    Viscosity

      -the larger the molecule the higher the viscosity  the slower the rate of penetration

iv)    Vacuum

      -serve to remove air bubbles trapped within the tissue –this will increase the contact

        with fluid to tissue

v)    Ultrasonic - not widely used

·                     Points to note

         -beakers  and wax bath must be filled to the correct fluid level

         -any spillage of fluid should be wiped

         -accumulations of wax must be removed

         -wax bath thermostats set at satisfactory level

         -checked - timing

                         -electrical plugs

         -changing of solution after using them for 2-3 days