Dr Piyush Tailor

Purpose of examination

Every disease affecting the cellular components of the blood can be visualized in the form of either cytologic changes in cell morphology or of altered distribution of cell types. Furthermore estimations of white blood cell, and platelet concentrations on blood smears provide an excellent check on the counts derived by either manual or electronic techniques. It is also useful tool for detection of parasites.

Principle of examination

Stained peripheral smears are examined for RBC, WBC, Platelet abnormalities and presence of parasites.

Performance specifications

Not applicable.

Sample type required

Anticoagulated whole blood (EDTA).

Stored at 2’C-8’C after analysis for 24 hrs.

Slide with thick and thin smear.

Preservatives needed

Sample collect In EDTA bulb .

Reagents required

Giemsa stain

Methanol (acetone free)

Calibration method

Not applicable

Detailed work bench instruction / programming steps:

In accordance with good laboratory practice, all samples should be considered potentially infectious and should be handled with care. The personnel should use protective laboratory coats, gloves, eye glasses & mask.

 Preparation of thin smear

• Take a clean & dry slide

• Transfer a small drop of blood near the edge of the slide

• Place a spreader slide at angle of 30 degree, pull back the spreader until it touches the drop of blood. Let the blood run along the edge of the spreader.

• Push the spreader forward to the end of the slide with a smooth movement.

• Dry the blood smear at room temperature.

• By using a lead pencil, write ID number on slide

 Preparation of Thick smear:

• Place a small drop of blood on the slide

• Spread the drop out with another slide to cover an area

About four times its original area

The correct thickness for a satisfactory thick film will have been achieved if, with the slide placed on a piece of newspaper, small print is just visible.

Allow the film to dry at least 30 minutes at 37 degree C before staining

 Precautions:

1. Slow movement of the spreader slide causes accumulation of larger white cells at the edges of the film.

2. Artefacts may be introduced due to irregular edges of the spreader slide.

 Qualities of a good smear

1. Smear must not be too thin

2. Tail should be smooth

3. There should be some overlap of the red cells diminishing to separation near the tail.

4. White cells in the body of the film should not be badly shrunken.

5. Usually neutrophils and monocytes predominate at the margins and the tail of the smear; Lymphocytes at the middle of the film.

 Fixation on thin smear

• Fix the thin smear by dipping in methyl alcohol for 1-2 second

• Dry the film after fixing in methyl alcohol

 Preparation of Giemsa stain

• Take 5 gm of giemsa powder and crush it properly, then mix it in 500 ml of glycerol and keep it for 48 hours.

• Add & well mix 500 ml of absolute methanol in the above mentioned solution and keep at room temperature for one week.

• After filtering it is stored in amber colored bottle at room temperature

 Staining procedure

• Add buffer( 6.8- 7.2 pH) in the filtered stain in 1:4 dilution

• Arrange thick/ thin smears on double rod stand.

• Pour the stain-buffer mixture on the arranged slides. Leave it for 10-15 minutes

• Stain over the slides should be mixed by blowing with pipette.

• After 10-15 minutes, wash the slides in slowely running tap water.

• Dry the washed slides in air at room temperature.

 Examination of the blood smear

• Using a light microscope the smear is first examined under 10 x objective to assess the adequacy of cellular distribution and staining.

• The slide is examined under 40 x to make an estimate of white blood cell count and scanned for abnormal cellular elements like blasts and nucleated RBC's as well as hemoparasites.

• Examine the slide under oil immersion to study the cell morphology details. A systematic evaluation of the blood smear is essential so that all cell types are examined and characterised.

Quality control procedure:

QUALITY CONTROL

• Maintain integrity of the Giemsa Wright stain by proper storage and frequent preparation.

• Check the stain for degenerative changes and stain deposits

• Filter prior to use.

• Check staining for every new lot prepared.

• Frequent checks of staining buffer pH

• Check for background staining. In these cases ensure proper preparation of the stain with emphasis on grinding with glycerol.

• External quality assessment: Participate in external quality assessment program and maintain data in C\Records\File\2\Results of EQA and interlaboratory comparison

• Internal Quality Control: Weekly one random PSCM slide is checked internally by three different observer and records of comparison are kept in control C\Records\File\9\Records of internal quality control records. For any nonconformance, Procedure for identification, correction and prevention of nonconformities is activated and records are filed in C\Records\File\12\Records of Nonconformities

Interferences 1. spreader must have smooth edges otherwise may lead to irregular distributrion of cells.

2. Slide must be clean and free of grease ,lint and dust .Failure to observe these precautions may prevent adequate spreading of blood specimen over the entire surface and thus introduces morphological artifacts.

3. Stains must be free of water even small amount can cause marked red cell artifacts.

4. Excessive blueing of slide: increase staining time, over alkalinity of buffer, old blood smear,overly thick smear.

5. Excessive reddening of slide: excessive acidity of buffer, decrease staining time,excessive washing.

Calculation of results

The differential count is expressed as % of each type of cell & results are reported in absolute numbers (x 109/l). For malarial parasite s, at least 100 oil immersion fields should be screened before reporting negative. Biological reference interval

RBC: Normocytic, normochromic

WBC: Normal count and morphology

Total count: 4000-10000/cmm

Neutrophils: 40-85%

Lymphocytes: 20-40%

Eosinophil: 1-6%

Monocyte: 2-10%

Basophils: 0-1%

Platelets: Normal count and morphology

No Abnormal cells seen

No hemoparasites seen

Reportable interval for examination results: Not Applicable

Critical values:

Acute myeloid & lymphoid leukemias

Very low platelet counts

Severe anemias

Interpretation by the laboratory

RBCs Morphology.

ANISOCYTOSIS: change in size of RBCs.

Microcytosis: MCV <80fl

Observed in iron deficiency anemia , various types of thalassaemias,severe cases of anemia of chronic disease.

Macrocytosis: MCV>100fl

Megaloblastic anaemia, aplastic anaemia, MDS, chronic liver disease.

POIKILOCYTOSIS: Change in shape of RBCs.

Types:

• Spherocytosis: HS, AIHA.

• Elliptocytosis: HE, macrocytic anaemias.

• Target cells: haemolytic anaemias, IDA, liver disease.

• Sickle cells: sickle cell disease.

• Schistocytes: MAHA, DIC.

• Stomatocytes: hereditary stomatocytosis, liver disease, Rh null phenotype.

• Acanthocytes: abetalipoproteinemia, liver disease.

• Burr cells: uraemia, drug induced haemolytic anaemia.

CHROMICITY:

• Hypochromia: IDA, thalassaemia.

• Hyperchromia: macrocytic anaemias, spherocytosis, irregularly contracted cells.

INCLUSION BODIES:

• Basophilic stippling: lead poisoning, megaloblastic anaemias, thalassaemias, sideroblastic anaemias, alcoholism.

• Pappenheimer bodies: sideroblastic anaemia, megaloblastic anaemia, post splenectomy, haemolytic anaemia.

• Howell-jolly bodies: megaloblastic anaemia, post splenectomy, acute haemolytic anaemias.

• Cabot ring: megaloblastic anaemia.

• Heinz bodies: exposure to oxidizing drugs or chemicals, G 6-PD deficiency.

Other abnormalities in RBC morphology:

• Rouleaux formation: multiple myeloma, kala azar, chronic inflammatory disease.

• Polychromasia: haemolytic anaemias, response to therapy in deficiency anaemias.

PLATELET MORPHOLOGY

• Adequate count: 7-20 platelets/ field in oil immersion

• Large platelets: increased platelet production, hyposplenism, severe immune thrombocytopenia.

• Giant platelets: Bernard soulier syndrome.

• Agranular /hypogranular platelets: grey platelet syndrome, myloproliferative disorders.

LEUKOCYTE MORPHOLOGY

NEUTROPHILS: GRANULATION

• Toxic granulation- septicaemia, aplastic anaemia, myelofibrosis.

• Hypogranulation - MDS, some forms of myeloid leukaemia.

• VACOULES: seen in severe sepsis, as an artefact, prolonged standing of blood before films are made.

• NUCLEUS: shift to left(less than 3 lobes)-sepsis, pregnancy

• Shift to right(6 or more lobes)- megaloblastic anaemia, uraemia, cytotoxic drug treatment.

LYMPHOCYTES:

• Reactive - lymphocytes-viral infection.

• Lobulated lymphocytes - HTLV-1 infection, adult T-cell leukaemia lymphoma, as a storage artefact.

EOSINOPHILS:

• Eosinopenia - prolonged steroid administration

• Eosinophilia-allergic conditions, parasitic infections, leukaemias.

BASOPHILS:

• basophilia- myeloproliferative disorders.

MONOCYTES:

• Monocytosis- TB, crohns disease, CML, kala azar, acute leukaemias with monocytic component.

Potential sources of variability

Preanalytical sources of variability:

1.spreader must have smooth edges otherwise may lead to irregular distributrion of cells.

2. Slide must be clean and free of grease ,lint and dust .Failure to observe these precautions may prevent adequate spreading of blood specimen over the entire surface and thus introduces morphological artifacts.

3.Stains must be free of water even small amount can cause marked red cell artifacts.

4.Excessive blueing of slide: increase staining time, over alkalinity of buffer, old blood smear, overly thick smear.

5.Excessive reddening of slide: excessive acidity of buffer, decrease staining time,excessive washing.