1. Romanowsky stains and Artefacts in blood films
Dr. R. Manchanda
Prof. & Director Pathology
KEM Hospital
Pune

2. History 1879 - Paul Ehrlich - used mixtures of acidic (fuchsine) and
basic (methylene blue) dyes to differentiate cells.
Dmitri Leonidovich Romanowsky Russian physician, developed
techniques using a mixture of eosin Y and  methylene
blue that produced a surprising hue, a beautiful distinctive
shade of purple.
1901 – 2 William Boog Leishman , James Homer Wright
introduced methanol as a solvent
Gustav Giemsa improved this technique by standardizing
the dye solutions and adding glycerol to increase stability

3. Romanowsky stains Giemsa Jenner Wright Field Leishman
May-Grunwald-Giemsa,
Wright-Giemsa,
Jenner-Giemsa,
Azure B-EosinY, etc.

4. Principle The main components of a Romanowsky stain are:
A cationic or basic dye (methylene blue or its oxidation products such as azure B), which binds to anionic sites and gives a blue-grey color to nucleic acids (DNA or RNA), nucleoproteins, granules of basophils and weakly to granules of neutrophils
An anionic or acidic dye such as eosin Y or eosin B, which binds to cationic sites on proteins and gives an orange-red color to hemoglobin and eosinophil granules.
pH value of phosphate buffer is very important.

5. TOO ACIDIC SUITABLE TOO BASIC

6. Excessively pink staining
Low pH of the buffer
Insufficient staining
Excessive washing / rinsing.
When exposed to air, formation of formic acid in methanol.
Excessively blue staining
Alkaline pH of the buffer
Prolonged staining
Insufficient washing
Thick films
Precipitate may form as a result of evaporation of methanol
Improper washing

8. artefact Year introduced: 1992
Any visible result of a procedure which is caused by the procedure itself and not by the entity being analyzed.
Common examples include histological structures introduced by tissue processing

9. Abnormalities on blood smears can either be
- pathological
- artefact.
It is important to correctly identify artefacts so that they are not incorrectly interpreted as a pathological process.
Can cause confusion and problems in diagnosis

10. Artefacts Fixation artefact
Occurs when there is water in the methanol used for fixation of the blood film.
This leads to refractile rings in red cells and makes it quite impossible to assess red cell morphology.

11. Excessive water on the slide or in the stain producing refractile edges on erythrocytes

12. Good smear

13. Tailing artefact
Poor distribution of leukocytes

14. Causes of hematologic artefacts are often mulit factorial
Can be classified as in vivo and ex vivo in nature

15. CAUSES OF ARTEFACTS IN BLOOD FILM
Causes Artifacts
In vivo factors
Antibodies to blood cells Agglutination of platelets, erythrocytes, and leukocytes
Increased plasma volume Pseudo anemia
Decreased plasma volume Pseudopolycythemia
Treatment-related Agglutination of platelets and pseudo–Pelger-Hue¨t anomaly
Monoclonal immunoglobulins Pseudothrombocytosis and pseudoleukocytosis
Ex vivo factors
Anticoagulant
EDTA Agglutination of leukocytes; agglutination, satellitism
and degranulation of platelets; and precipitation of proteins
Citrate, oxalate, and heparin Agglutination of leukocytes and platelets
Overfilling of tubes Pseudopolycythemia, pseudo thrombocytopenia, and
pseudoleukopenia
Prolonged storage of specimen Pseudo toxic changes, pseudoechinocytosis, and platelet
degranulation
Temperature of specimen Agglutination of platelets, erythrocytes, and leukocytes
Glass effect Formation of acanthocytes

16. POTENTIAL ARTIFACTS ON A BLOOD FILM
Classified by the cell type involved –
Platelets –
Platelet agglutination, satellitism, and pseudo thrombocytopenia
Pseudothrombocytosis
Pseudo–gray platelet syndrome
Pseudo–Bernard-Soulier syndrome

17. PLATELET AGGLUTINATION, SATELLITISM,AND
PSEUDOTHROMBOCYTOPENIA
more frequently in severely ill patients, and those with associated autoimmune rheumatoid arthritis,
Sjo¨gren’s syndrome
Guillain-Barre´ syndrome
neoplasms,
atherosclerotic disease
liver conditions

18. Storage artefact
Prolonged storage of blood before making the blood film, particularly storage at room temperature,leads to storage artefact.
White cells become fragile and may form smear cells [deep red arrow].
Neutrophil nuclei round up and form homogeneous round masses or a single mass [blue arrow].These cells have a resemblance to NRBC.
Red cells undergo an echinocytic change or crenation.

19. Leukocytes Pseudo–Pelger-Huet anomaly Pseudo–Chediak-Higashi granules
Leukoagglutination and pseudoleukopenia
Pseudoleukocytosis
Cytoplasmic fragments
Pseudo leukemia
Pseudo toxic changes

20. Nuclear lobulations, degeneration, pyknosis, rupture.
Cytoplasmic granulation, vacuolization.
Citrate-immediately
EDTA- after 2 hours

21. pyknotic and karyorhectic leukocyte is unidentifiable

23. Pseudoechinocytosis Erythrocytes Pseudo anemia Pseudopolycythemia
Satellitosis of red cells about neutrophils
Immunoglobulins
Cryoglobulin precipitation
Cryofibrinogen precipitation

24. Pseudoechinocytosis

25. Heat artefact
Heating of a blood sample e.g during transport in a hot car.
Red cells bud off vesicles.
Microspherocytes seen.
White cells disintegrate.
Proteins coagulate, producing
weakly basophilic particles,
which are similar in size to
platelets.

27. Poor slide- may miss! CAN LEAD TO SERIOUS MISTAKES!! Polychromasia
Agglutination/rouleaux
Poikilocytes, spherocytes, blister cells
Parasites
Degree of cytopenia/cytosis (tailing)
Rare blast (thick/ thin slide, distorted WBCs)
Inclusions
CAN LEAD TO SERIOUS MISTAKES!!