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

History 1879 - Paul Ehrlich - used mixtures of acidic (fuchsine) and basic (methylene blue) dyes to differentiate cells. 1891 - 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 1904 - Gustav Giemsa improved this technique by standardizing the dye solutions and adding glycerol to increase stability

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

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.

TOO ACIDIC SUITABLE TOO BASIC

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

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

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

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.

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

Good smear

Tailing artefact Poor distribution of leukocytes

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

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

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

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

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.

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

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

pyknotic and karyorhectic leukocyte is unidentifiable

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

Pseudoechinocytosis

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.

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!!