Stain of hematologyLeishmania stainReticulocyte stainIron stainPrepared by:

Nechirvan mustafaFarzad aliQahreman husseinHasan mamsofe

Supervised by:d.Bizavd.muna

leishman’s stain CompositionPolychromed methylene blueEosin azure leishman powderAcetone free absolute alcohol1.5gms leishman powder in 1 liter acetone free absolute alcohol

ProcedureMAKING A SMEAROne drop of blood on one end of slideSpreader slide is placed at 45°on the drop and moved along the slideIt is moved smoothly and once such that the blood film is thinCare should be taken to prevent the formation of air bubblesAir dry the smearMake identification mark on one edge

Staining a smearPlace the smear on the staining rackPour leishman stain to cover the smear completely allow to fix for 2-3 mienAdd water twice the amount of leishman’s stain allow to fix for 7-10 minsAppearance of golden scum or sheen on the surface of stainWash the stain off the slide with running waterWipe the back of slide and air dry the slide

Examination of the smearThe stained slide is placed on the microscope and is focused under low powerPlace a drop of oil on the slide and observe under oil immersion objectiveObserve RBC,platelet seriesWBC series is observed and a differential count is doneRecord the findings

Reticulocyte stainReticulocytes are young , premature, non nucleated red blood cells, contain reticular material (RNA) that stain gray blue. Reticulum is present in newly released blood cells for 1-2 days before the cell reach its full mature state

Continue….Reticulocytes are visualized by supravital staining (such as new methylene blue, Brilliant Cresyl Blue, Pure azure blue) that precipitate the RNA and organelles, forming a filamentous network of reticulumOn Wright stain. the Reticulocyte appears polychromatophilic or as a Macrocytic blue red cell.

Equipments requiredEDTA tubeStain :new methylene bluePipetteSlideMicroscopeCapillary tubeincubate

Procedure

Iron stain Introduction: Prussian Blue or Perls’ reaction is used to demonstrate ferric iron and ferritin. This is not a true staining technique rather, it is a histochemical reaction.

The protein is split off by the hydrochloric acid, allowing the potassium ferrocyanide to combine with the ferric iron. This forms the ferric ferrocyanide or Prussian Blue.

Fixation 10% Neutral Buffered Formalin or alcohol can be used. Sections should be cut at 4μ to 5μ, or blood or bone marrow smears.

Deparaffinize Slides should be deparaffinized through xylene or substitute to remove the paraffin from the section and descending grades of alcohol to water just prior to staining.

ProcedureMix equal amounts of Solution A, 4% Potassium Ferrocyanide, Aqueous with Solution B, 4% Hydrochloric Acid, Aqueous, for the working solution.

The standard room temperature staining will take 2 changes of the solution at 10 minutes each.Rinse in several changes of distilled water.Stain in Solution C, Nuclear Fast Red, for 2 to 5 minutes depending on the intensity of the counter stain required.

Continue….Rinse in running tap water for 1 minute. Dehydrate through ascending alcohols to xylene or substitute and coverslip with Poly Mount.

The stain will be removed in the alcohols and should be dehydrated as quickly as possible

result Ferric ion = blue Nuclei = red Background = pink