Reticulocyte StainsCHAPTER 1 I EXAMINATION OF BLOOD SAMPLES 13

Reticulocyte stains are commercially available. Those wishing to prepare theirown stain can do so by dissolving 0.5 g of new methylene blue and 1.6 g ofpotassium oxalate in 100 mL of distilled water. Following filtrations, equalvolumes of blood and stain are mixed together in a test tube and incubated atroom temperature for 10 to 20 minutes. After incubation, blood films are madeand reticulocyte counts are performed by examining 1,000 erythrocytes anddetermining the percentage that are reticulocytes." The use of a Miller's disc inone of the microscope oculars saves time in performing the reticulocyte count.The blue-staining aggregates or "reticulum" seen in reticulocytes (Fig. 4D)does not occur as such in living cells but results from the precipitation ofribosomal ribonucleic acid (RNA; the same RNA that causes the bluish colorseen in polychromatophilic erythrocytes) in immature erythrocytes during thestaining process.I I As a reticulocyte matures, the number of ribosomes decreasesuntil only small punctate (dotlike) inclusions are observed in erythrocytes(punctate reticulocytes) stained with the reticulocyte stain (Fig. 4E). To reducethe chance that a staining artifact would result in misclassifying a matureerythrocyte as a punctate reticulocyte using a reticulocyte stain. the cell inquestion should have two or more discrete blue granules that are visible withoutrequiring fine-focus adjustment of the cell being evaluated to be classifiedas a punctate reticulocyte.In normal cats. as well as in cats with regenerative anemia. the number ofpunctate reticulocytes is much greater than that seen in other species." Thisapparently occurs because the maturation (loss of ribosomes) of reticulocytes incats is slower than that in other species. Consequently, reticulocytes in cats areclassified as aggregate (if coarse clumping is observed) or punctate (if smallindividual inclusions are present). Percentages of both types should be reported.Based on composite results from several authors, normal cats generally havefrom 0% to 0.5% aggregate and 1% to 10% punctate reticulocytes when determinedby manual means. Higher punctate numbers of 2% to 17% have beenreported using flow cytometry."The percentages of aggregate reticulocytes in cats correlate directly withthe percentages of polychromatophilic erythrocytes observed in blood filmsIn contrast to those of the cat, most reticulocytes in other species are ofthe aggregate type. Consequently, no attempts are made to differentiate stagesof reticulocytes in species other than the cat. The percentage of reticulocytes inmost species correlates directly with the percentage of polychromatophilic erythrocytesobserved on routinely stained blood films.Heinz bodies are composed of denatured, precipitated hemoglobin. Theyare spherical, stain pale blue with reticulocyte stains, and are usually found atthe periphery of the erythrocyte.New Methylene Blue "Wet Mounts"A new methylene blue "wet mount" preparation can be used for rapid informationconcerning the number of reticulocytes, platelets, and Heinz bodiespresent. The stain consists of 0.5% new methylene blue dissolved in 0.85%NaCl. One mL of formalin is added per 100 mL of stain as a preservative. Thisstain is filtered after preparation and stored in dropper bottles. Alternately, thestain may be stored in a plastic syringe with a 0.2 IJ-m syringe filter attached sothat the stain is filtered as it is used. Dry unfixed blood films are stained byplacing a drop of stain between the coverslip and a glass slide. This preparationis not permanent and does not stain mature erythrocytes or eosinophil granules.Punctate reticulocytes are not demonstrated, but aggregate reticulocytes appearas erythrocyte ghosts containing blue to purple granular material (Fig. 4F).Platelets stain blue to purple, and Heinz bodies appear as refractile inclusionswithin erythrocyte ghosts. Although this staining method is not optimal fordifferential leukocyte counts, the number and type of leukocytes present can be

appreciated.

MANUAL RETICULOCYTE COUNT PROCEDURE

8A. PRINCIPLE:The reticulocyte is a non-nucleated immature red cell containing residual RNA. A supravital stain, new methylene blue, is used to precipitate the RNA into dark-blue filaments or granules to identify retics. B. SPECIMEN and REAGENTS: EDTA whole blood is the preferred anticoagulant; New Methylene Blue Staining solution, 12x75mm tubes, pipets, glass slides. C. PROCEDURE:1. Put 2 drops of new methylene blue in the bottom of a 12x75mm tube. Using a pipette, add 2 drops of well-mixed EDTA blood to the tube. 2. Mix blood/stain mixture. The mixture color should be smoky-gray. Adjust if needed, i.e., add more blood if mixture is too blue. 3. Incubate mixture at least 5 minutes but no longer than 10 minutes. 4. MIXsolution again....important! Prepare 2-4 good smears, LABEL and let dry. 5. Counting:Usingoil/100xpower, count 500 total red cells separating mature RBC's from retics (use two counter keys). Retics are greenish with blue precipitates of RNA. Two “dots” or more is a retic.Go from feather edge to body of smear, making sure you are not counting too thick. 6. Two techs count 500 RBC's on different retic smears for a total of 1000 RBC's counted. 7. Quality control:The number of retics/500 RBC's must agree +2 retics between techs to accept results or another slide is counted. Controls must read within the assayed range to accept results. 8. Both a relative percent retic and an absolute retic are reported: a. Relativenumber- # of retics in total of 1000 RBC's = percent (%). b. Absolutenumber- retic % x the RBC count/cmm = thousands/cmm. D. CALCULATIONS: 1. The relative reticulocyte count uses the sum of the two techs answers and the percent is reported to the nearest tenth (one decimal): # retics in 1000 RBCs= % OR# retics

x 100 = %.10 1000 RBCs 2. The absolute reticulocyte count is reported to the nearest thousand/cmm using the following calculation: # retics/cmm = # retics x RBC millions/cmm ORretic %x RBC/cmm 1000 RBCs 100 E. SOURCES OF ERROR:1. Inadequate mixing before making smears 2. Counting artifact or other inclusions as retics......black/shiny inclusions are “junk”. 3. Improper ratio of blood to stain. 4. Not counting all of the retics.....two blue “dots” or more is a retic. 5. Wrong calculati

EXAMINATION OF STAINED BLOOD FILMSAn overview and organized method of blood film examination are presentedhere. Descriptions and photographs of normal and abnormal blood cell morphology,inclusions, and infectious agents will be given in subsequent sections.Blood films are generally examined following staining with Romanowskytypestains such as Wright or Wright-Giemsa stains. These stains allow forexamination of erythrocyte, leukocyte, and platelet morphology. Blood filmsshould first be scanned using a low-power objective to estimate the totalleukocyte count and to look for the presence of erythrocyte agglutination (Fig.SA), leukocyte aggregates (Fig. SB), platelet aggregates (Fig. SC), microfilaria(Fig. SD), and abnormal cells that might be missed during the differentialleukocyte count. It is particularly important that the feathered end of bloodfilms made on glass slides be examined because leukocytes (Fig. SE) and plateletaggregates (Fig. SF) may be concentrated in this area. Aggregates of cells tendto be in the center of coverslip blood films rather than at the feathered edge.When examining a glass-slide blood film, the blood film will be too thickto evaluate blood cell morphology at the back of the slide (Fig. 6A) and toothin at the feathered edge where cells are flattened (Fig. 6C). The optimal areafor evaluation is generally in the front half of the smear behind the featherededge (Fig. 6B). This area should appear as a well-stained monolayer (a field inwhich erythrocytes are dose together with approximately one half touching eachother) of cells.

Hemoglobinometry Hemoglobin Determination Decrease in hemoglobin concentration beyond established normal ranges for age and sex is called “ anemia”, whereas increase in hemoglobin concentration beyond established normal ranges for age , sex, and geographical distribution is called “polycythemia”. So that, for correct diagnosis it is important to determine accurately and precisely hemoglobin concentration. Many methods are available for the determination of hemoglobin, but among them the relevant, and the recommended one is the Modified Drabkin’s Method.

ICSH (International Committee for Standardization in Hematology) consider this method as the reference method for hemoglobin determination. Drabkin’s solution contains the following:-1- Potassium Ferricyanide2- Potassium Cyanide.3- Non- ionic Detergent4- Dihydrogen Potassium Phosphate.Well mixed EDTA anticoagulated blood is diluted in Drabkin’s solution; non-ionic detergent will lyse the red cells to (1) liberate hemoglobin, and to (2) decrease the turbidity caused by red cell membrane fragments by dissolving them. Then, hemoglobin is oxidized and converted to methemoglobin (Hi) by potassium ferricyanide, this step is accelerated by the dihydrogen potassium phosphate, and requires approximately 3 minutes for total conversion. Potassium cyanide will provide cyanide ions to form cyanomethemoglobin (HiCN), which have a broad spectrum of absorption at 540 nm. The absorption can then be compared with a hemoglobin standard with a known hemoglobinconcentration, and by applying Beer’s law extract the hemoglobin concentration of the unknown (i.e. the patient).Hemoglobin + Potassium Ferricyanide Methemoglobin (Hi)Methemoglobin + Potassium Cyanide Cyanomethemoglobin