Practical Hematology Lab

Practical Hematology Lab
Blood Smear I- Preparation Of Blood Smear
There are three types of blood smears: The cover glass smear. The wedge smear . The spun smear. The are two additional types of blood smearused forspecificpurposes Buffy coat smearfor WBCs < 1.0109/L Thick blood smearsfor blood parasites . Wedge Blood Smear Specimen : EDTA blood within 2 to 3 hours &collected to the mark on tube. Note : May change RBCs morphology such asSpiculated (crenated) cells if : Excessive amount of anticoagulant to specimen Old blood - long standing. Warm environment (room temperature) mayhasten changes. Procedure placing a drop of blood from mixed sampleon a clean glass slide. Spreader slide using another clean glassslide at degree angle. Control thickness of the smear by changingthe angle of spreader slide Allow the blood film to air-dry completelybefore staining. (Do not blow to dry.Themoisture from your breath will cause RBCartifact.) Steps For Blood Film Procedural Notes Characteristics of a good smear
Thick at one end, thinning out to a smooth roundedfeather edge. Should occupy 2/3 of the total slide area. Should not touch any edge of the slide. Should be margin free, except for point ofapplication. 2.As soon as the drop of blood is placed on the glassslide, the smear should be made without delay.Anydelay results in an abnormal distribution of the whiteblood cells, with many of the large white cellsaccumulating at the thin edge of the smear. 3.The thickness of the spread
If the hematocrit is increased, the angle of the spreader slide should bedecreased. If the hematocrit is decreased, theangle of the spreader slide should beincreased 4. common causes of a poor blood smear
Drop of blood too large or too small. Spreader slide pushed across the slide in a jerky manner. Failure to keep the entire edge of the spreader slide againstthe slide while making the smear. Failure to keep the spreader slide at a 30 angle with theslide. Failure to push the spreader slide completely across theslide. Irregular spread with ridges and long tail: Edge ofspreader dirty or chipped; dusty slide Holes in film: Slide contaminated with fat or grease Cellular degenerative changes: delay in fixing, inadequatefixing time or methanol contaminated with water. Examples Of Unacceptable Smears
A: Blood film with jagged tail made from a spreader with a chipped end. B: Film which is too thick C: Film which is too long, too wide, uneven thickness and made on a greasy slide. D: A well-made blood film Examples Of Unacceptable Smears 5. Biologic Causes Of A Poor Smear
Cold agglutinin - RBCs will clump together. Warm the blood at 37 C for 5 minutes, and thenremake the smear. Lipemia - holes will appear in the smear.There isnothing you can do to correct this. Rouleaux - RBCs will form into stacksresembling coins.There is nothing you can do tocorrect this 6.Although this is the easiest and most popularmethods for producing a blood smear, it does notproduce a quality smear. The WBCs are unevenly distributed and RBCdistortion is seen at the edgesSmaller WBCs suchas lymphocytes tend to reside in the middle of thefeathered edge.Large cells such as monocytes, immature cells andabnormal cells can be found in the outer limits ofthis area.Spun smears produce the most uniform distributionofblood cells. Slide Fixation And Staining
Leishman'sStain II- Fixing the films To preserve the morphology of the cells, filmsmust be fixed as soon as possible after theyhave dried. Methyl alcohol (methanol) is the choice,although ethyl alcohol ("absolute alcohol") canbe used. To fix the films, place them in a coveredstaining jar or tray containing the alcohol for 2- 3 minutes. In humid climates it might benecessary to replace the methanol 2-3 times perday; the old portions can be used for storingclean slides. Notes It is important to prevent contact with water beforefixation is complete. The presence of water duringmethanol fixation produces refractile body artifacts (waterspots) in the erythrocytes. These water spots persist through staining of the smearand cover items of interest in the smear. Further, they aredistracting to the person evaluating the smear. In somecases, the water spots may interfere with diagnosis. To prevent the alcohol from becoming contaminated byabsorbed water, it must be stored in a bottle with a tightlyfitting stopper and not left exposed to the atmosphere,especially in humid climates. III. Staining the film Romanowsky staining: Romanowsky stains are universallyemployed for staining blood films and are generally verysatisfactory. There are a number of different combinations of these dyes,which vary, in their staining characteristics. May-Grunwald-Giemsa is a good method for routine work. 2. Giemsa stain is thought to produce more delicate stainingcharacteristics.Wright's stain is a simpler method. 4. Leishman's is also a simple method, which is especiallysuitable when a stained blood film is required urgently orthe routine stain is not available (e.g. at night). 5. Field's stain is a rapid stain used primarily on thin films formalarial parasites. Principle The main components of a Romanowsky stain are:
A cationic or basic dye (methylene blue or its oxidationproducts such as azure B), which binds to anionic sitesand gives a blue-grey color to nucleic acids (DNA orRNA), nucleoproteins, granules of basophils andweakly 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 anorange-red color to hemoglobin and eosinophilgranules. pH value of phosphate buffer is very important. Staining Procedure (Leishmans Stain)
Thin smear are air dried. Flood the smear with stain. Stain for 1-5 min. Experience will indicate theoptimum time. Add an equal amount of buffer solution and mixthe stain by blowing an eddy in the fluid. Leave the mixture on the slide for min. Wash off by running water directly to the centerof the slide to prevent a residue of precipitatedstain. Stand slide on end, and let dry in air. Staining Characteristics Of A Correctly Stained Normal Film
NucleiPurple Cytoplasm ErythrocytesDeep pink NeutrophilsOrange-pink LymphocytesBlue; some small lymphocytes deep blue MonocytesGrey-blue BasophilsBlue Granules NeutrophilsFine purple EosinophilsRed-orange BasophilsPurple-black MonocytesFine reddish (azurophil) PlateletsPurple Eosinophilic granules
Blue nucleus Basophilic granules Discussion The phosphate buffer controls the pH of the stain.
If the pH is too acid, those cells or cell parts taking up an acid dye stain will stain pinker and the acid components that stain with the basic dye show very pale staining. If the stain-buffer mixture is too alkaline, the red blood cells will appear grayish-blue and the white cell nuclei will stain very deeply purple. Therefore, to stain all cells and cell parts well, the pH of the phosphate buffer is critical. Causes & correction Too Acid Stain: insufficient staining time
prolonged buffering or washing old stain Correction: lengthen staining time check stain and buffer pH shorten buffering or wash time Too Alkaline Stain: thick blood smear prolonged staining insufficient washing alkaline pH of stain components Correction : check pH shorten stain time prolong buffering time too acidic suitable too basic Performing A Manual Differential And Assessing RBC Morphology Introduction When blood samples are evaluated by the use of automated hematology analyzers, this analysis includes automated differentials. Specific criteria pertaining to normal, abnormal, and critical values have been programmed into the analyzers by the institution, and if the differentials do not meet these criteria, verification is necessary. This is done by performing manual differentials and further evaluating the peripheral smear. Objective To determine the relative number of each type of white cell present in the blood by performing differential cell counts on five relatively normal blood smears and five sets of abnormal blood smears within a 15% accuracy of the instructor's values. To determine within one qualitative unit the red cell, white cell, and platelet morphology of each of the above blood smears. To determine within 20% accuracy an estimate of the white cell counts and the platelet counts of each of the above blood smears. Principle First, a differential white blood cell (WBC) count is performed to determine the relative number of each type of white cell present. Technologists/technicians must recognize and properly record the type(s) of white cell observed. Simultaneously, red cell, white cell, and platelet morphology is noted and recorded. Also, a rough estimate of platelets and WBC counts is made to determine if these numbers generally correlate with the automated hematology analyzer. Technologists/technicians must be proficient at recognizing red and white cell abnormalities, identifying them correctly, and quantifying them. Principle White Blood Cells Red Blood Cells, Examine for:
Check for even distribution and estimate thenumber present (also, look for any grossabnormalities present on the smear). Perform the differential count. Red Blood Cells, Examine for: Size and shape. Relative hemoglobin content. Polychromatophilia. Inclusions. Rouleaux formation or agglutination Principle Platelets Estimate number present.
Examine for morphologic abnormalities. Procedures Observations Under 10
Check to see if there are good counting areasavailable free of ragged edges and cellclumps. Check the WBC distribution over the smear. Check that the slide is properly stained. Check for the presence of large platelets,platelet clumps, and fibrin strands. Observations Under 40x : WBC Estimates
Usingthe 40 high dry with no oil. Choose a portion of the peripheral smear wherethere is only slight overlapping of the RBCs. Count 10 fields, take the total number of whitecells and divide by 10. To do a WBC estimate by taking the averagenumber of white cells and multiplying by 2000. Observations Under 100: Platelet Estimates
Platelet estimates are done under 100 with the RBCs barely touching, approximately 200 RBCs. This takes place under the 100 objective (oil). On average there are 8 to 20 platelets per field. Ten fields are counted using the zigzag method. This method of counting is done by going back and forth lengthwise or sidewise. Platelet Estimation Platelets per oil immersion field (OIF)
20 platelets/OIF =increased After the 10 fields are counted, the number of platelets is divided by 10 to get the average. The average number is now multiplied by a factor of 20,000 for wedge preparations. For monolayer preparations, use a factor of 15,000. PLATELETS Manual Differential Counts
These counts are done in the same area as WBC andplatelet estimates with the red cells barely touching. This takes place under 100 (oil) using the zigzagmethod. Count 100 WBCs including all cell lines from immatureto mature. Reporting results Results are expressed as a percentage of the total leukocytes counted. It is also helpful to know the actual number of each white cell type per L of blood.This is referred to as the absolute count and is calculated as follows: Absolute number of cells/l = % of cell type indifferential x white cell count Observing And Recording Nucleated Red Blood Cells (NRBCS)
If 10 or more nucleated RBC's (NRBC) areseen, correct the White Count using this formula: Corrected WBC Count = WBC x 100/( NRBC + 100) Example :If WBC = 5000 and 10 NRBCs havebeen counted Then 5,000100/110 = The corrected white count is Observing Direction: Observe one field and record the number of WBC according tothe different type then turn to another field in the snake-liked direction *avoid repeat or miss some cells 1- Normal Peripheral Blood Smear Characteristics Of Blood Cells
Erythrocyte: Shape & size: Biconcave disc , size like lymphocyte nucleus. Nucleus : lost. Cytoplasm: pinkish hue, small area of central pallor. Number in man varies between 5 and 5.5 million per cubic mm of blood. Platelet ( Thrompocytes) Nucleus: No nucleus. Cytoplasm: small amount bluish cytoplasm & contains reddish purple granules White Blood Cells White blood cells, or leukocytes, are classified into two main groups; granulocytes and non granulocytes (also known as agranulocytes). The granulocytes, which include neutrophils, eosinophils, and basophils, have granules in their cell cytoplasm. Neutrophils, eosinophils, and basophils also have a multilobed nucleus. As a result they are also called polymorphonuclear leukocytes or "polys." The nuclei of neutrophils also appear to be segmented, so they may also be called segmented neutrophils or "segs." The nongranulocytes white blood cells, lymphocytes and monocytes, do not have granules and have nonlobular nuclei. They are sometimes referred to as mononuclear leukocytes. Leukocytosis Leukocytosis, a WBC above 10,000 is usuallydue to an increase in one of the five types ofwhite blood cells and is given the name of thecell that shows the primary increase. Neutrophilic leukocytosis = neutrophilia 2. Lymphocytic leukocytosis = lymphocytosis 3. Eosinophilic leukocytosis = eosinophilia 4.Monocytic leukocytosis =monocytosis 5.Basophilic leukocytosis = basophilia Stab Neutrophil (Band)
Diameter:12-16 Cytoplasm : pink Granules: primary,secondary Nucleus: dark purpleblue dense chromatin Segmented Neutrophil Diameter: 12-16 Cytoplasm : pink
Granules: primary,secondary Nucleus: dark purple blue,dense chromatin, 2-5lobes. 1. Neutrophils Neutrophils are so named because they arenot well stained by either eosin, a red acidicstain, or by methylene blue, a basic oralkaline stain. Neutrophils are also known as "segs","PMNs" or "polys" (polymorphonuclear). They are the body's primary defense againstbacterial infection. Increased neutrophils count (neutrophilia)
Acute bacterial infection. Granulocytic leukemia. Decreased neutrophil count (neutropenia) Typhoid fever Brucellosis Viral diseases, including hepatitis, influenza,rubella, and mumps. Left-shift And Right-shift Of Neutrophil
Normally, most of the neutrophils circulating inthe bloodstream are in a mature form, with thenucleus of the cell being divided or segmented.Because of the segmented appearance of thenucleus, neutrophils are sometimes referred toas "segs. The nucleus of less mature neutrophils is notsegmented, but has a band or rod-like shape.Less mature neutrophils - those that haverecently been released from the bone marrowinto the bloodstream - are known as "bands" or"stabs". Left-shift: non-segmented neutrophil > 5% Right-shift: hypersegmented neutrophil >3% Shift to right Increased hypersegmented neutrophile.
Band Neutrophil Segmented Neutrophile Shift to left Increased bandsmean acute infection, usually bacterial. Shift to right Increased hypersegmented neutrophile. 2. Eosinophil Diameter: 14-16 Cytoplasm : full of granules
Granules: large refractileorange-red. Nucleus: blue, densechromatin, 2 lobes like apair of glass. The most common reasons for an increase in the eosinophil count are :
Allergic reactions such as hay fever, asthma,or drug hypersensitivity. Parasitic infection Eosinophilic leukemia 3. Basophil Diameter: 14-16 Cytoplasm : pink
Granules: dark blue black obscure nucleus Nucleus: blue The purpose of basophils is notcompletely understood. Basophile counts are used toanalyze allergic reactions. An alteration in bone marrowfunction such as leukemia maycause an increase in basophils. 4. Lymphocyte Diameter: small 7-9, large 12-16 Cytoplasm: medium blue
Granules: small agranular,large a few, primarygranules Nucleus: dark blue, round dense chromatin Lymphocytes Lymphocytes are the primary components ofthe body's immune system. They are thesource of serum immunoglobulin's and ofcellular immune response. Two types oflymphocytes: 1. B lymphocyte : Humeral immunity. 2. T lymphocyte : Cellular immunity. Lymphocytes increase (lymphocytosis) in:
Many viral infections Tuberculosis. Typhoid fever Lymphocytic leukemia. A decreased lymphocyte (lymphopenia) count of less than 500 places a patient at very high risk of infection, particularly viral infections. 5. Monocyte Tuberculosis Brucellosis Malaria Monocytic leukemia
Diameter: 14-20 Cytoplasm : grey blue Granules: dust-like lilac colorgranules Nucleus: blue, large irregularlyshaped and folded Diseases that cause a monocytosisinclude: Tuberculosis Brucellosis Malaria Monocytic leukemia Band Neutrophil Segmented Neutrophil Eosinophil basophil lymphocyte Monocyte Discussion 1. Do not count cells that are disintegrating
eosinophil with no cytoplasmicmembrane and with scatteredgranules Pyknotic cell (nucleus extremelycondensed and degenerated, lobescondensedinto small, roundclumps with no filamentsinterconnecting). smudge cells Basket cells smudge cells Basket cells 2- Abnormal Differentials
200 Cell diff: a. WBC > 15.0 (>20.0 for babies under 1 month andlabor unit) b. Three or more basophils seen. If more than five immature WBC's are seen (or anyblasts) let someone else diff slide and average results. Correct WBC for NRBC's if you seen ten or moreNRBCs/100 WBC. Always indicate number of cells counted on diff. If any cell type is extremely elevated (such as bands,monos, or eos > 20) indicate that you are aware of theabnormality by circling or checking on the card next tothe results. 3-Morphologic Changes Due To Area Of Smear
Thin area- Spherocytes which are really"spheroidocytes" or flattened red cells. Truespherocytes will be found in other (Good)areas of smear. Thick area - Rouleaux, which is normal insuch areas. Confirm by examining thinareas. If true rouleaux, two-three RBC'swill stick together in a "stack of coins"fashion.. TailBodyHead 4. A well-made and well-stained smear is essential tothe accuracy of the differential count. Theknowledge and ability of the cell morphologist iscritical to high-quality results. 5. Before reporting significant abnormalities such asblasts, malaria or other significant finding on apatients differential, ask a more experienced techto review the smear for confirmation.In clinicalsettings where a pathologist or hematologist ispresent, the smear is set aside for PathologistReview. 6. Never hesitate to ask questions concerningmorphology or the identification of cells.Thedifferential is one of the most difficult laboratorytests to learn.In fact, learning about cells andtheir morphology is a process that continues for aslong as you perform differentials.