|Bone marrow Differentials|
For the clinical laboratory professionals who are only familiar with peripheral blood morphology, the first few observations of bone marrow aspirate smears can be overwhelming. The difference in cellularity between the two sample types, not to mention the wider variety of cell types, can lead to mental and visual overload. It is important to step back and break it down into more manageable pieces, starting on low power. Use low power (10x) to look at the distribution on the slide and the quality of the stain. Find areas where the spread/distribution of cells are thin enough (monolayer) to read easily and where you like the color balance and intensity of the stain. Next, add oil and move up to 50x and/or 100x power on the microscope.* Remember that there are several different cell types that are normally present and develop in the bone marrow before heading out into the peripheral blood. Most hematology technologists are familiar with the myeloid maturation sequence from peripheral differentials, even if immature cells are less commonly seen. However, there are additional cell types that are not seen on the peripheral blood differential, since they reside only in the bone marrow. Becoming more familiar with these cell types and the maturation sequences of the myeloid, erythroid, and megakaryocytic cells found in normal bone marrows will make performing these differentials less intimidating.One important concept to grasp is the continuum of cellular maturation sequences. There is no such thing as a magical switch that flips causing cells to jump to the next "textbook photo stage" as cell lines mature. Rather, each cell matures at its own pace. The maturation and morphology will vary from cell to cell and bone marrow to bone marrow. Understanding both nuclear and cytoplasmic normal morphology can aid in the identification of cells. *As counter-intuitive as it sounds for most applications, higher magnification does not always help with morphology. Reserve 100x for ultra fine detail.
|Rules for Bone Marrow Differentials|
Bone marrow differentials have significant differences from peripheral blood differentials that need to be considered as they are reviewed and counted.One of the most important facts to consider is the large variability in cellularity and cell distribution depending on the type of preparation that is used. Choosing where to count and when to use which of the smear types available to you, takes time and experience and can be directed by a pathologist's preference.Regardless of how many, or what types of smears you have available to choose from, you will always start with a simple visual inspection of your smears. Begin by recording the patient identification information as well as date of sample, and any other mandatory patient identifying information necessary for your laboratory. Record aspiration site information when provided. Many patients will have bilateral bone marrow aspirates performed as part of a diagnostic or staging workup. Standard aspiration sites are: posterior iliac crest (PIC), anterior iliac Crest (ANT), sternum (S), spinous process (SP) and sometimes in very young children, bone marrow is obtained from the tibia (T). Be aware, that while a bilateral bone marrow aspirate usually involves an aspirate of the same site from opposite sides of the body, e.g., L-PIC and R-PIC, in some situations, a bilateral staging aspirate will be from two different compartments on the same side, e.g. R-AIC, R-PIC. Observe the appearance of the bone marrow smears. Do any have feather edges? Are there fragments or spicules present on any of the smears available? If so, they should be your first choice to view, since they are more representative of what the biopsy will show if one was obtained. Once you select your smears, scan using 10X magnification on the microscope. Are some of the fragments/smears so thick that you cannot see good morphology? If so, reject these areas/slides. Are some of the fragments/smears so thin that everything is smashed? These areas/smears cannot be used either. Are there areas in the vicinity of any of the fragments that have good staining characteristics as well as readable morphology? This is where you should begin your differential.
|Cellularity and Additional Information|
Depending on the institution and laboratory protocol, comments on the degree of cellularity, presence of megakaryocytes, and presence of tumor cells may be added to the report by the technologist who performs the differential count. The terms used to describe these features will be determined by the hematopathologist.Cellularity is usually rated as normal, increased, or decreased. However, other terms may be used as well, such as "slightly decreased," "markedly decreased," or "markedly increased," etc. When spicules/fragments are not present, terms like "hemodilute" can be used to note very dilute bone marrows or, it may simple be marked as "not evaluable". For megakaryocytes the common terms of quantitation may be: None Seen Rare Decreased Present Normal Increased The presence of tumor cells should be noted as well as the slide or site in which they were observed. The top image on the right demonstrates a bone marrow that is markedly hypocellular. Only fragments of the bone marrow structure are present, with very few bone marrow precursor cells observed. Compare this to the bottom image that depicts a bone marrow that is normocellular.
|Responsibility of the Technologist versus Hematopathologist|
Depending on the laboratory protocols and the hematopathologist's preference, the technologist's responsibilities in bone marrow aspirate processing and reviewing can vary from simply staining slides to complex tasks such as smearing, staining, counting and distributing samples based on laboratory standard protocols. While laboratory professionals may perform bone marrow differentials, it is the hematopathologist's responsibility to review, interpret, and verify those counts after evaluating all of the samples that were provided. This includes both the bone marrow aspirate and biopsy smears, as well as the flow cytometry data, if required. When differentiating and counting samples with increased numbers of blasts, the technologist may make a determination of cell line for the blasts, according to observations on a Wright or Wright-Giemsa stained slide, but the hematopathologist may modify this placement based on flow cytometry data and additional special stains.The laboratory technologist can assess cellularity, presence of megakaryocytes, and possible presence of tumor cells on the bone marrow smears. Ultimately, the hematopatholgist is responsible for interpreting the bone marrow differential results after meticulous review of all smears stained, as well as any biopsy sections available. It is also the hematopatholgist's responsibility to identify tumor cells that may be present.In laboratories that use a standard order set with sample drop off directly from bedside, it is the technologist's responsibility to split and distribute the marrow based on laboratory protocol. It is hematopathologist's responsibility to verify that standard order testing was actually sent and to add on any additional testing deemed necessary, based on clinical history and lab findings. Once you become comfortable with bone marrow morphology and more familiar with your laboratory's protocols, performing marrow differentials will become less intimidating and more of a collaboration between technologist and hematopathologist.
|Rules for Bone Marrow Differentials, continued|
It is important to note that not all smears will have good areas to perform a differential in the vicinity of the bone marrow fragment. When this occurs, you must keep looking on additional smears. This is one of the reasons that several smears are stained and prepared for possible review. It can take time to recognize from 10x magnification what will be countable on 50x magnification. The best tip is to be patient and do not fail to keep on looking! In fact, sometimes it may be necessary to stain additional smears/slides, if available, to obtain enough readable material. While you are checking the smears on 10x magnification for readable areas, you should take the time to evaluate and record the following: The cellularity of the bone marrow sample Presence and number of megakaryocytes Presence of tumor cells Anything else out of the ordinary, which should be noted on the report (such as evidence of hemophagocytosis, storage disease etc.).Once you have decided where to count the marrow, you will perform the differential count. Usually a 200-cell bone marrow differential is the minimum acceptable count. However, more cells may be required depending on your laboratory/pathology protocol. Remember, unlike peripheral differentials, all nucleated cells are included in the total count, including all maturation stages of the erythroid cell series.Cell counts are performed on 40 - 50x magnification with oil depending on the optics of your scope, moving up to 100x magnification with oil as needed for fine detail. Once oil is added to the smear, move systematically through your chosen area until the morphology/cellularity/stain quality is no longer acceptable, then move back to 10x power to find another good area in the vicinity of the fragment to continue your count. You may need to progress from one slide to the next to accumulate enough cells for your differential. In fact, if there is variability in cell distribution from one smear or fragment/spicule to the next, then the count should be split between more than one smear/fragment to avoid a biased final count.If there are no spicules, then the differential should be performed in any portion of the slide that demonstrates readable morphology. In pull preps and coverslip preps, this will usually be in the thin area near the edge of the smear. If differential-type (wedge) smears are available, then the usual feathered-edge area should be used. On any of these smears, be sure that you are in deep enough from the thin edge so that the numbers of stripped cells are kept at a minimum to avoid skewing the count, as some cell types are more fragile than others.The pathologist is ultimately responsible for the final sign-out and will change/adjust/return smears for recount if there is any disagreement over numbers and cell types.
|Rules for Bone Marrow Differentials, continued|
Bone marrow smears can be very cellular and it can be difficult to keep track of where you are on the smear while keeping your correct hand position on the keyboard . Having a good strategy to use when counting cells and performing differentials can make this less difficult. On peripheral blood differentials, it is easy to observe and count each cell individually as the stage is moved to bring the next field into view. However, with bone marrows, the total number of keys that need to be used on the differential counter is greater than the number that need to be used with a peripheral blood smear and the number of cells per field is also increased dramatically, making it easy to lose track of the cells on the smear or one's hand/keyboard placement. It can be simpler and less stressful to work on the quadrant system. There are two different ways to do this: Divide the field into quadrants. Count the individual cells in each quadrant separately. This decreases the number of cells into more manageable bites. However, you still have the increased number of cell types to deal with and possible keyboard frame-shifts. Divide your keyboard into quadrants. Search your field for a limited number of cell types and tally all you see before moving on to the next grouping of cell types. Once you tally all your groups then move on to the next field (e.g., lymphocytes, monocytes, macrophages, eosinophils, basophils, plasma cells, erythroids, segmented neutrophils, bands, etc). You can make these small groupings for any cells as long as you cover the entire list of cell types that your laboratory reports in its bone marrow differential protocol. Remember that blasts are identified by cell type and there will usually be a separate key for pronormoblasts, myeloblasts, lymphoblasts, and possibly monoblasts and plasmablasts. It is possible to combine both methods, using the keyboard quadrant technique with a restricted portion of the total microscope field. This is useful when you are getting close to your total tally and do not want to alter the balance by only counting one cell type for the last few cells.
|Which of the following smear techniques can be utilized when processing bone marrow aspirate samples? (Choose all that apply)||View Page|
The procedure your laboratory utilizes for bone marrow stains is determined by the type of stainer available to you. The stainer available may also dictate the type of smear preparations that your laboratory makes. There are several types of automated hematology stainers on the market today. Some stainers are simple continuous-feed stainers with limited programmability. Some are batch stainers that can have multiple programs, customizable to the sample type or stain preference of the user. Other stainers are dip stainers that automatically move a slide rack from bucket to bucket, or an inline corkscrew that moves slides down a platen and dispenses stain/solutions at fixed positions. Finally, there are centrifuge stainers that apply stains to a spinning slide tray during programmed intervals.Hema-tek® stainers, with a fixed stain area, require shorter preparations on long slides, so slide pull preps or differential smears would be the laboratory standard. Since the stain time and volume is fixed, bone marrow slides may need to be stained twice and sometimes even three times for extremely cellular bone marrows. When using this type of stainer, always check the stain quality before coverslipping.Automated dipping stainers can be used with either long slides or coverslips,when utilizing a coverslip basket, so the choice of smear type is driven by laboratory and pathologist preference. As with manual staining times, the laboratory should have a separate program for bone marrow staining that reflects the need for longer contact times. Wescor® hematology stainers are quite flexible. They are centrifugal stainers that are pre-programmed for rapid, Wright-Giemsa and May-Grunwald stains, as well as having programmable custom settings. Each stain type can be adjusted for color balance and intensity. They can be used with slides or coverslips when coverslip adapters are utilized. Since it is a centrifugal staining system, stain precipitate is minimized and it is very easy to change programs as you shift from peripheral bloods to fluids cytospins to bone marrows.
|The role of the laboratory technologist in processing bone marrow aspirates can vary depending on laboratory and clinician protocols. Which of the following roles may be performed by a laboratory technologist?(Select all that apply) ||View Page|
|Erythrocytic Cells: Introduction|
When performing bone marrow cell identification, it is necessary to differentiate the stages of erythrocyte development. This differs from a peripheral blood differential, where the term "nucleated red blood cells" ("NRBCs") is used to describe all stages of circulating normoblasts. As with the myeloid sequence, there is a continuum in the erythroid maturation process in terms of nuclear and cytoplasmic morphology. Becoming familiar with the range of variation in each nucleated erythrocyte stage will make bone marrow differentials less intimidating.The image to the right shows several different stages of erythroid maturation with several clusters of NRBCs all maturing together.
|When performing bone marrow differentials it is not necessary to distinguish the precursor forms of the erythroid sequence.||View Page|
Lymphocytes mature in the lymph nodes rather than in the bone marrow and therefore are not routinely assessed when deciding if a marrow has "trilinear" (myeloid, erythroid, megkaryocytic) maturation. However, they are normally present in the bone marrow and, when clustered in a lymphoid follicle, can be very prominent. Since lymphocytes mature in the lymph nodes, they will appear identical to peripheral blood lymphocytes when viewed in the bone marrow. They will have the same range of variation in size and cytoplasm and will demonstrate the same types of viral transformations noted in the peripheral blood. Viral/atypical lymphocytes are combined together with normal lymphocytes in a bone marrow differential count and not placed into their own category, as they are in a peripheral blood differential. However, the hematopathologist may include this information in the interpretation, if these changes are noted.Lymphocytes can be found scattered throughout the bone marrow and must be distinguished from early erythroid precursors, which they can closely resemble. Lymphocytes are frequently found in and around early NRBC clusters. In the top image on the right, notice the medium-sized lymphocyte (red arrow) next to the two basophilic normoblasts (blue arrow). The color and texture of the scant lymphoid cytoplasm is almost identical to the NRBC, which can be a bit confusing. However, observe the differences in the nuclei between the two cell types. The lymphocyte has a less distinct chromatin clumping pattern than the basophilic normoblasts and the lymphocyte does not have any "nuclear pores." Also, the lymphocyte has an irregularly-shaped nucleus that is hugging the cytoplasmic border, while the NRBC has a round and regular, centrally-placed nucleus. Identify the three lymphocytes circling the NRBCs in the second image (see red arrows). Notice the chromatin of the lymphocytes; the lymphoid smudgy/clumpy pattern is certainly not as dense and clumped as what is noted in the NRBCs. This nuclear difference becomes more pronounced as the erythroids mature. The cytoplasmic differences should be more apparent as well, since lymphocytes will never produce hemoglobin.
Plasma cells are terminally differentiated B-lymphocytes that have developed a characteristic morphology while actively producing and releasing immunoglobulins. While plasma cells have their origins in the bone marrow as B-cells, they usually leave the bone marrow to develop and mature in the lymph nodes or spleen. Plasma cells begin to produce immunoglobulins after being stimulated by T-cells and exposed to processed antigens.Under normal circumstances, plasma cells are not a large percentage of the lymphoid cells found in a marrow. They are usually placed in a separate category in the differential, unlike viral/atypical lymphs. There can be a relative increase in plasma cells in reactive marrows, and both plasma cells and their early precursors will be markedly increased in plasma cell disorders.While mature plasma cells somewhat resemble lymphocytes, there are a few important differences. The size of the cell is usually larger with more abundant cytoplasm. The nucleus is eccentrically placed and the overall shape of the cell generally resembles a wedge or comet with the nucleus leading the cytoplasm. The chromatin is just as thick and clumpy as a lymphocyte's but is aligned in a more "spokey" or "clockwork" pattern. The cytoplasm is usually more basophilic than the cytoplasm of a normal lymphocyte and will have a well-defined perinuclear halo or noticeable clearing in the golgi area. Vacuoles may or may not be present.Notice the size of the single plasma cell in the top image (see red arrow). It is larger than the neutrophil precursors surrounding it and is almost rectangular in shape. Observe that the nucleus leads the cytoplasm, causing the wedge or comet shape. Notice the prominent perinuclear halo. Find the two plasma cells in the upper left corner of the second image. There is much more cytoplasm in these plasma cells compared to the occasional lymphocyte present in the field. Notice the eccentric nuclear placement as well as the characteristic clearing in the golgi area.
|What are the cells that are indicated by the red arrows in the image on the right?||View Page|
Stromal cells are the cells that comprise the backbone of a bone marrow fragment. They provide the support matrix as well as some of the nutrients necessary for the growth of all cellular precursors found in the bone marrow.Stromal cells appear similar to macrophages and tend to be found in sheets. Usually, they are deep in the heart of a fragment. When the differential is counted in the areas adjacent to the fragments, they may not be noticed and should not be counted. While stromal cells may be present in clusters, it is important to recognize that these cells are normal bone marrow elements and are not tumor cells.In aplastic bone marrows, or bone marrows with decreased cellularity, they may be more apparent. However, since they are considered tissue cells, they are not included in the differential. The top image to the right demonstrates a typical stromal clump. The bottom image shows stromal cells mixed with phagocytic macrophages.
|Cerebrospinal fluid (CSF) specimens are usually collected by lumbar puncture and placed in numbered tubes starting with the first tube collected. They are delivered to the laboratory departments in a designated order. Which two collection tubes are commonly used for a CSF cell count and differential?||View Page|
|Which of the following cells are considered abnormal and clinically significant on a CSF differential?||View Page|
|Specimen Collection (continued)|
A syringe is used to remove 6 - 15 ml of spinal fluid. Less fluid is removed in babies and small children. The CSF sample is divided among 3 - 4 tubes, with 2 - 4 ml in each tube.
Glass tubes should be avoided due to cell adhesion which may affect the cell counts or differential. The tubes are numbered in the order in which the CSF is obtained.
|Collection Tubes (continued)|
The tubes used for CSF aspiration are generally pre-numbered 1 through 3 or 4, with tube 1 being the first tube filled. If possible, tube 1 should be reserved for non-routine studies. Tube 2 can be used for immunology and chemistry testing. Tube 3 can be used for microbiology testing. Hematology analysis is typically performed on the last tube collected (3 or 4) to assure that any peripheral blood that may have contaminated the sample during the lumbar puncture has cleared. When only three tubes are obtained:Tube 2 is often reserved for microbiology Tube 3 is shared, with the hematology testing performed first, followed by any chemistry or immunology testing. The first tube obtained should not be used for microbiology or hematology testing because of possible contamination from the lumbar puncture. However, in some health care facilities, a red blood cell (RBC) count may be requested on the first tube of CSF collected and the last tube collected as a means of differentiating a subarachnoid hemorrhage (SAH) from a traumatic tap. If the RBC counts in the first and last tubes are similar, an SAH is suspected. If the RBC count in the last tube is significantly less than the count in the first tube, a traumatic tap is suspected.One problem that could occur with this method is if a traumatic tap is overlying a subarachnoid hemorrhage and the presence of blood in the last tube is due to an SAH. The two-tube count can raise the suspicion of an SAH, but a question still remains whether this is the cause or is the residual blood due only to the traumatic tap, but the volume of CSF collected is insufficient to allow the CSF to clear. When a large number of RBCs are present in the last tube, but less than the number in the first tube, the physician must use additional clinical information to determine if an SAH is present.
|For best results, cell counts and differential smears should be processed within:||View Page|
|More on Undiluted Specimens|
In an undiluted specimen, count and differentiate red cells and white cells at the same time. You can count red cells on a hand counter and use the differential counter for white cells.
If you cannot differentiate white cells from red cells in the undiluted specimen, a plain capillary tube may be filled with crystal violet acetic acid diluent which is subsequently expelled from the tube. A very thin coating of the diluent will remain on the inside of the tube. CSF is drawn halfway up into the tube, which is then rocked back and forth to mix. The hemacytometer is then filled with the fluid containing stained white blood cells and lysed red cells.
If cells are numerous and overlapping and it is necessary to focus through several planes in order to see all of the cells, a dilution must be made.
When macroscopic appearance is turbid, milky or bloody, a significant dilution is usually necessary.
|Stained Cytospin Preparations of CSF|
All white cells present in a cerebrospinal fluid must be identified.
If more than 10 cells/mm3 are present or there is difficulty identifying the few cells that are present, make a cytospin, a filtration, or a sedimentation preparation, stain with Wright-Giemsa, and perform differential count.
Cytospins made with a cytocentrifuge are preferred since they are easiest to make and interpret, but filtration and sedimentation methods can also be used to prepare a slide for subsequent staining.
The cytospin technique uses a high speed centrifuge to concentrate the cells on a slide in a uniform monolayer 6 mm in diameter. The monolayer distribution enhances the morphological appearance of the cells present.Allow the slides to dry in air for several minutes and then stain them with Wright-Giemsa stain. Cytospin slides may be placed in an automatic stainer or stained manually.Perform a 100 or 200 cell differential and record the number of neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, and blasts cells.Pathologists must review any slide which has tumor cells, unidentified cells, or immature stages of cells, such as blasts.Since criteria for review may vary from one laboratory to another, be sure to check the requirements in your laboratory before reporting the differential.
|Mature Peripheral Blood Cells|
In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. Up to 2% neutrophils is also considered within normal limits when a cytospin smear is used for the differential. In children, normal CSF cells are 70% monocytes, up to 20% lymphocytes and up to 4% neutrophils. When any of these normal cell abundances are increased, the term pleocytosis is used. Neutrophil pleocytosis is an increase in neutrophils and usually indicates the presence of a bacterial infection.
|Which of the following statements are true regarding spinal fluid differentials?||View Page|
Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.html Accessed March 25, 2013.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, RochesterMN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.
|The cells included in the composite image were found in the peripheral blood smear of a patient with the following results:total WBC of 24.5 x 109/L. Differential count:myelocytes 1 metamyelocytes 4 band neutrophils 15 segmented neutrophils 40 monocytes 8 eosinophils 2 basophils 1 lymphocytes 29This hematologic picture is most consistent with:||View Page|
|Normal Bone Marrow|
Illustrated in the photograph is a normal bone marrow smear stained with Wright/Giemsa stain. Note the evenly distributed cells with normal maturation in both the myeloid and erythroid maturation sequences.An estimation of the percentage composition of cells can be made by experienced observers from scanning of multiple fields. In some instances a detailed differential count of 300 or more cells must be made.In normal bone marrows, the myeloid to erythroid ratio (M:E ratio)ranges from 1.2:1 to 5:1.A ratio of less than 1.2:1 indicates depressed leukopoiesis or erythroid hyperplasia. Ratios of 6:1 or greater usually indicates infection, erythroid hypoplasia, or chronic myelogenous leukemia.An assessment of the overall cellularity is also useful. In general, cellularity of less than 25% indicates hypoplasia; greater than 75% indicates hyperplasia.
|Criteria for Performing a Manual Review of the Smear|
Automated cell counters will flag abnormal findings. The following guidelines represent typical review criteria, although specific requirements vary between laboratories. Follow the criteria established by your laboratory's procedure:Total white blood cell count <3.0 X 109/L or >12.0 X 109/LNeutrophils >80% Lymphocytes >45% or <10% Monocytes >15% Eosinophils >10% Basophils >5%A morphology review may also be indicated if the platelet count is <100 X 109/L or >650 X 109/L.
|Evaluation Criteria: White Blood Cells and Platelets|
In most clinical hematology laboratories, an initial blood count is performed by an automated cell counting instrument. Additionally, most of these instruments also produce a five-part differential count, indicating the percentage of neutrophils, lymphocytes, monocytes, basophils, and eosinophils. Some instruments can also provide information about cellular immaturity and abnormal cellular morphologies.Occasionally, atypical cells, similar to those shown in the image to the right, would be flagged or counted as mixed cells, at which point a smear review would be required to make an identification. In cases where there are automated instrument differential flags, mixed cell count is high, or there are other indications that atypical cells may be present, a review of the smear is indicated.
May-Hegglin anomaly is an inherited dominant condition in which large (2 - 5 um) basophilic inclusions, resembling Döhle bodies, are present in granulocytes, including neutrophils, eosinophils, basophils, and monocytes. The inclusions are caused by accumulation of free ribosomes. A May-Hegglin body is indicated by the black arrow in the image on the right. Note that this inclusion is well-defined and there is no evidence of toxic granulation in the cytoplasm. When Döhle-like bodies are identified, May-Hegglin anomaly should be considered in the differential diagnosis, even though this entity is rare. Giant platelets containing few fine granules are also characteristic of May-Hegglin anomaly. The red arrow in the image on the right points to a giant platelet, observed in the same field as a neutrophil containing a May-Hegglin body. Sometimes the platelets have bizarre shapes and variable sizes. Variable degrees of thrombocytopenia complicated by mild bleeding problems and purpura may accompany the aberrant platelets.
A basophil and a small lymphocyte are compared in the same field in the upper image, while a single basophil is shown in the lower image.The cytoplasmic granules of the basophil are larger than the granules of toxic granulation.They contain chemical mediators of immediate hypersensitivity, and are found in the cytoplasm and overlying the nucleus (better seen in the lower image). Basophilic granules stain metachromatically with toluidine blue indicating the presence of acid mucopolysaccharide or proteoglycans, both thought to be heparin or heparin-like substances.Basophils are related to mast cells (tissue basophils), each involved in hypersensitivity responses and following anaphylactic episodes. Under the stimulation of complement components C3a and C5a, many mediators are released from the basophil granules, including histamine, heparin, and eosinophil chemotactic factors of anaphylaxis, or ECF-A.Basophils are the least common granulocytes in the peripheral blood, comprising 2% or less of the differential count. The presence of large granules of irregular size in basophils and the admixture of eosinophilic granules may indicate dysplastic changes associated with myelodysplastic disorders and leukemia. Basophils may be increased in:Myeloproliferative disordersChronic metabolic conditions Myxedema Diabetes mellitusHypersensitivity responses Tuberculosis
|A peripheral blood smear is observed during a manual differental review. The patient is a 10 year-old boy with symptoms suggesting appendicitis and an appendectomy is being considered. The total WBC is 18.5 X 1000/uL, RBC's = 5.45 X 1M/uL, hemoglobin = 16.0 g/dL, hematocrit 48.2%.WBC differential:Segs = 53%, bands = 42% (two of which are shown in the image) monocytes = 2% lymphocytes= 2% These findings support the diagnosis of appendicitis.||View Page|