| Which collection tube is used for a CSF cell count and differential? | View Page |
| Which of the following cells are considered abnormal 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.
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| Collection Tubes (continued) The samples from each tube are used for specific tests:The first tube may also be used for serological testing.The second tube is used for gram stain and culture.The third tube is used for the cell count and differential.The fourth tube is used for cytological examinations or other tests which may be needed to further characterize abnormal cells.
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| 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. | View Page |
| 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.
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| Cytospin Technique In the cytospin procedure, use 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, such as Hema-Tek, 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. | View Page |
| Mature Peripheral Blood Cells In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes.
Neutrophils abundance up to 2% 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. | View Page |
| Which of the following statements are true regarding spinal fluid differentials? | View Page |
| The reactions seen in the portion of the API strip shown in the photograph, effectively rules out Escherichia coli. | View Page |
| PYR Differential As mentioned before, the spot PYR test is commonly performed to separate Enterococcus species (positive reaction) from the Group D streptococci (S. bovis, S. equinus), which are negative.It should be remembered that Streptococcus pyogenes (group A) also produces PYR; therefore, additional characteristics such as beta hemolysis are important.Some species of Aerococcus and Gemella are also PYR-positive; however, they can be suspected if large cocci in tetrads or clusters are observed on gram stain.These species are rare isolates in most clinical practices. | View Page |
| Review 2 Citron DM. Appelbaum PC.:
How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay?
Clinical Infectious Diseases. 16 Suppl 4:S435-8, 1993Identification of anaerobic bacteria in specimens from sites of infection due to mixed organisms can be time-consuming and expensive. Laboratories should limit anaerobic workups by testing only those specimens that have been properly collected and transported to the laboratory.Use of selective and differential media for initial processing can provide rapid and relevant information to the clinician. Anaerobes isolated from normally sterile sites and sites of serious infection should always be completely identified. Group-or genus-level identifications may suffice in other instances.The Bacteroides fragilis group of organisms should always be identified because of their virulence and resistance to many antimicrobial agents.Some of the other organisms that warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); Clostridium ramosum, Clostridium innocuum, and Clostridium clostridioforme (which are resistant to antibiotics); Clostridium perfringens (a cause of myonecrosis and gas gangrene,potentially serious infection); anaerobic cocci (which may be resistant to metronidazole and clindamycin); and fusobacteria (which may be virulent and resistant to clindamycin and penicillin). | View Page |
| The test(s) which may be performed to establish a presumptive differential identification between group B streptococci and L. monocytogenes is/are: | View Page |
| Reticulocyte identification Reticulocytes are red blood cells prematurely released from the bone marrow. On a Wright-Giemsa stained blood smear, they appear as polychromatic macrocytes. Their presence in the peripheral blood may suggest hemolysis or bleeding. Their presence is expressed as a percentage of the red cell count: newly born= 3-7%; up to one week of age=1-3%; >one week =0.3-1.8%. Automated or manual methods may be used to enumerate reticulocytes. In clinical context, retics must be separated from debris, precipated stain, Pappenheimer bodies, Howell-Jolly bodies, and Heinz bodies. | View Page |
| Atypical smear: Case follow-up The patient whose blood smear is shown in the photograph was a 32-year-old female from Virginia who came to the high country of Colorado to ski. The day after arrival, she experienced shortness of breath, fatigue, and upper abdominal pain. She was seen in a medical center in the mountains where a working diagnosis of altitude sickness was made. A CBC revealed RBCs 5.1 x 1012/L, hemoglobin 12.8g/dL, MCV 60fL, hematocrit 40.9%, and normal total WBC, differential, and platelet count. The RDW was normal. Further questioning revealed a previous diagnosis of heterozygous beta-chain thalassemia. No other abnormal hemoglobins were found on hemoglobin electrophoresis, but HbA-2 was elevated to 5%, supporting the diagnosis of beta thalassemia. The patient's poikylocytosis and anisocytosis may be a clue to an underlying erythrocyte abnormality. Persons with iron deficiency anemia may experience various degrees of hypoxia upon arriving at high altitudes. Those with sickle cell disease and thalassemia minor (as in this case) may experience bone pain or other symptoms of "crisis" and/or alteration in the appearance of their erythrocytes upon sudden high altitude exposure. The classic teaching is that in differentiating iron deficiency anemia from thalassemia, increased RDW would favor iron deficiency; normal RDW favors thalassemia. | View Page |
| The cells included in the composite image were found in a peripheral blood smear with a total WBC of 24,500/mm3. The differential count was:
myelocytes 1
metamyelocytes 4
band neutrophils 15
segmented neutrophils 40
monocytes 8
eosinophils 2
basophils 1
lymphocytes 29.
This 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. | View Page |
| Criteria for requesting a hematologist's review of the smear. The following are suggested guidelines directed toward white blood cell data necessitating a hematologist's review:Total white blood cell count <3000/cumm or >12,000/cummNeutrophils >85%Lymphocytes >43% or <10%Monocytes >8%Eosinophils >6%Basophils >4%,.Mixed cells >8% on a 3-part automated differentialA morphology review may also be indicated if the platelet count is <100,000/cumm or >650,000/cumm.Thus, if the granulated cells illustrated in the photograph exceed 6% of the total WBC on a five-part differential or, in combination with monoctytes and basophils, exceed 8% of the total WBC on a three-part differential, a flag would alert the operator that a morphology review or manual differential is needed. | View Page |
| Criteria for evaluation of white blood cells and platelets In most clinical hematology laboratories, an initial blood count is performed by an electronic instrument. Some of these instruments also produce a differential blood count, and a platelet count. Instruments that provide a 3-part differential indicate the percentage of neutrophils, lymphocytes, and a mixed field group that includes monocytes, eosinophils, basophils, immature and atypical cells. Thus, the atypical cells shown in the photograph would be counted as mixed cells and a smear review would be needed to make an identification. Instruments providing a 5-part differential count include monocytes and eosinophils. In cases where the mixed cell count is high, or there are other indications that atypical cells may be present, a hematologist's review of the smear is indicated. | View Page |
| The May -Hegglin anomaly Illustrated in the upper photograph is a poorly defined cytoplasmic inclusion somewhat resembling a Doehle body. Note, however, that this inclusion is well defined and there is no evidence of toxic granulation in the cytoplasm.When Doehle-like bodies are identified, May-Hegglin anomaly should be considered in the differential diagnosis even though this entity is rare.The May-Hegglin anomaly is an inherited dominant condition in which large 2 - 5 um, basophilic and pyronophilic inclusions are present in granulocytes, including neutrophils, eosinophils, basophils, and monocytes.Similar to Doehle bodies, the May-Hegglin inclusions also are composed of RNA, probably derived from the rough endoplasmic reticulum. May-Hegglin anomaly includes giant platelets containing few fine granules (lower photograph).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. | View Page |
| Basophils A basophil and a small lymphocyte are compared in the same field of the upper photograph, A single basophil is shown in the lower photograph.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 photograph). 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 tissue mast cells, 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 neutrophils 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. | View Page |
| A peripheral blood smear is submitted for morphology 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 photograph), monocytes = 2%, and lymphocytes= 2%. These findings support the diagnosis of appendicitis. | View Page |