Differential Information and Courses from MediaLab, Inc.

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.

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.

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.

A screening test for bilirubin in the urine is included in most urine dipsticks and may be present when liver disease or damage is suspected. Bilirubinuria can be detected before other clinical symptoms such as jaundice are present or recognizable. The detection of small quantities is very important in early diagnosis of obstructive and hepatic jaundice. This test is also useful in the differential diagnosis of obstructive jaundice (positive for bilirubinuria) vs. hemolytic jaundice (negative for bilirubinuria).

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).

Examination of Wright-Giemsa stained bone marrow preparation involves examination under low power (10X objective) high power (40-50X objective )and oil immersion (100X objective). Low power examination: Assess quality of smear, assess number of megakaryocytes.Assess myeloid to erythroid ratio.Evaluate morphology and do differential count.

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.

Red cell morphology can be defined as the appearance of the erythrocytes on a Wright's stained smear.Careful examination of the red cells for the purpose of identifying abnormalities is part of the differential procedure. This examination is important because it may provide valuable diagnostic information to the physician, as well as provide a quality control mechanism to verify red cell indices values as determined by automated or manual methods.Evaluating red cell morphology involves differentiating normal morphology from abnormal and artificial morphology. The abnormal morphology covered in this unit may be seen in a variety of disorders.

Red blood cells can vary in size (diameter/volume) from smaller than normal, microcytes, to larger than normal, macrocytes. When red cells of normal size, microcytes and macrocytes are present in the same field, the term anisocytosis is used.Since the purpose of this unit is to acquaint you with the appearance (identification) of abnormal red cell morphology, percentages of abnormalities present will not be considered. It is important to be aware that rating red cell morphology for the purpose of reporting it is a skill which must be learned before you are able to complete this aspect of a differential count.

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.

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.