Quantitative Buffy Coat Analysis (QBC)

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GRANULOCYTOSIS GRANULOCYTOPENIA Granulocytes > 7.2 × 109/L Granulocytes < 1.8 × 109/L Possible causes of granulocytisis: Possible causes of granulocytopenia: 1. Acute bacterial infection, especially coccal. 1. Chemotherapy. Also in certain non-bacterial infections. 2. Certain infections such as typhoid, Quantitative Buffy Coat Analysis 2. Inflammation with tissue damage (myocardial paratyphoid, measles, infectious hepatitis, infarction, gout, burns, postoperative state). malaria and most rickettsial diseases. 3. Toxins, acidosis, uremia. 3. Overwhelming infection. A Pictorial Review and Reference Guide 4. Acute hemorrhage. 4. Adverse drug reactions, such as from 5. Malignancy. (Under some circumstances) aminopyrine, phenothiazines, sulfonamides, 6. Physiologic neutrophilia due to exercise or antithyroids, gold. Maxwell M. Wintrobe, M.D. | Stephen C. Wardlaw, M.D. | Robert A. Levine, M.D. stress. 5. Cachexia or debilitated state. 7. Steroid or epinephrine administration. 6. Anaphylaxis and early stages of reaction to * Wintrobe MM, p1285-1287, Table 54-1. foreign protein. A History of Buffy Coat Analysis.” The means than conventional data * Wintrobe MM, p1287-1291, Table 54-2. QBC™ Centrifugal Hematology presentation and also enhances Buffy Coat Analysis System, manufactured by QBC the user’s awareness of the The buffy coat has long interested Diagnostics, Inc. was developed pathophysiology. physicians, as “phlegm” was based upon their work. one of the four humors that Note: The QBC system is designed to dominated the medical thought The Significance of provide immediate data which can be used as a CBC in many circumstances. The of the ancients. In 1933, in a INCREASE IN LYMPHOCYTES/MONOCYTES DECREASE IN LYMPHOCYTES/MONOCYTES paper entitled “Macroscopic Absolute Counts data is not complete, however, and when clinically indicated or when samples show 9 9 Examination of the Blood”, Absolute leukocyte counts, Lymphocytes/Monocytes > 4.9 × 10 /L Lymphocytes/Monocytes < 1.7 × 10 /L values outside of the approved ranges, the Possible causes of increase: Possible causes of decreased lymphocytes/ Wintrobe1 was perhaps the which are obtained by simple results should be augmented or confirmed 1. Infectious Mononucleosis: Total WBC is 10- monocytes: first to suggest that quantitative multiplication, provide a more by an alternate method, including 20 × 109/L in 60-70%; may exceed 20 × 109/L in 1. Acute infections. examination of the buffy coat in accurate and more readily examination of a stained peripheral blood 15%. Increase is usually due to normal and atypical 2. Certain malignancies. centrifuged blood would provide comprehensible picture than the film. lymphocytes. Peripheral smear usually shows 3. Collagen vascular diseases. See package insert and operator’s atypical lymphocytes; heterophile or spot serology 4. Acquired Immunodeficiency Syndrome (AIDS). useful information. He stated total white blood count with the manual. For technical assistance, call may be positive after the first week of illness. 5. Steroid administration/ that its color and thickness were percentage leukocyte differential 866-265-1486. 2. Chronic Lymphocytic Leukemia: Most patients * Wintrobe MM, p1288,1302,1304. good guides for estimating the count. Although the information is 1. Wintrobe MM, Macroscopic have between 20-100×109/L lymphocytes. platelet and leukocyte counts. similar in both cases, presenting it Examination of the Blood, Am J Med Peripheral smear usually shows small, uniform Sci 185:58-71, 1933. “normal” lymphocytes. Mild anemia or Subsequently Bessis2 described a in the traditional percentage format thrombocytopenia may be present. way of separating the buffy coat may mask abnormalities in the 2. Bessis M. Une Methode Permettant L’isolement des Differents Elements 3. Certain acute infections; infectious hepatitis, into three layers. His work was absolute counts. Figures du Sang, Sang 14: 262-264, pertussis, cytomegalovirus infection. amplified by Davidson3 and by 1940. 4. Certain chronic infections; tuberculosis, Zucker and Cassen4. The importance of the absolute 3. Davidson E. The Distribution of Cells brucellosis, secondary congenital syphilis. 5. Primary hematopoietic disorder and lymphomas. counts is apparent when it is in the Buffy Layer in Chronic Myeloid (In acute leukemia the blasts will enlarge the Wardlaw, Levine and Massey in realized that the various leukocyte Leukemia, Acta Hematologica 23: 22- lymph/mono layer.) 28, 1960. 1977 developed the technique cell populations are separate 6. Certain protozoal infections. 4. Zucker RM, Cassen B, The Separation * Wintrobe MM, p1301-1303 & 1368-1372. Tables 54-5, 54-6. of expanding the buffy coat by entities and under separate control of Normal Human Leucocytes by using a high-precision plastic mechanisms; increases in one line Density and Classification by Size, float moving in a precision-bore may not be paralleled by increases Blood 34: 591-600, 1969. THROMBOCYTOSIS THROMBOCYTOPENIA 5. Wardlaw SC, Levine RA, Quantitative capillary tube. Their work5 in another. Thus, using the absolute 9 9 Buffy Coat Analysis, JAMA 249: 617- Platelets > 400 × 10 /L Platelets < 140 × 10 /L was reported in 1983 in a counts as indicators of a particular 620, 1983 Possible causes of thrombocytosis: (Bleeding is uncommon unless platelet count paper entitled “Quantitative process offers a more sensitive 1. Physiologic: result of stress and epinephrine drops below 50× 109/L.) release. Possible causes of thrombocytopenia: 2. Myeloproliferative syndromes. 1. Chemotherapy. 3. Regeneration after hemorrhage. 2. Drug reactions; diuretics, oral hypoglycemics, 4. “Rebound” thrombocytosis following ethanol, some antibiotics, anti-depressants, EXPECTED NORMAL HEMATOLOGICAL VALUES thrombocytopenia. gold, non-steroidal anti-inflammatory agents. 5. Asplenic state. 3. Idiopathic Thrombocytopenia (ITP). 6. Many acute and chronic infections. 4. Portal hypertension. 7. Many neoplasms. 5. Acute viral infections. 8. Trauma and surgery. 6. Bacterial septicemia. Reference ranges are a function of * Wintrobe MM, p1128-1134, Table 48-1 7. Disseminated Intravascular Coagulation (DIC). 8. Massive blood transfusion. age, sex, geographic location and * Wintrobe MM, p1090-1127. Tables 47-2, 47-3.

Plasma smoking history.

Fill Lines MYELOPROLIFERATIVE SYNDROME PANCYTOPENIA Decrease of all elements below reference Platelets Uncontrolled or disturbed release of cells from 140-400 the bone marrow. limits. This is not a disease entity but a triad 1. Polycythemia Vera: Increase hematocrit of findings that may be due to a number of not in response to stimuli such as hypoxia, causes. Lymph/Mono 1. Disorders associated with bone marrow 1.7-4.9 (22-55%) etc. Frequently associated with elevations in granulocyte and platelet counts. infiltration; neoplasm, myelofibrosis. † Total WBC 2. Chemotherapy. 4.3-10.0 2. Idiopathic Myelofibrosis: Increased platelets Granulocytes early in disease. Anemia with “tear drop” 3. Toxic exposure; benzene and derivatives, 1.8-7.2 (45-80%) RBCs and nucleated RBCs seen on peripheral ionizing radiation. smear Granulocytes may or may not be 4. Drug reactions; chloramphenicol, quinacrine, increased/ antithyroids, phenylbutazone, gold * Wintrobe MM, p1596-1630 compounds. (Counts × 10 /L) 5. Idiopathic aplastic anemia. 6. B12 or folate deficiency. 7. Overwhelming infection. † Red Blood Cell * Wintrobe MM, p698-700 & 1304-1305. Tables 28-1, 54-7 Fragment Line (May Hematocrit not be present in all samples) M 40-54% * Wintrobe MM, F 37-47% p50-52, 208, 356, Stopper 372, 1885.

NON-SEPARATION OF RBCs/GRANS RED BLOOD CELLS FRAGMENT LINE OTHER SAMPLE ABNORMALITIES

Blurred or absent red cell/granulocyte interface may Red blood cells that have been damaged tend to be Hyperbilirubinemia. Important occur when: lighter than the granulocytes and may form a small Inspection of the plasma layer may show increased observations A. Granulocyte adhesiveness increases; may occur with dark band between the granulocyte and lymph/mono plasma bilirubin. Generally, bilirubin >3 mg/dl can severe infection or inflammatory disorders.‡ Sample layers. be observed as a brownish coloration of the plasma; Inspection of QBC tube may incubation at 43˚C/ 5 min. destroys adhesiveness, may but, the yellow fluorescent dye in the tube may mask yield other valuable clinical improve separation. This band may be particularly large (up to the thickness it for some observers. Increased bilirubin causes the B. Densities of red cells and granulocytes overlap. Occurs of a normal lymph/mono layer) in asplenic patients and fluorescence of the granulocyte and platelet bands to information. in ~3% of ambulatory, up to 30% of hospitalized those who have recently received I.V. drugs that can appear green tinged. This does not affect the accuracy patients. Some causes are: damage red blood cells. of the measurement. 1. Hemoglobinopathies or diseases associated Static with microcytosis; e.g., iron deficiency anemia, Hyperlipidemia. thalassemia trait. Red cell fragmentation Increased lipids may show as an opalescence of the Syndromes, including microangiopathic anemia. plasma or as a small pellicle at the top of the plasma. In many of these conditions float sinks deeper into This also has no effect on the measurement. RBC layer. Cryoglobulinemia. 2. I.V. fluid or drug administration. Cryoglobulins may precipitate or form a gel at 0-15°C 3. Immediate postpartum state. and may show as opalescence of the plasma. A distinct layer of precipitated cryoglobulins may appear in the Note: Rarely, density overlap may be so great as to suggest plasma layer. the absence of granulocytes. ‡ Garvin JE: Factors influencing the adhesiveness of Spinning human leukocytes and platelets in vitro. J.Exp Med 114 Hyperbilirubinemia 51-52, 1961 Sample

INCORRECT FILLING/LEAKAGE OMISSION OF THE FLOAT WAVY BUFFY LAYERS

Technique error: Incomplete filling of the tube or Technique error: Omission of the float causes a lack Technique error: Wavy interfaces between the Proper technique specimen leakage during centrifugation will adversely of expansion and a marked apparent reduction of the buffy coat bands occur when they are disturbed effect the results. plasma level. The tiny buffy coat layers should not be following separation. The most common cause is ensures accuracy mistaken for thrombocytopenia and leukopenia. slippage of the closure, which can be prevented by Inspection of QBC tube prior to After centrifugation, ALWAYS check the plasma level to carefully drying the end of the tube prior to attaching ensure that it is within the fill lines. the closure. Overheating the centrifuge and operation reading is an important step in in ambient temperatures over 32°C (90°F) will also quality assurance. cause waviness.

NOTE: Because the tube is rotated, the waviness will Static be seen as an enlarged, blurred lymph/mono band. The waviness can be seen if the rotation is momentarily stopped.

Spinning

* Wintrobe MM, et al: Clinical Hematology, ed. 8, Philadelphia, Lea & Febiger, 1981. ©1984, Wintrobe MM, Wardlaw SC, Levine RA Drucker, Drucker Logo and Drucker Diagnostics are trademarks of Drucker Diagnostics, Inc. © 2005 Drucker Diagnostics, Inc. QBC system is covered by multiple U.S. and International Patents 4270-000-006 (Rev C) 11/2017 Printed in U.S.A.