Lec 3 Hematologic Exams PDF

PLASMA OUTLINE # Liquid part of the blood ○ Bulk of the blood HEMATOLOGIC EXAMS ○ Comprises 55% of the total whole blood vol...

PLASMA OUTLINE # Liquid part of the blood ○ Bulk of the blood HEMATOLOGIC EXAMS ○ Comprises 55% of the total whole blood volume I. HEMATOLOGY 1 Composed mainly of water (91%) A.Blood Components Dilute solution that contains components like dissolved 1. Plasma gasses and solids: 2. Red Blood Cells ○ Salts 3. White Blood Cells ○ glucose/carbohydrates 4. Platelets ○ Vitamins II. HEMATOLOGIC TESTS 2 ○ Urea A. Hemoglobin Tests ○ Protein 1. Methods Albumin 2. Interpretation of Results Antibodies 3. Physiologic factors affecting Hemoglobin Amino acids Levels ○ fats/lipids B. Hematocrit Determination ○ Electrolytes 1. Methods ○ Hormones 2. Results and Interpretation Has blood coagulation proteins essential for forming and 3. Hematocrit and Patient’s Health dissolving blood clots C. Complete Blood Count 1. Hemocytometer RED BLOOD CELLS 2. RBC Pipette and WBC Pipette 3. Dilutions and Cell Diluting Fluids Most numerous among the formed elements 4. RBC Count ○ Comprises 45% of the blood 5. Ways to Report RBC Count ○ 1 drop of the blood = 2.5 x 108 RBCs 6. Conditions Affecting RBC Count ○ 1 μL of blood = 5 x 106 RBCs 7. WBC Count Lifespan: around 120 days in circulation 8. Conditions Affecting WBC Count ○ Remain in the circulatory system vessels for its entire D. Red Cell Indices lifespan 1. Mean Corpuscular Volume (MCV) Old RBCs are removed by phagocytes in the spleen 2. Mean Corpuscular Hemoglobin (MC) ○ Components such as iron are recycled to produce 3. Mean Corpuscular Hemoglobin hemoglobin for new RBCs Concentration (MCHC) III. REVIEW QUESTIONS 7 OTHER IMPORTANT INFOS IV. APPENDICES 8 Donating blood removes RBC wastes. Consequently, the body replenishes by producing new RBCs. SOURCES Primary functions: Villanueva, S.Y.A.M. (2024). Hematologic examinations. ○ Transport oxygen to tissues Retrieved from ○ Transport carbon dioxide to the lungs https://drive.google.com/file/d/1U-5uV8U-D4-iEMB8Nlc96 Hemoglobin aKA61ShB_GH/ ○ Gives red color to the blood ○ Types: HEMATOLOGY Oxyhemoglobin Study of the cellular/formed elements of the blood and the Hemoglobin with bound oxygen blood-forming tissues Found in bright red arterial blood Study of blood and blood disorders Deoxyhemoglobin Branch of medicine concerned with the study of the cause, Hemoglobin with released oxygen prognosis, treatment, and prevention of diseases related to Found in dark red venous blood blood WHITE BLOOD CELLS BLOOD COMPONENTS Least numerous of the cellular components (100,000/mm3 ○ Thin air/reduced oxygen pressure stimulates increased High risk of committing production of RBCs leading to higher hemoglobin values visual errors (and RBC counts) of residents Color of glass standard may fade over time HEMATOCRIT DETERMINATION Measures the amount of space (volume) that RBCs take up in the blood Useful in the diagnosis of anemia and polycythemia vera (high RBC count) ○ May be a primary disease of known cause or a condition due to respiratory/circulatory disorder or cancer ○ Polycythemia vera Hematocrit is abnormally high and volume of Figure 3. Hemoglobinometer (left) and Sahli’s plasma is small Hemoglobinometer (right). Useful in screening potential blood donors, evaluation of anemia therapies and in estimating blood loss after hemorrhage or trauma Important in calculating blood indices The amount of hematocrit is three times the amount of hemoglobin OTHER IMPORTANT INFOS Figure 4. Sahli Method (left) and Cyanmethemoglobin Method Hematocrit and hemoglobin concentration do not always (right) follow the rule of 3. There are instances wherein one is high while the other is low. For example, pregnant women have low INTERPRETATION OF RESULTS hemoglobin but high hematocrit levels due to physiologic changes (Villanueva, 2023). Table 2. Hemoglobinometer reference ranges. Age/Gender g/dL Conversion g/l (SI Units) METHODS factor Automated Method Newborn 16-23 x10 160-230 Manual Wintrobe macromethod Children 10-14 x10 100-140 ○ Macromethod of determining hematocrit ○ Volume of packed red cells is read directly from the tube Adult males 13-17 x10 130-170 with your naked eye Adult females 12-16 x10 120-160 PH156:CLINICAL MICROSCOPY | GROUP A: MILLAN, REGUINDIN, RICAFORT, SIAT 3  HEMATOCRIT AND PATIENT’S HEALTH Check the appearance of normal blood samples, especially when they are centrifuged in a microhematocrit tube ○ Aside from looking at packed RBC for hematocrit determination Record color of plasma and appearance of buffy coat ○ Technician should be alert and report any abnormal findings Table 4. Condition and Appearance of Centrifuged Blood Sample Figure 5. Manual Wintrobe Macromethod. in a Hematocrit Tube Manual micromethod (microhematocrit method) Condition Appearance ○ Measures packed RBCs Normal Blood Sample Transparent, pale yellow blood ○ Speedy, accurate, and reproducible plasma ○ Preferred method ○ Buffy coat red cell interface is not always clear cut thus Narrow whitish-tan layer of buffy coat above red layer reading errors (minor) may occur rendering this method less reliable and less accurate for determining blood Elevated Bilirubin Levels Very yellow plasma indices Opaque plasma High fat (triglyceride) levels Prominent buffy coat Elevated WBC COMPLETE BLOOD COUNT (CBC) Also known as hematology profile blood test or full blood count (FBC) Examines blood components Provides vital information on the type, number, and appearance of cells in the blood ○ Especially RBCs, WBCs, and platelets Includes the following tests: ○ RBC Count ○ WBC Count ○ Hemoglobin ○ Hematocrit ○ RBC Indices ○ WBC Differential Count Figure 6. Microhematocrit Method. ○ Platelet Count or Platelet Estimate ○ Evaluation of blood cell morphology RESULTS AND INTERPRETATION Can be done using a machine or manually HEMOCYTOMETER Table 3. Hematocrit reference ranges. Blood cell counting chamber Average Range Tool used in conducting CBC Also used for sperm and bacterial count, as well as for the % L/L % L/L examination of cerebrospinal fluid (CSF) Adults ○ However, it is not called a hemocytometer for the above 47 0.47 42-52 0.42-0.52 purposes Males Females 42 0.42 36-48 0.36--0.48 ○ Only has a similar appearance to hemocytometer Children Newborn 56 0.56 51-56 0.51-0.56 1 year 35 0.35 32-38 0.32-0.38 6 year 38 0.38 34-42 0.34-0.42 Increased levels Figure 7. Hemocytometer (Improved Neubauer Chamber). ○ Pregnancy ○ Living at high altitudes Thick, rectangular glass slide ○ Polycythemia With two chambers separated by lateral ridges ○ Dehydration ○ Upper chamber and lower chamber with a ruled central ○ Low availability of oxygen part (see Figure 6) Low levels 9 equally-sized large squares ○ Anemia Each square = 1 mm wide x 1 mm long ○ Bleeding Entire ruled area is 9 mm2 4 large corner squares = 16 smaller squares (orange) OTHER IMPORTANT INFOS Large center square = 25 smaller squares (yellow) Newborns exhibit higher hemoglobin and hematocrit levels due to their higher affinity to oxygen for growth and development. PH156:CLINICAL MICROSCOPY | GROUP A: MILLAN, REGUINDIN, RICAFORT, SIAT 4  Table 5. Comparison of RBC pipette and WBC pipette RBC Pipette WBC Pipette Red bead White bead Graduation marks up to 101 Graduation marks up to 11 Larger bulb Smaller bulb Smaller lumen Larger lumen DILUTIONS AND CELL DILUTING FLUIDS Do not directly do the blood count on freshly extracted blood samples Blood samples must be diluted before counting ○ Degree of dilution and type of diluting fluid depend on the type of cell to be counted Table 6. Comparison of degree of dilution and diluting fluids used per type of cell Type of Cell Degree of dilution Diluting fluid RBCs 1:200 Isotonic Protects delicate RBCS from hemolysis Figure 8. Ruled center (hemocytometer). WBCs 1:100 using Ammonium oxalate LeukoChek system Lyses RBCs, leaving Counting chambers 1:20 using only WBC intact Leuko-TIC system Contains dye to make nuclei prominent for ease in counting Platelets 1:100 Diluted in fluids that lyse RBCs (e.g. ammonium oxalate which is commonly used) RBC COUNT Commonly used method or assay Usually a component of CBC Helps physician diagnose and treat many diseases ○ Significant decrease in RBCs can cause fatigue, weakness, headache, pallor, and increased heart rate Counted in the large center counting chamber using a Figure 9. Counting chambers of hemocytometer. high-power objective ○ Always look at the stage when adjusting the power ○ WBCs are counted in the 4 large corner squares objective (marked ‘W’ [left], encircled in blue [right]) ○ 5 squares in the large center counting chamber In counting, follow the direction noted in the upper 4 corners and center right corner square to avoid repeatedly counting the Count from left to right same cell ○ Cells in boundary lines should not be counted Snake-like direction from left to right, down to the Only count cells touching upper- and left-hand next row, then moving from right to left and so on lines to avoid double-counting ○ RBCs are counted in the large center square, in its 4 ○ corners and center (encircled in orange [right photo]) Follow the same direction in counting as stated for counting WBCs RBC PIPETTE AND WBC PIPETTE Used to suck blood and mix blood with diluent when conducting a CBC ○ Remember to exercise precaution when using the pipettes as you may swallow the blood ○ No mouth pipetting ○ Figure 11. Diagram of counting for RBC (zoomed in). WAYS TO REPORT RBC COUNT RBC/mm3 ○ Average of the total number of RBCs counted in upper and lower chamber x dilution factor x depth factor x area Figure 10. RBC pipette and WBC pipette. factor Ave. # of cells counted x 200 x 10 x 5 Ave. # of cells counted x 10,000 PH156:CLINICAL MICROSCOPY | GROUP A: MILLAN, REGUINDIN, RICAFORT, SIAT 5  RBC/L CONDITIONS AFFECTING WBC COUNT ○ N x D/A X 10X106 Leukocytosis - increase in WBC count N = total number of RBCs counted ○ Pathologic: infection or leukemia or polycythemia D = dilution of blood ○ Physiological: exercises, exposure to sunlight, obstetric A = total area counted (in mm2; 0.2 per square of 25 labor stress, anesthesia squares in large central counting chamber) Immune system is stimulated to produce WBCs 10 = factor to convert volume (in μL) Explains why those with active lifestyle have higher 105 = factor to convert count per μL to count per WBCs liter Which means they are more protected against pathogens Leukopenia - decrease in total number of WBCs Table 7. Normal values for RBC counts ○ Other term is leukocytopenia Sex Normal RBC Count ○ Viral infections (e.g. HIV) ○ Ionizing radiation Male 4.5-6.0 x 10612RBC/mm3 ○ Certain chemicals 4.5-6.0 x 10 RBCs/L ○ Chemotherapy drugs Female 4.0-5.5 x 10612RBC/mm3 4.0-5.5 x 10 RBCs/L Table 8. Reference ranges for WBC count Average Range CONDITIONS AFFECTING RBC COUNT Age Conventional SI units Conventional SI units After conducting the count, the clinician is also expected to units (cell/L) units (cell/L) think of possible reasons why a patient’s RBC count does (cells/μL) (cells/μL) not fall in the normal range Newborn 18,000 1.8 x 1010 9,000 - 30,000 9.0 - 3.0 x 109 ○ Decreased RBC count may be due to: Iron-deficiency anemia One year 11,000 1.1 x 1010 6,000 - 14,000 6.0 - 14.0 x 109 Sickle cell anemia Six years 8,000 8.0 x 1010 4,500 - 12,000 4.5 - 12.0 x 109 Vitamin B12 anemia Adult 7,400 7.4x 1010 4,500 - 11,000 4.5 - 11.0 x 109 Folic acid deficiency Acute or chronic blood loss ○ Increased RBC count may be due to: RED CELL INDICES Polycythemia vera Characteristics of individual red cells Living in high altitude ○ Mean corpuscular volume (MCV) ○ Mean corpuscular hemoglobin (MCH) WBC COUNT ○ Mean corpuscular hemoglobin concentration (MCHC) WBCs are round, slightly iridescent structures with a definite Aids in the diagnosis of anemia outline and well-define structures ○ Determining type of anemia in therapy depending on Count all leukocytes (WBCs) lying within the 4 large corner calculated values squares Normal values for MCV, MCH, and MCHC do not differ ○ Using boundary rule among ages or sexes Count in both the upper and lower chambers of the Neubauer Table 9. Normal values of the characteristics of individuals RBCs Dilution factor = 20 Characteristic Normal values Area counted = 4 mm2 MCV 80 - 100 fl or μm3 MCH 27 - 32 pg or μg MCHC 33.0 - 38.0% or g/dL MEAN CORPUSCULAR VOLUME (MCV) Volume of the average red cell in a given sample of blood Expressed in femtoliters (fl) Average size of the RBC Determines if the RBC is: ○ Microcytic ○ Normocytic ○ Macrocytic Hematocrit is three times the amount of hemoglobin FORMULA Figure 12. Neubauer counting chamber for WBCs 𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 (%) 𝑋 10 WBC/mm3 = average of the total number of cells counted in 𝑀𝐶𝑉 = 12 3 2 chambers x dilution factor x depth factor in 4 squares 𝑅𝐵𝐶 (10 /𝐿) 𝑜𝑟 (𝑚𝑚 ) 𝑁 𝑋 20 𝑋 10 ○ 4 ○ 𝑁 𝑋 50 6 MEAN CORPUSCULAR HEMOGLOBIN (MCH) WBC/L = 𝑁 𝑋 𝐷/𝐴 𝑋 10 𝑋 10 ○ N = total number of WBCs counted Amount of hemoglobin by weight in average cell of a ○ D = dilution of blood blood sample ○ A = total area counted (in mm2) Expressed in pictograms (pg) or micrograms (ug) ○ 10 = factor to convert to volume (in μL) MCH is three times less than the MCV ○ 106 = factor to convert count per μL to count per liter ○ MCH is 1⁄3 of MCV When counting, make sure it is done left to right, right to left PH156:CLINICAL MICROSCOPY | GROUP A: MILLAN, REGUINDIN, RICAFORT, SIAT 6  b. Only the second statement is true FORMULA c. Both statements are true d. None of the statements are true 𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 (𝑔/𝑑𝐿) 𝑋 10 𝑀𝐶𝐻 = 8. How are RBCs counted using a hemocytometer? 12 3 𝑅𝐵𝐶 (10 /𝐿) 𝑜𝑟 (𝑚𝑚 ) a. In the 4 large corner squares b. In the 4 corners of the large center square c. In the 4 corners and center of the large center square MEAN CORPUSCULAR HEMOGLOBIN d. In the 4 corners and center of the upper-left CONCENTRATION (MCHC) corner square Concentration of hemoglobin in the RBCs relative to their 9. Which of these cells should not be included when counting size the number of RBCs? May Indicate whether the cells are: a. Cells touching upper-hand lines ○ Normochromic b. Cells in boundary lines ○ Hypochromic c. Cells touching left-hand lines Expressed in g/L or %g/dL d. All of the above 10. Mean Corpuscular Hemoglobin (MCH) is the amount of FORMULA hemoglobin by volume in an average cell of a blood sample. MCH is expressed in femtoliters (fl) 𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 (𝑔/𝑑𝐿) 𝑋 100 a. Only the first statement is true 𝑀𝐶𝐻𝐶 = 𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 (%) b. Only the second statement is true c. Both statements are true d. None of the statements are true REVIEW QUESTIONS ANSWER KEY 1. Hematology is the study of cellular/formed elements of the blood. Plasma is the liquid part of the blood and is composed 1) A Correct: Plasma is 6) D. Can be done either as mainly of gasses and solids. composed mainly of water individual test or as part of CBC a. Only the first statement is correct. 2) C 7) A Diagnosis of anemia and 3) A polycythemia vera, not detection b. Only the second statement is correct. 4) D of blood loss and polycythemia c. Both statements are correct. 5) A. Venous blood is typically vera. d. Both statements are incorrect. collected 8) C 2. The life span of RBC; WBC; and platelets are: 9) B a. RBC: 10 days; WBC: few days to several years; 10) D platelets: ~120 days Amount of hemoglobin by b. RBC: ~120 days; WBC: 10 days; platelets: few days to weight, not volume. several years Pictograms (pg) or micrograms (ug), not c. RBC: ~120 days; WBC: few days to several years; femtoliters (fl) platelets: 10 days d. RBC: few days to several years; WBC: 10 days; platelets: 120 days 3. This type of white blood cell exhibits a very potent phagocytic activity a. Neutrophils b. Eosinophils c. Basophils d. Monocytes 4. Which method is commonly used for performing hemoglobin tests in office practice and laboratory classes? a. Specific gravity method b. Cyanmethemoglobin c. Azide methemoglobin d. Acid hematin method 5. Platelets are fragments of cytoplasm released into the circulating blood. Capillary blood is typically collected in tubes with anticoagulants for hematology tests. a. Only the first statement is true b. Only the second statement is true c. Both statements are true d. None of the statements are true 6. All of the statements about the hemoglobin test are true EXCEPT: a. It does not directly evaluate the oxygen-carrying capacity of the blood. b. It can be done through capillary blood c. It can be performed using a hemoglobin instrument. d. It cannot be done as part of CBC. 7. Normal blood sample appears transparent, pale yellow blood plasma with a narrow whitish-tan layer of buffy coat above a red layer. Hematocrit determination is useful in the detection of blood loss and polycythemia vera. a. Only the first statement is true PH156:CLINICAL MICROSCOPY | GROUP A: MILLAN, REGUINDIN, RICAFORT, SIAT 7 APPENDICES Table 10. Summary table of all hematologic tests Hematologic Test Assays Interpretation Other Remarks Hemoglobin Test Specific Gravity High hemoglobin Indirect evaluation of ○ Increased Chemical methods RBC the oxygen-carrying Cyanmethemoglobin production capacity of the blood Azide methemoglobin ○ Lung disease Hemoglobin is ⅓ the Acid hematin method ○ People living Sahli in high value of hematocrit hemoglobinometer altitude Detects blood loss and ○ Dehydration, anemia smoking, bone marrow Monitors treatment of disorders certain RBC disorders Low hemoglobin ○ Anemia due to pregnancy ○ Blood loss ○ Liver damage ○ Iron deficiency Hematocrit Determination Manual Wintrobe Increased levels Measures amount of space (macromethod) ○ Pregnancy (volume) taken up by RBCs ○ Living at high altitudes in the blood Microhematocrit ○ Polycythemia Useful in diagnosis of ○ Dehydration anemia, screening donors, ○ Low availability of evaluating therapies for oxygen anemia Normal values depend on Low levels age and sex ○ Anemia Rule of thumb: 3x the value ○ Bleeding of hemoglobin Complete Blood Count (CBC) RBC Count Decreased RBC count Provides information to ○ Iron deficiency anemia physicians that are useful Provides vital information ○ Sickle cell anemia for management and on the type, number, and ○ Vitamin B12 deficiency treatment of patients appearance of cells in the ○ Folic acid deficiency Normal values depend on blood, especially RBCs, Acute or chronic blood the sex of patient WBCs, and platelets loss Increased RBC count ○ Polycythemia vera ○ Living in high altitude WBC Count Leukocytosis Provides information on the ○ Pathologic: infection or immune status of an leukemias or individual polycythemia Normal values depend on ○ Physiological: age exercises, exposure to sunlight, obstetric labor stress, anesthesia Leukocytopenia ○ Viral infections (e.g., HIV) ○ Ionizing radiation ○ Certain chemicals ○ Chemotherapy drugs Red Cell Indices Mean Corpuscular Volume Formula: (MCV) Characteristics of 𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 (%) 𝑋 10 individual cells 𝑀𝐶𝑉 = 12 3 𝑅𝐵𝐶 (10 /𝐿) 𝑜𝑟 (𝑚𝑚 ) Useful in diagnosing anemia Mean Corpuscular Formula: Hemoglobin (MCH) 𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 (𝑔/𝑑𝐿) 𝑋 10 𝑀𝐶𝐻 = 12 3 𝑅𝐵𝐶 (10 /𝐿) 𝑜𝑟 (𝑚𝑚 ) Mean Corpuscular Formula: Hemoglobin 𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 (𝑔/𝑑𝐿) 𝑋 1 Concentration (MCHC) 𝑀𝐶𝐻𝐶 = 𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 (%) PH156:CLINICAL MICROSCOPY | GROUP A: MILLAN, REGUINDIN, RICAFORT, SIAT 8  Figure 1. Composition of Blood Figure 2. Hematocrit Tube PH156:CLINICAL MICROSCOPY | GROUP A: MILLAN, REGUINDIN, RICAFORT, SIAT 9