Universal Precaution: "All blood and body fluids are treated as if they're infectious" Always Remember!!! 1.

Correct patient identification 2. Correct specimen identification 3. Gloves must be worn all the times 4. Clean the puncture site thoroughly 5. Dispose all sharp objects in puncture-resistant containers Factors to be considered before blood collection: 1. Type of test 2. Age of patient 3. Condition of patient 4. Skill of phlebotomist General Methods for Obtaining Blood 1. Skin puncture/ Capillary puncture 2. Venipuncture 3. Arterial puncture Skin Puncture Mixture of arterial and venous blood Increased pressure in arterioles yield a specimen rich in arterial blood Also contains interstitial and intracellular fluids Application of Microsample Collection Infants less than 6 months In young children In adult with poor veins o Extreme obesity o Severe burns o Geriatric patients o Patients with thrombotic tendencies Sites of Puncture Palmar surface of the 3rd or 4th finger Margins of the earlobe Plantar surface of the heel (fleshy part) or big toe Advantages of skin puncture using the finger Easily accessible to the medtech Less intimidation Easy to manipulate Ideal for peripheral blood smears Advantages of skin puncture using the earlobe Less painful due to lesser nerve endings Lesser tissue juice contamination More free flow of blood due to thinner skin

Ideal when searching for abnormal cells (ex. Histiocytes in bacterial endocarditis)

Disadvantages of skin puncture Less amount of blood can be obtained Blood obtained by skin puncture hemolyses easily Additional and repeated test cannot be done Sites to Avoid in Skin Puncture Cold and cyanotic areas (less blood flow) Inflamed and pallor areas (increased WBC due to chemotaxis) Congested and edematous area (increased tissue fluids) Scarred and calloused areas (cannot reach the capillary bed) Things to remember in doing the skin puncture Recommended depth of puncture o for children and adults : 2.4-3.0 mm o for infants: 1.6 mm Avoid excessive massaging Thumb, earlobe, and big toe should not be used as sites of puncture in adults Fingers must be dry before the test is performed Collect platelet count and blood smear first Venipuncture Three Factors Involve in a Good Venipuncture The phlebotomist The patient and his veins The equipment needed Methods of Collection Syringe Method Vacutainer/ Evacuated Tubes Butterfly infusion set Advantages of Vacutainer Method over syringe method: Safer method of blood collection as samples are taken directly into labeled tubes Offers a wide range of tube size and contains anticoagulant Avoidance of glass syringe breakage Commonly Used Anticoagulants EDTA o generally available as disodium, dipotassium or tripotassium salt of ethylenediaminetetraacetic acid o prevents coagulation by binding to calcium

prevents formation of artifacts and can be used in preparing blood smears 2-3 hours after collection o excess EDTA results to: shrinkage of RBCs = decrease spun Hct, increased MCHC, falsely low ESR (no effect on Hgb) degenerative changes in WBCs :; breaking up of platelets (falsely high platelet count) o After 3 hours of EDTA at room temp WBC vacuolation of cytoplasm, more homogenous nuclei, irregularly or poorly defined cytoplasmic borders, devt of irregularly shaped nuclei Platelets slowly swell and disintegrate o After 6 hours of EDTA at room temp RBC swell= increase MCV, increase OFT, decrease ESR Sodium Citrate o Used for coagulation studies in concentration of 1 part 0.109 M (3.2%) sodium citrate to 9 parts of whole blood o Binds calcium in blood, helps platelets retain their functional capabilities o Buffered sodium citrate (0.109 M + citric acid) may increase the stability of factors V and VIII Heparin o May be used in concentrations of 15-30 units/ml of whole blood o Interacts with antithrombin III and subsequently inhibits thrombin o Causes clumping of WBCs and platelets o Causes blue coloration in a blood smear after staining with Wrights Stain

Advantages: stable, prominent, less painful, not movable, bigger lumen, palpable

Advantages of Venipuncture 1. Large amounts of blood can be obtained for a variety of tests. 2. Blood can be transported and stored for future use. 3. Additional and repeated test can be done. 4. Fastest method of collecting blood samples from a large number of patient. 5. Blood collected is ideal for blood chemistry and other serological test. Disadvantages of Venipuncture 1. Requires more time and skill on the part of the phlebotomist. 2. Requires more equipment. 3. More complications may rise 4. Difficult to do on infants, children and obese individuals. Areas to be Avoided in Venipuncture 1. Hematomas 2. Burns 3. Scars 4. Edema 5. Side which mastectomy was performed 6. In patients arm receiving an intravenous infusion Complications in Venipuncture Local Immediate Complication 1. Hemoconcentration 2. Failure of blood to enter the syringe due to collapsed vein, which may be due to: Nervousness excessive pull of the plunger hitting past the wall of the vein in case of sclerotic and movable vein or hitting the vein through and through 3. Circulatory 4. Fainting Local Delayed Complication 1. Hematomoa- discoloration and inflammation of surrounding tissues due to extravasation of blood 2. Thrombosis of the vein- formation of small blood clots at the site of puncture 3. Thrombophlebitis- inflammation of vein due to trauma wherein thrombus is present General Delayed Complication 1. Serum hepatitis

Sites of Venipuncture: In newborn infants up to 18 months old o External jugular vein o Temporal vein (scalp vein) o Superior longitudinal sinus In older children, 18 months up to 3 years old o Femoral vein o Long saphenous vein o Popliteal vein o Ankle vein In 3 years old to adult life o Wrist vein o Dorsal veins of the hand o Veins on the antecubital fosa Median basilica vein Median cephalic vein

2. AIDS Storage of Specimens 1. Specimens that cannot be examined immediately should be stopped and placed in the refrigerator (prevent decreased in pH) Ex. For plasma Hemoglobin- plasma should be separated from other blood components before it is refrigerated 2. Blood specimen for platelet count, ESR, coagulation tests (PT, aPTT, etc.) should not be stored longer than 2 hours. If possible, it should be done immediately. 3. Stored anticoagulated blood must be thoroughly mixed on blood rotators at least 2 minutes (or 60 inversions) after it has reached the room temperature. Do not shake 4. Serum to be tested for agglutinins should be separated as soon as possible and should not be stored in the refrigerator in contact with RBC. EXERCISE 3-6: Blood Diluting Fluids, Hemocytometry, RBC and WBC Count Complete Blood Count 1. RBC Count 2. WBC Count 3. Hemoglobin 4. Hematocrit 5. WBC differential Count 6. Qualitative Platelet Count Importance of CBC 1. Screening Procedure 2. Drug and Radiation Therapy monitoring 3. Indicator of patients progress in certain disease states Hemocytometry - the estimation of the number of blood cells in a known volume of blood Methods: 1. Turbidimetric Method o an unknown sample is compared to a set up of different solutions of turbidity; each having a specified number of blood cells 2. Manual/Microscopic o for cell counts of body fluids, RBC, WBC, and platelets o counts expressed on cubic millimeter due to the linear dimensions of the counting chamber

Pipet 1. Unopette- disposable diluting pipette which consist of a uniform bore glass capillary pipette capable of measuring accurately and precisely various volumes of blood 2. manual pipette (RBC and WBC pipette) Comparison of RBC ad WBC Pipette RBC Size of bulb Color of bead Volume in bulb Size of bore Calibration Principal use Larger Red 100 Smaller 0.5, 1.0, 101 RBC count, direct platelet count, WBC count (leukemia) WBC Smaller White 10 Larger 0.5, 1.0, 11 WBC count, eosinophil count, CSF and other body fluids

Diluting Fluids - used to dispersed blood cells to facilitate counting Properties of an Ideal Diluting Fluid 1. It should by isotonic for RBC and hypotonic for WBC 2. Easy to secure and prepare 3. Cheap and economical 4. Stable 5. With preservative action 6. With buffer action 7. with high specific gravity 8. non-allergenic and non corrosive RBC Diluting Fluids 1. Hayems fluid 2. Gowers fluid 3. Toissons 4. Bethels 5. Formol Citrate (Dacies Fluid) 6. Normal Saline Solution 7. 3.8% Sodium Citrate WBC Diluting Fluids 1. 2-3% Glacial Acetic Acid 2. 1% HCl 3. Turks Solution

Computation of Cell Counts

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Where: Avg = average number of cells/mm3 D (mm) = depth factor DF = dilution factor A (mm3) = area counted

Advantages of Manual Counting Do not require expensive reagents and equipments No constant calibration and quality control Less expensive No special laboratory setting needed Automated Hemocytometry 1. Optical- Blood cells are counted as deflection of light beams as they pass through darkfield area of the machine which are converted to electrical impulses by a photomultiplier tube ex. Fisher autocytometer, Frommer-Sanborn Autocytometer, Technicon Autoanalyzer 2. Electrical- cell passing through an aperture causes changes in electrical resistance which are counted as voltage pulses and will be recorded as voltage impulses in a photomultiplier tube and appears on . Results are interpreted in a digital print-out at 25 microsecond ex. Coulter counter, Celloscope, Sysnex o Electronic Particle Counter determines directly the concentration of: WBC, RBC, Hgb, MCV calculates PCV or Hct, MCHC, and MCH Errors in Automated Counting Low RBC and WBC count- repeat counts and use of the second value Increase WBC count- due to turbidity of the solution Increased Hgb, MCV, and Hct- white cells are counted and sized as RBC Cold agglutinins in high titer tend to give spurious macrocytosis and low RBC count with high MCV and MCHC Low WBC count in leukemic patients- WBC appears to be fragile and escape being counted Anemic and patients receiving immunosuppressive drugs will give a low WBC count Erroneous PCV occurs if there are sever electrolyte and serum protein abnormalities Advantages of Electronic Cell Counted Over Hemocytometer Generally faster and more prcised Human error avoided- provided machine is properly calibrated and subjected to constabt quality control Elimination of visual fatigue of the medtech EXERCISE 7: Hematocrit Determination Hematocrit PCV, packed cell volume; ratio of RBC volume to that of the whole blood

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Multiply by 106 if reported in SI units Allowable difference in cell counts per chamber is 15-16 cells

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Multiply by 106 if reported in SI units Even distribution of cells in all four large squared (not more than 10-12 variation between squares) 10% agreement in repeat counts

Sources of Errors in Blood Counting 1. Collection-bad sampling of blood to an adequate flow from skin ouncture o prolonged used of tourniquet o insufficient mixing of venous blood which has settled after collection 2. Pipetting- inaccurate pipetting of blood and diluting fluid o presence of bubbles 3. Inadequate mixing of blood cell suspension 4. Faulty filling of counting chamber o overfilling or underfilling of counting chambers o pressure of trapped bubble 5. Presence of clots in the dilutions 6. Failure to discard the first few drops before filling the chamber 7. Presence of yeast cells in the diluting fluids 8. Uneven distribution of cells 9. Careless counting of cells within the chambers 10. Incorrect computation and reporting of final counts 11. Faulty calibrated pipette 12. Uneven surface of coverslip and counting chamber 13. Chipped or broken pipette 14. Defective microscope

Reflects the concentration of the red blood cells, not the red cell mass Types of Hematocrit 1. Body Hematocrit - 9% higher than venous blood 2. Venous Hematocrit ratio of red cells to plasma in venous blood measures the proportion of RBC to plasma in the peripheral blood

if anticoagulated blood is used, several layer are formed: a. red cell layer- bottom layer; reticulocytes can be found on the topmost layer b. Buffy Coat Layer- thin, yellowish white layer which consist of: 1. platelets 2. white blood cells 3. nucleated RBCs 1 mm buffy coat layer represent about 10,000 WBC/mm3 Plasma Layer- normally pale yellow and fairly clear excessive hemolysis results in cherry red color jaundice produce a deep yellow color Fatty layer- normally barely visible, in the presence of lipidemia, the layer is several mm thick

Advantages: less amount of blood needed less error due to less plasma trapped less time involved which makes the method very reliable from the clinical standpoint fairly accurate and gives about 2% variation only when different technologist read the same hematocrit preparation 3. Automated Hematocrit Method o Coulter Counter o Autoanalyzer o Yellow Springs Instrument Factors Affecting Hematocrit Determination relative centrifugation force o Speed of centrifugation 5000 rpm = 30 min 10,000 rom = 10 min 15,000 rpm = 5 min o radius of centrifugation 22.5 cm 3000 rpm 30min 15 cm 3500 rpm 30-45 min o length of centrifugation red blood cell count red cell size Sources of Error in Hematocrit Reading Centrifugation- adequate duration and speed 10-12 thousand rpm for 5 minutes; 1-3% of column is trapped plasma Greater amount of trapped plasma-macrocytic anemia, spherocytosis, hypochromic anemia, sickle cell anemia Sample: posture, muscular activity, and prolonged tourniquet inadequate blood for fixed amount of EDTA: Hct falsely low due to cell shrinkage; Hgb and cell count not affected Immediately after blood loss Dehydration EXERCISE 8: Hemoglobin Determination Hemoglobinometry an estimation of the mount of hemoglobinin a known amount of blood used as as creening test for disease associated with anemia and following the course of disease in the treatment Methods of Determination: 1. Gravimetric Method

Methods of Hematocrit Determination 1. Macrohematocrit Method Anticoagulant Used Wintrode Method Haden's modification Van Allen's Sanford and Magath Bray's Method
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1.1% Sodium oxalate 1.6% Sodium oxalate 1.3% Sodium oxalate Heparin

no longer used large amount of blood needed time consuming higher percentage of trapped plasma centrifuge at 2-3 thousand rpm in 30 minutes 2. Microhematocrit Method o uses heparinized capillary tubes; the tubes are centrifuge for 5 minutes and the length of the packed red cell is measured by the hematocrit reader

the specific gravity of blood is the ratio of the weight of the volume of blood to the weight of the same volume of water at room temperature o method for screening blood donors Copper Sulfate Method o A set up of tubes containing CuS04 solution of different but known specific gravities is prepared. A drop of patients blood is placed in each tube . The tube wherein the drop of blood remained suspended for 10-12 seconds as they are being coated with copper proteinate, after which the drop of blood may either sink of float is the specific gravity value. Normal range= 1.048-1.057; Male= 1.056, Female= 1.053 2. Gasometric Method o indirect method of hemoglobin determination based on the oxygen content of blood o based on the hemoglobin capacity of 1.34 ml oxygen Van Slykes Method (improved method of Haldane and Smith) o determine oxygen and carbon dioxide and is measured by the amount of mmHg displaced o sample is hemolyzed with saponin, and oxygen is liberated and measured o used to calibrate instruments for Hgb determination and not for clinical work o correction is done for temperature and pressure o Formula: 3. Chemical Method o indirect method of hemoglobin determination based on the total iron content of blood regarded as bound to hemoglobin o based on the hemoglobin iron content of 0.3467g/ 100 g of Hb or 3.47 mg of iron per 1 gm Hb 4. Automated Coulter Counter 5. Electrophoresis 6. Colorimetric Method Direct Visual Colorimetric Method 1. Tallquist Method- uses absorbent pads with lithographed color scale representing values ranging from 10-100 %. Patients undiluted blood is absorbed into the absorbent pad and the color of blotted blood is compared with the colored scale. This method gives as much as 50% error

2. Dares Hemoglobinometer- blood is drawn by capillary action between 2 glass plates, and the color of glass is matched with a rotating disc of tinted glass varying in thickness and in depth of red color. Gives as much as 30% error 3. Acid Hematin Methods- hemoglobin in known quantity of blood , in the presence of acid like 0.1N HCl, is converted into acid hematin which is a brown solution. Acid hematin is diluted with water drop by drop until the color matches that of the standard 4. Alkaline Hematin Methods- hemoglobin in known quantity of blood , in the presence of an alkali, is converted into alakaline hematin which is true and relatively stable solution. Alkali hematin is also diluted with water drop by drop until the color matches that of the standard solution not ideal for use in infants and children since HbF is resistant to denaturation by alkali Cyanmethemoglobin - recommended by ICSH Advantages: o Cyanmethemoglobin pigment is stable in dilute solution o Measures all forms of hemoglobinderivatives except sulfhemoglobin o Certified hemoglobin standards are available o Spectral Curve allows the use of different types of spectrophotometer Principle: The diluents (Drabkins solution) contains potassium ferricyanideand potassium cyanide Ferricyanide + hemoglobin= Methemeoglobin Methemoglobin + KCN = stable hemoglobin

The resulting solution is then read spectrophotometrically and the equivalent hemoglobin is determined from the calibration curve or table Tube Cyanmethemoglobi Drabski Hemoglobin Labelin n Standards (mL) n Concentratio g Solution n (g/dL) (mL) Blank 1 2 3 4 5 0 1 2 3 4 5 5 5 4 3 2 1 0 4 8 12 16 20

the standard is used to build a standard covering the concentration range and to prepare quality control samples at low, moderate and high concentration to monitor assay performance Hemoglobin Values are affected by: age, sex, altitude, pregnancy

Rule of Three 3 x RBC = Hb (g/dL) 3 x Hb = Hct + 3 (%) Red Blood Cell Indices commonly used parameters to assess anemia mean corpuscular volume o average volume of a single RBC in a given blood volume o reference values 80-100 = normal > 100 = macrocytic < 80 = microcytic dimorphic RBCs yield a normal MCV increased reticulocytes increases MCV
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color index amount of Hb in each RBC as compared w/ the average size of a normal RBC CI= % Hb / % RBC Normal value = 0.9-11 volume index average size of RBC as compared w/ the average size of normal RBC VI = % Hct/ % RBC Normal value = 0.9-1.1 saturation index average amount of Hemoglobin per unit volume of cell SI = CI/VI or %Hb/%Hct Normal Value: 0.8-1.2

mean corpuscular hemoglobin concentration o average concentration of Hb in the RBC o reference values 31-37 = normochromic > 37 = hyperchromic < 31 = hypochromic o at most 37 g/dL of Hb
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mean corpuscular hemoglobin o average weight of hemoglobin in each RBC o reference values 26- 34 = normal
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other indices: o red cell distribution width variation in mean diameter of RBC Normal = 11.14% macrocytosis = increased MCV, normal RDW IDA = decreased MCV, increased RDW mixed = increased RDW o mean corpuscular avergae thickness average height/ thickness of RBC MCAT = MCV/ MCD