Practical Hemostasis & Thrombosissite.iugaza.edu.ps/akhudair/files/Hemosta… · Web view · 2018-01-28Practical Hemostasis & Thrombosis 2016. Practical Hemostasis & Thrombosis

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Collected by : Ibtisam

H Al-Aswad Alaa M Khudair

The Islamic University Of Gaza Faculty Of

Health Science

2016

2016Practical Hemostasis & Thrombosis

1 - Laboratory Safety .................................................................................. 2

2 - General Approach In Investigation Of Hemostasis ............................. 5

3 - Bleeding Time Test .............................................................................. 16

4 - Coagulation Time Of Whole Blood (Clotting Time) .......................... 23

5 - Coagulation Assay – Prothrombin Time Test .....................................32

6 - Prothrombin Time (Continue) – ISI & INR .......................................42

7 - Activated Partial Thromboplastin Time (APTT) ................................47

8 - Mixing Study ........................................................................................52

9 - Lupus Anticoagulant ...........................................................................59

10 - D-dimer Test ......................................................................................67

11 - Tests To Measure Fibrin Clot ..........................................................69

12 - One Stage Factor V Assay ................................................................76

13 - Platelet Aggregation Study ...............................................................81

14 - Coagulation Instruments .................................................................95

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CONTENTS

Laboratory SafetyLaboratories that handle chemicals and biological samples are potentially hazardous places. In recent years, there has been an increasing appreciation of the importanceof safe working practices in industry, for both health and environmental reasons. This awareness leads to greater stress on issues such as safety documentation, staff training, and risk assessment.Employers have a responsibility to provide the necessary protective clothing and equipment, and they are required to provide training in safe working practices.If safe working practices are in place, the probability of serious injury to yourself, your colleagues, and members of the public should be greatly reduced.

Safety ManualThere should be a comprehensive safety manual that covers all aspects of safe working practices for the whole department.All staff members must read the manual and sign a declaration to indicate that they have understood it.Copies should be kept with the safety officers and also made available in places that are easily accessible to all members of the staff.

Safety Measures: Universal PrecautionsThe system of universal precautions requires that any danger of infection fromany source will be avoided or minimized by good working practices.All blood samples, blood products (including plasma-based reagents and kits), and other human body materials should be regarded as posing a possible danger of infection. The fullest possible protective measures should always be taken when working with any material. No other classification of risk should be made. All body fluids and materials other than blood, whether collected or brought into the unit for testing or any other purpose, should be handled with the same care as that given to blood.

The LaboratoryThe laboratory should always be clean and tidy. Paperwork should be kept separate from laboratory testing areas. Try not to use the laboratory for storage of bulk items. Try to ensure that everyone participates in keeping the laboratory orderly.

Protective ClothingEveryone who enters the laboratory, including visitors, should wear a laboratory coat. They should immediately replace the coat if it becomes contaminated.

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Disposable GlovesMany people do not like to use gloves, but every sample handled in the laboratory is potentially hazardous. Gloves should always be worn when handling toxic material.Gloves and coats will obviously not protect against a needle stick-type accident, but they will prevent, for example, HIV positive serum or a toxin coming into contact with any cuts or abrasions on your skin. Always replace gloves immediately if they are broken or punctured.

Eye WashingMany infections can be easily acquired by contact with the mucous membranes of the eyes.Wash your eyes immediately with lots of cold running water if contact with a possible infectious material may have occurred.

SharpsSharps, in the form of needles and broken glass, present a great danger: use asharp box capable of containing sharps without being punctured. There have been cases of workers becoming infected as a result of needle-stickinjuries.

AerosolsAvoid all practices in the open laboratory that may cause splashing or the release of airborne droplets or dust. Operations that cause aerosols must always be carried out in a suitable fume cupboard, and safety glasses must be worn. All spills should be cleaned up immediately, using bleach or a neutralizing agent as necessary.

Toxic and Flammable SubstancesToxic or flammable materials must always be contained within a fume cupboard or suitable safe box.

Electrical EquipmentTake special care with any equipment that uses liquids, such as electrophoresis tanks and water baths. Always leave installation, servicing, and repairs to qualified personnel.

Personal Possessions and BehaviorNever take personal items, such as pens, bags, and combs, into the laboratory.Avoid bringing your hands into contact with your face or mucosae (eyes, nose,and mouth) while in the laboratory but if you must do this, always wash yourhands first. Food, cigarettes, and cosmetics must never be brought into the laboratory. Never mouth a pipette.Always wash hands thoroughly before leaving the laboratory.

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AccidentsAll accidents should be reported immediately and should be recorded in an accident book kept by the unit Safety Officer. This is particularly important in relation to needle stick injuries. In these situations, follow local hospital systems for recording and reporting, along with any locally recommended or mandated actions.

The Control Of Substances Hazardous To Health (Coshh)

This legislation, used in U.K. laboratories, is a useful guide in identifying risksand hazards.

Hazard and RiskThe hazard presented by a substance is its potential to cause harm. The risk from that substance is the likelihood of its harming someone under the actual conditions of use.

Identification of HazardsThe identification of hazards is an essential prerequisite of risk assessment.The time spent in identifying the hazards will vary according to the substance.

Risk AssessmentConsider the following facts:

The hazards The conditions of use The amounts to be used The likely routes or sites of exposure (inhalation, ingestion, skin, or eyes)

The outcome of the risk assessment will determine: The storage conditions The handling procedures The disposal procedures The requirement of monitoring and health surveillance The emergency procedure

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Lab 1: General Approach in Investigation of Hemostasis

Objective

Highlighting contributory elements and providing suggestions on how problems can be minimized or prevented.

Improving the likelihood that reported test results actually represent the true clinical status of the patient rather than that of an inappropriate sample

Introduction

Hemostasis is a complex interaction between vessels, platelets and coagulation proteins that, when working properly, stops bleeding while maintaining blood flow in the vessel. Specific tests are available to evaluate platelet function, coagulation proteins, natural occurring inhibitors and fibrinolysis.

Pre-Analytical Variables

the sample that has reached the laboratory and been entered into the analytical process, actually reflects what is happening in the patient. If this simple requirement is not satisfied by the sample we analyze, then the results that are produced will be meaningless, no matter how well quality controlled the testing process or how robust our means of transmitting the information to the end-user are, and the clinical decision making process will be, potentially disastrously, flawed.

To ensure that the laboratory analysis actually reflects the patient’s state we must consider what factors may influence the state of the sample and from that we may determine mechanisms to properly regulate these factor

Pre-analytical Variables including

1. Sample Collection.

2. Site Selection.

3. Storage Requirements.

4. Transportation of Specimen.

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Sample Collection

Proper sample collection is of outmost importance for reliable test results to evaluate the bleeding patient, thrombosis or fibrinolysis (pre-analytical phase), all these tests are influenced by sample collection, sample processing and sample storage. Any sample that is not obtained quickly with an immediately successful venipuncture should be discarded because of possible activation of coagulation when blood is withdrawn from a vessel, changes begin to take place in the components of blood coagulation. Some occur almost immediately, such as platelet activation and the initiation of the clotting mechanism dependent on surface contact.The blood should not be passed back through the needle after collection into a syringe. The needle should be removed before passing the blood from the syringe into the container with anticoagulant. There should be no delay between collection and mixing with anticoagulant.Once blood and anticoagulant are mixed, the container should be sealed andmixed by gentle inversion five times. Avoid vigorous shaking. The laboratory will not evaluate samples that are hemolyzed, clotted, contain fibrin strands or improperly stored.Reference Laboratory Services will immediately notify the client of any problems with the sample.

Anticoagulant of choiceThe blood should be mixed with 3.8% or 3.2% sodium citrate anticoagulant in theproportion 9 parts blood: 1 part anticoagulant.3.2 % Preferred as the standard measure due to stability and closeness to the plasma osmolality.Exact amount of blood must be drawn. No short draws are acceptable, this will falsely increase results due to presence of too much anticoagulant CLSI guideline is +/- 10 % of fill line.Purpose of the anticoagulant is to bind or chelate calcium to prevent clotting of specimen. Other anticoagulants, including oxalate, heparin, and EDTA, are unacceptable. The labile factors (factors V and VIII) are unstable in oxalate, whereas heparin

and EDTA directly inhibit the coagulation process and interfere with end-point determinations.

Additional benefits of tri-sodium citrate are that the calcium ion is neutralized more rapidly in citrate, and APTT tests are more sensitive to the presence of heparin.

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If the patient has a reduced hematocrit, or particularly if the hematocrit is raised, results can be affected. According to the latest CLSI (formerly NCCLS) guideline on coagulation testing, it is important to adjust the sodium citrate volume when a patient’s hematocrit is greater than 55%.Examples of patients who may have elevated hematocrit values are newborns or people with polycythemia vera. NCCLS recommends adjusting anticoagulant ratio for patients with hematocrits exceeding 55% as high hematocrits may cause falsely prolonged test results due to an over- anticoagulated sample.Formula correction achieves a 40% hematocrit. The volume of anticoagulant should be adjusted to take account of the reduced plasma volume.

Volumes of blood and anticoagulant required for a 5 ml samples with abnormal hematocrit

Hematocrit Volume of Anticoagulant Volume of Blood

20% 0.7 ml 4.3 ml25% 0.65 ml 4.35 ml30% 0.61 ml 4.39 ml55% 0.39 ml 4.61 ml60% 0.36 ml 4.64 ml65% 0.31 ml 4.69 ml70% 0.27 ml 4.73 ml

Alternatively, the anticoagulant volume of 0.5 ml can be kept constant and the volume of added blood varied accordingly to the hematocrit. The volume of blood to be added (to 0.5 ml of 0.109M citrate) is calculated from the formula:X = (100–PCV) × vol.

(595–PCV)

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Where:

X= volume of sodium citrate Vol = volume of whole blood drawn

PCV= patient’s hematocrit

Examples:

Patients Hct= 60%, V= 5 mL

X = (100-60) × 5 = 40 × 5 / 535 = 0.34 ml

(595-60)

Patient Hct = 25%, V=5 ml

X = (100-25) × 5 = 75 × 5 / 570 = 0.65 ml

(595-25)

Site SelectionCollections from venous lines should include a process for flushing and/or discarding the initial collection volumes. Size and type of needle used may also influence results and too large (less than 16 gauge) or too small a needle bore (greater than 25 gauge) should be avoided, and heparinized needles (sometimes used for blood gas collection) not used.

Untraumatic venipuncture is required

Traumatic venipunctures release tissue factor and initiate coagulation

Finger sticks / Heel sticks are not allowed

Indwelling IV line draws are discouraged

Contain heparin & diluted blood

Falsely increased results

Order of Draw

Evacuated tube system

Blue top is 2nd

If 2nd tube drawn, 1st top must be anticoagulant free (i.e. red top).

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Storage Requirements

Prothrombin Time: PT

Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or 18-24 oC must be tested within 24 hours of collection.

Activated Partial Thrombin Time: APTT

Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or 18-24 oC must be tested within 4 hours of collection.

Other Assays

Fibrinogen, Thrombin Time, Factor Assays.

Centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or 18-24 oC must be tested within 4 hours of collection.

TESTPlasma Stability At Rt

Centrifuge To Prepare

Platelet-Free Plasma

Refrigeration (Or transport on

ice)

Freeze Platelet-FreePlasma

PT 24 hours Do not refrigerate If >24 hour delay in

testing

PTRX 24 hours Do not refrigerate If >24 hour delay in

testing

PTT 4 hours If >4 hour delay in testing

PTTRX 2 hoursWithin one hour of collection

If >2 hour delay in testing

TT 4 hours If >4 hour delay in testing

OTHERASSAYS

4 hoursWithin one hour of collection

Within one hour of collection

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Other general notes

Perform coagulation tests As Soon As Possible. Specimen may deteriorate rapidly (especially factors V and VIII).

If the testing is not completed within specified times, plasma should be removed from the cells and placed in a frost free freezer.

-20 oC for two weeks. -70 oC for six months.

Transportation of Specimen

Samples should be transported non-refrigerated in as short a time as possible, or can be transported on ice. Extremes of temperature (ie, both refrigerated or high) should be avoided. Delays in transport may affect in particular the labile factors (FV, FVIII), leading to prolonged clotting times and in vitro loss of factor activity. In such cases, local centrifugation and separation of plasma followed by freezing and frozen transport of the plasma should be considered.

Centrifugation

Platelet-rich plasma (PRP)

Platelet-rich plasma (PRP) for platelet function tests is prepared by centrifugation of anti-coagulated blood at room temperature at 150 g–200 g for 10 minutes. The supernatant is removed and kept at room temperature in a stoppered vial during use for a time not exceeding two hours.

Platelet-poor plasma (PPP)

Platelet-poor plasma (PPP) is used for most tests of coagulation and is necessary for coagulation testing to prevent activation of platelets and release of PF4, a heparin inhibitor. The plasma platelet count must be < 10,000 /mm3.

Why is PPP essential?

Contains platelet factor 4 (heparin neutralizer).

Contains phospholipids (affects lupus anticoagulant and factor assay testing).

Contains proteases (affect testing for vWF).

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To prepare platelet-Poor plasma The blood sample should be centrifuged at a minimum of 1700 g for at least 10

minutes. This can be at room temperature provided this does not exceed 25°C, in which case a refrigerated (4°C) centrifuge should be used.

The 2/3 of PPP from the first centrifugation is transferred to a plastic stoppered tube and centrifuged a second time. Care is taken not to use the bottom part of the plasma after the second centrifugation, since it may contain any platelets that remained after the first centrifugation.

remove the top ¾ of the plasma to a plastic aliquot tube with a fresh plastic pipette. Freeze the specimen within one hour of collection.

Samples For Immediate TestingSamples should be tested within four hours of sample collection when possible.More prolonged storage should be avoided for screening tests and clotting factorassays, although it has been shown that whole blood samples stored at roomtemperature may be stable for prothrombin time measurements. Samples for screening tests and assay of factor VII should be maintained at room temperature to avoid the possibility of cold activation.

Deep-Freezing PlasmaSamples can be stored deep frozen for testing at a later stage. Storage at -70°C or lower is preferable. Clotting factors are stable at this temperature for at least six months. Short-term sample storage at -35°C is adequate for most tests. Storage at -20°C is usually inadequate. Double centrifugation should be used if samples are deep-frozen prior to analysis for lupus anticoagulant.Freezing and thawing is best avoided before APTT determinations, since results obtained by some techniques can be affected. Any frozen plasmas must be transferred immediately to a 37°C water bath, thawed for four to five minutes, and mixed by gentle inversion prior to analysis. A slow thaw at lower temperature should be avoided to prevent the formation of cryoprecipitate, which reduces the FVIII, VWF, and fibrinogen content of the supernatant plasma.

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Common Collection Problems

Error Consequence CommentShort draw<2.7 mL

PT/PTT falsely prolonged

Anticoagulant to blood ratio exceeds 1:9

Failure to mix specimen after collection


Blood clots form when anticoagulant & blood do not mix

Excess vigorous mixing

PT/PTT falsely shortened

Hemolysis and platelet activation cause start of cascade

Hemolysis PT/PTT falsely shortened

Reject specimen

Improper storage: wrong temperature or held too long


Must follow storage requirements

Chilling in refrigerator or placing on ice

PT falsely shortened Chilling to 4 oC activates factor VII.

Inadequate centrifugation

PTT loses sensitivity for lupus anticoagulant and heparin.

Factor assays inaccurate

Prolonged tourniquet application

Falsely elevates vWF, factor VIII

Tourniquet causes venous stasis,

Drawing coagulation tube after to other anticoagulant tubes

PT/PTT falsely affected

Contamination

Probing the vein PT/PTT falsely shortened

Tissue thromboplastin is released activating coagulation

Heparin contamination from line draw

PTT falsely prolonged

Heparin keeps the blood from clotting

Lipemia Test may not work Photo-optical methods affected

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Principles of Laboratory Analysis

The more detailed investigations of coagulation proteins also require caution in their interpretation depending on the type of assay performed. These can be divided into three principal categories, as described in the following sections.

1. Immunological Assays

Immunological Include immuno-diffusion, immuno-electrophoresis, radioimmunometric assays, latex agglutination tests, and tests using enzyme-linked immunosorbent assays (ELISA).

Fundamentally, all these tests rely on the recognition of the protein in question by polyclonal or monoclonal antibodies. Polyclonal antibodies lack specificity but provide relatively high sensitivity, whereas monoclonal antibodies are highly specific but produce relatively low levels of antigen binding.

latex agglutination kit

Latex microparticles are coated with antibodies specific for the antigen to be determined. When the latex suspension is mixed with plasma an antigen–antibody reaction takes place, leading to the agglutination of the latex microparticles. Agglutination leads to an increase in turbidity of the reaction medium, and this increase in turbidity is measured photometrically as an increase in absorbance.

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Usually the wavelength used for latex assays is 405 nm, although for some assays a wavelength of 540 or 800 nm is used. This type of assay is referred to as immuno-turbidimetric.

Notes:

Do not freeze latex particles because this will lead to irreversible clumping.

An occasional problem with latex agglutination assays is interference from rheumatoid factor or paraproteins. These may cause agglutination and overestimation of the protein under assay.

2. Assays Using Chromogenic Peptide Substrates (Amidolytic Assays)

Chromogenic, or amidolytic, methodology is based on the use of a specific color-producing substance known as a chromophore. The chromophore normally used in the coagulation laboratory is para-nitroaniline (pNA), which has an optical absorbance peak at 405 nm on a spectrophotometer.

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3. Coagulation Assays

Coagulation assays are functional bioassays and rely on comparison with a control or standard preparation with a known level of activity. In the one-stage system optimal amounts of all the clotting factors are present except the one to be determined, which should be as near to nil as possible. The best one-stage system is provided by a substrate plasma obtained either from a patient with severe congenital deficiency or artificially depleted by immuno-adsorption.

Coagulation techniques are also used in mixing tests to identify a missing factor in an emergency or to identify and estimate quantitatively an inhibitor or anticoagulant. The advantage of this type of assay is that it most closely approximates the activity in vivo of the factor in question. However, they can be technically more difficult to perform than the other types described earlier.

4. Other Assays

Using snake venoms (The Taipan venom time employs a reagent isolated from the venom of the Taipan snake (Oxyuranus scutellatus) that directly activates prothrombin in the presence of phospholipid and calcium.)

Aassay of ristocetin cofactor (used to diagnose von Willebrand disease ) The clot solubility test for factor XIII. DNA analysis is becoming more useful and more prevalent in coagulation.

However, this requires entirely different equipment and techniques.

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Lab 2 : Bleeding Time Test

Objective

State and compare the four types of bleeding time methods. This test helps diagnose bleeding problems.

Introduction

When blood vessels are damaged, Hemostasis (clot formation) will arrest bleeding. This process is divided into three phases:

I. Vascular Phase - Cutting or damaging blood vessels leads to vascular spasm of the smooth muscle in the vessel wall. This produces a vasoconstriction which will slow or even stop blood flow. This response will last up to 30 minutes and is localized to the damaged area.

II. Platelet phase - Damaged endothelial cells lining the blood vessel release von Willebrand's Factor. This substance makes the surfaces of the endothelial cells "sticky". This condition may, by itself, be enough to close small blood vessels. In larger blood vessels, platelets begin to stick to the surfaces of endothelial cells. This effect is called Platelet Adhesion.The platelets that adhere to the vessel walls now begin to secrete Adenosine diphosphate (ADP) which is released from "stuck" platelets. This material causes the aggregation of nearby free platelets which attach to the fixed platelets and each other. This aggregation of platelets leads to the formation of a platelet plug.

III. Coagulation Phase - Begins 30 seconds to several minutes after phases I and II have commenced. The overall process involves the formation of the insoluble protein Fibrin from the plasma protein Fibrinogen through the action of the enzyme Thrombin. Fibrin forms a network of fibers which traps blood cells and platelets forming a thrombus or clot.

This phase followed by : Clot Retraction - After 2 or 3 days, the clot begins to contract. Platelets in the

clot contain contractile proteins. These proteins pull the edges of the wound together and reduces the chance of further hemorrhage. This activity also assists the repair processes.

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Fibrinolysis - Dissolution of the clot. The breakdown of the clot is due to the production of a powerful proteolytic enzyme Plasmin. Plasmin is formed through the same chemical pathway that produces thrombin. These reactions demonstrate that materials which induce clot formation (Thrombin and Factor XII) will eventually assist in the breakup of the clot.

Coagulation Tests1. Tests of the Vascular Platelet Phase of Haemostasis:

Bleeding Time (BT)2. Tests of the Coagulation Cascade:

Clotting Time ( CT) or Coagulation time Activated Partial Thromboplastin Time (APTT). Prothrombin Time (PT).

3. Tests of Fibrinolysis and the Mechanisms That Control Hemostasis: Fibrin Degradation Products (FDP)

Principle:

The bleeding time test is a useful tool to test for platelet plug formation and capillary integrity. Occasionally, the bleeding time test will be ordered on a patient scheduled for surgery. The bleeding time is dependent upon:

The efficiency of tissue fluid in accelerating the coagulation process

Capillary function.

The number of blood platelets present and their ability to form a platelet plug.

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Prolonged bleeding times are generally found when The platelet count is below 50,000/µL When there is platelet dysfunction.

Four procedures are currently in use for determining the bleeding time: 1. The Duke method.2. The Ivy Method.3. The Mielke Method.4. The Simplate or Surgicutt Method.

Duke Method

With the Duke method, the patient is pricked with a special needle or lancet, preferably on the earlobe or fingertip, after having been swabbed with alcohol. The prick is about 3–4 mm deep. The technician then wipes the blood every 30 seconds with a filter paper.

1. The test ceases when bleeding ceases. 2. The usual time is about 1–3 minutes. 3. No repeat testing is allowed due to space.4. The test causes nervousness in the patient.5. This test method is the easiest to perform, but is the least standardized and has

the less precision and accuracy.

Ivy Method

A blood pressure cuff is used to maintain constant pressure within the capillaries to help standardize the procedure. The cuff is inflated to 40 mm Hg on the upper arm to control capillary tone and to improve the sensitivity and reproducibility -this will maintain constant pressure within the capillaries and help standardize the procedure.

1. The forearm is the bleeding time site used.2. A sterile, disposable blood lancet is used to make a shallow incision that is

1 millimeter deep on the underside of the forearm.3. Every 30 seconds, filter paper is used to draw off the blood.4. The length of time required for bleeding to cease is recorded.5. The test is finished when bleeding has stopped completely.

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http://en.wikipedia.org/wiki/Fingertip

http://en.wikipedia.org/wiki/Earlobe

6. A prolonged bleeding time may be a result from decreased number of thrombocytes or impaired blood vessels. However, it should also be noted that the depth of the puncture or incision may be the source of error.

7. Normal values fall between 2 – 7 minutes depending on the method used.8. The greatest source of variation in this test is largely due to difficulty in

performing a standardized puncture. This usually leads to erroneously low results.

Mielke Method

Modification of the Ivy Method. A Bard-Parker or similar disposable blade is used, along with a rectangular polystyrene or plastic template that contains a standardized slit. The blade is placed in a special handle containing a gauge in order to standardize the depth of the incision. The same procedure as described for the Surgicutt method is employed.Advantages of this method include:

a. That the surgical incision more closely approximates the patient’s hemostatic response to surgery, when compared to the puncture in the Ivy Method.

b. The depth of the incision can be controlled.Disadvantages of this method include:

a. Cost- scalpel and template required sterilization after each use.b. Patient apprehension, due to unconcealed scalpel.c. Small scars might form.

Simplate/Surgicutt Method: Preferred Method

Modification of the Ivy Method. The first bleeding time device introduced was the Simplate. The Simplate device has a trigger and spring method for the blade. The blade has a depth of 1.0 mm and a width of 5.0 mm. Another brand name is the Surgicutt.Advantages of this method include:

a. Instrument is a sterile, standardized, easy to use device that makes a uniform incision.

b. Instrument is a spring activated surgical steel blade which is housed in a plastic unit. This eliminates variability of blade incision.

c. This method is the most standardized method of all the bleeding time procedures.

d. Inexpensive

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Disadvantages of this method include:a. Slight scarring can occur so patient should be informed.

1Preparation of the Patient

After proper greeting and identification of the patient, explain that a "bleeding time" has been ordered by his/her doctor. This test is used to see how long it will take for the patient to stop bleeding and a clot to form after a puncture is made in the forearm. Unless the patient is an infant, outline to the patient how the test will be done. Explain that the test will take approximately 10 to 20 minutes to complete. Let the patient know that he may experience some discomfort from the incision and the cuff of the sphygmomanometer. After explaining the procedure to the patient, stop and ask if they have any additional questions. The technician may respond to any questions the patient has concerning the procedure ONLY. If the patient inquires about the significance of the test relative to his/her condition, he/she should be referred to the physician who ordered the test. The patient's physician can interpret test results within the context of each patient's condition.

It is critical that you ask the patient whether or not they have taken aspirin, aspirin containing compounds (many over the counter medications contain aspirin) or blood thinners such as Heparin or Coumadin recently. These drugs will cause a falsely abnormal bleeding time and the test should not be done.

1Overview of Procedure

Select a site on the patient’s forearm approximately three fingers widths below the bend in the elbow that is free of visible subcutaneous veins. Avoid surface veins, scars, bruises and edematous areas. Apply the blood pressure cuff and inflate to 40 mm Hg. Perform the incision after the cuff has been in place 30-60 seconds. Apply the bleeding time device to the arm in a horizontal position, do NOT apply undue pressure as this will falsely increase the depth of the incision. Start the stop watch as soon as the incision is made. The blood is wicked onto the filter paper at 30 second intervals taking great care that the wound is NOT directly touched with the filter paper. Once blood no longer stains the filter paper the timing is stopped. The bleeding time is reported out to the nearest 30 seconds.

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Procedure:1. Greet and identify the patient.2. Explain the procedure to the patient.3. Obtain a history about aspirin or aspirin containing compounds taken within last

7 - 10 days.4. Select a site on the patient's forearm approximately three fingers widths below

the bend in the elbow that is free of visible subcutaneous veins.5. Cleanse the outer surface of the patient's forearm by moving the alcohol pad in

concentric circles from the incision site outward; allow to air dry.6. Place a blood pressure cuff on the patient's arm above the elbow. Turn the knob

on the bulb of the sphygmomanometer until it stops. Squeeze the bulb to inflate the sphygmomanometer. Inflate the cuff and maintain pressure at 40 mm Hg.

7. Remove the Surgicutt device from the blister pack, being careful not to contaminate or touch the blade-slot surface. Remove the safety clip.

8. Depress the “trigger” on the bleeding time device. The puncture must be performed within 30 to 60 seconds of inflation of the blood pressure

cuff. Simultaneously start the stopwatch.9. After 30 seconds have passed blot (do not wipe) the blood with the filter paper.

The filter paper must not touch the wound on the arm. Blot the site at regular thirty second intervals. Rotate the filter paper after each 30 seconds.

10. When bleeding ceases and blood no longer is drawn to the filter paper, stop the watch and release the blood pressure cuff by turning the knob next to the bulb in the opposite direction used to inflate the cuff. Remove the blood pressure cuff.

11. Record the bleeding time. Bleeding time is determined to the nearest 30 seconds.

12. If bleeding continues for more than 15 minutes, the procedure should be discontinued, and pressure applied to the wound sites.

13. After ensuring that the bleeding has stopped, carefully bandage the site.14. Appropriately discard all used materials and wash hands.

Normal Values : 1.0 – 9.0 minutes

Quality ControlQuality control is accomplished through the standardization of the procedure. The wound should be a standard length and depth and a constant pressure of 40 mmHg should be maintained throughout the procedure.

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1Sources of Error/Troubleshooting

1. If the patient has taken aspirin or aspirin-containing compounds 7 to 10 days prior to the procedure, the bleeding time may be prolonged. The technician must determine the patient's history concerning aspirin ingestion to ensure quality results. Aspirin blocks the formation of thromboxane and prostacyclin in both platelets and endothelial cells. This results in abnormal aggregation of the platelets. Damage from aspirin effects the platelets for its entire life span.

2. Results may be affected by an improperly performed puncture. A puncture that is too shallow, too deep, or in an inappropriate location will adversely affect test results.

3. The alcohol must be completely dried before making the puncture. If residual alcohol is on a puncture site, the bleeding time will be erroneously prolonged.

4. If the technician does not initiate timing of the procedure simultaneously with the puncture, the results will be adversely affected.

5. If the technician allows the filter paper to touch the wound, the platelet clot may be dislodged, causing falsely elevated results.

6. If the stopwatch has not been appropriately calibrated, it may keep incorrect time. Stopwatches should be calibrated on a regular basis as a part of the quality assurance program.

7. The direction of the incision should be consistent. A horizontal incision gives a longer bleeding time than a vertical incision.

8. The bleeding time is prolonged in thrombocytopenia, hereditary and acquired platelet dysfunctions, von Willebrand's disease, afibrinogenemia, severe hypofibrinogenemia, and some vascular bleeding disorders.

9. Certain drugs like Plavix and Ticlid, which are platelet inhibitors can falsely prolong the bleeding time.

NOTE:

The critical Value for bleeding time is > 15 min. If bleeding continues for more than 15 minutes, the procedure should be discontinued, and pressure applied to the wound sites.

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Lab 3 : Coagulation Time Of Whole Blood (Clotting Time)

Objective

The clotting time (CT) is a test that is used primarily to monitor high doses of unfractionated (standard) heparin therapy.

To be familiar with the methods used to test the clotting tendency.

Introduction

Clotting Time is the time required for blood to form a clot in vitro. The basis for the test is that whole blood will form a solid clot when exposed to a foreign surface such as a glass tube. It is a rough measure of all intrinsic clotting factors in the absence of tissue factors. Variations are wide and the test sensitivity is limited. As the test is the least effective test in the diagnosis of actual haemostasis failure, it has been replaced by PTT. Clotting time was used as a screening test to measure all stages in the intrinsic coagulation system and to monitor heparin therapy . It is however, a time-consuming test, has poor reproducibility, is sensitive only extreme factor deficiencies. It is therefore, of limited use in today’s laboratory . It is a complex process involving over 30 substances

Conditions accompanied by increased Clotting Time:1. Factors V, VII, VIII, IX, XI, XII Deficiencies.2. Hemorrhagic disease of Newborn3. Vitamin K deficiency.4. Heparin Therapy.5. Presence of Circulating antibodies (inhibitors)6. Anemia and leukemia. 7. Afibinogenemia and Pneumonia.8. heparin.

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Methods of Testing

Capillary Method.

Slide Method.

Tube Method.

Clotting Time - Capillary method

Material

Sterile disposable pricking needle or lancet.

Stop watch

Dry glass capillary tube (narrow diameter top 2 mm, minimum 10 cm long.)

Cotton Swab of absorbent cotton.

Spirit wetted, cotton swab.

70 % v/v ethyl alcohol

Procedure

1. Apply alcoholic 70 % v/v to the clean finger with cotton swab. Allow it to dry naturally.

2. Prick the finger with usual aseptic precautions. Remove the first drop.

3. Dip one end of the capillary into blood drop gently without pressure. (use 3-4 capillary tubes)

4. The Timer is started when the first blood start to enter the first capillary tube.

5. Allow to fill the capillary with blood by lowering the end of fitted capillary. (Do not suck the blood) around ¾th of its length undipped.

6. After every 30 seconds, using stopwatch, break a small piece of capillary.

7. Repeat breaking at regular time intervals, till fibrin thread appears at the broken end of capillary tube. Do not pull away the cut pieces ling apart and bristly.

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8. Record time interval between pricking finger and first appearance of fibrin thread at the broken ends of capillary tube. That is clotting time of blood.

Don't forget to dispose of the broken tube in the SHARPS CONTAINER.

Clotting Time - Slide Method

The surface of the glass tube initiates the clotting process. This test is sensitive to the factors involved in the intrinsic pathway

The expected range for clotting time is 4-10 min.

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Tube Method (Lee-White method)

Reagent & equipment

Water bath, 37O C.

Glass test tube (10 x 75 mm)

Stopwatch.

Plastic syringe and 20-gauge needle.

Specimen

Fresh whole blood , 4 ml .

Procedure

1. Label 3 glass test tube with patient name and number them, 1, 2, and 3.

2. Perform a clean, Untraumatic venipucture using a 20-gauge needle and drawn 4 mL of blood.

3. Remove the needle from the syringe, and fill each of the three tubes with 1 ml blood.

The last 1 ml of blood may be discarded.

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Start the stopwatch as soon as the blood enters the syringe.

4. Place the three test tubes in a 37°C water bath.

5. At exactly 3 min., Remove the first tube form water bath and tilt gently to a 45° angle to see whether the blood has clotted.

6. If Blood not clotted return it to the water bath and examine it at 30 second intervals.

7. After the blood in the first tube has clotted, examine the second tube immediately.

8. Then examine the 3rd one.

9. Record the time it took the blood in the 3rd test tube to clot.

Normal Range : 5 – 12 Minutes

Discussion

1. Poor venipucture technique, causing hemolysis or tissue thromboplastin to mix with the blood, shortens the clotting time.

2. Bubbles entering the syringe when the blood sample is being obtained increase the rate of coagulation.

3. Always tilt the tube in the same direction and at the same angle so that the blood is moving in the same pathway up the side of the tube each time.

4. Excessive agitation of blood (occur during the transfer of the blood from the syringe to the test tube). The blood should be allowed to flow gently down the inside of the test tube and not forcefully squirted into the test tube.

5. At the completion of the clotting time, one tube should remain in the 37°C water bath to be checked for clot retraction. Also, this same tube may be allowed to remain in the water bath overnight and checked the next day for clot lysis.

6. Dirty Test Tubes will affect the result.

Coagulation will be retarded by the following:

27


Temperature Below 35O C.

Temperature above 45O C.

Diameter of the test tube (the smaller the diameter, the more rapid the clot formation is).

ACT (Activated clotting Time)

Fresh whole blood is added to a tube containing negatively charged particles and timed for the formation of a clot.

The type of negatively charged particle affects the "normal" length of ACT

Celite = Diatomaceous Earth: normal is 100-170 seconds

Kaolin: normal is 90-150 seconds

Glass particles: normal 110-190 seconds

Type of machine affects normal and therapeutic values

Daily calibration checks are imperativee

Therapeutic Range : 180-240 seconds.

Hemochron®

Tests measured

Heparin monitoring with or without Aprotinin.

Sample and volume requirements

Fresh whole blood- 2mL. Elite: 200µL to fill cuvette well.

Controls

Electronic QC

Liquid quality control- normal and abnormal

Reference Ranges

Normal range- healthy donors

RFTCA 510 tubes: 105 – 167 seconds

FTK-ACT tubes: 91 – 151 seconds

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Normal range- cardiopulmonary bypass patients

HRFTCA 510 tubes: 86 - 147 seconds

FTK-ACT tubes: 89 - 153 seconds

Clot retraction (Obsolete test)

This test measures the amount of time it takes for a blood clot to pull away from the walls of a test tube (Shrinking). The edges of the blood vessel wall at the point of injury are slowly brought together again to repair the damage. It is used to evaluate and manage blood platelet disorders, including Glanzmann's thrombasthenia , So Clot retraction depends primarily on the number and activity of the blood platelets. This test is reliable only when the concentration of fibrinogen within normal limits. Normally a blood clot will begin to retract from the walls of the tube and express serum within one hour.

Glanzmann's thrombasthenia

Is an abnormality of the platelets, it is an extremely rare coagulopathy in which the platelets lack glycoprotein IIb/IIIa. Reduced glycoprotein IIb/IIIa causes reduced platelet aggregation and clot retraction - no fibrinogen bridging can occur, and bleeding time is significantly prolonged);

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Clot retraction test

This is a measure of platelet function. The test is done in blood to which no anticoagulant has been added and is allowed to clot. Clot retraction is then looked for.

Clot retraction becomes abnormal in conditions like

Fibrinogen deficiency (congenital or acquired)

Thrombocytopenia < 1ooooo

Thrombosthenia

Polycythaemia

Reduced clot formation: Glanzmann thrombasthenia , DIC, hypofibrinogenemia, dysfibrinogenemia (small clot with increased red blood cell “fall-out”)

Procedure

1. Obtain 2 ml of blood by venipucture. Place one ml in a glass test tube in a 37oC water bath.

2. Inspect clot at 1, 2, 4 and 24 hours after clotting. Observe for retraction, quality, and lysis of the clot.

3. Remaining 40-60% consists of serum and red blood cell “fall-out” from clot

4. A visual examination of the clot is made. Usually the clot retracts and expresses serum within two hours.

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Interpretation of results

The clot will retract from the walls of the tube until the red cell mass occupies 50% of the total volume of blood in the tube

There is a variable degree of retraction or there is no retraction at all. Grades as follows:

0 : no serum extruded

1+ : 5-10% serum extruded




Results are reported as the length of time it took for the clot to retract

2-4 hrs is reported as normal

After 4 hrs is reported as poor

After 24 hrs is reported as no retraction

Clot lysis test

The whole blood clot lysis time is used to detect increase fibrinolysis. This test is only able to detect high increase in fibrinolytic activity. Clot lysis testing is a measure of the presence of the clot after 24 hours (blood fibrinolysis).

Procedure

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1. At the completion of the clotting time one tube should remain in the 37 water bath

2. A second tube is placed in the refrigerator as soon as clotted as a control

3. The tube is test for the disappearance of clot after 4, 8 and 24 hrs

4. If the sample becomes fluid in less than 48 hrs , the blood is poured out onto a piece of filter paper to be sure for clot disappearance

Interpretation

The disappearance of clot before 48 hrs means increase fibrinolysis

After 48 hrs clot may be

CommentRefrigerated tube370 C tubeLow fibrinogenDisappearDisappearAbnormal fibrinolysis(No clot lysis after 48 hrs)

Still intactStill intact

Nomal fibrinolysisStill intactDisappear

Lab 4 : Coagulation Assay – Prothrombin Time Test

Objective

to help diagnose the cause of unexplained bleeding or inappropriate blood clots.

used for monitoring of warfarin(cuomadin) dosage.

Introduction

Prothrombin time is the time required for the plasma to clot after an excess of thromboplastin and an optimal concentration of calcium have been added. Although the PT was originally described as a specific, one-stage assay of prothrombin (FII),

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it is sensitive to a quantitative or qualitative abnormalities of any of the factors involved in the extrinsic and common pathways of the coagulation system (Factors II, V, VII, X, and Fibrinogen).

The PT used to determine the clotting tendency of blood, in the measure of warfarin dosage, liver damage, and vitamin K status. Occasionally, the test may be used to screen patients for any previously undetected bleeding problems prior to surgical procedures.

When is it ordered

Used to monitor oral anticoagulant therapy (Warfarin / Coumadin).

When a patient who is not taking anti-coagulant drugs has signs or symptoms of a bleeding disorder

When a patient is to undergo an invasive medical procedure, such as surgery, to ensure normal clotting ability.

An elevated Prothrombin time may indicate the presence of :

1. Vitamin K deficiency

2. DIC,

3. Liver disease,

4. Presence of FSP’s

5. A deficiency in one or more of the Concerning factors: Factor I (Fibrinogen), Factor II (Prothrombin), Factor V (Proaccelerin, Labile Factor), Factor VII (Proconvertin, Stable Factor, Factor X (Stuart-Prower Factor, Factor XIII (Fibrin Stabilizing Factor)

6. In addition, inhibitors can cause prolonged PT’s.

The mechanism of coagulation (intrinsic & extrinsic pathways)

33


34


The Standard operating Procedure of the test

Procedure name

Clinical significance

Principle of method

Specimen of choice

Reagents and equipments

Procedure

Reference values

Comments

References Principle

Principle

� When reagent thromboplastin-to which calcium has been added-is mixed with plasma (derived from sodium citrated whole blood), the time (in seconds) it takes for the formation of a clot is reported as the Prothrombin time (PT).

� Calcium is necessary for the correct orientation and binding of a number of complexes including : tissue factor-VIIa, IXa-VIIIa, and Va-Xa.

PT Reagent :

Thromborel® S Reagent : lyophilized human placental thromboplastin, calcium chloride, stabilizers.

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Thromboplastin may be obtained from other sources like Rabbit brain or lung tissue

Neoplastine® C1 Plus : lyophilized fresh rabbit brain thromboplastin with a specific heparin inhibitor hydrated with a solvent containing calcium with stabilizers, polybrene, buffer and preservatives.

(Tissue Factor, coagulation factor III ) protein-lipid complex found in tissues outside blood vessels. It is the combination of both phospholipids and tissue factor, both needed in the activation of the extrinsic pathway.

Recombinant thromboplastin has been produced using human tissue factor in Escherichia coli. These synthetic phospholipids do not contain any other clotting factors such as Prothrombin, factor VII, and factor X.

Therefore they are highly sensitive to factor deficiencies and oral anticoagulant–treated patient plasma samples and have an International Sensitivity Index (ISI) close to 1.

Calibration of Reagent

� Each Thromboplastin Reagents must be calibrated against standard PT reagent established by the WHO. {ISI = International Sensitivity Index}.

� ISI is assigned by the manufacturer for each lot of reagent using reference material from WHO.

� The lower the ISI the more sensitive the Reagent

1. ISI of 1.8 to 2.4 = Low sensitivity

2. ISI of 1.4 to 1.8 = Average sensitivity

3. ISI 1.0 to 1.4 = High Sensitivity

Specimen

Citrated plasma: 1 part of sodium citrate solution (0.11 mol/ L) to 9 part of venous blood, avoiding the formation of foam.

Control: normal plasma (Commercial, Pooled Plasma).

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Pooled normal plasma collection Minimum 20 normal healthy individuals not taking medications that interfere

with clotting factors and coagulation reaction. It is acceptable to include women taking oral contraceptives. An approximately equal number of males and females is desirable.

The age range should be 20 to 50 years.

Equipments

100 μL micropipettes (0.10 mL)

200 μL micropipettes (0.20 mL)

Stop Watch.

Thromborel® S Reagent

Racks

Test tubes

Waterbath (37ºC)

Waste containers/ Biohazard bags

Procedure

Reconstitute tissue thromboplastin according to instructions. Label the thromboplastin with the time, date and initials. The thromboplastin reagent is stabile for 7 days after reconstitution. Allow to sit 10-15 minutes, then invert gently several times.

Mix well prior to pipetting any of this reagent at any step in this procedure.

Prewarming of the reagent by pipetting 1-2 mls, using a plastic pipette, of the tissue PT reagent into a glass test tube and place in a 370 C water bath incubator.

1. Pipette 100 µL of normal control, Patient PPP into each of the test tubes.

2. Allow at least one (1) minute to reach 37°C.

3. Pipette 200 µL of PT reagent into the tube containing the control. Start the stop watch simultaneously.

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4. Mix the tube and leave in the water bath for a minimum of 7-8 seconds. Then remove, wipe the exterior, tilt back and forth gently until a visible clot is formed. As the clot forms, the mixture will gelatinize and may turn cloudy.

5. Stop the stop watch and record the result. If the results from run 1 and run 2 are within + 10% second from each other, average the two results and report with appropriate units.

6. If results are not within required limits, a third run should be performed and average the two that match within acceptable limits.

Be sure and cross out any values you are not using for the final calculation. Include measurement unit of seconds on report sheet.

7. Carry out 1 significant figure passed the decimal point. For example, if your result is 12.23 seconds, report as 12.2 seconds.

8. Repeat the procedure for the samples and Record the time.

Interpretation of Result

Results are expressed as the mean of the duplicate reading in:

1. Seconds

2. Prothrombin ratio

3. International Normalized Ratio (INR)

A Normal Plasma is used to evaluate routine result.

The INR is not used to evaluate Routine PT results.

For Patients who are on oral anticoagulant therapy such as Coumadin INR result must be reported.

Standardization of Report from lab to lab, by using INR results.

Patients with lupus anticoagulants are not be requested for PT as they have antiphospholipid

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Reference ranges

PT: 11.0 – 13.0 seconds.

Therapeutic levels are at a P/C ratio of 2.0 – 3.0

Critical Values

PT critical value changed to > 42.1 seconds. INR changed to > 4.5.

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Interfering Factors

Diet: ingestion of excessive green, leafy vegetables will increase the absorption of vit-K, which promotes blood clots.

Alcoholism, Prolonged PT levels.

Diarrhea and vomiting decrease PT because of dehydration.

Quality of Vein puncture.

Medication : Antibiotics , Aspirin, Cimethidine….

Prolonged Storage of plasma at 4o C.

Sources of Error

1. Associated with specimen (Preanalytical)

a. Inappropropriate ratio of anticoagulant to blood

b. Failure to correct citrate volume if hematocrit > 55%

c. Clotted, hemolyzed or lipemic samples

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d. Lack of PPP

e. Delay in testing or processing

f. Inappropriate storage

2. Associated with Reagent (Analytical)

a. Incorrect preparation of reagents

b. Use of reagents beyond reconstituted stability time or expiration date

c. Contaminated reagent.

3. Associated with procedure (Analytical)

a. Incorrect temperature

b. Incorrect incubation times

c. Incorrect volumes of sample, reagents or both

Notes If thromboplastin reagent does not contain calcium, the test procedure is ml

plasma, 0.1 ml thromboplastin, and clot with 0.1 ml pre-warmed 25mM calcium chloride.

Activation of FIX by tissue factor:FVII occurs in vivo. Under the conditions of most PT tests, FX is so strongly activated that the assay is insensitive to deficiency of FIX or FVIII.

Clotting times are normally influenced by the use of different coagulometers, depending on how and when the end point is detected. This further emphasizes the importance of establishing normal ranges for the method currently in use in the laboratory.

In the presence of mild deficiencies of factor II, V, VII, or X, the degree of prolongation may be minimal. In the case of FII deficiency, the PT may be within the normal range.

Some PT reagents can be affected by the presence of lupus anticoagulants/anti-phospholipid antibodies, and some rare types of antibody may prolong the PT without any prolongation of APTT. Reagents with lower phospholipid concentrations are more likely to be affected, including some reagents that are constructed by lipidating recombinant tissue factor.

The presence of activated FVII, either following therapy with recombinant VIIa or when native FVII has been activated, can shorten the PT. The effect is

41


dependent on the tissue factor reagent used. Reagents containing bovine tissue factor are particularly susceptible to this effect.

Blood samples should not be stored at 2°C–8°C prior to determination of PT, since cold activation of FVII may occur.

PTs determined with reagents containing human tissue factor may be different from those obtained with reagents containing tissue factor from other species, such as rabbit. In such cases, the result obtained with human tissue factor reagents may be more indicative of bleeding risk.

Heparin Extracted Prothrombin Time to better serve dialysis patients on oral anticoagulant therapy, Spectra Laboratories has validated the use of heparin extraction reagent to neutralize heparin-contaminated samples for PT testing. An anion exchange resin is added to the plasma and binds the heparin. The heparin-cellulose complex is removed from plasma by centrifugation. Prothrombin time results equal to or greater than 60 seconds on initial testing, will be retested utilizing the heparin extraction procedure. This procedure effectively absorbs and removes up to 2U of heparin per ml of plasma, producing accurate PT results without residual heparin interference.

Lab 5 : Prothrombin Time (Continue)- ISI & INR

Objective

used to monitor individuals who are being treated with the blood-thinning medication (anticoagulant) warfarin (Coumadin®).

Introduction

The Prothrombin Time (PT) will vary with the type of thromboplastin (e.g. rabbit, human, bovine etc) used in the assay. This difference in sensitivities is known as the sensitivity index. Individual thromboplastins can be calibrated against an international WHO reference thromboplastin (International Reference Preparation or IRP) to assign them an International sensitivity index or ISI. The first WHO reference thromboplastin was assigned an ISI of 1.0

Calculating the ISI

42


https://labtestsonline.org/glossary/anticoagulant/

The calibration of a thromboplastin must be against a reference thromboplastin of the same species e.g. human against human, rabbit against rabbit etc. Prothrombin Times are performed in duplicate for each thromboplastin and the mean for each pair of tests derived.

Tests are performed on 20 normal donors not on anticoagulants and 60 patients who have been on oral anticoagulant treatment for at least 6 weeks. If there is more than a 10% difference in the clotting times between duplicate samples, the tests on that plasma sample should be repeated.

The mean of each pair of PT results are plotted on double-log paper with the reference sample on the Y axis and the test plasma on the X-axis. A best fit line is drawn with points above the highest recorded PT and the lowest PT. The slope of the line is calculated and this represents the ISI.

Slope = A ــ C B ــ C

Example:

Distance A to C = 125mm and distance B to C = 110mm

So the ISI of the test thromboplastin is calculated from the formula:

So if the ISI of the Reference Thromboplastin is 1.1 and the ISI derived from the Slope is 1.14, the ISI of the Test Thromboplastin is 1.1 x 1.14 = 1.25.

Effects of Sensitivity Index:

Thromboplastins should be chosen with an ISI close to 1.0

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The next table show this by Using a thromboplastin with an ISI of 1.0 the PT can range from 15s - 35s but the INR is still in the therapeutic range. In contrast if the ISI of the thromboplastin is 2.0 an increase in the PT from 15s to 30s results in an INR outwith the therapeutic range.

Patient PT [s]

Thromboplastin 1 [ISI 1.0]



15 1.25 1.39 1.5420 1.67 2.15 2.7825 2.08 3.00 4.330 2.5 3.95 6.2535 2.91 4.98 8.51

The previous table illustrates a number of patients with varying PTs and in whom the INR was calculated using three Thromboplastins with varying ISIs. The simplest way to calculate the INRs is to use an electronic calculator.

Local Calibration

Use of different coagulometers for the PT and the differing methods of end-point detection can lead to significant variations in PT. For these reasons, a local calibration of thromboplastins is recommended. This involves testing a set of plasma samples with known INRs with the a laboratory-specific thromboplastin and on the coagulometers which will be used to derive the PT.

Reporting of Result

Time (Seconds)

Ratio (PTpatient/PTcontrol) PTr

Activity Percentage PTp

INR

44


45


PT Activity percentage

The result reported as percent activity of normal instead of seconds as the unit of measurement of the Quick test. A study show that the results were expressed in PTp but not INR, PTs and PTr is valid for comparison of patients with liver disease. In patients on oral anticoagulant therapy, only INR standardize PT results.

Pooled, normal plasma that is assumed to be 100% of normal activity is diluted with any variety of diluents (barium-sulfate-adsorbed human plasma, barium-sulfate-adsorbed beef plasma, 0.85% saline, imidiazole-saline buffer).

Prothrombin activity assayed by on stage assay.

To obtain PTp expression, a saline dilution curve was constructed with normal pool plasma and the patient result was expressed as the percentage of normal plasma yielding the same PT in seconds.

1:801:401:201:10 Pooled Plasma

12.5%25%50%100%Percentage

The International Normalised Ratio

The International Normalised Ratio (INR) is the PT ratio of a test sample compared to a normal PT (of 20 normal donors) corrected for the sensitivity of the thromboplastin used in the test. It is the value for the Prothrombin Time Ratio which has been obtained using the first WHO Reference Thromboplastin with an ISI of 1.0

Example; for patient on warfarin and his PT 23 and the Normal PT is 12 with ISI 1.2

46


http://ukpmc.ac.uk/abstract/MED/19330428/?whatizit_url_Chemicals=http://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:50249

http://ukpmc.ac.uk/abstract/MED/19330428/?whatizit_url=http://ukpmc.ac.uk/search/?page=1&query=%22liver%20disease%22

Lab 6 : Activated Partial Thromboplastin Time (aPTT)

Objective

Monitoring heparin therapy (unfractionated heparin). Screening for certain coagulation factor deficiencies. Detection of coagulation inhibitors such as lupus anticoagulant, specific factor

inhibitors, and nonspecific inhibitors.Introduction

Also known as: Partial Thromboplastin Time (PTT); Kaolin Cephalin Clotting Time (KCCT). Is a performance indicator measuring the efficacy of both the "intrinsic"

47


(the contact activation pathway) and the common coagulation pathways. APTT is the most widely used method for monitoring intravenous heparin anticoagulant therapy (Heparin acts to accelerate Antithrombin which inhibits the actions of thrombin). The PTT evaluates the coagulation factors XII, XI, IX, VIII, X, V, II (prothrombin), and I (fibrinogen) as well as prekallikrein (PK) and high molecular weight kininogen (HK). It measures deficiencies mainly in factors VIII, IX, XI, and XII, but can detect deficiencies of all factors except III and VII. The PTT can also detect Nonspecific inhibitors, such as lupus anticoagulant and anticardiolipin antibodies, which associated with clotting episodes and with recurrent miscarriages, especially those that occur in the second or third trimester.

When is it ordered

Unexplained bleeding or bruising(The PTT may be ordered along with other tests such as a PT)

A thromboembolism like DIC or Liver Disease. Recurrent miscarriages or a thrombotic episode , the PTT may be ordered as

part of an evaluation for lupus anticoagulant or anticardiolipin antibodies. A person is on intravenous (IV) or injection heparin therapy (Monitoring

Therapy). (the Therapeutic range should be 1½ to 2½ times normal). When someone is switched from heparin therapy to longer-term warfarin

(COUMADIN®) therapy, the two are overlapped and both the PTT and PT are monitored until the person has stabilized.

Pre-surgical evaluation for bleeding tendencies.

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Clinical Significance

When the partial thromboplastin time is used in combination with the prothrombin time, most procoagulant disorders can be classified.

Principle

Patient platelet poor plasma (PPP) is incubated at 37°C then phospholipid (cephalin) and a contact activator (e.g. Kaolin) are added followed by the calcium (all pre-warmed to 37°C). Addition of calcium initiates clotting and timing begins for a fibrin clot to form.

Kaolin is used as a surface activator. It binds directly to FXII resulting in its activation to XIIa. XIIa cleaves FXI to XIa but in the absence of calcium, activation of the subsequent factors does not occur.

Cephalin is a phospholipid substitute that replaces platelet phospholipid in the test (remember the test uses platelet poor plasma and so requires a source of

49


phospholipid for coagulation to occure).

Procedure

1. Pre- warm a sufficient quantity of calcium chloride reagent to 37oC for at least 10 min.

2. Pipet 100 µL of Sample into a labeled test tube, incubate 1-2 min.

3. Into each test tube, add 100 µL of partial thromboplastin reagent.

4. Incubate the mixture at 37oC for a minimum of three minutes. (optimum activation of contact factors)

5. Forcefully add in100 µL of calcium chloride into mixture and start the stop watch immediately.

6. Mix the tube once, immediately after adding the calcium reagent. Allow the tube to remain in the water bath, approximately 20 seconds, mixing occasionally.

50


7. After 20 seconds, remove the tube from the water bath/heat block. Wipe off the outside of the tube. Gently tilt the tube back and forth until a visible clot forms. Stop the stop watch immediately and record the time in seconds.

8. Carry out 1 significant figure passed the decimal point. For example, if your result is 30.31, report as 30.3 seconds.

9. The result must be run in duplicate.

10. Repeat the steps using Normal Control.

Reference Range

Reference Range: 27-35 seconds.

Critical Value: > 70 seconds

Sources of Error

1. Associated with specimen (Preanalytical)

a. Inappropropriate ratio of anticoagulant to blood

b. Failure to correct citrate volume if hematocrit > 55%

c. Clotted, hemolyzed or lipemic samples

d. Lack of PPP

e. Delay in testing or processing

f. Inappropriate storage

2. Associated with Reagent (Analytical)

a. Incorrect preparation of reagents

b. Use of reagents beyond reconstituted stability time or expiration date.

c. Contaminated reagent.

3. Associated with procedure (Analytical)

a. Incorrect temperature.

b. Incorrect incubation times.

c. Incorrect volumes of sample, reagents or both.

Notes

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The activator for the APTT e.g. kaolin, silica etc should be selected to ensure that the reaction is sensitive to mild deficiencies of factors VIII, IX and XI (i.e. levels of 0.35-0.4 IU/ml).

If an optical density method is used to monitor clot formation, falsely normal APTTs may occur if the patient’s plasma is turbid (e.g. hyperbilirubinaemia, hyperlipidaemia).

A deficiency of factor XIII does not prolong the APTT or the PT.

When selecting a phospholipid for the APTT it is important to choose a reagent that is sensitive to deficiencies in clotting factors. Some laboratories may choose APTT reagents that are insensitive to a Lupus Anticoagulant to prevent it (if present) in interfering with factor assays. In such cases, an alternative reagent may be necessary if the APTT is used as a screening test for a lupus anticoagulant.

The APTT can be made more or less sensitive to specific clotting factors by varying the incubation time. A short incubation time e.g. 2 minutes makes the test very sensitive to the levels of contact factors whereas a long incubation period make it very insensitive to these factors. Many laboratories employ a 5 minute incubation period. If a contact factor deficiency is suspected, then comparing a short and long incubation time may be useful.

In patients receiving very high concentrations of unfractionated heparin e.g. during cardiopulmonary bypass, the APTT will be unclottable. In these cases, monitoring of the degree of heparinisation is undertaken using a different assay - usually the Activated Clotting Time (ACT).

New anticoagulants are used now Ribaroxaban and Dabigatran

Lab 7 : Mixing Studies

Objective

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A mixing study is instrumental to determine the underlying cause for the unexplainable prolongation of aPTT or PT in plasma samples of patients in clinical settings.

Introduction

Plasma samples found to have abnormal screening tests (i.e. PT/APTT) may be further investigated to define the abnormality by performing mixing tests. Mixing studies are tests performed on blood plasma used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII.

� Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal Prothrombin time (PT) or Partial Thromboplastins time. Mixing studies can help determine the appropriate next steps to take to diagnose the cause of an abnormal APTT or PT.

Principle

� The patient plasma is mixed 1:1 with Normal pooled plasma that contains 100% of the normal factor level results in a level ≥ 50% in the mixture (say the patient has an activity of 0%; the average of 100% + 0% = 50%).

� Therefore, correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor.

�

Some inhibitors are time dependent. The clotting test performed immediately after the specimens are mixed may show correction because the antibody has not had time to inactivate the added factor (false positive). A test performed after the mixture is incubated for 2 hours at 37°C will show prolongation.

53


� Nonspecific inhibitors like the lupus anticoagulant usually are not time dependent; the immediate mixture will show prolongation.

� Many specific factor inhibitors are time dependent, and the inhibitor will not be detected unless the test is repeated after incubation (factor VIII inhibitors are notorious for this).

� Reagents and Equipment

Pooled Plasma - platelet-poor plasma from 20 or more healthy, male and female adult donors.

DO NOT use a single-sourced normal plasma. Pooled plasma must be used to ensure approximately 100% of all factors are

present. Do Not Use Lyophilized Normal Control. Other reagents required to perform the screen test(s) (i.e., PT or PTT).

Quality Control

The pooled plasma must be evaluated for the test to be performed and results must fall within the reference range or testing is repeated with a fresh aliquot of the pooled plasma.

Procedure

1. Prepare a 1:2 dilution of patient plasma using pooled plasma as the diluents, by mixing equal volumes of each of the plasmas. (make sufficient quantities to run the test in duplicate)

2. Label two test tubes for each test plasma to be re-tested (Mixture, NPP)

3. Add 0.1 ml of patient plasma to 0.1 ml of NPP in one of the two labeled tube

4. Carefully mix the plasmas using the pipette, aspirating and expelling the solution several times (avoid making bubbles).

5. Transfer 0.1 mL of the diluted patient plasma to the second labeled test tube.

6. Measure the APTT or PT for the mixed and incubated tube, and the control tube.

In cases where time and temperature dependent inhibitors are suspected, repeat testing should also be performed on incubated mixes: patient plasma – pooled plasma mix incubated for 1 to 2 hours at 37° C prior to testing.

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1. Mix patient plasma with pooled normal plasma in equal volumes in a plastic test tube. In two separate tubes, pipet a volume of patient plasma and a volume of pooled normal plasma.

2. Incubate all 3 tubes for 1 to 2 hours at 37°C.

3. Combine the incubated patient plasma tube and the incubated pooled normal plasma and use as the control tube.

4. Measure the APTT or PT for the mixed and incubated tube, and the control tube.

Expected Values

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Prolonged PTT

Correction

Incubation 2 hours in (370c)

Correction

Factor defeciency

No correction

Tube D (control )

Correction

Specific inhibitor

No correction

Labile factors are affected

No Correction

Inhibitor (non specific)

Interpretation

The first step when evaluating unexpected prolonged PT or PTT results is to rule out preanalytical interference, e.g., presence of contaminating heparin.

If the APTT or PT is corrected by normal plasma, a factor deficiency is indicated.

If the APTT or PT is not corrected by the addition of nor-mal plasma immediately, a strong inhibitor is indicated.

A weak or time-dependent inhibitor is indicated by a prolonged APTT or PT following incubation at 37°C for 1 to 2 hours ( factor VIII inhibitor).

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Table: A Differentiation of Factor Deficiency and Inhibitors By Mixing Studies

1:1 Mixing Study ResultsNot incubated Incubated

Factor deficiency Correction Correction

Immediate acting inhibitor No correction No correctionTime/temperature dependent inhibitor Correction (Falsely) No correction

Possible Interpretations

Coagulation Screen Results:PT prolongedPT mixing study results:PT correctsMost likely interpretation:Factor VII deficiency

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Probable cause(s):Early response to warfarin, early vitamin K deficiencyRare cause:Congenital factor VII deficit

Coagulation Screen Results:PTT prolongedPTT mixing study results:PTT correctsMost likely interpretation:Factor deficit

Probable cause(s):Factor VIII or IX (male) deficiency, or von Willebrand Disease (female)

Possible causeFactor inhibitor

Coagulation Screen Results:PTT markedly prolonged (>200 seconds)PTT mixing study results:PTT correctsMost likely interpretation:Severe Contact Factor deficitProbable cause(s):Factor Prekallikrein, HMWK, XI, or XII

Coagulation Screen Results:PT and PTT prolongedPT & PTT mixing study results:PT and PTT correctMost likely interpretation:Acquired, multiple factor deficiencyProbable cause(s):DIC, Liver Disease, Vitamin K deficiencyPossible cause:Warfarin therapy

Coagulation Screen Results:PTT slightly – moderately prolongedPTT mixing study results:No correctionMost likely interpretation:Immediately reacting antibody inhibitorProbable cause(s):Lupus anticoagulant

Comment

The antibody that inhibits factor VIII is most often a specific IgG antibody (temperature and time dependent) , which will cause only a slightly prolonged APTT on initial testing.

If a factor VIII inhibitor is present, it is important to determine the initial level of factor activity because the development of an inhibitor complicates the management of a patient with hemophilia A when therapy involves AHF* concentrates. These should be monitored periodically.

Repeating the mixing study with 4 parts patient sample and 1 part normal pooled plasma may increase the chance of detecting a weak inhibitor.

Notes

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Be careful when thawing the pooled plasma because prolonged incubation at 37°C will selectively decrease Factor V, prolonging the results and making interpretation of the 1:1 mix test results difficult.

The pooled normal plasma is stable for ~2 hours at room temperature. Initial test results for the pooled normal plasma must be within the reference range or the mix should be repeated with a fresh aliquot of pooled normal plasma.

Lab 8 : Lupus Anticoagulant

Introduction

Anti-phospholipid antibodies are a heterogeneous family of autoantibodies that react with epitopes on proteins that are themselves complexed with negatively charged phospholipids. The blood vessel problems can then lead to complications such as stroke, heart attack, and miscarriage.

The two most commonly measured kinds of Antibodies are:

Lupus anticoagulant Anticardiolipin antibody

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Lupus Anticoagulants (lupus antibody, LA, or lupus inhibitors)

Lupus anticoagulants (LA) are heterogeneous IgG or IgM autoantibodies which inhibit phospholipid-dependent assays of blood coagulation by binding to plasma phospholipid-binding proteins such as beta 2-glycoprotein I (β2-GPI) or prothrombin.

Lupus Anticoagulant was found in patient with SLE. Most patients with a lupus anticoagulant do not actually have lupus Erythematosus, and only a small proportion will proceed to develop this disease. It is a prothrombotic agent, that is, presence of Lupus anticoagulant antibodies precipitates the formation of thrombi in vivo. In vivo, it is thought to interact with platelet membrane phospholipids, increasing adhesion and aggregation of platelets; thus it's in vivo prothrombotic characteristics.

The term "anticoagulant" accurately describes its function in vitro, but in vivo, it is now known that it functions as a coagulant. Lupus anticoagulants impair the in vitro phospholipid dependent activation of factor X, factor IX, and Prothrombin.

Presence of these antibodies causes an increase in aPTT. The initial workup of a prolonged PTT is a mixing test, if the mixing test indicates an inhibitor, diagnosis of a lupus anticoagulant is then confirmed with phospholipid-sensitive functional clotting testing, such as the dilute Russell's viper venom time, or the Kaolin clotting time

The presence of an LA is usually not associated with a bleeding problem unless accompanied by

Thrombocytopenia,

Factor II deficiency,

Platelet dysfunction

Or drug administration (e.g., aspirin).

The ISTH(International Society on Thrombosis and Haemostasis ) recommends that the laboratory diagnosis of lupus anticoagulants (LA) should be carried out on double-centrifuged plasma following a four-step procedure adhering to these principles:

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Prolongation of a phospholipid-dependent coagulation test. Extrinsic (dPT), Intrinsic (aPTT, dilute aPTT, KCT, colloidal silica clotting time), Final common pathway (dRVVT, Taipan venom time, Textarin and Ecarin time)

Evidence of inhibitory activity on mixing tests. Evidence of phospholipid dependence.

Correction of the prolonged coagulation time after addition of excess phospholipid or platelets

Lack of specificity for any one coagulation factor Lack of specifi c inhibition of one coagulation factor (such as

FVIII:C,FIX:C, or FXI).

Anti-Phospholipid Antibody Syndrome

APS is present if at least one of the clinical criteria and one of the laboratory criteria that follow are met:

Clinical criteria: vascular thrombosis or pregnancy morbidity

Laboratory criteria:

1. Lupus anticoagulant present in plasma on two or more occasions at least 12 weeks apart.

2. Anticardiolipin antibody, IgG or IgM positive in medium or high titer on two or more occasions at least 12 weeks apart

3. Anti-β2GPl antibody, IgG or IgM present on two or more occasions at least 12 weeks apart.

The positive laboratory criteria and the clinical criteria should occur within 5 years of each other.

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Anti-Cardiolipin Antibodies

There is many classes of anticardiolipin antibody, abbreviated as IgG, IgM, and IgA. The IgG type of anticardiolipin antibody is the type most often associated with complications. Some lupus patients with very high IgM anticardiolipin antibody have a problem called hemolytic anemia, in which their immune system attacks their red blood cells. The anticardiolipin antibody is measured in an ELISA test.

Laboratory Investigations

Immunological Assays

Individuals with Lupus anticoagulants may also show the presence of other anti-phospholipid antibodies. The most frequent finding is the presence of Anti- cardiolipin antibodies.

The commonly employed method is the ELISA technique where the solid phase is coated with cardiolipin and β-2 GPI (as a cofactor). The ELISA method detects IgM, IgG and IgA class of anti-cardiolipin antibodies.

An important point to note is that Lupus anticoagulants and anti-cardiolipin antibodies differ in their phospholipid-binding characteristic hence detection of anti

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cardiolipin antibodies is not specific for the presence of Lupus anticoagulants though they may be present in the same patient.

Clot based assays

1. APTT ( Activated Partial Thromboplastin Time)

2. TTI (Tissue Thromboplastin Inhibition test)

3. KCT (Kaolin Clotting Time)

4. PNP (Platelet Neutralization procedure)

5. Hexagonal Phase Phospholipids (Staclot-LA)

APTT ( Activated Partial Thromboplastin Time)

The specificity of the APTT to LA is reduced due to:

Different reagents have varying sensitivity for the presence of Lupus anticoagulants on the basis of their phospholipid composition ( variability of the test)

The APTT test is affected by the presence of inhibitors to Factor VIII, IX and XI.

The APTT test is also the test of choice for monitoring heparin therapy.

TTI (Tissue Thromboplastin Inhibition test)

The Tissue Thromboplastin Inhibition test utilizes a diluted PT reagent. The results are expressed as ratio of patient values to normal control values.

The TTI test is affected by numerous variables:

Species and tissue origin of thromboplastin can affect the test results as different sources of thromboplastin have varying sensitivity and responsiveness.

Choice of “Normal” reference plasma is the most critical variable, because depending on the laboratory the choice of reference plasma could be lyophilized plasma, a frozen plasma pool or fresh plasma. The ratio of patient to normal can therefore change according to the choice of “normal” plasma.

Some IgM Lupus anticoagulants do not prolong the TTI test

KCT (Kaolin Clotting Time)

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KCT is similar to APTT, the difference being that KCT reagent is devoid of phospholipids and incorporates Kaolin as contact activator. The test is performed on a range of mixture of normal and patient’s plasma. Different patterns of response are obtained indicating the presence of Lupus anticoagulants or the deficiency of one or more coagulation factors.

The KCT test though sensitive is not specific for LA, additionally:

It cannot be automized

The test shows prolonged results with factor VIII, IX, XI & XII deficiency or corresponding inhibitors

The test is also highly sensitive for the presence of heparin.

PNP (Platelet Neutralization procedure)The platelet neutralization procedure (PNP) is based on the ability of platelets to significantly correct in vitro coagulation abnormalities. The disrupted platelet membranes present in the freeze-thawed platelet suspension neutralize phospholipid antibodies present in the plasma of patients with LA.

After the patient plasma is mixed with the freeze-thawed platelet suspension, the APTT will be shortened when compared with the original baseline APTT. But if an inhibitor is directed against specific coagulation factor, the clotting time is not shortened. A correction of the baseline APTT of a defined amount of time (i.e., 3 to 5 seconds or more) by the platelet suspension as compared with the control is indicative of the presence of an LA.

Due to limited stability the platelet preparations loose their activities on storage hence do not show reproducible results.

Hexagonal Phospholipid Neutralization Assay

The hexagonal phospholipid neutralization assay uses the same principle as the PNP assay, normalization of the aPTT in the presence of added phospholipid, but this assay specifically uses a phospholipid in a hexagonal conformation, Neutralization by this hexagonal form in an assay with a very lupus-anticoagulant sensitive aPTT reagent, is a more sensitive confirmation test than the PNP. Comment: Specimen collection, centrifugation, and processing are critical

when testing for the presence of an LA.

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Confirmatory Tests (for Lupus Anticoagulants)Confirmatory tests to identify an LA include those that utilize a low concentration of phospholipid in the test system, thereby increasing the LA effect such as The tissue thromboplastin inhibition test (TTIT), Dilute Russell's viper venom time (dRVVT), And the kaolin clotting time (KCT), or Those that increase the phospholipid, thereby neutralizing the LA effect, such

as the platelet neutralization

Dilute Russell’s Viper Venom Time (DRVVT)dRVVT : The test of choice for screening and confirmation of LA Indicating the phospholipid dependence of LA Achieving maximum sensitivty for the precence of LA’s.

In general dRVVT based tests comprise of: SCREENING REAGENT, containing limited amount of phospholipids with

RVV (Russell’s Viper Venom) CONFIRMATION REAGENT, containing additional phospholipids with same

amount of Russell’s Viper Venom, to confirm the presence of phospholipid dependent Lupus anticoagulants.

Principle of dRVVT for LA Detection

Russell’s Viper Venom directly activates Factor V and X in presence of phospholipid and calcium ions, bypassing Factor VII of the extrinsic pathway and the contact and antihaemophilic factors of the intrinsic pathway.In normal plasma, in the absence of lupus anticoagulants, Factor V and X is directly activated by Russell’s Viper Venom, which in presence of phospholipid and calcium ion leads to clot formation.

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In patients with LA, autoantibodies bind the epitopes of reagent phospholipids thereby preventing the activation of prothrombinase complex. This results in a prolongation of clotting time with SCREENING reagent. The CONFIRMATION reagent incorporates additional phospholipids to neutralize the LA. Once LA are neutralized clot formation proceeds relatively uninterrupted achieving a lower clotting time, to prove the phospholipids dependence of the autoantibodies.

Interpretation Of Results With drvvt Test If SCREEN TIME is prolonged, to confirm the presence of lupus

anticoagulants the plasma sample is tested with CONFIRMATION REAGENT.

If CONFIRM TIME results shows a lower clotting time as compared to SCREEN TIME, it indicates the presence of phospholipid dependant Lupus Anticoagulants.

Also the results can be expressed as ratio.

The results expressed, as ratio is further useful in classifying the patient as

normal, moderate, high and very high LA. If results of the ratio are borderline, mixing studies may be done further with the sample specimen. The mixing studies should be carried out with a 50:50 mixture of test plasma and normal plasma.

D-Dimer Test

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Objective used to help rule out the presence of an inappropriate blood clot (thrombus).

Introduction

D-dimer (Fragment D-dimer; Fibrin degradation (fragmentD-dimer is a fibrin degradation product (or FDP), a small protein fragment present in the blood after a blood clot is degraded by fibrinolysis. It is so named because it contains two crosslinked D fragments of the fibrinogen protein.D-dimer test used to diagnose thrombosis, disseminated intravascular coagulation.

The circulating enzyme plasminncleaves the fibrin gel in a number of places. The resultant fragments, "high molecular weight polymers", are digested several times more by plasmin to lead to intermediate and then to small polymers (fibrin degradation products or FDPs). The cross-link between two D fragments remains intact, however, and these are exposed on the surface when the fibrin fragments are sufficiently digested. The typical D-dimer containing fragment contains two D domains and one E domain of the original fibrinogen molecule

the D-dimer assay depends on the binding of a monoclonal antibody to a particular epitope on the D-dimer fragment. Several detection kits are commercially available;

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all of them rely on a different monoclonal antibody against D-dimer. Of some of these it is known to which area on the D-dimer the antibody binds. The binding of the antibody is then measured quantitatively by one of various laboratory methods.

D-dimer testing is of clinical use when: There is a suspicion of deep Venous Thrombosis (DVT) Pulmonary Embolism (PE) Disseminated Intravascular Coagulation (DIC). It can also rise Postoperatively. It is under investigation in the diagnosis of Aortic Dissection

Interpretations of Results A very high score, or pretest probability, a D-dimer will make little difference

and anticoagulant therapy will be initiated regardless of test results, and additional testing for DVT or pulmonary embolism may be performed.

For a moderate or low score, or pretest probability A negative D-dimer test will virtually rule out thromboembolism: the

degree to which the D-dimer reduces the probability of thrombotic disease is dependent on the test properties of the specific test used in the clinical setting: most available D-dimer tests with a negative result will reduce the probability of thromboembolic disease to less than 1% if the pretest probability is less than 15-20%

If the D-dimer reads high, then further testing (ultrasound of the leg veins or lung scintigraphy or CT scanning) is required to confirm the presence of thrombus. Anticoagulant therapy may be started at this point or withheld until further tests confirm the diagnosis, depending on the clinical situation.

False positive readings can be due to various causes: liver disease, high rheumatoid factor, inflammation, malignancy, trauma, pregnancy, recent surgery as well as

advanced age[citation needed] False negative readings can occur

if the sample is taken either too early after thrombus formation or if testing is delayed for several days. Additionally, the presence of anti-coagulation can render the test negative

because it prevents thrombus extension. Likelihood ratios are derived from sensitivity and specificity to adjust pretest

probability.

Lab 9 : Tests to measure fibrin clot

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Objective The fibrinogen test aids in the diagnosis of suspected clotting or bleeding disorders c

aused by fibrinogen abnormalities. Detecting increased or decreased fibrinogen (factor I) concentration of acquired or

congenital origin

Introduction

The fibrinogen test is a blood test that evaluates the blood clotting mechanism, and used to evaluate fibrinogen, a protein that is essential for blood clot formation. When there is an injury and bleeding occurs, the body forms a blood clot through a series of steps. In one of the last steps, soluble fibrinogen is converted into insoluble fibrin threads that crosslink together to form a net that stabilizes and adheres at the injury site until the area has healed.

There are different assays that measure fibrinogen levels:

Thrombin Time TT Reptilase Time Fibrinogen Activity assay

Thrombin Time (TT) Also called Thrombin Clotting Time (TCT)The thrombin time (TT) is the time required for thrombin to convert fibrinogen to an insoluble fibrin clot. It does not measure defects in the intrinsic or extrinsic pathways. The test is affected by Abnormal levels of fibrinogen (usually less than 100 mg/dl.) and

dysfibrinogenemia The presence of antithrombins such as heparin and direct thrombin inhibitors

such as hirudin and FDPs.PrincipleThe thrombin time involves only the addition of bovine or human thrombin to platelet poor plasma. It, therefore, reflects the conversion of fibrinogen to fibrin but is also sensitive to the presence of inhibitors that may be present in the plasma e.g. heparin. Thrombin cleaves fibrinogen, releasing fibrinopeptide A (FpA) and fibrinopeptide B (FpB).

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https://labtestsonline.org/glossary/protein/

Commercially prepared bovine thrombin reagent at 2 NIH units/mL cleaves fibrinopeptides A and B from plasma fibrinogen to form a detectable polymer

1 WHO unit = 0.56 NIH unit 1 NIH unit = 0.324 +/- 0.073 µg

Reptilase (Atroxin)TimeThe Reptilase time is a modification of the thrombin time in which the purified enzyme Reptilase is used to replace thrombin. It is a thrombin-like enzyme, isolated from the venom of Bothrops atrox, that catalyzes the conversion of fibrinogen to fibrin in a manner similar to thrombin. Reptilase cleaves fibrinogen releasing fibrinopeptide A (FpA) generating fibrin. In contrast thrombin cleaves both fibrinopeptide A and fibrinopeptide B from fibrinogen to generate the fibrin clot.

Interpretation All the congenital dysfibrinogenemias have an infinite reptilase time. The reptilase time is also infinitely prolonged in cases of congenital

afibrinogenemia. In states of hypofibrinogenemia, the reptliase time may be variable, depending

on the levels of fibrinogen present. The reptilase time is moderately prolonged in the presence of FDPs and is

unaffected by heparin.

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Comment In the presence of heparin, thrombin is inhibited through the interaction of

antithrombin (AT-III). However, heparin does not interfere with the ability of reptilase to cleave fibrinopeptide A from fibrinogen

Ancrod a similar enzyme from Agkistrodon rhodostoma can also be used to replace thrombin in the thrombin clotting time test.

Reptilase TimeThrombin TimeNormal↑Presence of unfractionated

heparinNormalMay show some

prolongationPresence of LMWH

Normal↑Presence of direct thrombin inhibitors

NormalNormalWarfarin↑↑Decreased/absent fibrinogen,

Dysfibrinogenaemia↑↑DIC, Liver disease

Normal↑Heparin-like anticoagulants↑↑Parproteinaemias,

Thrombolytic therapy,Neonate, Amyloid

↑↑Hypoalbuminaemia

FibrinogenFibrinogen concentration can be measured in 3 ways. Fibrinogen concentration is usually reported in milligrams per deciliter (mg/dl). Heat precipitation:(Heating at 56o C will precipitate fibrinogen only) Clotting method - thrombin clot time: Immunologic assays: Fibrinogen antigen levels may be assayed by means of

radial immunodiffusion (RID) or nephelometry

Fibrinogen activity test PrincipleFibrinogen assays are quantitative techniques to measure the amount of functional fibrinogen present in the plasma. The assay is based on the Clauss assay, which is the reference method. This fibrinogen assay measures the time required for thrombin to convert fibrinogen to fibrin.

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The procedure is a determination based on fibrinogen activity, but results are converted to concentration (mg/dL) by comparison with control plasma results. In the fibrinogen procedure, thrombin is added to various dilutions of known concentrations of fibrinogen to produce a thrombin-clotting time in seconds. The clotting times are then plotted on a graph, with the known concentrations on the x-axis, versus the clotting time on the y-axis. The clotting times are performed using controls and the patient sample at a 1:10 dilution. An excess amount of thrombin reagent is added and the time it takes for the specimen to clot is recorded in seconds. This time is then converted to mg/dL of fibrinogen by comparing these results to results obtained on a fibrinogen standard curve. Patient results may be read directly off of the standard curve graph, or off of a data chart prepared from the graph that already converts time in seconds to mg/dL. The time it took for the specimen to clot is inversely proportional to the fibrinogen concentration in mg/dL. For instance, a prolonged fibrinogen clotting time means the fibrinogen level (mg/dL) is low.

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Reagents and Equipment

Test tubes

Commercial fibrinogen determination kit:

Thrombin, 100 National Institutes of Health (NIH) units/mL, bovine lyophilized (reconstitute with 2 ml Distilled water)

Fibrinogen standard

Imidazole buffer,

Control (with a known fibrinogen concentration)

Procedure

1. Collect blood by clean venipuncture technique according to recommended procedures previously described.

2. Process and store plasma samples following recommended guidelines.

3. Reconstitute the thrombin reagent according to the manufacturer's directions.

4. This assay is commonly performed on a coagulation analyzer.

Preparation of Calibration Curve

The calibration curve is prepared from the reference standard .

Make dilutions of the fibrinogen standard with Imidazole buffer as follows: 1:5, 1:10, 1:20 and 1:40. Make all transfers from the first test tube.

Calibrator = 317 mg/dlDilutionBuffer

(mL)Fibrinogen standard

(mL)Tube

63.41:50.80.2131.71:100.40.4 of tube 1 (mixed)215.851:200.40.4 of tube 2 (mixed)37.9251:400.40.4 of tube 3 (mixed)4

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Perform determinations on each dilution of the fibrinogen standard as follows:

A. Incubate 0.1 mL of fibrinogen standard dilution at 37°C for at least 2 minutes but no more than 5 minutes.

B. Add 0.05 mL of thrombin reagent.

C. Measure the clotting time. performed in duplicate, and average the results.

Sample Assay

1. Prepares a 1: 10 dilution of each patient PPP and control with Imidazole buffer.

2. Incubate 0.1 ml of the patient dilution at 37°C for at least 2 minutes but no more than 5 minutes.

3. Add 0.05 mL of thrombin reagent.

4. Measure the clotting time. run in duplicate.

Interpretation

Reference range: 200-400 mg/dL

Prolonged clotting times may indicate either

A low fibrinogen concentration

The presence of inhibitors such as heparin or circulating FDPs.

Some manufacturers include a heparin neutralizer in the fibrinogen reagent that will negate any interference by therapeutic levels of heparin.

The effect of heparin may also be excluded by

treatment of the sample with a heparin-digesting enzyme

performing the reptilase time, because reptilase is unaffected by heparin.

Clinical Significance

There are several causes for a deficiency of fibrinogen.

Severe hemorrhaging may result in any case.

congenital deficiencies may be due to

1. Afibrinogenemia (a lack of fibrinogen)

2. a dysfibrinogenemia (abnormal fibrinogen)

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Acquired deficiencies may be due to

1. liver disease

2. disseminated intravascular coagulation (DIC)

3. fibrinolysis

High fibrinogen levels are seen

During pregnancy

In women taking oral contraceptives.

In patients in a hypercoagulable state such as with thrombosis.

Fibrinogen is considered an acute-phase reactant, and, therefore, high levels may be seen in states of acute infection, neoplasms, collagen disorders, nephrosis, and hepatitis along with other conditions causing physical stress.

NOTE

For fibrinogen values out of the linearity range (46-700 mg/dL for this fibrinogen standard curve) a 1:10 dilution of the plasma will not work and a different dilution must be used.

For extremely high fibrinogen levels (>700 mg/dL) a 1:20 dilution of the plasma is used for the procedure. However, due to the change in dilution, the result read off of the fibrinogen data table must be multiplied by a factor of 2 (since our 1:20 dilution is 2 times the 1:10 dilution originally meant for the data table).

For extremely low fibrinogen levels (<46 mg/dL) a 1:5 dilution of the plasma is used for the procedure. The result read off of the data table must then be divided by a factor of 2 (since our 1:5 dilution is half of the 1:10 dilution originally meant for the data table).

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Lab 10 : One Stage Factor V Assay

Objective

used for the measurement of factors V.

Introduction

When Prothrombin time (PT) and activated partial Thromboplastins time (APTT) are prolonged, a one-stage assay is used to detect a deficiency of factor II, factor V, or factor X. If PT is abnormal but APTT is normal, factor VII may be deficient. The percentage of factor activity is determined by the amount of correction of the PT when specific dilutions of patient plasma are added to the factor-deficient plasma. These results are obtained from an activity curve made using clotting times of dilutions of normal reference plasma and specific factor deficient plasma.

Principle

The assay of a clotting factor relies upon measuring the degree of correction of the Prothrombin time (PT) when plasma is added to a plasma sample specifically deficient in the factor to be measured.

Reagents and Equipment

PT / PTT Reagent

Specific factor-deficient plasma (II, V, VII, and X)

Imidazole buffered saline, pH 7.3 ± 0.1

Normal reference plasma (commercial reference plasma with known factor levels)

Note

It is recommended that the factor-deficient plasma utilized be verified as having less than I% activity for the specific factor being measured and close to 100% activity of all other factors.

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Procedure

Preparation of the activity curve.

Procedure for testing patient plasma.

Preparation of the activity curve

1. Prepare 1:10, 1:20, 1 :40, 1:80, 1:160, 1:320, 1:640, and 1:1280 serial dilutions of the normal reference plasma with Imidazole-buffered saline.

The 1: 10 dilution is considered 100% factor activity.

It is recommended that at least five dilutions be used to prepare the factor activity curve, although it is common to use seven or eight dilutions (Table).

% of FactorDilutionBuffered

Saline(mL)

Amount of plasma (mL)

Tube No.

1001:100.90.11501:200.50.5 of tube no. 12251:400.50.5 of tube no. 23

12.51:800.50.5 of tube no. 346.251:1600.50.5 of tube no. 453.131:3200.50.5 of tube no. 561.561:6400.50.5 of tube no. 670.781:12800.50.5 of tube no. 88

2. Warm Thromboplastins to 37°C.

3. Perform the following test procedure on each dilution.

1) Add 0.05 mL of specific factor-deficient plasma to 0.05 mL of the diluted normal reference plasma and warm to 37°C for the 2 min.

2) Add 0.2 mL of commercial Thromboplastins to the sample and determine the clotting time.

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4. Plot results on 2 x 3 cycle log graph paper, with percent factor activity on the x axis and seconds on the y axis. Draw a best-fit line.

The curve will demonstrate a plateau at the least concentrated dilutions and should be plotted as such, demonstrating the end of sensitivity for the assay.

If using an automated analyzer, the curve is generally constructed internally and stored for a specified length of time.

A graph shows the results of a 1-stage PT-based factor X assay with varying factor X levels - note the parallel lines. The reference plasma is shown in red. The axes are both logarithmic and the dilutions are plotted on the X-axis and the clotting time in seconds on the Y axis.

Procedure for testing patient plasma

1. Warm Thromboplastins to 37°C.

2. Prepare I: 10 and I :20 dilutions of citrated patient plasma with Imidazole-buffered saline.

If a third dilution is desired, Prepare 1:5 Dilution for test plasma expected to have reduced level and 1:40 dilution for test plasma expected to be normal

3. Add 0.05 mL of specific factor-deficient plasma to 0.05 mL of diluted patient plasma.

4. Add 0.2 mL of Thromboplastins to the sample and determine the clotting time.

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5. Repeat steps 3, 4, and 5 on the 1 :20 and .1:40 dilution of patient plasma, multiplying the measured result by 2 or 4 respectively to correct for the dilution ratio when compared with the I:10 dilution. The results of the 1: 10, 1 :20 and 1:40 dilutions should agree within 15%. Report the average of the results.

Read the percent activity directly from the activity curve.

A “Blank” should also be tested as follows:

0.1 ml buffer , 0.1 ml Factor V deficient plasma

The clotting time for the blank reflects the quality of the deficient plasma and should be equivalent to less than 1%.

Note

Inhibitors will often have a "dilutional" effect, demonstrating nonparallel curves with increasing dilutions.

This should be considered if the results of the 1: 10, 1 :20 and 1:40 dilutions do not agree within 15%. In this case, results should not be averaged, but further dilutions such as 1 :80, and I: 160 performed until results of two consecutive dilutions match within 15% and measure within linearity of the calibration curve.

Specific volumes required for adding factor-deficient plasma, diluted patient plasma, and Thromboplastins reagent may vary depending on the automated analyzer used.

Reference Ranges

The reference ranges for the factors are as follows:

factor II: 80% to 120% of normal factor V: 50% to 150% of normal factor VII: 65% to 140% of normal factor X: 45% to 155% of normal.

Laboratories should establish their own reference ranges although it is probable that many do not and choose to use either published ranges or those provided with a commercial standard.

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Discussion

If you find a low FV result (and similarly if you find a low FVIII result) you should request a FVIII (or FV) assay to exclude these rare autosomally inherited disorders.

If you find a low FVII result make sure that the test was performed using human TF in the PT. Some FVII gene mutations e.g. FVII Padua can give rise to varying factor levels depending upon the source of TF used in the PT. Wherever possible, human recombinant TF should be employed as this gives a result that more closely relates to the situation found in vivo.

If you find a low level of a vitamin K dependent clotting factor - consider the possibility that that the patient is on warfarin, has true vitamin K deficiency or may have a mutation within the genes involved in encoding the proteins involved in the vitamin K cycle.

Remember - PT-based factor assays will be reduced in patients with vitamin K deficiency and in patients on oral vitamin K antagonists such as warfarin.

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Lab 11 : Platelets aggregation

Objective

The platelet aggregation test aids in the evaluation of bleeding disorders by measuring the rate and degree to which platelets form a clump (aggregate) after the addition of a chemical that stimulates clumping (aggregation).

Introduction

Platelets are disk shaped blood cells that are also called thrombocytes. They play a major role in the blood clotting process. The platelet aggregation test is a measure of platelet function.

Studying Platelets Disorders

Numbers

CBC

PLT count

PLT morphology

Function

Bleeding Time (BT)

Platelet Aggregation

Whole blood aggregation

Platelet-rich plasma aggregation

Platelets contribute to Hemostasis in two main ways:

Primary haemostatic plug :

Adhesion

Secretion

Aggregation

Secondary Haemostatic plug:

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Procoagulant activities are generated

Platelet Plug Formation:

Adhesion

Platelets bind to exposed adhesive subendothelial connective tissue

Collagen

vWF

Fibronectin

Mechanism components

vWF: links PLT to endothelial binding site

PLT receptor GPIb

Collagen fibers

Actin contracts & pseudopods form

REVERSIBLE

Facilitates activation

Platelets Aggregation

Platelet-Platelet interaction

Mechanism components

ATP

Ionized calcium

Fibrinogen

PLT receptor GPIIb/IIIa

Initial aggregation – REVERSIBLE

Secondary aggregation – IRREVERSIBLE* = white clot, also known as platelet plug formed.

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Why Platelet aggregation testing

Evaluation of suspected hereditary and acquired disorders of platelet function.

Platelets normally contain three major types of granules .

The alpha granules contain fibrinogen, PF4, factor V, von Willebrand factor….

The dense granules contain ADP or ATP, calcium and serotonin.

lambda granules – similar to lysosomes and contain several hydrolytic enzymes.

Hereditary platelet function disorders includes

Rare defects of adhesion (Bernard Soulier syndrome)

Rare defects of aggregation (Glanzmann thrombasthenia)

More common defects of secretion (alpha or dense granule deficiency, aspirin-like defects, or other primary secretion defects).

Acquired platelet function disorders are more common than the hereditary disorders and include drug-induced platelet dysfunction (including aspirin, NSAID’s, clopidogrel, antibiotics, various cardiovascular and psychotropic drugs), uremia, and myeloproliferative disorders.

NSAIDs: Nonsteroidal anti-inflammatory drugs, like Ibubrufin, asprin, Naproxen are drugs with analgesic (also known as a painkiller) and antipyretic (fever-reducing) effects and which have, in higher doses, anti-inflammatory effects. Clopidogrel is an oral, thienopyridine class antiplatelet agent used to inhibit blood

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clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease. Uremia is a term used to loosely describe the illness accompanying kidney failure .

Principle

Using Aggregating agents to induce platelet aggregation or cause platelets to release endogenous ADP, or both. Platelet aggregation is studied by means of a platelet aggregometer, Used Principle:

1. Photo-optical Method

2. luminescence technology (Platelet Lumiaggregometry)

3. Electrical Impedance Method

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Aggregating Agents

Arachidonic acid: used to assess the viability of the thromboxane pathway.

Thrombin: reacts with several membrane sites to induce full aggregation and secretion of organelle contents independent of the prostaglandin or ADP pathways.

ADP: binds to a specific platelet membrane receptor and causes platelet activation and release of dense granule stored ADP. Shows biphasic aggregation.

Epinephrine: binds to specific receptor and causes ADP secretion, but does not cause aggregation in storage pool disorder or release defects.

Collagen: Shows no primary wave of aggregation and depends on intact membrane receptors, membrane phospholipase pathway integrity and normal cyclooxygenase and thromboxane pathway function.

Ristocetin: requires vWF and intact surface membrane including a functional vWF receptor site (GPIb).

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Electrical Impedance Method

These types of analyzers may use citrated whole blood, as the test sample. As platelets aggregate, the coat an electrode, impeding the electrical current through the analyzer.

Luminescence technology (Platelet Lumi aggregometry)

The lumi-aggregometer may be used to simultaneously measure platelet aggregation and secretion. The instrument records both aggregation and secretion of dense-granule ATP. The ATP is measured by its reaction with firefly luciferin to give chemiluminescence. The resulting light emission is detected, amplified, and recorded by the instrument. This modification of aggregation is particularly sensitive to ATP release, and is as sensitive measure of platelet activation. Performed by using whole blood or PRP.

[A] + [B] → [AB◊] → [Products] + light

Firefly luciferin is the luciferin, or light-emitting compound, found in many firefly (Lampyridae) species, a yellow light emission from. As with all other luciferins, oxygen is required to elicit light; however, it has also been found that ATP and magnesium are required for light emission.

Photo-optical Aggregometer (PLT Aggregometry Using PRP)

The Platelet-rich plasma, which is turbid in appearance, is placed in a cuvettes, warmed to 37°C in the heating block of the instrument, and stirred via a small magnetic bar. Baseline light transmittance through the platelet-rich plasma is recorded. The addition of an aggregating agent causes the formation of larger

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platelet aggregates with a corresponding increase in light transmittance, because of a clearing in the platelet-rich plasma. The change in light transmittance is converted to electronic signals and recorded as a tracing by the chart recorder.

Patient Sample – 3.2% citrated WB

Approximately 20 ml of blood is needed for a full aggregation study.

Test Sample – PRP ( Platelets count fall (200–600 × 109/L)

Principle – photometry: optical density of PRP warmed to 37° C is determined before and after the addition of various aggregating agents

Issues

Sample quality is critical

Fibrinogen levels are important

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Agonists must be prepared fresh daily

Thrombocytopenia makes result interpretation difficult

Complete patient history is essential

Figure 1 - Platelet-rich plasma in an optical aggregometer. Platelet count is approximately 200 × 109/L, and platelets are maintained in suspension by a magnetic stir bar turning at 1000 rpm. (Courtesy of Kathy Jacobs, Chronolog, Inc., Havertown, PA.)

Figure 2 – Five possible phases of PLT aggregation:

1. baseline, 2. agonist addition and shape change,

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Figure 2

3. primary wave,4. secretion, and 5. secondary wave

Sample Quality for PRP PLT Aggregometry

Clean venipunture

o Hemolysis = increased plasma ADP = prematurely activated platelets

Lipemia may obscure OD during PLT aggregation

PRP contact with glass = prematurely activated PLTs

Keep samples capped to prevent loss of CO2 – change sample pH ( PRP PH should be maintained within the range 7.7–8.0)

Store samples @ room temp – prevent inhibition of PLT aggregation

Perform testing within 3 hours of sample collection

Procedure

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1. Switch on aggregometer to warm to 37°C. Select AGGR TEST

2. Select the type of agonist (ADP, AA, RISTO, THR, COLL, ADR), ADP should placed in ICE

3. Place 0.27 ml PRP in a test cup and put in the incubation channels. Put beads into the cups, wait 1 minute to warm.

4. Transfer the cups to testing channels.

5. Press PPP, it will appear the status <PPP>

6. Add 0.03 ml of agonist to bottom of cuvettes and press start key then monitor optical density changes for three minutes.

7. Repeat this procedure for each agonist

8. Curve Display.

PRP Aggregometry Agonist & Patterns

ADP (at appropriate concentration) Biphasic curve: 1o and 2o waves (requires intact

prostaglandin pathway) Note: if ADP is added at too low or too high

concentration, it will not get biphasic response

Epinephrine Biphasic curve; requires intact prostaglandin

pathway

Collagen Lag phase followed by 2o wave only

Ristocetin A biphasic however, often only a single broad

wave Binds to vWF/GPIb/IX complex and results in agglutination Evaluates adhesion reaction

Thrombin

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Biphasic curve. Irreversible aggregation only (does not require cyclooxygenase)

Arachidonic acid 2o wave only; assesses cyclooxygenase pathway

Serotonin A primary wave of aggregation with a maximum of 10% to 30%

transmittance followed by disaggregation.

Interpretation

Platelet aggregation occurs as a two-step process, known as primary and secondary waves of aggregation.

The primary wave of aggregation is observed when platelets adhere to one another in the presence of an external agent (agonist) such as ADP, epinephrine, or ristocetin.

Secondary aggregation is characterized as the aggregation that occurs after the platelets have been stimulated to secrete the substances contained in their organelles.

It should be noted that some agonists will stimulate primary aggregation and some will stimulate secondary aggregation. Others will stimulate both primary and secondary aggregation, yielding a "biphasic" aggregation curve.

In addition, different concentrations of the same agonist can produce varying patterns of primary and secondary aggregation. For example,

Low concentrations of ADP induce biphasic aggregation (i.e., both a primary and a secondary wave of aggregation).

Very low concentrations of ADP (l.5 ug/ml. final concentration) induce a primary wave followed by disaggregation.

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And high concentrations of ADP (10 ug/ml, final concentration) induce a single, broad wave of aggregation.

A biphasic aggregation response to ADP will not be seen in patients with platelet release disorders.

Patients with Glanzrnann's thrombasthenia show incomplete aggregation with ADP regardless of the final concentration.

In patients with severe von Willebrand disease, aggregation to ristocetin is characteristically absent. Decreased to normal aggregation to ristocetin can be seen in patients with mild von Willebrand disease. Correction of the abnormal ristocetin aggregation curves can be seen by the addition of normal, platelet-poor plasma to the patient's platelet-rich plasma.

Abnormal ristocetin-induced platelet aggregation may also occur in patients with

1. Bernard-Soulier syndrome,

2. Platelet storage pool defects

3. Idiopathic thrombocytopenia purpura (ITP).

Glanzmann thrombasthenia

Normal PLT count, but abnormal clot retraction

Absence of secondary aggregation to ADP, epinephrine, collagen, (thrombin)

Normal response to ristocetin

Bernard-Soulier syndrome

Platelet aggregation test

Failure to aggregate in the presence of ristocetin

Aggregation by other agonists (ADP, collagen, epinephrine): normal

Response to low-dose thrombin: may be delayed

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Platelet storage granule defects

Dense (δ) granule defects ~ storage pool deficiency

α granule defects ~ gray platelet syndrome

Heterogeneous group of disorders

Mild to moderate bleeding diathesis

Abnormalities in platelet aggregation

Precautions Prior To Studying Platelet Aggregation

Aspirin-containing compounds should be excluded for at least 10 days prior to testing, as Aspirin interferes with the release reaction.

Ingestion of other drugs known to influence platelet function should also be avoided for at least the time required for their elimination from the circulation. These include certain antihistamines, antibiotics, and anti-depressants. A check should be made of any drugs being prescribed before performing platelet function testing.

Chylomicrons can interfere with the measurement of platelet aggregation, studies should not be carried out shortly after a fatty meal.

Many other “normal” dietary constituents, including alcohol, onions, garlic, peppers, and ginger, may also inhibit platelet aggregation.

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Chilling activates platelets, and so the blood is processed at 20°C –25°C.

Comment

In evaluating patients with suspected platelet disorders, the aggregating agents most commonly used are ADP in varying concentrations, collagen, epinephrine, and ristocetin.

Aspirin, aspirin compounds, and anti-inflammatory drugs inhibit the secondary wave of aggregation by inhibiting the release reaction of the platelet.

The intensity of platelet aggregation may be estimated by recording the change in absorbance as a percentage of the difference in absorbance between platelet-rich and platelet-poor plasma.

This has limited usefulness because absorbance is dependent on the size and density of platelet clumping and the number of platelets that aggregate.

Drugs and PLT Function

Aspirin:

Acetylsalicyclic acid Irreversibly inhibits Cyclooxygenase

Clopidogrel :

Plavix Irreversibly inhibits P2Y12

Dipyridamole:

inhibits Thromboxane synthase

Abciximab

ReoPro inhibits GP IIb/IIIa

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Lab 12 : Coagulation Instruments

Introduction

Evaluation of Coagulation tests:

Manual Method:

Coagulometers:

Semiautomated Method.

Automated Method.

Manual methods were used at the begining, then semi-automatic equipments appeared based on photometric or mechanical principles to detect fibrin. More recently, fully automated instruments have become common in modern laboratories.

Today, new equipment connected to specific data processing systems can undertake clotting, Chromogenic, and immunological tests.

Automation has contributed to improvements in standardization and facilitating tests – improve lab efficiency and repertoire

Manual Method

All reagents and samples are added manually by the operator.

Temperature is maintained by a waterbath or heat block;

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May require external measurement by operator, most often using a stopwatch.

Semi Automated Method.

All reagents and samples are added manually by the operator.

Usually contains a device for maintaining' constant 37°C temperature, Analyzer may or may not internally monitor temperature .

Has mechanism to automatically initiate timing device upon addition of final reagent and internal mechanism for detecting Clot formation.

Automated Method

All reagents are automatically pipetted by the instrument. Samples may or may not be automatically pipetted.

Contains monitoring devices and internal mechanism to maintain and monitor constant 37οC temperature throughout testing sequence.

Timers are initiated and clot formation detected automatically

Methods of Endpoint Detection

Mechanical

Optical

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Manual Method Semi-automated Method Automated Method.

Photo-optical

Nephelometric

Chromogenic

Immunologic

Electrochemical

Mechanical

Two primary methodologies are utilized for mechanical detection of clot formation.

Electromechanical (Impedance) Method.

Magnetic, Steel Ball Method.

Electromechanical Method.

When coagulation process takes place, the concentration of clotting factors (charges) and inorganic ions will change along the time and the measured impedance or conductance will be also changed correspondingly at the same time.

During the reaction, one probe moves in and out of the solution at constant intervals. The electrical circuit between the two probes is not maintained as the moving probe rises in and out of the solution.

When a clot (fibrin) is formed in the solution, the fibrin strands maintain electrical contact between the two probes when the moving probe leaves the solution, which stops the timer.

Magnetic, Steel Ball Method

Mechanical clot detection involves monitoring the movement of a steel ball within the test solution using a magnetic sensor. As clot formation occurs, the movement of the ball changes, which is detected by the sensor.

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There are two variations of this principle used in current instrumentation.

1. A change in the movement of the steel ball may be detected when there is increased viscosity of the test solution, changing its range of motion,

2. Or by a break in contact with the magnetic sensors when the steel ball becomes incorporated into a fibrin clot as the cuvette rotates.

Photo-optical Method

Detection of clot formation measured by a change in OD of a test sample is the basis of photo-optical instrumentation, which is also known as turbidometric methodology:

When a light source of a specified wavelength is passed through a test solution (plasma), a certain amount of light is detected by a photodetector or photocell located on the other side of the solution.

The amount of light detected is dependent on the color and clarity of the plasma sample and is considered to be the baseline light transmission value.

When soluble fibrinogen begins to polymerize into a fibrin clot, formation of fibrin strands causes light to scatter, allowing less light to fall on the

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photodetector (i.e., the plasma becomes more opaque, decreasing the amount of light detected.

When the amount of light reaching the photodetector decreases to an exact point from the baseline value as predetermined by the instrument, this change in OD triggers the timer to stop, indicating clot formation.

Nephelometric

Quantifying plasma proteins based on the specific reaction of the protein being measured with highly specific antisera.

Precipitants are antigen-antibody complexes, which show up in solutions as turbidity and scatter incident light.

The nephelometer uses a light -emitting diode at a high wavelength (usually >600 nm) to detect variations in light scatter as antigen-antibody complexes are formed. When the light rays encounter insoluble complexes such as fibrin strands, they are scattered in both forward (l80-degree) and side (90-degree) angles.

Instruments employing this type of measuring system detect the amount of agglutination of particles by reading the increasing amount of light scattered at a 90-degree angle as agglutinates are formed. The timer is triggered to stop when the amount of light scatter reaches a specific predetermined level.

This method of endpoint detection is in contrast to the photo-optical systems, which sense decreased light transmission at 180 degrees due to the opaqueness of the sample in a cuvette when fibrin is formed.

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Chromogenic

Chromogenic, or amidolytic, methodology is based on the use of a specific color-producing substance known as a chromophore. The chromophore normally used in the coagulation laboratory is para-nitroaniline (p-nitroaniline or pNA), which has an optical absorbance peak at 405 nm on a spectrophotometer.

Immunologic Method

Immunologic assays are based on antigen-antibody reactions.

Latex Microparticles are coated with a specific antibody directed against the analyte (antigen) to be measured.

A beam of monochromatic light is then passed through the suspension of microlatex particles. When the wavelength of light is greater than the diameter of the particles in suspension, only a small amount of light will be absorbed by the particles.

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When the latex microparticles coated with specific antibody come in contact with the antigen present in the solution, the antigen attaches to the antibody and forms bridges between the particles, causing them to agglutinate.

As the diameter of the agglutinates becomes larger and closer to the wavelength of the monochromatic light beam, the greater the amount of light that is absorbed.

The increase in light absorbance is proportional to the size of the agglutinates, which, in turn, is proportional to the antigen level present in the sample, which is read from a standard curve.

Electrochemical

INRatio Meter (Hemosense) - Near Patient Testing Devices

The INRatio single-use test strip is made of laminated layers of transparent plastic. Each test strip has a sample well where blood is applied, three channels through which the blood sample flows to reach the testing areas, reagents to start the coagulation process, and electrodes that interface with the INRatio meter. The device detects a change in electrical resistance when blood clots.

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