routine laboratory evaluation of coagulation

8/11/2019 Routine Laboratory Evaluation of Coagulation

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ROUTINE LABORATORY

EVALUATION of COAGULATION

REPORTER #8 TIMBASAL, FATIMA ALLIN

TUBAING, SHEENA MARIEYBIO, AMIE


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Routine Laboratory Evaluation ofCoagulation

REFERENCE RANGESTEST FOR THE INTRINSIC AND COMMONPATHWAYS

1. Lee and White Whole Blood Coagulation Tim e

2. Plasma Recalcification Time3. Activated Clotting Time4. Partial Thromboplastin Time

TEST FOR THE EXTRINSIC AND COMMOMPATHWAYS1. ProthrombinTime2. Other tests
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Test for the Intrinsic and CommonPathways

Lee and White Whole Blood Coagulation Time- described the procedure for the whole blood

coagulation time that was a modification of thenumerous methods described on literature time.

L-W Test- Base on the fact that when venous blood is put

into a glass tube, it will form a solid clot.- The time required for this response is a measure

of the overall intrinsic and common pathways of

coagulation


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Plasma RecalcificationTime- A modification of the L-W,- using citrated plasma instead of whole blood.- Glass or siliconized tubes- Platelet-rich Plasma (PRP)- Platelet-poor Plasma (PPP)

This test is based on the fact that except for calcium, normal

PRP contains all the components of the coagulationmechanism necessary for generating a fibrin clot.REFERENCE RANGE

PRP 100 150 seconds

ppp 130 240 seconds


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Plasma RecalcificationTime- Disadvantage-

- 1. Are the difficulty in standardizing the number ofplatelet in the PRP

- 2. Length of time necessary to perform the test, whichmoreover is insensitive to moderate factor deficiencies.

- 3. Errors in collection technique can significantly affectresults.

It is important that the same size tube always be used fortesting and that the specimen be tilted uniformly.


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Plasma Recalcification Time

The sensitivity of this test is somewhat improved bydiluting the plasmaThis accomplishes three things:

1. It adjust the PRP closer to in vivo platelet count

2. It increases the sensitivity of the test system tofactor deficiencies.

3. It dilutes the natural inhibitors to coagulation thatare present

A normal control should run with each test.


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ACTIVATED CLOTTING TIME TEST

Heparin-monitoring procedure

Uses diatomaceous earth (diatomite) as an activator

of the contact factor.

Requires to be kept warmed to a constant 37C bytaking a special incubator to the patients bedside.

The most reliable and rapid screen of the intrinsic andcommon pathways.


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PRINCIPLE- Whole blood contains all the components

necessary to produce a clot when removed from theveins and put into a glass tube.

- By adding an activator and keeping the blood at aconstant 37C.

REAGENTS- 2 evacuated tubes containing 12mg of diatomite are

needed


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ACTIVATED CLOTTING TIME TEST PROCEDURE

1. Two tubes containing diatomite are brought to 37C in aheat block at the patients bedside.2.Using good venipuncture technique, at least 2 ml of blood isdrawn into a tube and discarded.

3.The tourniquet is removed, and the 1st

tube of diatomite isattached to the needle.4. When blood starts to flow into the tube, the stopwatch isstarted.5. The tube is filled, mixed and placed in the heat block.

6. The procedure is repeated with the 2nd

tube, and 2nd

stopwatch is started.7. After 60 seconds, the 1 st tube is observed by tilting it at 5-second interval until clot is formed, same procedure with testtube 2.


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REFERRENCE RANGE- 75 to 120 secondsThe target range during heparin therapy is

- 140 to 185 seconds

INTERPRETATION- Prolongation of the ACT is indicative of one or more

factor defects in the intrinsic or common pathways or thepresence of circulating anticoagulant such as heparin.


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PARTIAL THROMBOPLASTIN TIMEHemophiliacs have a prolong bleeding time.

This test is more sensitive to abnormalities in the early stagesthan were previous test in intrinsic system.

An important refinement of the PTT was the addition ofnegatively charged activators to the system, resulting insignificantly shorter clotting time.

Modification is APTT(Activated Partial ThromboplastinTime)

- test of choice to screen for factor deficiencies of thecommon and intrinsic pathways and also for monitoring

heparin therapy.


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Activated Partial Thromboplastin Time

PRINCIPLE- The APTT measures all factors except VII and XII.Maximum activation of the contact factor is accomplishedby addition of the activator. Phospholipid is supplied to

substitute for platelet factor 3 (PF3).- APTT is essentially same as a recalcification time of

plasma.

REAGENTS1. Phospolipid with activators2. 0.025 M CaCl 2 (as recommended by the manufacturer)


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Activated Partial Thromboplastin TimeCONTROLS

Commercial lyophilized controls are available in midrange,and extended ranges.

It is recommended that a normal control and at least onabnormal control can be used.

In-house preparation of pooled/frozen plasma may be

used as controls.

Each laboratory must specify when controls are to betested, what the satisfactory control limits are, and, ifduplicates are run, how closely the values should agree.


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PROCEDURE1. Platelet-poor plasma (0.1ml) is added to 0.1mL of APTT

reagent and incubated at 37C for the period of timespecified by the reagent manufacturer ( approximately 3

to 5mins)2. After incubation, 0.1mL of warmed CaCl2 is added, and

the time for clotting to occur is recorded.

REFERRENCE RANGEThe ranges differ according to the reagent, method andinstrument used. May extend from lower limit of 20seconds to an upper limit of 45 seconds


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INTERPRETATION

A prolonged APTT in the absence of heparin useindicates a factor deficiency, an acquired circulating

anticoagulant such as lupus inhibitor, or an antibody to aspecific factor such as factor VII.

Most commercial reagents will demonstrate a prolongAPTT if any factor measured by the APTT is less than40% to 50% of normal.


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The APTT is also known as:

Kaolin Cephalin Clotting Time (KCCT)Partial Thromboplastin Time with Kaolin

(PTTK).


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Prothrombin Time

Is a blood test that measures thetime it takes for the liquid portion(plasma) of your blood to clot.

A prothrombin time test can be usedto check for bleeding problems. PTis also used to check whethermedicine to prevent blood clots isworking.


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TEST FOR THE EXTRINSIC AND COMMONPATHWAYS

PROTHROMBIN TIME

PRINCIPLEThe PT referenc e interval varies from site to

site depending on the patient population, type of thromboplastin used, type of instrument used, and pH andpurity of the reagent diluent. Each center must establish itsown range for each new lot of reagents or at least once ayear.

This may be done by testing a sample of at least 20specimens from healthy donors of both sexes spanning theadult age range over several days and computing the 95%confidence interval of the results. A typical PT referenceinterval is 12.6 to 14.6 seconds.


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PROTHROMBIN TIMEREAGENTS1. Thromboplastin/CaCl 2 (PT reagent)2. Controls (discussed in APTT) are needed.

PROCEDURE1. Aliqouts of control and patient plasma are warmed

according to method being used.2. The PT thromboplastin reagent is warmed by

incubating it at 37C for 3 to 5 mins. , and 0.2mL of PTreagent is added to 0.1mL of plasma (patient orcontrol)

3. The clotting time is recorded.


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PROTHROMBIN TIMEREFERENCE RANGE

Normal values differ with the reagent and method.- 10 to 12 seconds (photo-optical systems)- 12 to 14 seconds (manual methods)

INTERPRETATIONProthrombin times may be reported in several ways;

1. Patient time (in seconds) with reference range.

2. Patient time with control time (in seconds) withreference range .3. Prothrombin ratio

(PT of the patient mean of reference range x 100)


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Prothrombin Time Quality ControlThe medical laboratory scientist tests normal andprolonged control PPP specimens at the beginningof each 8-hour shift or with each change of reagent.

Although lyophilized control PPPs are commerciallyavailable, the laboratory manager may choose tocollect and pool PPP specimens from designatedsubjects to make home -brew controls. In this case,the specimens must be collected and managedusing the same tubes, anticoagulant, and protocolthat are used for patient plasma specimen collection.The samples are pooled, tested, and aliquoted.Regardless of whether commercial or locallyprepared controls are used, the control is testedalongside patient specimens using the sameprotocol as for patient PPP testing


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The normal control result should be within thereference interval and the prolonged control result

should be within the therapeutic range for warfarin. If the control results fall within the stated limits in thelaboratory protocol, the test results are consideredvalid.

If the results fall outside the control limits, thereagents, control, and equipment are checked; theproblem is corrected; and the control and patientspecimens are retested. The operator records all theactions taken. Control results are recorded andanalyzed at regular intervals to determine the long-term validity of results


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Use of Prothrombin Time as a Diagnostic Assay

The PT is performed diagnostically when anycoagu lopa thy is suspected. Acquired multipledeficiencies, such as disseminated intravascular

coagulation (DIC), liver disease, and vitamin Kdeficiency, all affect factor VII activity and aredetected through prolonged PT results. The PT isparticularly sensitive to liver disease, whichcauses factor VII levels to become rapidlydiminished


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Reporting of Prothrombin Time Results andthe International Normalized Ratio

The medical laboratory scientist reports PT resultsto the nearest tenth of a second along with the PTreference interval. If the PT assay is performed induplicate, the results are averaged, and the

average is reported.In view of the inherent variations among

thromboplastin reagents, most laboratories reportthe international normalized ratio (INR) for patientswith a stable anticoagulation response using thefollowing formula.


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Factors That Interfere with the Validity of Prothrombin Time (PT) Results


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Limitations of Prothrombin Time

Anticoagulant volume must be adjusted when thehematocrit is greater than 55% to avoid falseprolongation of the results.Specimens must be inverted five times

immediately after collection to ensure goodanticoagulation, but the mixing must be gentle.Clotted and visibly hemolyzed specimens are

rejected, because they give unreliable results.Plasma lipemia or icterus may affect the results

obtained with optical instrumentation.


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Heparin may prolong the PT. If the patient isreceiving therapeutic heparin, it should be noted

on the order and commented on when the resultsare reported. The laboratory manager selectsthromboplastin reagents that are maximallysensitive to oral anticoagulant therapy and

insensitive to heparin. Many reagentmanufacturers incorporate Polybrene into theirthromboplastin reagent to neutralize heparin.The medical laboratory scientist may detectunexpected heparin by using the TCT test, whichis described subsequently


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Other Tests for Extrinsic and CommonPathway

1. Stypven time

2. Prothrombin-proconvertin time


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OTHER TESTSSTYPVEN TIME

- Utilizes the powerful coagulant properties of Russellsviper venom , obtained from the snake Vipera ruselli .

- This venom is capable of bypassing the action of factorVII and directly activating factor X to Xa.


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OTHER TESTS. Prothrombin-proconvertin time

- Based on the earlier observations that minor deficienciescan be more pronounced when the plasma is diluted.

- Today, the test has little value as on overall screen forextrinsic system because of its insensitivity to factor Vand fibrinogen

- Also, with the current use of sodium citrate as theanticoagulant of choice, the instability of factor V in nolonger a great problem.


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END

routine laboratory evaluation of coagulation

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