About Lifelong Learning - Contact Us - DonateFree-Ed.Net Home   Bookmark and Share

Return to
List of  Lessons

Lesson 8-4
Coagulation Studies

INTRODUCTION

The activated partial thromboplastin time, prothrombin time, thrombin time (or quantitative fibrinogen), bleeding time, platelet count, and clot retraction constitute a satisfactory coagulation screen to perform on the bleeding patient or on one who is suspected of having a bleeding disorder. The activated partial thromboplastin test is the most useful single laboratory test available. This procedure detects deficiencies present in the intrinsic coagulation system, except for platelets and factor XIII. The prothrombin time is the method of choice for detecting disorders in the extrinsic system. The thrombin time is useful as a test for functional fibrinogen and to test the conversion of fibrinogen to fibrin. It is also sensitive to the presence of thrombin-inhibitors such as heparin. The most widely used screening tests for platelets are the platelet count, the bleeding time, and clot retraction.

Once it has been established through screening procedures that a coagulation disorder exists, a systematic approach should be followed in identifying the exact deficiency or disorder. Specific tests to measure the various coagulation functions are listed in table 2-2.

Table 2-2. Diagnostic coagulation tests.


BLOOD COLLECTION

Principle

There are general rules for using a "Vacutainer-type" blood collection system. Tests requiring sterility are always drawn first (blood culture). Next, obtain those tubes for specimens which do not require anticoagulants. This is followed by those specimens which do require anticoagulants. Coagulation specimens come first.

Two-Tube Technique. To avoid contamination of blood by tissue juices, the two-tube (Vacutainer-type) venipuncture is usually employed for specialized coagulation tests using venous blood. The technique is as follows:

  1. Place a tourniquet on the arm, no longer than 1 minute.
  2. Observe the area for the most accessible vein.
  3. Cleanse the site with 70 percent isopropyl alcohol and allow to dry.
  4. Insert the needle and remove the tourniquet. (Quick removal of the tourniquet prevents stasis.)
  5. Obtain specimens for all tests that require no anticoagulants first. If only coagulation work is specified, use a "red" top tube to withdraw about 5 ml of blood, and dispose of the "red" top tube.
  6. Draw off the required specimen using a "blue" top coagulation tube. The ratio of blood to enclosed anticoagulant is 1:10. Allow the tube to fill to its capacity, do not remove until filling stops completely.
  7. Blood samples needing anticoagulation should be mixed immediately following withdrawal from the needle.

Anticoagulants. Anticoagulants for coagulation studies can be obtained in commercially-prepared vacuum tubes or prepared in the laboratory. The preparation and use of anticoagulants is as follows:

  1. Sodium citrate, 3.2 percent. Dissolve 3.2 g of sodium citrate in 100 ml distilled water. Refrigerate. As an anticoagulant, combine one part 3.2 percent sodium citrate with 9 parts blood.
  2. Sodium oxalate, 0.1 M. Dissolve 1.34 g sodium oxalate in 100 ml distilled water. Refrigerate. As an anticoagulant, combine one part 0.1 m. sodium oxalate with 9 parts blood.
  3. EDTA. Dissolve 10 g of EDTA salt in 100 ml distilled water. Pipet 1 ml of this solution into a suitable test tube. Allow to dry in the oven at low temperature. For use as an anticoagulant, add 5 ml of venous blood and mix well.

Glassware. All glassware for coagulation studies must be scrupulously clean. Used glassware should be free of chemicals and any traces of human blood components. All glassware should be cleaned in detergents free of organic solvents and rinsed several times with distilled water. The use of disposable syringes and needles eliminates the need for siliconizing glassware.

CAUTION: Never use test tubes or pipets that are damaged.

TEMPLATE BLEEDING TIME (MIELKE MODIFICATION)

The bleeding time is an in vivo measurement of platelet participation in small vessel hemostasis. The introduction of the template bleeding time technique has improved the reproducibility and sensitivity of the test by controlling the length and depth of the incision. Control of the depth is important so that only the smaller vessels (capillaries) are incised.

The platelets initially adhere to the cut surface of the vessel wall; aggregation takes place. The platelets interdigitate and release the contents of their granules. This is followed by the formation of fibrin that stabilizes the hemostatic plug.

The purpose of the bleeding time test is to provide a measure of such platelet functions in small vessel hemostasis. A prolonged bleeding time does not in itself diagnose underlying platelet disorders, either qualitatively or quantitatively. It indicates the need for more quantifiable testing.

Reagents and Materials.

  • 1 Surgicutt.
  • 1 70 percent alcohol pad.
  • Sphygmomanometer.
  • Stopwatch.
  • Filter paper (2 to 3 sheets).
  • Band-Aid or other bandage.

Procedure

  1. Position the patient's arm with the volar surface exposed.
  2. Select a site avoiding surface veins, bruises, and edematous areas.
  3. Place the sphygmomanometer on the upper arm.
  4. Cleanse the test area with the 70 percent alcohol pad and let air-dry.
  5. Inflate the cuff to 40 mm Hg., and hold at this exact pressure for the duration of the test.
NOTE: The time between inflation and incision should be 30 to 60 seconds.
  1. Open the sterile package and gently rest the Surgicutt surface on the patient's forearm.
  2. Apply minimal pressure so that both ends of the instrument lightly touch the skin.
  3. Gently push the trigger, starting the stopwatch simultaneously.
  4. Remove the Surgicutt immediately after triggering.
  5. After 30 seconds, wipe the flow of blood with the filter paper. (Bring the paper close to the incision, but do not touch the paper directly to the incision, so as not to disturb the formation of the platelet plug.)
  6. Wipe the blood every 30 seconds thereafter, until no blood stains the paper.
  7. Stop the timer when only clear fluid is absorbed onto the filter paper. The bleeding time is determined to the nearest 30 seconds.
  8. Release the pressure of the sphygmomanometer.
  9. Record the bleeding time.

Sources of Error.

  • A horizontal incision, approximately 5 cm below the antecubital crease, 2.4MM depth, gives the best reproducibility.
  • If body hair will interfere, lightly shave the area.
  • Patient should be advised of a potential to produce a scar. This can usually be avoided by the use of a butterfly bandage applied for 24 hours.
  • Aspirin and aspirin-containing products may cause a prolonged bleeding time for up to two (2) weeks.
  • A standardized cut is necessary for valid results. Too little pressure on the device and the wound will be shallow or nonexistent. Too much pressure and the wound will be too deep. This is the one area where standardization has not been completely controlled.
  • Low skin temperature produces a constriction of the capillary vessels, resulting in decreased blood flow.

The Template Bleeding Time is the method of choice because the blood pressure on the vessels is constant, the incision is uniform, and the arm offers an area for multiple determinations.

The bleeding time depends primarily on extravascular and vascular factors and, to a lesser degree, on the factors of coagulation. The chief factor controlling bleeding from a small cut is the constriction of the minute vessels following injury. Accuracy in this test is enhanced by blotting the drops of blood at shorter intervals of time as the drops of blood become progressively smaller.

Thrombocytes play an important part in the formation of the hemostatic plug that seals off a wound. In thrombocytopenic purpura there is a decrease in platelets resulting in a prolonged bleeding time due to a defective platelet plug. An additional factor prolonging the bleeding time in this condition is a defect in capillary contraction. It can be hereditary and acquired platelet dysfunction.

In hemophilia, the bleeding time is normal. This is explained by the fact that there are no vascular or extravascular abnormalities. However, the test should not be performed on a known hemophiliac, for delayed oozing of blood is a real hazard.

Range of Values.

Normal values: 2 to 9 minutes.

Critical values:

  • Less Than -- Not applicable
  • Greater Than -- 15 minutes

WHOLE BLOOD COAGULATION (CLOTTING) TIME (LEE-WHITE)

The whole blood clotting time is a rough measure of all intrinsic clotting factors in the absence of tissue factors. Variations are wide and the test sensitivity is limited. Whole blood, when removed from the vascular system and exposed to a foreign surface, will form a solid clot. Within limits, the time required for the formation of the solid clot is a measure of the coagulation system.

Reagents and Materials.

  • Stop watch equipment for collection of blood.
  • 2 plastic syringes.
  • 3 clean, dry glass test tubes (10 x 75 mm).
  • Water/dry bath (at 37oC).

Procedure

  1. Label glass tubes #1, #2, and #3.
  2. Collect at least 1 to 2 ml of blood in a plastic syringe. Discard this blood. (This prevents tissue thromboplastin from entering the blood sample.) Change syringes.
  3. Collect at least 5 ml of blood in the second plastic syringe.
  4. Approximately 1 ml of blood is placed in each of the three glass test tubes. (#3 first, then #2, then #1)
  5. The stopwatch is started as soon as the blood enters the first tube #3.
  6. All tubes are placed into the 37C water bath.
  7. Gently tilt tube #3 (45 angle) every 30 seconds, until the blood in it clots.
  8. Thirty seconds after tube #3 clots, proceed with tube #2, tilting every 30 seconds, until a clot is formed.
  9. Thirty seconds after tube #2 is clotted, tube #1 is tilted until no flow of blood is observed on tilting.
  10. Record the time. The coagulation time is the time required for the blood to clot in the last tube. (Tube #1)
NOTE: This range should be between 5 to 10 minutes.

Range of Values.

  • Normal values: 5 to 15 minutes.
  • Critical value:
Less Than -- Not applicable
Greater Than -- 15 minutes
Special notes:
  • The following variables tend to decrease the clotting time: rough handling of the blood specimen, presence of tissue fluids (traumatic venipuncture), frequent tilting of the tube, and unclean tubes.
  • The following variables tend to increase the clotting time: extreme increases in temperature, variation in pH, and performance of the test at room temperature.
  • This test is of value primarily as it was used to follow heparin therapy. Its use as a screening procedure is limited due to its poor sensitivity.
  • The whole blood clotting time is affected mainly by defects in the intrinsic pathway factors and by defects in fibrin and fibrinogen. It is not sensitive to platelet abnormalities.
  • A prolonged clotting time immediately indicates impaired coagulation, but a normal clotting time does not exclude many serious clotting defects.
  • One disadvantage of the whole blood clotting time is its relative lack of reproducibility.
  • This procedure has been replaced in most laboratories with the APTT, which is more reproducible and easily controlled.
  • The coagulation time is normal in thrombocytopenic purpura. This is explained by the fact that only a small number of thrombocytes need be present for normal coagulation to take place.

CLOT RETRACTION TEST

When blood coagulation is complete, the clot retracts and expresses serum as the clot becomes denser. Thrombosthenin, released by the platelets, is responsible for clot retraction. The number of platelets present also affects the clot retraction time.

Reagents and Equipment.

  • Venous specimen, approximately 3 ml, using the two-tube technique.
  • 12 x 75 mm glass test tubes, 3.
  • 37C waterbath.
  • Timer.
  • 1 ml Pipettor or volumetric pipet.

Procedure

  1. Withdraw 3 ml of venous blood using the two-tube technique.
  2. Place 1 ml of blood into each of 3 glass test tubes and immediately incubate in a 37C water bath.
  3. Set a timer for 1 hour.
  4. At 1 hour, observe the clot and record results.
  5. Inspect the tubes at 2, 4, and 24 hours, observe and record results.
  6. Examine the tubes for retraction after incubation. Separation of the clot from the test tube is complete retraction (+4).

Calculations: Approximate amount of shrinkage of clot. Either in percentage or using (+1 to +4) grading.

Sources of Error.

  • Shaking or jarring of the tube of blood should be avoided. This may lead to a shortened clot retraction time.
  • Certain anemic patients with a low hematocrit value show increased clot retraction due to the formation of a small clot. Polycythemia vera may also affects results.

Poor clot retraction occurs in thrombocytopenia, qualitative platelet deficiency, and in cases of increased red cell mass. The clot retraction is normal in hemophilia since there are a normal number of platelets. However, the onset of contraction is often delayed in blood samples from hemophiliac patients. Clot retraction varies inversely with the plasma fibrinogen concentration. That is, if the plasma fibrinogen level is elevated, clot retraction may be poor.

Generally, there is a small amount of what is termed red cell fallout during clot retraction. This is seen as a few red cells at the bottom of the tube that have fallen from the clot. The significance of an increased amount of red cell fallout is not known. Whenever red cell fallout is increased, a notation on the patient's report should be made. Tubes from a completed Lee-White clotting time can be used to perform this test.

Normal Values. Results are reported as the length of time it took for the clotted blood to retract. A normal clot retracts from the sides and bottom of the test tube within 1 to 2 hours.


TOURNIQUET TEST

The fragility of capillaries is determined under increased pressure due to a sphygmomanometer. The pressure partially obstructs the venous return from the arm and increases intracapillary pressure. The number of petechial hemorrhages reflects the degree of capillary fragility.

Equipment: Sphygmomanometer.

Procedure:

  1. Place a blood pressure cuff on the patient's arm.
  2. Inflate it to a point midway between the systolic and diastolic pressure.
  3. Maintain this pressure for 5 minutes.
  4. Remove the cuff and wait 2 minutes.
  5. Examine a representative area (a circle about 2.5 centimeters in diameter) on the hand or arm for the presence of petechiae.

Calculations:

Grade the number of petechiae as follows:

0 -10 = 1+

10 -20 = 2+

20 -50 = 3+

over 50 = 4+

Sources of Error:

  1. Mistaking skin blemishes for petechia increases the number.
NOTE: Check for skin blemishes before the test.
  1. Capillary fragility varies at different sites.
  2. Maintaining the pressure too long causes false positives.

Normal Values: 0-10 petechiae per 2.5 cm area.

Increased petechiae are observed with vascular purpura.

Platelet disorders also cause increased petechial formation.

The tourniquet test is a crude test to determine the ability of blood vessels to withstand trauma and should not be used as a screening test for surgery.

Increased vascular fragility is sometimes found in qualitative and quantitative platelet defects, vitamin C deficiency, dietary ascorbic acid deficiency, and in the various purpuras. The term "purpura" is not specific but applies to a number of affections characterized by bleeding into tissue.

The tourniquet test is most often performed by the physician.


PARTIAL THROMBOPLASTIN TIME (ACTIVATED)

Normal citrated plasma contains all clotting factors except calcium ions and platelets. Calcium ions and a partial thromboplastin (platelet-like substance, a phospholipid) are added to the plasma and the clotting time recorded. An activator (such as ellagic acid, celite, or kaolin) is added to make activation of the plasma independent of the surface contact of the tube. As a result of optimal activation of the contact factors, the activated partial thromboplastin time is shorter and less variable than the partial thromboplastin time. The time required for the plasma to clot is the activated partial thromboplastin time.

NOTE: The tube must have the exact required blood volume. A "short" draw will affect results. Ratio of blood to anticoagulant must be1:10. Most tubes draw 4.5 ml of whole blood to be mixed with 0.5 ml anticoagulant, sodium citrate

Reagents and Equipment.

  • Blue top, sodium citrated Vacutainer tube.
  • Kontact APTT (Curtin Matheson Supply (CMS)).
  • Liquid reagent calcium chloride, CMS.
  • Thromboscreen controls.
  • Levels I & II MLA 800 cuvettes 0.1 ml pipettor.
  • Pipettor tips.
  • MLA 800 automatic coagulation timer.

Procedure

  1. Obtain the coagulation specimen by the two-tube method.
  2. Centrifuge the specimen, at 2500 RPM, for 5 minutes, to obtain a platelet poor plasma (PPP) specimen.
  3. Remove the specimen and if not tested, place in refrigerator.
  4. Make sure that the correct reagents on set up on the instrument, since it performs both PT and APTT testing, using the same pumps.
  5. Pump #2 should contain the activated partial thromboplastin reagent.
  6. Pump #1 should contain the calcium chloride reagent.
  7. Place a cuvette in the first well.
  8. Place pipette into wells A and B, 0.1 ml of the same patient's PPP specimen.
  9. Place the instrument in the APPT mode.
  10. Press run on the instrument's touch panel.
  11. Print out the results on both the screen and hard copy from wells A and B, in addition to the average, following the incubation period that the instrument is set up for, either three or five minutes.

Calculations are all done by the instrument.

Sources of Error.

  • Plasma must be prepared with care without disturbing the buffy coat.
  • If plasma is left at room temperature, the test must be performed within 45 minutes. Otherwise plasma can be stored at 4C for three hours.
  • Accurate pipetting is essential for valid results.
  • Do not use sodium oxalate as an anticoagulant. Sodium citrate is a better preservative and activates plasma faster.
  • Paired APTT values must be within plus or minus 10 percent of each other in the normal range. If not, instrument will beep and flag the results.

This test is an overall screening test for all three stages of coagulation with the exception of factor VII, calcium, and platelet factor 3. The partial thromboplastin time is prolonged in all deficiencies of prothrombin and factor V, as well as deficiencies of all the plasma factors in the intrinsic system. To exclude an abnormality in the extrinsic system, a prothrombin time test should be performed on all abnormal plasma.

In conjunction with the prothrombin time the following deficiencies can be determined:

  • PTT PT Deficiency
  • Abnormal
  • Normal
  • Stage I: factors VIII, IX, XI, XII
  • Abnormal
  • Abnormal
  • Fibrinogen, prothrombin, factor V, and X
  • Normal
  • Abnormal
  • Factor VII

A differential partial thromboplastin time is performed to detect the specific deficiency.

The partial thromboplastin test is much more sensitive to coagulation factor deficiencies than is the whole blood clotting time.

Range of Values

Both Normal and Abnormal controls should be run on a daily or shift basis. Controls should come in within 2 S.D.'s of the established mean.

(1) Normal range: 25 to 40 seconds.

(2) Critical values:

Less Than Greater Than

APTT 20 secs. 100 secs.


ONE-STAGE PROTHROMBIN TIME

When optimal amounts of thromboplastin, calcium, and citrated plasma are mixed under carefully controlled conditions, fibrin strands will normally form within a matter of seconds. The interval during which timed reaction occurs is the prothrombin time. This test measures the overall prothrombin activity of plasma. The prothrombin time measures the integrity of the extrinsic system. It is the most widely used measurement for monitoring patients on coumarin (oral anticoagulant) therapy.

Reagents and Equipment.

  • Blue top, sodium citrated Vacutainer tube thromboscreen.
  • Thromboplastin (CMS),liquid thromboscreen controls, levels I & II MLA 800 cuvettes, and 0.1 ml pipettor pipettor tips.
  • MLA 800 automatic coagulation timer.
  • One tube of citrated blood (blue top tube), drawn by the two-tube method.
NOTE: The tube must have the exact required blood volume. A "short" draw will affect results. Ratio of blood to anticoagulant must be 1:10. Most tubes draw
  • 4.5 ml of whole blood to be mixed with 0.5 ml anticoagulant, usually sodium citrate. Tube should be centrifuged as soon as possible, or, preferably centrifuged within one hour.

Procedure

  1. Obtain the coagulation specimen by the two-tube method.
  2. Centrifuge the specimen, at 2500 RPM, for 5 minutes, to obtain a platelet poor plasma (PPP) specimen.
  3. Remove the specimen and place in an ice bath or cold block.
  4. Make sure that the correct reagents on set up on the instrument, since it performs both PT and APTT testing, using the same pumps.
  5. Pump #1 should contain the thromboplastin reagent.
  6. Pump #2 may contain the activated partial thromboplastin reagent, it does not matter. It will not be used during this Procedure
  7. Place a cuvette in the first well.
  8. Pipette into wells A and B, 0.1 ml of the same patient's PPP specimen.
  9. Place the instrument in the PT mode.
  10. Press run on the instrument's touch panel.
  11. Following the two minute incubation period that the instrument is set up for, the results will be printed out on both the screen and on a hard copy.
  12. The instrument will print out both results, from wells A and B. In addition, it will print out the average plus a ratio from a stored normal range value.

Calculations.

All done by the instrument.

Source of Error.

  • Avoid traumatic venipuncture.
  • Tourniquet application must not be prolonged.
  • The test must be accomplished within three hours after collection of the blood specimen. If the test is not run immediately, refrigerate the plasma and thromboplastin. Refrigerated plasma is stable for a maximum of three hours.
  • The blood must be added to the anticoagulant immediately to avoid clot formation.
  • The use of sodium oxalate as anticoagulant decreases the activity of factor V. Use sodium citrate as stated in the Procedure
  • Hemolyzed plasma must not be used.

Paired PT values must be within plus or minus 0.5 secs of each other in the normal range, or the instrument will beep and flag the results. The prothrombin activity may also be reported as a ratio as well as in seconds by applying the patient's prothrombin time (in seconds) to a stored normal range prothrombin value.

The prothrombin activity of a patient's plasma has important diagnostic and prognostic significance in diseases of the liver, in vitamin K deficiency, specific coagulation deficiencies, and in the use of coumarin drugs as therapeutic anticoagulants.

Patients receiving coumarin drugs for thromboembolic disorders generally have prothrombin times of 20 to 30 seconds or 1.5 to 2.5 times their normal prothrombin time.

Prolongation of the one-stage prothrombin time does not measure prothrombin deficiency alone but rather indicates some failure of conversion of prothrombin to thrombin. Specifically, the test detects deficiencies of factors I, II, V, VII, or X. Varying reductions in any one, or combination, of these factors prolong the one- stage prothrombin time.

This test is well adapted for fibrometer use also. Prothrombin time Variation

  • 12-20 seconds 0.5 second
  • 20-30 seconds 1.0 second
  • Over 30 seconds 2.0 second

Range of Values. Both normal and abnormal controls should be run on a daily or shift basis. Controls should come in within 2 S.D.'s of the established mean.

  • Normal value: 12 to 14 seconds.
  • Critical value:
    • Less Than -- PT Not applicable
    • Greater Than -- 40 secs.

PROTHROMBIN CONSUMPTION TIME

Prothrombin is partially used up in the normal clotting process. The amount of prothrombin remaining in serum indicates the adequacy of the thromboplastin complex. The time required for a clot to form when optimal amounts of thromboplastin, fibrinogen, factor V, calcium, and serum are mixed is the prothrombin consumption time. The test is less sensitive than either TGT or the APTT. The test is not sensitive enough to exclude minor defects in the intrinsic pathway as for mildly deficient patients. Thrombocytopenia and certain other platelet defects will cause the PCT to be abnormal.

Reagents and Specimen Required.

  • Thromboplastin, liquid thromboscreen controls, levels I & II MLA 800 cuvettes, and 0.1 ml pipettor pipettor tips.
  • MLA 800 automatic coagulation timer.
  • One tube of citrated blood (blue top tube), drawn by the two-tube method.
  • Normal adsorbed plasma reagent.
    • Mix 9 parts of freshly collected, normal blood with 1 part 3.2 percent sodium citrate.
    • Centrifuge at 2,000 rpm for 5 minutes and remove supernatant plasma.
    • For each ml of plasma, add 100 mg of chemically pure barium sulfate (see Federal Supply Catalog).
    • Mix well for 3 minutes and allow to stand at room temperature for an additional 2 minutes to complete adsorption.
    • Centrifuge the material and the upper three-quarters of the plasma.
    • Perform a prothrombin time on the adsorbed plasma. The prothrombin time should be greater than 60 seconds; if not, repeat the absorption.
NOTE: Barium sulfate-adsorbed plasma is available commercially. Also available is a reagent containing fibrinogen, factor V, thromboplastin, and calcium.

Procedure

  1. Draw 5 ml of blood by a nontraumatic venipuncture.
  2. Place the blood in a test tube and allow it to clot at room temperature.
  3. Place the clotted blood in a 37C water bath for 1 hour.
  4. Centrifuge the specimen at 3,000 rpm for 3 minutes and transfer the serum to another test tube.
  5. Place the serum, thromboplastin reagent, and barium sulfate- adsorbed plasma in a 37C water bath for 5 minutes to equilibrate to that temperature.
  6. Pipet 0.1 ml of serum, 0.1 ml of barium sulfate-adsorbed plasma, and 0.2 ml of thromboplastin reagent into a Kahn tube in that order, Once items are added, start a stopwatch.
  7. Determine the time for the clot to form as in the one-stage prothrombin time.
  8. Repeat the procedure, commencing with step 6, for two other determinations.
  9. The three determinations should agree as follows:

Time Variation

  • Over 30 seconds 2.0 seconds
  • 20-30 seconds 1.0 second
  • 12-20 seconds 0.5 second

Sources of Error

  • Patients having decreases or defects in the thromboplastin complex (thrombocytopenia, hemophilia, deficiency in plasma thromboplastin antecedent or component) will not use up normal amounts of prothrombin in the clotting process. The excess of residual prothrombin in the serum will result in a serum prothrombin time of less than 20 seconds. If the content of any of the factors in the first phase is low, the rate will be decreased and, therefore, the prothrombin will not be consumed as rapidly as normal.
  • Administration of heparin will serve to shorten the prothrombin consumption time as well as prolong the "Lee-White" clotting time. Coumarin, on the other hand, will have no effect upon the serum prothrombin time, even while prolonging the plasma prothrombin time. This indicates that coumarin has no influence upon the thromboplastin complex.
  • Normal results are considered valid only if the one-stage prothrombin time is normal.
  • The test is abnormal in platelet deficiencies and deficiencies of factors VIII, IX, X, XI, and XII.
  • It is important that the barium sulfate-adsorbed plasma be deprothrombinated sufficiently to give a prothrombin time of over 1 minute. This reagent is a source of fibrinogen and factor V since they are used up in the formation of a clot.

Range of Values

(1) Normal value: Longer than 30 seconds.
(2) Doubtful: 20 to 30 seconds.
(3) Abnormal: Less than 20 seconds.

THROMBOPLASTIN GENERATION TIME

Principle

A potent thromboplastin is generated when platelets, prothrombin- free plasma, serum, and calcium are mixed. After generation of the thromboplastin, all factors necessary to produce a clot are present except for factor I and factor II. When these factors are added to a normal thromboplastin generation mixture, a clot is detected within 7-16 seconds. With an abnormal time, deficient factors are identified by substituting adsorbed patient plasma or aged patient serum. This procedure, which is quite complicated, tests the ability of the patient to generate adequate prothrombin activation through the intrinsic system pathway.

Reagents and Specimen Required.

  1. Normal plasma substrate. Available from the Federal Supply Catalog. This control is used as the source of factors I, II, V, VII, and X.
  2. Partial thromboplastin (platelet-like substance). Available from commercial sources.
  3. Normal adsorbed plasma reagent. See paragraph 6-17b(2). The adsorbed plasma is the source of factors V, VIII, XI, and XII. Dilute normal adsorbed plasma 1:5 in saline (0.85 percent NaCl) and allow to stand for 1 hour at 4C or place in an ice bath before use.
  4. Normal aged serum. Add 2 ml of freshly-drawn blood to a clean test tube and allow to clot at 37C for 4 hours. After the incubation, centrifuge and remove the clot. Dilute the serum 1:10 with 0.85 percent NaCl (saline) and allow to stand for 1 hour before using.
  5. Calcium chloride, 0.025 M. Add 0.277 g anhydrous calcium chloride to a 100 ml volumetric flask. Dilute to the mark with distilled water.
  6. Patient's adsorbed plasma reagent. Prepare in the same manner as normal adsorbed plasma, substituting patient's plasma for normal plasma.
  7. Patient's aged serum reagent. Prepare in the same manner as normal aged serum substituting patient's serum for normal serum.

Procedure for Standardization of Control Reagents

  1. Pipet 0.1 ml of 0.025 M calcium chloride into three 12 x 75 mm test tubes.
  2. Place test tubes of the following reagents in a 37C water bath:
  • Partial thromboplastin reagent.
  • Normal plasma substrate.
  • Normal adsorbed plasma.
  • Normal aged serum.
  • 2.0 ml of 0.025 M calcium chloride.
  • Four tubes containing 0.1 ml of 0.025 M calcium chloride.
  1. Prepare a generation mixture by adding the following reagents to a clear 12 x 75 mm test tube:
  • 0.2 ml partial thromboplastin reagent.
  • 0.2 ml normal adsorbed plasma reagent.
  • 0.2 ml normal aged serum reagent.
  • 0.2 ml of 0.025 M calcium chloride, simultaneously starting a stopwatch or automatic timer.
  1. At 2 minutes, pipet 0.1 ml of the generation mixture into the first tube containing 0.1 ml of 0.025 M calcium chloride. Immediately add 0.1 ml of normal plasma substrate to the tube, simultaneously starting a stopwatch.
  2. Check for the first formation of a clot using the tilt-tube method. (If available, substitute a Fibrometer for detection of clot formation.)
  3. Repeat steps 4 and 5 at 4, 6, and 8 minutes.
  4. The normal range for the control reagents is 7 to 16 seconds for clot formation. This range is obtained within the 2 to 8 minute generation time.

Procedure for Test for Patient Deficiencies

  1. Repeat the procedure for standardization of control reagents, substituting patient's aged serum for normal aged serum and patient's adsorbed plasma for normal adsorbed plasma.
  2. If there is a marked difference between the normal generation time and the patient's generation time, repeat the test, substituting normal aged serum and normal adsorbed plasma, one at a time, for the patient's reagent in the generation mixture.

Interpretation

See chart below.

Sources of Error

  1. All reagents must be freshly prepared each day.
  2. Tubes of plasma substrate left at room temperature are unreliable.
  3. All U.S.P. barium sulfate preparations are not standard in reaction. Factor IX at times may be incompletely removed.
  4. Antithromboplastin activity in blood prolongs TGT, and accounts for some double deficiencies.
  5. This is not a routine laboratory procedure; considerable technologist skill is required.
  6. A clot can form when the generation mixture is prepared. This is removed with a wooden applicator stick.
  7. Other sources of error can be found in paragraphs 2-15d and 2-16d.

Discussion

  1. A platelet deficiency can be detected by the TGT. This is done by preparing platelet-rich plasma and substituting it for the partial thromboplastin reagent. An abnormal generation time is noted in a patient with platelet deficiency.
  2. The TGT reveals abnormalities essential to the development of thromboplastin activity in the intrinsic blood system.
  3. The TGT should be correlated with the PT and PTT to determine the specific abnormality.
  4. Adsorbed plasma contains factors V, VIII, XI, and XII. If the TGT is prolonged when patient's adsorbed plasma is substituted for normal adsorbed plasma, a deficiency in one or more of these factors is indicated.
  5. Aged serum contains factors IX, X, XI, and XII. If the TGT is prolonged when patient's aged serum is substituted for normal aged serum, a deficiency in one or more of these factors is indicated.
  6. Factors XI and XII are present in adsorbed plasma and aged serum. A deficiency in either of these factors prolongs the TGT. Determination of the specific factor deficiency is done by the physician on the basis of clinical grounds.

Normal Values

Clot formation time of 7 to 16 seconds within 8 minutes generation time.


THROMBIN TIME

Principle

A known amount of thrombin is added to plasma and the time required for clot formation is recorded. Results are affected by abnormal fibrinogen levels, heparin, and antithrombins. This is useful in the diagnosis of DIC.

Reagents

  1. Thrombin, 5000 units per ml. Available from the Federal Supply Catalog.
  2. Stock thrombin solution, 500 units per ml. Reconstitute the thrombin with 10 ml of saline (0.85 percent NaCl).
  3. Working thrombin solution, 10 Units per ml. Dilute the stock solution 1:50 with saline (0.85 percent NaCl).
  4. Control plasma. Available from the Federal Supply Catalog.

Procedure

  1. Obtain 4.5 ml of venous blood and add to 0.5 ml sodium citrate. Mix well.
  2. Centrifuge and separate the plasma.
  3. Incubate patient plasma, control plasma, and thrombin solution in 37C water bath for 3 minutes.
  4. Pipet 0.1 ml of control plasma and 0.1 ml of thrombin solution into a clean test-tube, simultaneously starting a stopwatch. Observe for clot formation.
  5. Repeat step (4) for the patient's plasma.

Discussion

(1) The thrombin time is prolonged in fibrinogen levels below 100 mg per dl, presence of fibrinolysins, and presence of circulating anticoagulants.

(2) Low concentrations of heparin in the patient's plasma may not be observed by this test. To detect small amounts of heparin, thrombin is diluted, and normal patient's plasma thrombin times are determined. A prolongation of the patient's thrombin time over that of the normal at some dilution indicates an antithrombic substance.

(3) To perform the antithrombin test, dilute the working thrombin 1:2, 1:4, 1:8, 1:16, and 1:32 with saline. Then follow the procedure for the thrombin time test using the dilutions.

Normal Values.

11 to 15 seconds.


FIBRINOGEN ASSAY (SEMIQUANTITATIVE)

Principle

Fibrinogen, a plasma globulin formed in the liver, is salted out by ammonium sulfate and measured with a spectrophotometer.

Reagents

  1. Parfentjev reagent. Add 133.33 g ammonium sulfate, 10.0 g NaCl, and
  2. 0.025 g merthiolate to a 1-liter volumetric flask. Dilute to the mark with distilled water.
  3. Saline (0.85 percent NaCl). Add 8.5 g sodium chloride to a 1-liter volumetric flask. Dilute to the mark with distilled water.
  4. Fibrinogen, U.S.P. Available from the Federal Supply Catalog.
  5. Fibrinogen stock standard, 500 mg per dl. Add 500 mg of fibrinogen to a 100-ml volumetric flask. Dilute to the mark with saline.

Calibration Curve

  1. Prepare the following dilutions of the stock standard:

  1. Set up the following cuvettes for each standard: Blank Cuvet Test Cuvette

  1. Three minutes after addition of the Parfentjev reagent, shake the cuvettes vigorously, and read the absorbances of the test cuvettes at 510 nm with the blank set at zero absorbance.
  2. Plot absorbance versus concentration on linear graph paper.

Procedure

  1. Draw 4.5 ml of fresh venous blood and add to a test tube containing 0.5 ml of 3.8 percent sodium citrate.
  2. Centrifuge and separate the plasma.
  3. Set up the following cuvettes for each unknown plasma:

  1. Three minutes after addition of the Parfentjev reagent, shake the cuvets vigorously, and read the absorbances of the test cuvettes at 510 nm with the blank set at zero absorbance.
  2. Obtain the fibrinogen concentration from the calibration curve.

Sources of Error

  1. The Parfentjev reagent deteriorates after two weeks of storage. Prepare fresh every 2 weeks.
  2. The fibrinogen has a potency for 60 months. Do not use outdated fibrinogen to prepare standards.
  3. The procedure is limited as it is dependent on time, temperature, anticoagulant, and concentration of other proteins.

Discussion

  1. Although the procedure is limited, a rapid determination can be obtained when adequate fibrinogen levels are present.
  2. Screening procedures, in kit form, are available commercially.
  3. Fibrinogen is essential to clot formation in stage 3 of the clotting mechanism. Bleeding is encountered when the plasma fibrinogen level falls below 75 mg per dl.
  4. A colorimetric fibrinogen procedure is outlined in AFM 160-49.

Normal Values

200 to 400 mg per dl.


PLASMA RECALCIFICATION TIME

Principle

Calcium chloride is added to plasma and the clotting time recorded.

Reagents

  1. Calcium chloride, 0.025 M
  2. Sodium citrate, 3.8 percent, is available from the Federal Supply Catalog.
  3. Sodium chloride, 0.85 percent (w/v).

Procedure

  1. Draw 4.5 ml of fresh venous blood and add to 0.5 ml of 3.8 percent sodium citrate.
  2. Centrifuge at 2,500 rpm for 20 minutes and separate the plasma.
  3. Incubate, at 37C for 2 to 3 minutes prior to each test, each of the following, in separate test tubes:
  • Patient's platelet-poor plasma.
  • Normal platelet-poor control plasma.
  • Calcium chloride, 0.025 M.
  • Sodium chloride, 0.85 percent.
  1. Into a 13 x 100 mm test tube, in the 37C water bath, pipet 0.1 ml 0.85 percent sodium chloride and 0.1 ml of patient's plasma. Mix.
  2. Blow in 0.1 ml 0.025 M calcium chloride and simultaneously start a stopwatch.
  3. Allow the tube to remain in the 37C water bath for 90 seconds, tilting the tube gently every 30 seconds.
  4. After 90 seconds, remove the tube from the water bath and gently tilt. Stop the watch as soon as a clot forms, and record the results.

Sources of Error

  1. Platelet-poor plasma must be used in the test. Platelet-rich plasma shortens the recalcification time.
  2. The test is dependent on the platelet count, concentration of plasma clotting factors, time of storage in glass, and the presence of circulating anticoagulants.

Discussion

  1. This test is the basis for other coagulation procedures.
  2. A decrease in any of the clotting factors present in the intrinsic system will cause a prolonged clotting time.

Normal Values

90 to 250 seconds.


DETECTION OF A CIRCULATING ANTICOAGULANT

Principle

An abnormal recalcification time is not corrected by the addition of normal plasma if a circulating anticoagulant is present.

Reagents

  1. Calcium chloride, 0.025 M
  2. Sodium citrate, 3.8 percent, is available from the Federal Supply Catalog.
  3. Sodium chloride, 0.85 percent (w/v).

Procedure

  1. Obtain citrated plasma from a normal donor and from the patient.
  2. Set up the following mixture in 12 x 75 mm test tubes:

  1. Add 0.2 ml of 0.025 M. CaCl2 to each tube and determine the plasma recalcification time on each tube.

Interpretation

  1. The abnormal recalcification time is corrected by normal plasma if no circulating anticoagulant is present.
  2. A circulating anticoagulant present in patient's plasma prolongs the recalcification time of normal plasma.

DETECTION OF FACTOR XIII DEFICIENCY

Principle

Factor XIII converts a loosely-linked, fibrin clot (in the presence of calcium ions) into a tough gel. The well-formed clot is insoluble in 5 M urea. In the absence of factor XIII, the clot lyses within 3 hours.

Reagents

  1. Sodium citrate, 3.8 percent. Available from the Federal Supply Catalog.
  2. Calcium chloride, 0.05 M. Add 0.555 g anhydrous calcium chloride to a 100 ml volumetric flask. Dilute to the mark with distilled water.
  3. Urea solution, 5 M. Add 30 g of urea to a 100-ml volumetric flask. Dilute to the mark with distilled water.

Procedure

  1. Draw 4.5 ml of fresh venous blood and add to 0.5 ml of 3.8 percent sodium citrate.
  2. Centrifuge at 2,500 rpm for 5 minutes and separate plasma.
  3. Pipet 0.5 ml of patient's plasma into each of two test tubes. Repeat, pipeting 0.5 ml of normal control plasma into each of two additional tubes.
  4. Add 0.5 ml of 0.05 M calcium chloride to the four tubes.
  5. Incubate the resulting fibrin clots at 37C for 30 minutes.
  6. Loosen the clots from the sides of the test tubes by gently tapping the sides of the tube.
  7. Transfer one of the patient's clots and one of the normal control clots to respective tubes containing 5 ml of 5 M urea. Transfer both the remaining patient clot and the normal control clot to a third tube containing 5 ml of 5 M urea.
  8. Allow the mixtures to stand at room temperature.
  9. Check the clots at the end of 1 hour, 2 hours, 3 hours, and 24 hours, and note if the clots have dissolved.

Interpretation

A clot that lyses within 3 hours is deficient in factor XIII.


PLATELET COUNT (UNOPETTE)

Principle

Unopette Test is a stable in vitro diagnostic reagent system for the enumeration of leukocytes and platelets in whole blood. Whole blood is added to diluent, the tonicity of which lyses red cells but preserves platelets, leukocytes and reticuloytes.

Reagent

Unopette reservoir- containing 1.98 ml of diluent mixture. 11.45 gm of ammonium oxalate. 1.0 gm sorensens phosphate buffer. 0.1 gm thimersat and purified water. Unopette capillary pipette.

Procedure

  1. Puncture diaphram in unopette reservoir
  2. Remove shield and draw blood into the unopette capillary tube.
  3. Add the whole blood in capillary tube to diluent in reservoir, squeeze gently two to three times, let stand for 10 minutes, the diluted specimen is good for 3 hours at room temperature
  4. Prepare a moist chamber by placing a moist piece of filter paper in the top of a Petri dish. Paper should be moist enough to adhere to the Petri dish.
  5. Discharge a few drops from the pipet. Then charge both chambers of a hemacytometer.
  6. Allow the platelets to settle for 10 minutes. To prevent drying, place the moist chamber over the hemacytometer.
  7. Under 10x magnification, leukocytes will appear refractile, under 40x magnification platelets appear oval to round.
  8. Count all (9) large squares of counting chamber.

Calculations

Add 10 percent of count to total number of cells counted. Multiply by 100 to get total leukocyte count. The formula for calculation of indirect platelet count is as follows.

Sources of Error

  1. Platelet agglutinates invalidate the count. This is prevented by using scrupulously clean glassware. Therefore, the following precautions are recommended:
  • Boil counting chambers and coverslips for 2 hours in distilled water to which a small amount of sodium bicarbonate has been added.
  • Pipets must be very carefully cleaned.
  • The diluting fluid must be frequently filtered or centrifuged and stored at 2o to 4oC.
  1. Other sources of error as listed under red and white blood cell counts also apply, including pipetting errors. These procedures should be consulted.
  2. More accuracy may be obtained through use of the phase contrast microscope.

Discussion

  1. No one method for the enumeration of thrombocytes is satisfactory in every respect. In experienced hands the direct procedure is more accurate than the indirect method, and venous blood samples are more representative and preferable to those obtained by capillary puncture. Direct procedures, however, require greater skill and experience for proper performance and interpretation. The error in a single count has been estimated to be about 10 percent and this margin of error can be reduced by multiple counts.
  2. A major disadvantage of the direct thrombocyte count is that the platelets must be counted under high-power rather than oil-immersion magnification as is used in the indirect method. Because of the extremely small size of blood platelets, it is a common error to confuse them with yeasts, debris, and precipitated stain.
  3. Due to the inherent errors in this procedure, it is recommended that a thrombocyte count be performed on a normal person as a control.

Normal Values

150,000 to 350,000 platelets per cu mm.

NOTE: This range is generally acceptable, but it must be realized that there is a great variance in normal values which differ with each technique, laboratory, and technician.

PLATELET COUNT (PHASE MICROSCOPY)

Principle

Blood is diluted with ammonium oxalate, and the diluted specimen is introduced into a counting chamber. The platelets appear round or oval, pink, purple, or even black under a phase condenser.

Reagent

Ammonium oxalate, 1%. Add 1g of ammonium oxalate to a 100-ml volumetric flask. Dilute to the mark with distilled water. Store in the refrigerator and filter before use.

c. Procedure

  1. Draw 5 ml of venous blood and immediately place in a test tube containing EDTA. (If capillary blood is used, immediately fill a red blood cell diluting pipet to the 1 mark.)
  2. Fill a red blood cell diluting pipet to the 1 mark and then draw ammonium oxalate to the 101 mark.
  3. Shake the pipet 10 to 15 minutes.
  4. Discard from 1/3 to 1/2 the volume from an RBC pipet, then charge a special flat bottom-phase counting chamber. Place the counting chamber in a Petri dish containing moist gauze and let stand 15 to 20 minutes to ensure complete settling of platelets.
  5. Count the platelets in all 25 squares of the area normally used for the red cell count. Multiply the results by 1,000.
  6. Both sides of each chamber are filled with the same sample; the count from either side should not deviate more than 10% from the other count. If a greater than 10 percent deviation occurs, repeat the count using a fresh dilution and a second chamber.
  7. Report the average of the two sides counted.

Sources of Error

  1. The diluting fluid must be fresh and free of bacterial contamination.
  2. Platelet clumping occurs if there is delay in adding blood to the anticoagulant or if the mixing is inadequate. Platelet clumps cause invalid results.
  3. Occasionally extraneous material is mistaken for platelets.
  4. Glassware that is not scrupulously clean causes platelets to attach to the debris on the glassware.
  5. Platelet agglutinates invalidate the count. This is prevented by using scrupulously clean glassware. Therefore, the following precautions are recommended:
  • Boil counting chambers and cover slips for 2 hours in distilled water to which a small amount of sodium bicarbonate has been added.
  • Pipettes must be very carefully cleaned.
  • The diluting fluid must be frequently filtered or centrifuged and stored at 2 to 4 C.
  1. Other sources of error as listed under red and white blood cell counts also apply, including pipetting errors. These procedures should be consulted.
  2. More accuracy may be obtained through use of the phase contrast microscope.

Discussion

  1. Platelets sometimes show dendritic processes. Structures such as dirt, crystals, and WBCs are refractile. Platelets are not. The white cells are normally lysed; however, with patients that demonstrate excessive high white counts some cells will be observed in the platelet counting prep.
  2. Ammonium oxalate ensures clearing of the background by hemolysis.
  3. This method is more accurate than the Rees-Ecker. The main drawback to this method is that special equipment is required.
  4. No one method for the enumeration of thrombocytes is satisfactory in every respect. In experienced hands the direct procedure is more accurate than the indirect method, and venous blood samples are more representative and preferable to those obtained by capillary puncture. Direct procedures, however, require greater skill and experience for proper performance and interpretation. The error in a single count has been estimated to be about 10 percent and this margin of error can be reduced by multiple counts.
  5. A major disadvantage of the direct thrombocyte count is that the platelets must be counted under high-power rather than oil-immersion magnification as is used in the indirect method. Because of the extremely small size of blood platelets, it is a common error to confuse them with yeasts, debris, and precipitated stain.
  6. Due to the inherent errors in this procedure, it is recommended that a thrombocyte count be performed on a normal person as a control.

Normal Values

150,000 to 350,000 per cu mm.


MACROGLOBULIN DISTILLED WATER SCREENING TEST

Principle

 Macroglobulins are precipitated when brought into contact with distilled water.

Procedure

  1. Collect 5 ml of blood from the patient by venipuncture.
  2. Set the blood aside for 1 hour to clot, and then separate the serum by centrifuging at 2,000 rpm for 5 minutes.
NOTE: Oxalated, citrated, or heparinized plasma is also satisfactory for the performance of this test.
  1. Place 5 ml of distilled water in a Wassermann tube.
  2. With a 1 ml pipet, draw up 0.5 ml of patient's serum or plasma. Holding the pipet tip close to the meniscus of the water, add 0.2 ml of the patient's serum or plasma to the distilled water.
  3. Interpret results.

Interpretation

  1. Observe the behavior of the drop of serum or plasma sinking in the water and the modifications of color of the solution.
  2. If macroglobins are present:
  • (a) The drop will sink leaving a smoky trace.
  • (b) The entire distilled water medium will promptly assume a whitish color, due to the condensation of transparent, slimy masses forming a precipitate which quickly sinks to the bottom.
  1. The separated precipitate will dissolve well when transferred to a test tube containing normal saline.

CRYOGLOBULIN SCREENING TEST

Principle

Cryoglobulins in serum or plasma are precipitated at 4C and dissolved by temperature elevation to 37C.

Procedure

  1. Collect 10 ml of blood by venipuncture and place a 5 ml portion in a tube containing anticoagulant (citrate, oxalate, or heparin) and place the other 5 ml portion into a clean, sterile test tube.
  2. Incubate both tubes immediately for 1 hour at 37C.
  3. One hour later, separate the supernatant plasma from the tube containing the anticoagulant and the serum from the clot in the untreated tube.
  4. Transfer aliquots of 1 ml of serum into 2 clean sterile Wassermann tubes.
  5. Transfer aliquots of 1 ml of plasma into 2 clean sterile Wassermann tubes.
  6. Incubate 1 serum specimen and 1 plasma specimen at 4C (refrigerator temperature) for 4 hours.
  7. Incubate 1 serum specimen and 1 plasma specimen at 37C for 4 hours.
  8. Read results.

Interpretation

  1. The test is positive if the serum or plasma incubated at 37C shows no changes and the serum or plasma incubated at 4C shows the following changes:
  • (a) The serum or plasma appears clotted in toto.
  • (b) The plasma or serum appear divided into two layers; the upper one containing normal plasma or serum, faintly stained by bilirubin; the lower one represented by the cryoglobulin, whitish in color, which has precipitated to the bottom of the test tube.
  1. The test is confirmed if the serum or plasma incubated at 4C is brought back to a temperature of 37C and its appearance returns to normal.

FIBRIN/FIBRINOGEN DEGREDATION (SPLIT) PRODUCTS

Principle

. In the normal production of clot formation, fibrinogen is acted upon by thrombin, breaking off small molecules from the fibrinogen molecule. These small molecules are fibrinopeptides D and E (fibrinogen split products). Once these molecules are broken off, the main molecule that remains is the fibrin monomer (A & B). It is this monomer that polymerizes to form the fibrin threads of a clot. In addition to the split products from the action of thrombin on fibrinogen, additional split products are produced by the action of fibrinolysin on fibrinogen and plasma in both fibrinogen and fibrin clots. When large amounts of FSP's are produced or are present, prior to adsorption antifibrinogen antiserum with an affected individual's serum, they may interfere with polymerization of fibrin monomers, preventing clot formation (DIC).

Specimen and Reagents

  1. (Venous blood in a special blue top vacutainer-type tube does not contain the anticoagulant found in blue top tubes, but a clot activator, specific for FDP assay. The tube contains thrombin to promote clotting, and soybean trypsin inhibitor to prevent in vitro fibrinolysis. This is sufficient to collect 2 ml whole blood or urine.
  2. Use an FDP Assay Kit

Procedure

  1. Prepare dilutions of serum samples. Take 2- 12 x 75 mm test tubes and label them #1 and #2.
  2. Mark 2 of the rings on the glass slide #1 and #2.
  3. Using the graduated dropper provided with the bottle of buffer solution in the kit, place 0.75 ml of glycine buffer into each test tube.
  4. Using 1 of the plastic droppers (provided with the kit), with the teat provided (blue), aspirate clear serum.
  5. Place 5 drops of serum into test tube #1 and 1 drop of serum into test tube #2.
  6. Mix the contents of each test tube:
Dilution #1 = 1:5.
Dilution #2 = 1:20.
  1. Rinse the plastic dropper with glycine buffer.
  2. Transfer 1 drop from test tube #2 to position #2 on the slide.
  3. Now take 1 drop from test tube #1 and place it in position #1 on the slide.
NOTE: Since you're going from a lesser to greater concentration, you must pipette in this order.
  1. Mix the latex suspension by shaking the container vigorously for a few minutes.
  2. Add 1 drop of latex suspension to each position on the slide.
  3. Stir the serum/latex mixture with a wooded applicator, spreading the mixture to fill the entire circle.
  4. Rock the slide gently back and forth for a maximum of 2 minutes, while observing for agglutination. Agglutination means that there is at least 2 <μg/ml present.

Calculations

(1) If agglutination is present, there is at least 2 μg/ml present in that position.

(2) If agglutination occurs in #1, concentration is greater than 10 ug/ml. If agglutination occurs in #2, concentration is greater than 40 μg/ml.

NOTE: Additional serial dilutions may be made and tested accordingly.

Controls

Controls should be run with each batch of tests. Positive control should contain 5 to 10 μg/ml FDP. Negative control should contain less than 2 μg/ml of FDP.

Range of Values

Normal values are below 10 μg/ml. In cases of DIC, levels of FDP exceed 10 μg/ml, and in severe cases can exceed 40 μg/ml.


FACTOR V ASSAY (SIMILAR FOR FACTORS VII & X)

Principle

The patient's plasma is mixed with a commercially known deficient plasma. It is then tested, by doing a Prothrombin Time test on the mixture, and compared on a logarithmic chart. Factors V, VII, and X are synthesized in the liver and are decreased in severe liver disease.

Material and Equipment Required

  1. Citrated plasma from a blue top, vacutainer-type tube.
  2. Commercially prepared factor (specific) deficient plasma and Log-Log chart paper.
  3. Any device for performing prothrombin time testing.

Procedure

  1. Prepare the graph from normal citrated plasma (see chart below).

  1. Set up a series of five 10 x 75 mm test tubes.
  • 1st tube -- 0.01 ml undiluted plasma.
  • 2nd tube -- 0.01 ml 50% dilution.
  • 3rd tube -- 0.01 ml 25% dilution.
  • 4th tube -- 0.01 ml 10% dilution.
  • 5th tube -- no normal plasma (optional).
  1. To the first 4 tubes, add 0.09 ml factor V (or VII or X) deficient plasma, and mix.
  2. Proceed with performing a regular prothrombin time analysis.
  3. The above mixtures are in a ratio of 1:9, where 1 is normal and 9 is deficient.
  4. Record the results on a double-logarithmic (Log-Log) scale.
  5. For each patient to be analyzed, use 100% and a 50% dilution.
  6. Read the results off the graph.

Controls

Any normal coagulation controls will do. Any abnormal coagulation controls will do.

Normal Values

Normal patients should show values from 80 to 120 of the normal control.

NOTES:
  • Refrigeration preserves factor V, but after 24 hours, even at 4C, factor V is greatly reduced.
  • Factor VII is a vitamin K dependent factor.

FACTOR VIII ASSAY (SIMILAR FOR FACTORS IX, XI, & XII)

The patient's plasma is mixed with a commercially known deficient plasma. It is then tested, by doing a activated partial thromboplastin test on the mixture, and compared on a logarithmic chart. Factors VIII, IX, XI, and XII are analyzed using this Procedure

Materials, Equipment, and Specimen Required.

  • Citrated plasma from a blue top, vacutainer-type tube.
  • Commercially prepared factor (specific) deficient plasma and Log-Log chart paper.
  • Any device for performing activated partial thromboplastin time testing.

Procedure

  1. Prepare the graph from normal citrated plasma (see chart below).

  1. Set up a series of five, 10 x 75 mm test tubes.
  • 1st tube -- 0.01 ml undiluted plasma.
  • 2nd tube -- 0.01 ml 50% dilution.
  • 3rd tube -- 0.01 ml 25% dilution.
  • 4th tube -- 0.01 ml 10% dilution.
  • 5th tube -- No normal plasma (optional).
  1. To the first 4 tubes, add 0.09 ml factor VIII (or IX, XI, or XII) and deficient plasma. Mix.
  2. Proceed with performing a regular activated partial thromboplastin time analysis.
  3. The above mixtures are in a ratio of 1:9, where 1 is normal and 9 is deficient.
  4. Record the results on a double-logarithmic (Log-Log) scale.
  5. For each patient to be analyzed, use 100% and a 50% dilution.
  6. Read the results off the graph.

Control

Any normal coagulation controls will do. Any abnormal coagulation controls will do.

Normal Values

Normal patients should show values from 80 to 120 percent of the normal control.

 

David L. Heiserman, Editor

Copyright   SweetHaven Publishing Services
All Rights Reserved

Revised: June 06, 2015