METHODS: Using Danish healthcare registries from 1995 to 2020, we obtained data on cancer patients with venous thromboembolism and

 

ABSTRACT

BACKGROUND: Despite recent improvements in the treatment of cancer, little is known about the long-term survival in patients with cancer and venous thromboembolism. We aimed to examine the five-year mortality of venous thromboembolism in cancer patients in a large population-based cohort study.

METHODS: Using Danish healthcare registries from 1995 to 2020, we obtained data on cancer patients with venous thromboembolism and comparison cohorts of cancer patients without venous thromboembolism, matched in terms of cancer type, age, sex, and year of cancer diagnosis, and adjusted for level of comorbidity and frailty using the Charlson Comorbidity Index Score and Hospital Frailty Risk Score, marital status, use of selected medications, and recent surgery (<90

FINDINGS: During the study period, 886,536 patients were diagnosed with cancer. Of 1882 cancer patients diagnosed at the time of their venous thromboembolism, 44.4% (835/1882) had distant metastases. In this cohort, the one- and five-year mortality cumulative incidences were 68% (1284/1882) and 84% (1578/1882), respectively, in contrast to 38% (2135/5549) and 67% (3653/5549) in the comparison cohort. The mortality rate ratio was 4.34 (95% confidence interval [CI], 3.95-4.78) for the first year of follow-up and 3.44 (95% CI 3.17-3.73) for the five-year follow-up period. Of the 23,366 patients diagnosed with venous thromboembolism after cancer diagnosis, 18% (4183/23,366) had distant metastases at the time of cancer diagnosis. The cumulative incidence of death at one year was 45% (10,465/23,366; mortality rate ratio 3.48, 95% CI 3.37-3.60) and at five years 69% (15,669/23,366; mortality rate ratio 2.57, 95% CI 2.50-2.63).

INTERPRETATION: Despite improved cancer treatment, venous thromboembolism in cancer patients is strongly associated with a poor prognosis.

FUNDING: The study was supported by grants from the Independent Research Fund Denmark (record no. 3101-00102B) and the Karen Elise Jensen Foundation.

PMID:37809052 | PMC:PMC10558815 | DOI:10.1016/j.lanepe.2023.100739

07:05

PubMed articles on: Cancer & VTE/PE

A framework to characterise the reproducibility of meta-analysis results with its application to direct oral anticoagulants in the acute treatment of venous thromboembolism

Res Synth Methods. 2023 Oct 17. doi: 10.1002/jrsm.1676. Online ahead of print.

s and clinically relevant non-major bleedings were merged to "clinically relevant bleedings". Risk factors were estimated by odds

 

ABSTRACT

BACKGROUND: Incidence of and risk factors for bleeding in cancer patients with venous thromboembolism (VTE) treated with apixaban are poorly described.

METHODS: We analyzed data from the prospective CAP study where 298 cancer patients with any type of VTE received 5 mg apixaban twice daily for 6 months, and then 2.5 mg apixaban twice daily for 30 months. For most analyses major bleedings and clinically relevant non-major bleedings were merged to "clinically relevant bleedings". Risk factors were estimated by odds ratios (OR) and 95% confidence intervals (CI).

RESULTS: The incidence of clinically relevant bleedings was 38% per person year during the first 6 months of treatment, 21% per person year from 7 to 12 months, and between 4% and 8% per person year from 13 to 36 months. Clinically relevant bleedings were associated with age above 74 years (OR 2.0, 95% CI 1.0-4.1), BMI below 21.7 (OR 2.3, 95% CI 1.1-4.8), and hemoglobin at baseline below 10.5 for females (OR 2.8, 95% CI 1.1-7.3) and 11.1 for males (OR 3.3, 95% CI 1.3-8.4) during the first 6 months. Gastrointestinal (GI) or urogenital cancer were not associated with clinically relevant bleedings compared with other cancers. Among patients with luminal GI-cancer, non-resected cancer had increased risk of bleeding (OR 3.4, 95% CI 1.0-11.6) compared with resected GI-cancer.

CONCLUSION: It was very few bleedings while patients were on low-dose apixaban. Factors associated with bleeding in patients treated with full-dose apixaban were high age, low BMI, and low hemoglobin, and probably non-resected luminal GI-cancer.

PMID:37816388 | DOI:10.1055/a-2188-8773

07:05

PubMed articles on: Cancer & VTE/PE

Impact of venous thromboembolism on the mortality in patients with cancer: a population-based cohort study

Lancet Reg Health Eur. 2023 Sep 28;34:100739. doi: 10.1016/j.lanepe.2023.100739. eCollection 2023 Nov.

 

and the severity of the bleeding is proportional to the degree

of deficiency. In order to treat the hemorrhagic condition,

it is important to identify and quantify the deficient factor.

Fibroscreen reagent is one such test reagent, which can

identify the deficiency of factor I (fibrinogen). The reagent

is used as a source of thrombin to determine the qualitative

reactivity of fibrinogen.

Reagent

Fibroscreen reagent is a lyophilized preparation of bovine

thrombin of 50 NIH/mL. Reconstitute with 1 mL of distilled

water; wait for 5 minutes, do not shake and mix gently by

swirling till the solution attains homogeneity. Further keep

aside for 10 minutes to attain equilibrium. Gently swirl the

vial while drawing the reagent for use. Once reconstituted

it is ready to use reagent for the thrombin time test.

Storage and Stability

1. Store the unopened reagent vials at 2–8°C. Do not

freeze.

2. The shelf-life of the reagent is as per the expiry date

mentioned on the reagent vial label and carton label.

3. Once reconstituted the Fibroscreen reagent is stable

for 6 days when stored at 2–8°C and for 4 hours

at room temperature (20–25°C), provided it is not

contaminated. Extreme care has to be taken to maintain

aseptic precautions while reconstituting, retrieving

and handling reagents to prevent contamination. The

Fibroscreen reagent vial must be replaced at 2–8°C

immediately upon retrieving the reagent for the day’s

work.

Principle

When a known quality and concentration of Fibroscreen

reagent is added to citrated plasma, by observing the time

required for clot formation and the quality of clot formed,

a qualitative estimation of fibrinogen in the sample can be

obtained.

Note

1. In vitro diagnostic reagent for laboratory and professional use. Not for medicinal use.

2. The reagent contains 0.1% sodium azide as preservative.

3. Fibroscreen thrombin reagent is not from a human

source, hence contamination due to HBsAg, HIV and

HCV is practically excluded.

4. It is very important that absolutely clean and dry

micropipettes be used to aspirate and dispense the

reagent.

5. Avoid exposure of the reagent to elevated temperatures,

direct light and contamination. Immediately replace

cap after use and store at recommended temperature.

Quality Control

A known normal control should be run in parallel with

each batch of tests. This control may be Tulip plasma

coagulation control Plasmatrol-I or freshly drawn normal

plasma.

298 Concise Book of Medical Laboratory Technology: Methods and Interpretations Sample Collection and Preparation

No special preparation of the patient is required prior to

sample collection by approved techniques. Withdraw

blood without undue venous stasis and without frothing

into a plastic syringe fitted with a short needle of 19 to

20 SWG. The venipuncture must be a ‘clean’ one and,

if there is any difficulty, take a new syringe and needle

and try another vein. Transfer the blood into tubes, after

detaching the needle from the syringe. Mix nine parts of

freshly collected blood with one part of sodium citrate

(0.109-M mol/L, 3.2%). Centrifuge immediately for fifteen

minutes at 3000 rpm (approximately 2000 g) and transfer

the plasma into a clean test tube. Plasma must be tested

within 3 hours of collection.

Additional Material Required

10 ×75 mm glass test tubes, 0.2 mL precision pipettes, stopwatch, distilled water, fresh plasma.

Procedure

Bring all the reagents and samples to room temperature

before testing.

Manual Method

Testing should be done in duplicate at room temperature

(20-25°C):

1. To a clean and dry 10 × 75 mm test tube add 0.2 mL

of plasma to be tested and 0.2 mL of the reconstituted

Fibroscreen reagent.

2. Start a stopwatch simultaneously with the addition of

the Fibroscreen reagent.

3. Shake the tube gently to mix the contents and then tilt

the tube back and forth.

4. Note the time at the first appearance of the clot and for

the remaining portion of 60 seconds for consistency

and character of the clot formed.

Interpretation of Results

Normal plasma begins to show clot formation within 15

seconds after Fibroscreen reagent has been added. Because

time of clot formation may be influenced by additional factors

1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.

2. The source material used for preparation of the reagent

is screened by third generation assays for HBsAg, HCV

and HIV antibodies and are found to be non-reactive.

However, handle the material as if it is infectious, as no

known test method can assure that infectious agents

are absent.

Preparation of the Reagent

1. Reconstitute the control plasma with exactly 1 mL

of bi-distilled water. Avoid using water-containing

preservatives.

2. Re-stopper the vial and allow to stand until, the

hydration is complete (usually 5–7 minutes).

3. Mix by gently swirling and inversion, avoiding froth

formation. Do not shake.

4. Allow to stand and equilibrate for a further 15 minutes

before use.

5. Use the reconstituted plasma within 3 hours of reconstitution.

Test Procedure

1. Use the reconstituted Plasmatrol controls in the same

manner as freshly prepared titrated platelet poor

plasma from a patient.

2. Use the procedure as laid out in the Uniplastin,

Liquiplastin, Liquicelin-E, Fibroscreen, Fibroquant

pack inserts.

Expected Values

1. The expected value of specific assays are provided on

the assay value sheet accompanying each kit, and are

lot specific.

Clinical Hematology: Bleeding Disorders 297

2. The expected values are obtained using replicate assay

of each manufactured lot of Plasmatrol, manually and

using mechanical coagulometers such as Hemostar,

Hemostar XF.

3. The individual laboratory values should fall within the

expected values.

4. It must however be noted that each laboratory should

establish its own normal values and reference range

according to GLP.

Remarks

1. When used appropriately, Plasmatrol controls are

subjected to the limitations of the assay system

deployed.

2. If proper values are not obtained it may indicate

problems with one or more variables of the assay

system.

3. Stability of the reagent is dependent on storage and

handling conditions. Since these can vary between

laboratories, each laboratory should determine

the stability of the reagent under usual operating

conditions.

4. Incorrect mixing of control plasma and reagent, insufficient preparation of plasma/reagent, contaminated

reagents and glassware, etc. are a potential source of

error.

5. Due to interlaboratory variations in techniques,

standardization of test procedures and calibration

of equipments, some variation from assigned mean

values may be expected.

FIBROSCREEN THROMBIN TIME TEST FOR

QUALITATIVE ESTIMATION OF FIBRINOGEN

FIBROSCREEN®

(Courtesy: Tulip Group of Companies)

Summary

At present there are known to be at least eleven factors in

circulating blood, which are required for normal hemostasis.

Deficiency in any of these factors viz. Factors I, II, V, VII, VIII,

IX, X, XI and XIII results in a notable hemorrhagic condition,

 

Calibration Curve Method

Heparin concentration in the test sample can be directly

obtained from the Liquicelin-E calibration curve by

interpolating the test plasma clotting time against the

heparin concentration in U/mL.

Expected Values

Normal values using Liquicelin-E reagent are between

21 and 29 seconds at 3 minutes activation time. Between

manual and turbodensitometric instrument results a

variation of 1-2 seconds may be expected. For photo-optical

instruments, it is recommended that each laboratory must

establish their own normal range.

Remarks

1. Due to inter and intralaboratory variations users must

establish their own normal population range as well

as normal and abnormal range.

2. It is recommended that controls with known factor

activity should be run simultaneously with each test

series routinely.

3. Incorrect mixture of blood and trisodium citrate,

insufficient prewarming of plasma and reagent,

contaminated reagents, glassware, etc. are potential

source of errors.

4. Incorrect dilutions of heparin is also a potential source

of error.

5. Oxalated plasma may induce prolonged clotting times.

6. Clotting time of patients on anticoagulant therapy

depends upon the type and dosage of anticoagulant

and also the time lag between the specimen collected

and the last dose.

7. Abnormalities of coagulation factor VII, factor XIII and

platelets are not detected by this test procedure.

8. For automated equipment, it is strongly recommended

that the equipments manufacturer’s methodology is

strictly adhered to.

9. In heparin monitoring time of collection of blood

sample is important since the in vivo half-life of

heparin is approximately 1.5 hours. When it is

administered intravenously, it has an immediate

anticoagulant effect but its efficacy decreases rapidly

with time.

10. Platelet factor IV, a heparin-neutralizing factor can

be released due to platelet aggregation or damage.

In order to prevent this phenomenon in vitro the

specimen should be collected with a minimum of

trauma.

11. Decrease in APTT time is observed in males under

estrogen therapy and oral contraceptive administration

in females.

Clinical Implications of APTT

1. The APTT is prolonged in all coagulation defects of

stage I (includes platelet activity and thromboplastin).

2. The APTT is usually prolonged in Willebrand’s disease

and is accompanied by a consistently diminished

factor VIII level.

3. The APTT and PT will detect 95% of coagulation

abnormalities. When APTT is performed in conjunction

with a prothrombin time (PT), a further clarification of

296 Concise Book of Medical Laboratory Technology: Methods and Interpretations coagulation defects is possible. For example, a normal

PT and abnormal APTT means that the defect lies in

the first stage of the clotting mechanism.

Causes of prolonged APTT

¾ Hemophilia

¾ Vitamin K deficiency

¾ Liver disease

¾ Presence of circulating anticoagulants

¾ DIC disease (chronic or acute).

Shortened APTT occurs in:

¾ Extensive cancer, except when liver is involved

¾ Immediately after acute hemorrhage

¾ Very early stages of DIC.

Circulating Anticoagulants

Usually occurs as an inhibitor of a specific factor (e.g. factor

VIII). Most commonly seen in the development of antifactor VIII or anti-factor IX in 5 to 10% of hemophiliacs.

Anticoagulants that develop in the treated hemophiliac

are detected by prolonged APTT. Circulating anticoagulants also can be detected in some cases:

¾ Following repeated plasma transfusions

¾ Drug reactions

¾ Tuberculosis

¾ Chronic glomerulonephritis

¾ Systemic lupus erythematosus

¾ Rheumatoid arthritis.

NORMAL AND ABNORMAL CONTROL PLASMAS

FOR COAGULATION ASSAYS PLASMATROL H-I/II®

(Courtesy: Tulip Group of Companies)

Summary

Tulip Plasmatrol H-l and Plasmatrol H-ll are two level

human plasma controls that are suitable for use as normal

and abnormal control plasma for PT, APTT, TT and

fibrinogen testing using clot based methods. Coagulation

controls provide a means of day-to-day quality control

in the hemostasis laboratory for control of accuracy and

precision.

Reagent

Plasmatrol is a stabilized and freeze dried preparation

of selected human plasma with values determined and

assigned for specific clot based tests, which are lot specific.

The plasma controls are assayed using Tulip coagulation

reagents.

Reagent Storage and Stability

Unopened vials should be stored at 2–8°C and are stable

up to the expiry date mentioned on the vial labels. After

reconstitution the shelf life of the control plasma is 3 hours

at 25–30°C and 8 hours when stored at 2–8°C.

Principle

The properties of the control plasma are similar to those

of pooled fresh plasmas. Since, the plasma controls have

assigned values, when substituted in place of a sample, in

clot based coagulation assays, they can be used for laboratory quality assurance.

Note

 

Reagent

Liquicelin-E is a liquid ready to use activated cephaloplastin reagent for the determination of activated partial

thromboplastin time. It is a phospholipid preparation

derived from rabbit brain with ellagic acid as an activator.

Each batch of reagent undergoes rigorous quality

control at various stages of manufacture for its sensitivity

and performance.

294 Concise Book of Medical Laboratory Technology: Methods and Interpretations Reagent Storage and Stability

a. Store the reagent at 2-8°C. Do not freeze.

b. The shelf-life of the reagent is as per the expiry date

mentioned on the reagent vial label. The reagent is

stable for: 1 year at 2–8°C, 1 week at 18–25°C, 2 days

at 37°C.

Principle

Cephaloplastin activates the coagulation factors of the

intrinsic pathway of the coagulation mechanism in

the presence of calcium ions. APTT is prolonged by a

deficiency of one or more of these clotting factors of the

intrinsic pathway and in the presence of coagulation;

inhibitors like heparin.

Note

1. In vitro diagnostic reagent for laboratory and

professional use only. Not for medicinal use.

2. Liquicelin-E, reagent is not from human source hence,

contamination due to HBsAg and HIV is practically

excluded.

3. Reagent contains 0.01% thimerosal as preservative.

4. It is very important that clean and dry micropipette

tips be used to dispense the reagent.

5. Avoid exposure of the reagent to elevated temperatures

and contamination. Immediately replace cap after use

and store at recommended temperatures only.

Sample Collection and Preparation

No special preparation of the patient is required prior to

sample collection by approved techniques. Withdraw

blood without undue venous stasis and without frothing

into a plastic syringe fitted with a short needle of 19 to 20

SWG. The venipuncture must be a ‘clean’ one and, if there

is any difficulty, take a new syringe and needle and try

another vein. Transfer the blood into tubes, after detaching

the needle from the syringe.

Mix exactly nine parts of freshly collected blood

with one part of Trisodium citrate (0.11 mol/L, 3.2%)

or Profact available from Tulip; Centrifuge immediately

for 15 minutes at 3000 rpm (approximately 2000 g) and

transfer the plasma into a clean test tube. Plasma must be

tested within three hours of blood collection. For heparin

determination, platelet deficient plasma should be used,

hence higher centrifugation time is required.

FNP Collection

Prepare a plasma pool (FNP) of freshly collected blood

from at least five normal healthy donors and process as

above. Plasma must be tested within three hours of blood

collection.

Additional Material Required*

12 × 75 mm test tubes; 0.1 mL, 0.2 mL and 2.0 mL precision

pipettes; Stopwatch; Water bath or heating block 37°C;

Fresh normal pooled plasma; CaCl2 (0.02 mol/L).

*Available from Tulip Diagnostics.

Test Procedure

Manual Method

1. Before use, the reagent should be mixed well by gentle

swirling. Do not shake.

2. Aspirate from the reagent vial enough reagent for

the immediate testing requirement in a thoroughly

clean and dry test tube. Bring this reagents to room

temperature before prewarming at 37°C for testing

purposes.

3. Separate test tubes containing Liquicelin-E and

Tulip’s calcium chloride solution should be brought

to 37°C (depending on volume, approximately 5 to 10

minutes required). Do not incubate the test plasma.

4. To a 12 × 75 mm test tube, add 0.1 mL test plasma

and 0.1 mL Liquicelin-E. Shake tube briefly to mix

the reagent and plasma, place tube at 37°C for 3 to 5

minutes.

5. Following incubation period, add forcibly 0.1

mL prewarmed calcium chloride into the plasma

and Liquicelin-E mixture, simultaneously start a

stopwatch. Shake tube briefly to mix contents, keep

at 37°C for 20 seconds.

6. Following 20 seconds incubation, remove the tube,

gently tilt back and forth until a gel clot forms, stop

the watch, record time.

7. Repeat steps 2–4 for a duplicate test using the same

test plasma.

8. Find the average from the duplicate test values. This

is the activated partial thromboplastin time (APTT of

patient plasma).

9. Similarly repeat steps 2-4 twice, and record duplicate

values using FNP in place of test plasma (APTT of

FNP).

If a coagulation instrument is being used to perform the

tests, the instrument manufacturer's instructions must be

strictly adhered to.

Calibration Curve Method (For determination of

heparin concentration):

1. Dilute heparin (as used for treatment) with

physiological saline to a concentration of 10 U/mL.

2. Mix 0.2 mL of 10 U/mL diluted heparin with 1.8 mL

of FNP to give a heparin standard of 1 U/mL concentration.

3. Dilute the heparin standard as prepared above

(1 U/mL) with FNP as follows :

Clinical Hematology: Bleeding Disorders 295

Test tube No. 1 2 3 4 5 6 7

Heparin standard

(1 U/mL) in mL

0.5 0.4 0.3 0.2 0.1 0.1 -

FNP in mL - 0.1 0.2 0.3 0.4 0.9 0.5

Heparin concentration (U/mL)

1 0.8 0.6 0.4 0.2 0.1 0.0

4. Pipette 0.1 mL each of the seven heparin dilutions into

clean test tubes.

5. Add 0.1 mL Liquicelin-E reagent to each test tube.

6. Mix well and incubate each test tube at 37°C for exactly

3 minutes before testing.

7. Forcibly add 0.1 mL calcium chloride (prewarmed at

37°C) to each test tube, one by one and simultaneously

start the stopwatch.

8. Gently tilt the tube back and forth and stop the

stopwatch as the first fibrin strand is visible and the

gel/clot formation begins. Record the time in seconds.

9. Repeat steps 4–8 for each dilution for duplicate test,

and find the average of the duplicate test values.

10. Plot the mean of the double determination in ‘seconds’,

against each heparin concentration using Liquicelin-E

graph paper.

11. Clotting times (APTT) of test specimens can be

interpolated against the heparin concentration to

determine the heparin concentration of the sample in

U/mL.

Calculation and Reporting of Results

Manual Method

a. The result may be reported directly in terms of the

mean of the double determination of the APTT of the

test plasma.

OR

b. As a ratio R as follows:

APTT of patient plasma (in seconds)

R = _________________________________

APTT of FNP (in seconds)

 

¾ The correct use of the formula to compute the INR.

¾ Uniform understanding of the INR system by clinicians

as well as laboratorians.

Patient Variables in PT/INR Testing

There are many factors that can influence the results of the

PT/INR tests so that they do not reflect the patient’s usual

coagulation state. Coagulation tests are susceptible to

errors introduced by suboptimal specimen quality because

of a number of factors such as blood collection technique,

labile state of several coagulation proteins, and laboratory

transportation factors. In order to get acceptable accuracy

it is important to understand and control these factors as

much as possible.

Factors that Influence Coagulation Test Results

Age and Gender

Age specific reference ranges are critical for correct

interpretation of coagulation data. Bleeding time declines

with age and many coagulation factors increase with age

as do markers of coagulation activation. Age and gender

can also influence platelet function. Females tend to have

longer bleeding times than males.

Blood Type

Type O individuals have significantly lower von

Willebrand factor and factor VIII activity than subjects

with type A, B, or AB. This causes increased bleeding and

dotting times.

Within Day Variation

Incidences of platelet activation are highest in the

mornings, resulting in increased coagulation activation.

Seasonal Variation

Increased coagulation activity has been described in cold

weather.

Intraindividual Variability

Many coagulation analytes are less precise than other

analytes and thus can give variable results within the same

individual.

Diet, Alcohol and Smoking

Cardiac risk factors can increase coagulation factor level/

activation. Smoking elevates plasma fibrinogen. Von

Willebrand factor, thrombin generation and platelet

activation may all have an effect causing variability.

Moderate ethanol intake inhibits platelet reactivity and

increases fibrinolysis and INR.

Medications

A number of other medications, including hormone

replacement therapy, selective estrogen receptor,

modifiers and oral contraceptives can alter coagulation

and raise the INR. In addition, non-steroidal antiinflammatory drugs, antibiotics and fluoroquinolones

can also alter the INR.

Menstrual Cycle, Pregnancy

Significant hormonally determined changes in coagulation

factors, inhibitors, fibrinolysis and activation markers

must be considered as interpretation of the results.

Diseases

States, which lead to anemia, polycythemia or hemolysis

or uremia, can also interfere with coagulation tests.

Physical and Emotional Stress

These are commonly associated with increased coagulation

and platelet activation.

Clinical Hematology: Bleeding Disorders 293

Posture

Values can change from supine to upright positions

due to the shift of water and subsequent reduction in

plasma volume. Hence, standardization of posture is

recommended.

Venous Occlusion

Traumatic or prolonged phlebotomy accentuates the

hemostatic activation, producing artificially altered

coagulation times.

Vitamin K

Certain fat substitutes in some snackitems contain

unspecified amount of vitamin K. Green, leafy vegetables

and green tea also contain high levels of vitamin K. This

can have an impact on serum vitamin K levels and the INR

can drop as a result. Alternative medicines: According to

the AANA (American Association of Nurse Anesthetists)

some sources, certain herbal drugs can cause interference

in coagulation cycles, falsely elevating the INR.

Anticoagulant Therapy

It is of utmost importance to bear in mind that patients on

heparin will show inaccurate INR results.

While certain pre-analytical factors are not entirely

controllable, every effort must be made to ensure that most

conditions have been stable for a period of time. Patient

preparation and blood collection should be standardized

according to the guidelines.

Prothrombin Determination (Two-stage Method)

Principle

Prothrombin in the presence of optimal procoagulants

and calcium will form thrombin. The amount of thrombin

formed can be calculated by determining the dilution of

plasma that will clot a standard fibrinogen reagent in a

specific period of time. The amount of thrombin formed

is a measure of the amount of prothrombin present in the

starting sample.

The test consists of two stages. In the first stage,

prothrombin is incubated with a standard mixture

containing thromboplastin, calcium, a buffer and a source

of procoagulants. In the second stage, samples of the

incubating mixture are added to a standard fibrinogen

solution and the clotting time is determined.

Results

1. The object of the procedure is to determine the dilution

of plasma from which will evolve one unit of thrombin

under optimal conditions. A unit of thrombin is defined

as that amount which will form a clot of 1 mL of fibrinogen in 15 seconds under standard conditions.

2. If varying amounts of thrombin are added to standard

amounts of fibrinogen the clotting time of the mixture

is an index of the thrombin concentration within a

specific range. When thrombin concentrations are

plotted against clotting times, the results describe

a hyperbolic curve. With thrombin concentrations

between 0.80 and 1.34 units, there is good correlation

between thrombin concentration and clotting time.

With greater amounts of thrombin, there is little

change in the speed of clotting, with relatively large

changes in thrombin concentration. With lesser

amounts of thrombin, small changes in thrombin

concentration result in large changes in the speed of

clotting.

APTT/PTTK CEPHALOPLASTIN REAGENT FOR

PARTIAL THROMBOPLASTIN TIME (APTT)

DETERMINATION USING ELLAGIC ACID AS

ACTIVATOR LIQUICELIN-E®

(Courtesy: Tulip Group of Companies)

Summary

The arrest of bleeding depends upon primary platelet plug

formed along with the formation of a stable fibrin clot.

Formation of this clot involves the sequential interaction of

a series of plasma proteins in a highly ordered and complex

manner and also the interaction of these complexes with

blood platelets and materials released from the tissues.

Activated partial thromboplastin time is prolonged by a

deficiency of coagulation factors of the intrinsic pathway of

the human coagulation mechanism such as factor XII, XI,

IX, VIII, X, V, II and fibrinogen. Determination of APTT helps

in estimating abnormality in most of the clotting factors of

the intrinsic pathway including congenital deficiency of

factor VIII, IX, XI and XII and is also a sensitive procedure

for generating heparin response curves for monitoring

heparin therapy.

 

A PT ratio is obtained by dividing the patient PT in

seconds by the “mean of the normal range” (MNPT). This

ratio is then “normalized” by raising the results to the

power of the ISI of the PT reagent used. Lower the ISI of

the reagent used, closer will be the INR to the observed PT

ratio. Ideally, when the ISI of the reagent is 1.0 then the INR

is a simple PT ratio since (R)1.0 = R.

Currently many coagulation instruments are available

that can perform this exponential calculation by entering

the ISI of the reagent in use. Alternatively a table is provided

by reagent manufacturers for reading off “INR” directly for

the given patient PT ratio, corresponding to the ISI value of

the reagent used.

Recommended Therapeutic Ranges for Oral Anticoagulant Therapy

Indications INR Intensity

• Prophylaxis of venous thrombosis

(high risk surgery)

• Treatment of venous thrombosis

• Treatment of pulmonary embolism

• Prevention of systemic embolism

– Tissue heart values 2.0–3.0

– Acute myocardial infarction

(to prevent systemic embolism)

– Valvular heart disease

• Atrial fibrillation

• Mechanical prosthetic values

(high risk)

• Prevention of myocardial

infarction recurrent 2.5-3.5 High

Clinical Hematology: Bleeding Disorders 291

INR CONVERSION TABLE

ISI

R 1.00 1.05 1.10 1.15 1.20 1.25 1.29

1.0 1.00 1.00 1.10 1.00 1.00 1.00 1.00

1.1 1.10 1.11 1.11 1.12 1.12 1.13 1.13

1.2 1.20 1.21 1.22 1.23 1.24 1.26 1.27

1.3 1.30 1.32 1.33 1.35 1.37 1.39 1.40

1.4 1.40 1.42 1.45 1.47 1.50 1.52 1.54

1.5 1.50 1.53 1.56 1.59 1.63 1.66 1.69

1.6 1.60 1.64 1.68 1.72 1.76 1.80 1.83

1.7 1.70 1.75 1.79 1.84 1.89 1.94 1.98

1.8 1.80 1.85 1.91 1.97 2.02 2.08 2.13

1.9 1.90 1.96 2.03 2.09 2.16 2.23 2.29

2.0 2.00 2.07 2.14 2.22 2.30 2.38 2.45

2.1 2.10 2.18 2.26 2.35 2.44 2.53 2.60

2.2 2.20 2.29 2.38 2.48 2.58 2.68 2.77

2.3 2.30 2.40 2.50 2.61 2.72 2.83 2.93

2.4 2.40 2.51 2.62 2.74 2.86 2.99 3.09

2.5 2.50 2.62 2.74 2.87 3.00 3.14 3.26

2.6 2.60 2.73 2.86 3.00 3.15 3.30 3.43

2.7 2.70 2.84 2.98 3.13 3.29 3.46 3.60

2.8 2.80 2.95 3.10 3.27 3.44 3.62 3.77

2.9 2.90 3.06 3.23 3.40 3.59 3.78 3.95

3.0 3.00 3.17 3.35 3.54 3.74 3.95 4.13

3.1 3.10 3.28 3.47 3.67 3.89 4.11 4.30

3.2 3.20 3.39 3.59 3.81 4.04 4.28 4.48

3.3 3.30 3.50 3.72 3.95 4.19 4.45 4.67

3.4 3.40 3.61 3.84 4.09 4.34 4.62 4.85

3.5 3.50 3.73 3.97 4.22 4.50 4.79 5.03

3.6 3.60 3.84 4.09 4.36 4.65 4.96 5.22

3.7 3.70 3.95 4.22 4.50 4.81 5.13 5.41

3.8 3.80 4.06 4.34 4.64 4.96 5.31 5.60

3.9 3.90 4.17 4.47 4.78 5.12 5.48 5.79

4.0 4.00 4.29 4.59 4.92 5.28 5.66 5.98

4.1 4.10 4.40 4.72 5.07 5.44 5.83 6.17

4.2 4.20 4.51 4.85 5.21 5.60 6.01 6.37

4.3 4.30 4.63 4.98 5.35 5.76 6.19 6.58

4.4 4.40 4.74 5.10 5.50 5.92 6.37 6.76

4.5 4.50 4.85 5.23 5.64 6.08 6.65 6.96

4.6 4.60 4.96 5.36 5.78 6.24 6.74 7.16

4.7 4.70 5.03 5.49 5.93 6.40 6.92 7.36

4.8 4.80 5.19 5.62 6.07 6.57 7.10 7.56

4.9 4.90 5.31 5.74 6.22 6.73 7.29 7.77

5.0 5.00 5.42 5.87 6.37 6.90 7.48 7.97

5.1 5.10 5.53 6.00 6.51 7.06 7.66 8.18

5.2 5.20 5.65 6.13 6.66 7.23 7.85 8.39

5.3 5.30 5.76 6.26 6.81 7.40 8.04 8.60

5.4 5.40 5.88 6.39 6.95 7.57 8.23 8.81

5.5 5.50 5.99 6.52 7.10 7.73 8.42 9.02

5.6 5.60 6.10 6.65 7.25 7.90 8.61 9.23

5.7 5.70 6.22 6.78 7.40 8.07 8.81 9.44

5.8 5.80 6.33 6.91 7.55 8.24 9.00 9.66

5.9 5.90 6.45 7.05 7.70 8.41 9.20 9.87

6.0 6.00 6.56 7.18 7.85 8.59 9.39 10.09

Other Factors Influencing the INR

The variability in the responsiveness of the PT reagents,

is corrected through the “ISI” calibration, however, three

additional technical factors influence the INR:

¾ Derivation of MNPT

¾ Magnitude of difference in the ISI value of test

thromboplastin and IRP (ISI=1.0)

¾ Method of clot detection employed during PT test.

MNPT

MNPT is a critical requirement in the derivation of INR.

Ideally each laboratory must derive its own MNPT from

20 or more normal patients for a given PT reagent and lot

under use. This corrects within laboratory test variables

that influence PT results. If “normal control plasmas”

are used in place of patient plasma for arriving at the

MNPT it can effect the evaluation of the patients level of

anticoagulation. For example,

Reagent ISI=2.5 Test Day 1 Test Day 2 Test Day 3

Patient PT (sec)

Normal Control

(10.4–12.3 sec)

INR Formula

[R]ISI

Resulting INR

16.0

11.5

16.02.5

______

2.27

16.0

10.4

l6.02.5

______

2.89

16.0

12.3

16.02.5

______

1.92

If the control time is greater than the mean normal

range (MNPT), the PT ratio for any patient PT will be

smaller, potentially leading to over coagulation. If the

control time is lesser than MNPT the ratio for any patient

PT will be greater, leading to under coagulation.

On the other hand MNPT for a particular laboratory

using the same combination of methodology, reagent and

instrument would remain constant.

ISI Value of PT Used and Method of Clot Detection

INR loses some precision when comparisons are made

with thromboplastins with markedly different ISI values

as against the IRP (ISI=1.0) and different methods of clot

detection, e.g. manual, mechanical, optical, etc.

Therefore, manufacturers must provide ISI values

adapted to the method used for clot detection. Also the

reagent used for reporting results should be ideally as

close to 1.0 as possible.

Advantages of the INR system

¾ Major advantage of the INR system is that it helps

alleviate confusion in the interpretation of PT

results. Usually laboratory changes like change in

thromboplastin and/or equipments could go unnoticed

292 Concise Book of Medical Laboratory Technology: Methods and Interpretations by the attending physicians. the INR remains constant

even with such changes.

¾ INR system affords comparison of PT results between

laboratories.

¾ INR system provides a more accurate and convenient

mean of monitoring patients who travel extensively.

¾ INR therapeutic ranges for different clinical conditions

are based on international collaborative studies. Usage

of standardized dosage reduces the risk of thrombotic

episodes or secondary bleeding.

Disadvantages of the INR System

¾ The prothrombin time test is always a part of the

preoperative screening panels. It is also frequently used

to evaluate other hemostatic disorders, such as liver

disease, DIC, LA, hereditary factor deficiencies and

acquired vitamin K deficiency. Since these disorders

have been excluded from the derivation of the ISI,

INR has a diagnostic and therapeutic value mainly

applicable for patients stabilized on oral anticoagulants.

Therefore, laboratories may prefer to report both the

INR and patients time in seconds depending on clinical

application.

¾ The INR systems effectiveness would still depend on

the calibration of the coagulation instruments as well as

thromboplastin reagents used.

¾ Derivation of the correct MNPT and use of the mean

normal range in each laboratory.

¾ Usage of thromboplastin reagents with ISI of preferably

1.0 or as close to 1.0 as possible.