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Laboratory Quality Assurance Program College of Physicians & Surgeons of Saskatchewan

Laboratory Guidelines General Anatomic Pathology Chemistry Hematology Transfusion Medicine

2014 Edition

Laboratory Guidelines 2014 Edition Page 1

LABORATORY GUIDELINES - 2014

SUMMARY OF CHANGES The following GUIDELINES have been revised, added, or deleted in this edition of the document. REVISED: Differential Performance and Referral Practice Guidelines D-dimer Reporting Units ADDED: DELETED: Erythrocyte Sedimentation Rate (ESR)


Table of Contents

General Retention Guideline 4 Recommended Reference Textbook List for Laboratories 7

Anatomic Pathology Specimens Exempt from all Gross &/or Microscopic Pathology Laboratories 10 Surgical Pathology Reports 12 Removal of Tissue, Blocks or Slides from the Original Hospital Site 13 Performance of Non-Gynecological Cytology 15 Follow-up Reports for Gynecological Cytology 16 Follow-up Program for Cytology 17

Chemistry Estimated Glomerular Filtration Rate – eGFR 19 Cholesterol/Triglyceride/Lipid Testing 20 Urinalysis 21 Reporting Sperm in Urine 21 Quality Control 22 Diagnosis and Monitoring of Thyroid Disease 23 Presence of Small Amounts of Albumin 26 Crosscheck/Validation Guideline for Those Facilities with Multiple Chemistry Instruments 27 Procedure/Method Statistical Work-up/Validation Study Guidelines 28

Hematology Principles for Hematology Practice 31 Hematology Films/Labelling of Slides 32 Morphology of Lymphocytes 33 Differential Performance and Referral Practice Guideline 35 Red Blood Cell Morphology Reporting Guideline 37 Differential Quality Control 39 Smudge Cells 42 Flow Cytometry for the Diagnosis of Lymphoma/Leukemia 43 Malaria 44 3.2% Na Citrate Anticoagulant Recommended vs. 3.8% for Coagulation Studies 45 Vitamin B12 & Folate 46 Bleeding Time 48 Crosscheck Validation Guideline for Facilities with Multiple Hematology Instruments 49 Procedure/Method Statistical Work-up/Validation Study Guidelines 50 Protocol for Validation of Linearity on Automated Hematology Analyzers 52 Procedure for WBC Estimate 55 Establishing Conversion Factor for WBC Estimation 57 Procedure for Platelet Estimates 59 Establishing Conversion Factor for Platelet Estimation 61 Indirect Platelet Count 63 International Sensitivity Index (ISI) Verification 65 Verifying or Establishing a Normal Reference Range for Routine Coagulation Testing 66

Transfusion Medicine Retention of Transfusion Medicine Records 70 Procedure/Method Statistical Validation/Work-up Guidelines – Trans. Med. 71


GENERAL


RETENTION GUIDELINE

Laboratories vary in size, facility and extent of services provided. Clinical laboratories must

maintain thorough, accessible records that can demonstrate an acceptable standard of care and

compliance with the accreditation requirements.

The Laboratory Quality Assurance Program of the College of Physicians and Surgeons urges

laboratories to retain records, materials, or both for a longer period of time than specified for

educational and quality improvement needs.

Laboratories must establish policies that meet or exceed the following minimum requirements

for retention of documents and specimens as established by professional and/or regulatory

organizations.

References include: CSTM, CSCC, CAP, CSA, ISO, CPSS


Record Retention Storage Time

Hematology Biochemistry Microbiology Cyto-

pathology

Surgical

Pathology

Accession record 1 year 1 year 1 year 2 years 2 years

Worksheets 1 year 1 year 1 year 6 months 6 months

Instrument print-outs 1 year 1 year 1 year n/a n/a

Paper copy of patient reports 3 months 3 months 3 months indefinite indefinite

Quality control/PT documents 2 years 2 years 2 years 2 years 2 years

Maintenance records 2 years 2 years 2 years Life of the instrument, plus 2

years

Service records Life of the instrument, plus 2 years

Method /instrument evaluation 2 years after the method has been discontinued

Procedure Manual 2 years after procedure has been discontinued

Technologist ID & initials

log/computer

1 year 1 year 1 year 1 year 1 year

Telephone logs 3 months 3 months 3 months 3 months 3 months

Requisition 3 months 3 months 1 year 5 years 5 years

Laboratory Information Systems

Records

- Validation records, including

transmission of results and

calculations

- Database changes

- Hardware and software

modifications

- LIS downtime and corrective action

2 years 2 years 2 years 2 years 2 years

Biomedical Waste Manifests must be retained for a minimum of 1 year


Specimen Retention Hematology Chemistry Microbiology Cytopathology Surgical

Pathology

Peripheral Blood

Smear

-7days

Peripheral

Blood Smear

reviewed by a

pathologist

-1 yr

Semen smears

- 3 months

Bone Marrow

Slides/Reports

- 20 yrs (adults)

- 50 yrs

(children)

Whole

blood/Plasma

- 24 hrs after

report has been

finalized

Body fluids

- 48 hrs after

report has been

finalized

Urine

- 24 hrs after

report has been

finalized

Whole blood,

serum &

plasma

- 48 hrs after

report has been

finalized

Body fluids

- 48 hrs after

report has been

finalized

Urine - routine

- 24 hrs after

report has been

finalized

24hr Urines

- samples

discarded 48

hrs after report

has been

finalized

Swabs or

specimens

- 24 hrs after

report has been

finalized

Positive Blood

Culture

- 5 days after

reporting

Gram Stain

- one week or

until final report

is sent

Ova & Parasite

slides

- one month

Slides neg/unsatisfactory

- 5 years

Slides

suspicious/pos

- 20 years

Fine-needle

aspiration slides

- 20 years

Cytology

Consultation/

Requisition

- Indefinitely

Male fertility

slides

- 1 year

Cytology

paraffin blocks

- 20 years

Blocks & slides

- 20 yrs (adults)

- 50 yrs (children)

Autopsy

- 20 yrs

Gross specimen

- min. 8 weeks

after issue of

report

Wet Autopsy

Tissue

- 8 weeks after

issue of report

Bone Marrow

Slides/ Reports

- 20 yrs (adults)

- 50 yrs (children)

Photographic

Transparencies –

indexed and kept

indefinitely


RECOMMENDED REFERENCE TEXTBOOK LIST FOR LABORATORIES

Microbiology

1) Manual of Clinical Microbiology 10th

Edition; American Society for Microbiology; Murray,

Patrick R.

2) Bailey & Scott's Diagnostic Microbiology 13th

Edition, Mosby, Inc.; Forbes, Betty A., et al.

3) Clinical Microbiology Procedures Handbook 3rd

Edition; Issenburg

Transfusion Medicine

1) Circular of Information, Canadian Blood Services (most recent version)

2) Canadian Standards Association Blood and Blood Component Z902 (most recent version)

3) Canadian Society for Transfusion Medicine (most recent version)

4) Modern Blood Banking and Transfusion Practices 6th

Edition; Harmening, Denise M.

5) Canadian Medical Association Journal Guidelines for Red Blood Cell and Plasma

Transfusion for Adults and Children; supplement to CAN MED ASSOC J 1997;

156 (11)

6) American Association of Blood Banks Technical Manual - most recent edition

7) Bloody Easy 3: Blood Transfusions, Blood Alternatives and Transfusion Reactions 3rd

Edition; Ontario Regional Blood Coordinating Network

Hematology

1) Color Atlas of Hematology, Hematology and Clinical Microscopy Resource Committee,

CAP; Glassy, Eric F.

2) Clinical Hematology: principles, procedures, correlations 2nd

edition; Stiene-Martin,

Lotspeich-Steininger, Koepke

3) Hematology: Clinical Principles and Applications 4

th Edition; Rodak, Fritsma, Doig

4) Clinical Hematology Atlas 4

th Edition; Carr/Rodak

Chemistry

1) Tietz Fundamentals of Clinical Chemistry 6th

Edition, W. B. Saunders Company; Burtis,

Ashwood, Bruns


2) Clinical Chemistry – Principles, Procedures, Correlations 6th

Edition; Bishop

Urinalysis

1) Graff’s Textbook of Urinalysis and Body Fluids 2nd

Edition; Mundt, Shanahan

Anatomic Pathology

1) Histotechnology: A Self Instructional Text 3rd

Edition; Carson & Hladik

2) Principles of Anatomy & Physiology 13th

Edition; Tortora & Grabowski

Safety

1) Transportation of Dangerous Goods Act and Regulations

Supplement Canada Gazette, Part II [www.tc.gc.ca/eng/tdg/clear-tofc-211.htm]

2) CSMLS Guidelines – Laboratory Safety, 7th

Edition

Competency Evaluation

1) Canadian Society of Medical Laboratory Science

PO Box 2830 LCD 1

Hamilton, ON L8N 3N8

website www.csmls.org

Certification - Competency Profiles

http://www.csmls.org/


ANATOMIC

PATHOLOGY


SPECIMENS EXEMPT FROM ALL GROSS &/OR MICROSCOPIC

PATHOLOGY LABORATORIES

Irrespective of the exemptions listed below, gross &/or microscopic examinations will be

performed whenever the attending physician requests it, or at the discretion of the pathologist

when indicated by gross findings.

SPECIMEN TYPE DISCRETIONARY GROSS &/OR

MICROSCOPIC

Abdominal pannus

Accessory Digits

Amputation

Bone or cartilage removed from the arthritic

joints during joint replacement surgery

Bone segments removed as part of corrective

orthopedic procedures (for example: rotator

cuff, synostosis repair, spinal fusion)

Bunions and hammer toes

Calculi (renal bladder etc.), are sent for

chemical analysis and description

(By Chemistry Dept.)

Disc Materials

Extraocular muscle from corrective surgical

procedures (strabismus repair)

Femoral head removed for prosthesis (if

straight forward)

Foreign bodies, such as bullets or

medicoloegal evidence that is given directly

to law enforcement personnel

Gangrenous and traumatized limbs

Intrauterine contraceptive devices without

attached soft tissue

Loose bodies (joint)

Medical devices such as catheters,

gastrostomy tubes, myringotomy tubes,

stents and sutures that have not contributed

to patient illness, injury or death

Middle ear ossicles

Nasal bone and cartilage from rhinoplasty or

septoplasty

Orthopedic hardware and other radioopaque

mechanical devices provided there is an

alternative policy for documentation


SPECIMEN TYPE DISCRETIONARY GROSS &/OR

MICROSCOPIC

Placentas which do not meet the criteria for

examination

Prosthetic breast implants

Prosthetic cardiac without attached tissue

Rib segments or other tissue removed only

for the purpose of gaining surgical access

from patients who do no have a history of

malignancy

Saphenous vein segments harvested for

coronary artery bypass

Skin or other normal tissue removed during

cosmetic or reconstructive procedures

(blepharoplasty, cleft palate repair,

abdominoplasty, rhinectomy or syndactyly

repair) that is not contiguous with a lesion

and that is taken from a patient who does not

have a history of malignancy

Teeth without attached soft tissue

Therapeutic radioactive sources

Tonsils and adenoids if clinically not

suspicious

(under 10 yrs

old)

(over 10 yrs)

Torn menicus

Varicose veins


SURGICAL PATHOLOGY REPORTS

Timeliness of reports is critical to providing quality of care. Guidelines for surgical pathology

reporting include:

(i) Routine Surgical Pathology reports

Complete within 2 working days.

Where additional procedures are being performed, an extension of 24

hours is appropriate.

(ii) Autopsy Reports

Written initial reports of gross pathological findings within 72 hours.

Final Report – 30 days for routine cases, 90 days for complicated cases


REMOVAL OF TISSUE, BLOCKS OR SLIDES FROM THE ORIGINAL HOSPITAL SITE

Anatomic Pathologists are charged with the responsibility of keeping and guarding the integrity

of the ever-growing number of tissue containing paraffin blocks and slides derived from surgical,

cytological and autopsy diagnostic services. Documentation and maintenance (tracking) of the

continuous care shall ensure quality practice. These specimens must be maintained in orderly

files to ensure ready access. There are inevitably increasing demands for slides, blocks or tissues

to be retrieved from the original site.

*A release form must be provided and retained on file at the original institution for permanent

release.

Summary

Follow HIPA guidelines

Ensure there is sufficient material for further work-up

Indicate reason for request

Return all material as soon as possible

The lab is the custodian of tissue, blocks or slides collected.

The source of material remains the property of the patient.

These are the various suggested categories to be considered:

In-Province Consultation

Request may be initiated by the primary physician, surgeon or oncologist for review by local

or out-of-district pathologist.

Request may be initiated by the original signing out pathologist who is responsible for

maintaining records and assuring return of the material.

Note: The return of materials to the original site must be documented and a consult report sent

to the original pathologist as well as the requesting pathologist.

Out-of-Province (recommended slides only)

Request from an originating pathologist to seek out-of-province consultation for diagnostic

purpose.

Research or national study groups request specimen be referred to another institution for

treatment.

If blocks are requested, cut a set and send the cut set. The originals should be maintained at

the processing site.

Educational Consultation

Requests for educational rounds should be restricted to slides; to ensure integrity of patient

property.

Request should indicate “for rounds” and materials returned promptly to ensure ongoing

patient care.


Research

Regulations require pathologists to obtain patient authorization and/or an Institutional

Review Board (Ethics Committee) waiver of informed consent when using any identifiable

patient health information for research purposes.

Requests must ensure the integrity of the patient material.

All materials that have critical diagnostic, prognostic or medical-legal implication may be

retained at the discretion of the releasing institution.

Return all materials as soon as possible.

References:

Guardians of the Wax…and the Patient. Editorial.; American Journal of Clinical Pathology 1995 104 p 356-7

Use of Human Tissue Blocks for research. Association of Directors of Anatomic and Surgical Pathology. Human Pathology

1996.27 p 519-520


PERFORMANCE OF NON-GYNECOLOGICAL CYTOLOGY

Non-gynecological cytology comprises of fine needle aspiration biopsies (FNAB) of

organs/tissues such as lungs and other visceral lesions, effusion cytology of pleural, peritoneal,

pericardial fluids, cytology of urine, CSF, sputum, broncho-alveolar lavage (BAL) and brush

biopsies of endoscopic procedures, (gastrointestinal tract, etc). Scrapings of open lesions and

nipple discharges may also be included along with cytology of transplant organs to test for

rejection (kidneys), or for cyclosporin toxicity. BAL and transplant cytology is usually done in

specialized centers as it requires specific interpretation and often special tests. Most of the other

samples can be handled and processed in a routine surgical pathology/cytology lab equipped

with basic facilities including a biological safety cabinet (fume hood), cyto-centrifuge and

staining capability for H & E and PAP stains.

In contrast to gynecological cytology, non-gynecological cytology (NGC) does not necessarily

require a screening step. If adequate diagnostic material is present the focus is on diagnosis of

and interpretive correlation with the clinical setting. If cell block or cytospin samples are

available, further testing with special procedures could be performed.

Some aspects of NGC require a rapid turnaround time such as FNAB performed under CT-scan

or ultrasound guidance and intra-operative cytology requests.

It is important for institutions with CT scanner facilities to be able to provide cytology service in

house. However, it is acceptable, if the lab does not have a cytology department, the

technologists in the histology lab are trained in processing the specimens. Such training is easily

obtained and can be provided by short courses provided on site and documented.

Most NGC procedures are performed on patients who are in-patient residents in a hospital/health

care institution or are required to come in for a day procedure/ambulatory care. Due to the time

factor involved in patients’ institution stay; a rapid turnaround time becomes a key factor in

availability of the service. On the other hand, the patient in an acute care setting may have an

infectious process or malignancy requiring rapid diagnosis and treatment.

The final interpretation and reporting of non-gynecological cytology shall be made by a

pathologist.


FOLLOW-UP REPORTS FOR GYNECOLOGICAL CYTOLOGY

To ensure a quality cytology service, a follow-up mechanism must be in place to provide reports

to the primary and/or consulting physician.

In an attempt to eliminate the potential for “LOST – TO FOLLOW” reporting situations, the

following require follow-up letters to the primary and/or consulting physician:

1. A repeat smear was requested at the time of reporting and 3 months have lapsed since the

date of request.

2. A diagnosis is rendered requiring follow-up and none has occurred. For example:

HSIL required follow-up a.s.a.p. and if this has not occurred within 3 months, a letter

is required.

LSIL (ASCUS, AGUS) within 6 months requires a letter in 9 months.

A malignant diagnosis with no apparent followup.

3. A follow-up letter has been previously issued with no reply. These letters should be

automatically generated by the computer system and then replies must be recorded and

reviewed quarterly.

4. A minimum laboratory requirement of a computer system used to report gynecological

cytology shall have the ability to generate automatic follow-up letters that are linked to

diagnostic codes.


FOLLOW-UP PROGRAM FOR CYTOLOGY

A follow-up mechanism shall be in place to ensure that actions appropriate for abnormal findings

are implemented.

a) All patients who are reported to have a significant abnormality should be followed up by

the laboratory or other agency to which this task may be delegated, to obtain final clinical

or preferably tissue confirmation of the diagnosis.

b) Statistical data should be maintained which would include the number of cases screened

annually in each category, and all correlative follow-up data available. Discrepancies, if

any, should be included with this information.


CHEMISTRY


ESTIMATED GLOMERULAR FILTRATION RATE - eGFR

The glomerular filtration rate is the estimated volume of glomerular filtrate that moves from

renal glomerular capillaries into the Bowman’s capsule per unit time. Evidence-based clinical

practice guidelines suggest that an estimate of GFR (eGFR) provides the best clinical tool to

gauge kidney function. The Canadian Society of Nephrology has recommended that laboratories

report eGFR routinely for adult patients, in order to detect chronic kidney disease.

Serum creatinine results can vary significantly and tend to be ineffective in general practice as an

early marker. 24 hr. collection for creatinine clearance is impractical and prone to error. eGFR

should be reported on outpatients over the age of 18 years. eGFR has not been validated for use

in hospitalized patients and therefore, is not recommended for reporting on inpatients.

eGFR is less reliable in,

patients with near normal eGFR

unstable serum creatinine

acute illness

extremes of body composition (eg. obesity, cachexia)

unusual muscle mass (e.g. marked muscularity, muscle disease, amputation)

pregnancy

age under 18 years or over 70 years

drugs with significant renal toxicity or clearance

drugs affecting creatinine metabolism or clearance

unusual dietary intake (e.g. vegetarians)

other serious comorbid conditions

Summary:

Reporting of eGFR is becoming the standard of care in helping identify, stage and monitor

patients with chronic kidney disease.

eGFR > 60 Normal or slightly decreased kidney function (stages 1 or 2)

eGFR 30-59 Moderately decreased kidney function (stage 3)

eGFR 15-29 Seriously decreased kidney function (stage 4)

eGFR < 15 Kidney failure (stage 5)

eGFR is frequently used for DRUG DOSING using the Cockroft-Gault equation. eGFR-MDRD has not been

validated for this purpose.

eGFR-MDRD assumes “steady state”. For rapidly changing kidney function, monitor serum creatinine.

(MDRD: Modification of Diet in Renal Disease)

The reported eGFR shall be multiplied by 1.21 for patients of African descent.

NOTE: This information is intended for clinicians, patients and allied health professionals.

References: www.jasn.org, www.csnscn.ca, www.renal.org

http://www.kidney.org/professionals/kdoqi/gfr_calculator.cfm

http://www.jasn.org/

http://www.csnscn.ca/

http://www.renal.org/

http://www.kidney.org/professionals/kdoqi/gfr_calculator.cfm


CHOLESTEROL / TRIGLYCERIDE / LIPID TESTING

Cholesterol results are based on optimal performance of testing. Considerations to be included:

a) treatment/preparation of the patient

b) the emergent or non-emergent nature of the test

c) appropriate technical equipment

d) adequate quality control

The accomplishment of treatment goals also demands accurate cholesterol measurements. This

requires standardization of all cholesterol measurement for accuracy to minimize the method-

specific biases. This can be achieved ONLY by standardizing the cholesterol measurements and

ensuring accuracy that is traceable to the National Reference System for Cholesterol

(NRS/CHOL), National Cholesterol Education Program.

Clinical protocols are well established and should be followed by all testing sites.

Laboratories testing for lipids shall be capable of performing the entire profile, to include:

Cholesterol, Triglycerides, HDL and LDL, for diagnosis and assessment. All lipid

measurements should be performed by the same methodology.

Only instrumentation capable of maintaining intralaboratory precision that is less than or equal to

3 % (C.V.); and can demonstrate an accuracy bias of less than 3 % from the true value may be

used for cholesterol analysis.


URINALYSIS

Complete urinalysis shall include microscopic examination if the following are present:

leukocyte esterase, blood, protein, turbidity and nitrites.

The microscopic examination should be completed within four hours of collection. (Note:

specimens not tested within 2 hours should be refrigerated.)

Report semi-quantitative results in SI units.

REPORTING SPERM IN URINE

Sperm are not normally present in urine and if presence is detected, it is usually considered a

contaminant.

When sperm appear in a microscopic exam, they shall be reported as present. However, prior to

reporting, results should be discussed with the attending physician, in order to avoid errors (ie.

mislabeling, etc.).


QUALITY CONTROL (QC)

The purpose of QC is to detect the problems early enough to prevent their consequences.

QC emphasizes statistical control procedures, but may also include non-statistical check

procedures, such as linearity checks, reagent and calibration checks, etc.

Two or three different materials should be selected to provide concentrations that monitor

performance at different levels of medical decision-making.

For quantitative tests, the use of two levels of control material shall be run each day of use, as a

minimum.

For qualitative tests that include built-in controls, a positive and negative control shall be

performed a minimum of once per month and upon initiation of a new lot number and shipment.

For those that do not include a built-in control, known positive and negative external controls

shall be tested each day of use.


DIAGNOSIS AND MONITORING OF THYROID DISEASE

Thyroid function tests are among the most commonly ordered laboratory tests in the province. In

the past, investigations of thyroid disease required more than one test. Sensitive thyroid

stimulating hormone (TSH) is the initial test in the diagnosis of hypothyroidism (TSH elevated

or above normal) and hyperthyroidism (TSH is suppressed or below normal). Free T4 (FT4) is

preferred over total T4 (TT4) measurement to confirm the diagnosis of hypothyroidism or

hyperthyroidism. FT4 is 0.02 – 0.04% of total T4. FT4 is the metabolically active form of TT4

and is a better indicator of thyroid status than TT4 because it is unaffected by protein binding

abnormalities such as pregnancy and oral contraceptives. Free T3 (FT3) is mainly of value in

diagnosing T3 toxicosis, in determining the T3 response to therapy, and clarifying protein binding

abnormalities. It can also be of use in the early progression of subclinical hyperthyroidism to

overt thyrotoxicosis when FT4 is normal and TSH is suppressed. FT3 is often the first to be

increased. FT3 is approximately 0.2 – 0.5% of total T3.

American Association of Clinical Endocrinologists (AACE) recommends that the TSH reference

range run from 0.3 - 3.0, versus the old range of 0.5 - 5.5. Keep in mind that there is

disagreement among practitioners.

Limitations:

1. These guidelines do not apply to neonates.

2. TSH is not reliable in the investigation of hypothalamic or pituitary disease.

3. TSH may be an unreliable indicator of thyroid status in patients with acute severe non-

thyroidal illness (e.g. CCU and ICU patients) and the test is only recommended when there

are clinical indicators of possible pre-existing thyroid disease.

4. Medications such as lithium, amiodarone, glucocorticoids, and dopamine affect TSH and

may also affect the individual’s thyroid status.

Clinical Aspects of Testing:

1. Screening asymptomatic, apparently healthy patients for thyroid disease is not considered

indicated at this time.

2. Testing is indicated in the presence of symptoms or signs that are suggestive of thyroid

disease especially in high risk populations.

3. High-risk groups include women over 50, the ambulatory elderly, postpartum, individuals

with a strong family history of thyroid disease and other autoimmune diseases such as Type I

diabetes.

Symptoms and Signs of Hypothyroidism: Cold intolerance, lethargy, depression, constipation,

menstrual disorders, dry skin, weight gain. There will be slow growth in children.

Symptoms and Signs of Hyperthyroidism: Palpitations, fatigue, weakness, increased appetite,

heat intolerance, usually enlarged thyroid, weight loss, warm moist skin, tremor and tachycardia.

Restlessness, sleep disturbances, difficulty maintaining attention and concentration occur in

children.


Recommended Testing Algorithm

If TSH screen is abnormal, a FT4 will be done on the same sample reducing the need to call back

the patient for subsequent testing. If the TSH is less than 0.3mU/L a FT4 and FT3 will be done

on the same sample. Free T3 is a better indicator of the degree of thyrotoxicosis in most patients.

Further Testing Recommendations:

1. Follow-up for Primary hypothyroidism or replacement therapy: TSH should be performed 6

– 8 weeks after start of therapy or dosage adjustment. After normal results have been

achieved, TSH should be done annually, unless the clinical condition changes or unless the

clinical condition warrants re-testing. In children under one year of age the TSH and FT4

should be measured every 3 months and every 6 months for children under six years of age.

Also rapidly growing adolescents should have a TSH checked once every 6 months.

2. After Radioactive iodine treatment: A FT4 should be done at 4-6 weeks interval for the first 6

months or until normal. At 6 months and then annually a TSH should be done to detect

hypothyroidism. In most children and young people following thyroid ablation the FT3 is a

better indicator of control.

3. Antithyroid drugs for Hyperthyroidism: Patients should be monitored by means of FT4

monthly until controlled and then at least every 3 months while on medication. If clinical

signs and symptoms are present a FT3 may be indicated. In cases of T3 toxicosis a FT3

should be ordered. In patients with thyrotoxicosis the TSH may not recover for quite some

time after euthyroidism has been achieved and sometimes requires a period of

hypothyroidism before recovery.

4. Suppressive doses of thyroxine: Designed to support a neoplasm or goiter. TSH and FT4

every 2 months until TSH has reached a level of suppression acceptable to the clinician.

5. Subclinical hypothyroidism: Borderline results are fairly common in elderly patients and

individuals with an autoimmune mechanism are more likely to progress to a hypothyroid

state. Observation and monitoring by TSH at 6-12 month intervals is recommended.

TSH

Increased Normal Decreased

FT4 low If clinical suspicion

is high for secondary

hypothyroidsm

order FT4 as initial

test

FT4 high FT4 normal

Primary hypothyroidism

hyperthyroidism FT3 if clinical

suspicion is high


6. Subclinical hyperthyroidism: Patients with low or suppressed TSH but thyroid hormones in

the normal levels with minimal or no symptoms are followed by FT4 and/or FT3 at 6 –12

months to gauge the progression of their condition.

References:

1. HSURC guidelines. Nov 1992

2. Ontario Association of Medical Laboratories. Guidelines for the use of serum tests to detect thyroid dysfunction.

May 1987

3. Ontario Association of Medical Laboratories. Guidelines for the use of serum testing in the management of

primary hypothyroidism. June 1998

4. Alberta Medical Association. Laboratory Testing Guidelines for Investigation of Thyroid Dysfunction. May 1999

5. Protocol Steering Committee B.C. for the use of Thyroid function tests in the diagnosis and monitoring of patients

with thyroid disease. Aug 1997

6. The College of Physicians and Surgeons of Manitoba. Investigation of Thyroid Disease. February 1995

7. National Academy of Clinical Biochemistry. Laboratory Support for the Diagnosis and Monitoring of Thyroid

Disease. November 2000

8. Vanderpump MPJ, Ahlquist JAO, Franklyn JA et al 1996. Consensus statement of good practice and audit

measures in the management of hypothyroidism and hyperthyroidism. BMJ 313: 539-44.

9. Singer PA, Cooper DS, Lewy EG et al 1995. Treatment guidelines for patients with hyperthyroidism and

hypothyroidism. JAMA 273: 808-12.

10. Ladenson PW, Singer PA, Ain KB et al 2000. American Thyroid Association Guidelines for detection of thyroid

dysfunction. Arch Intern Med 160: 1573-5.


PRESENCE OF SMALL AMOUNTS OF ALBUMBIN

Presence of small amounts of albumin in urine is considered an early predictor of the

development of glomerular damage in the absence of overt nephropathy. Patients with diabetes

and hypertension are the primary risk groups.

The Canadian Diabetes Association recommends testing for small amounts of albumin once/year

after the onset of diabetes.

The presence of small amounts of albumin in urine is detectable by dipstick methodologies, and

is approved as a screen for renal damage in the known diabetes patient.

All positive results must be confirmed by quantitative analysis.


CROSSCHECK/VALIDATION GUIDELINE FOR THOSE FACILITIES WITH MULTIPLE CHEMISTRY/HEMATOLOGY INSTRUMENTS

PERFORMING THE SAME TEST PROCEDURE

Proficiency testing registration is mandatory for each analytical ‘test’.

Rotating proficiency testing result submission between analyzers is not a requirement, but is suggested where appropriate (e.g. Blood Gases).

Where there are multiple analyzers performing the same test procedure in larger facilities, it may be more appropriate for proficiency testing

submission to be consistently submitted from the same analyzer for tracking purposes.

An internal cross-check/validation protocol is required to ensure that there is correlation between all analyzers providing the same test result

in the same facility.

If this protocol is not followed, then each analyzer must be registered in the external proficiency testing program as mandated by LQAP.

This procedure is recommended every six months.

Chemistry/Hematology High Volume Analyzers (e.g. Electrolytes/CBC)

Validation/Crosscheck

Element: Requirement:

Frequency/Data Points:

Patient correlation Regularly scheduled intervals

Whenever criteria for recalibration/validation is met:

- change of manufacturer for reagents or equivalent

- after maintenance or service as per manufacturers

recommendations

- as required for purposes of troubleshooting

/validation of reagent lot # changes or as indicated

by quality control data

Minimum of 20 patient specimens/2 times per year or

equivalent

(i.e. 10 patient specimens/4 times per year or on-going data

collection as appropriate)

As necessary per recalibration/validation event

Chemistry/Hematology Low Volume Analyzers (e.g. Fibrinogen)








recommendations




Minimum of 5-10 patient specimens/2 times per year or

equivalent



PROCEDURE/METHOD STATISTICAL WORK-UP/VALIDATION STUDY GUIDELINES: CHEMISTRY/HEMATOLOGY

Work-up guidelines and definitions (change in method/instrument):

Work-up

Element

Definition: CLIS or International Federation of Clinical

Chemistry (IFCC)

Minimum Data Requirements (where

appropriate):

Applicability:

Qualitative Quantitative

1. Imprecision

within run

between run

The variation in analytical results demonstrated when a particular

specimen of aliquot is analyzed multiple times or on multiple days.

Imprecision is expressed quantitatively by a statistic such as standard

deviation or coefficient of variations.

Within run – use preferably a patient

sample or pool close to the decision levels

with a minimum of 10 data points.

Between run – 20 results from 20 separate

runs on 2 levels over a 10-day minimum

time period using appropriate QC material.

2. Patient

Correlation

The correlation coefficient is a means to look for a relationship, not

agreement, between pairs. Two methods may have a perfect

correlation throughout the measuring range but may not agree in

value (i.e. one may be double the value of the other).

40 data points are recommended with a

minimum of 20 having 50% of the data

points outside the reference intervals, if

possible. Correlations should involve

comparison with an acceptable reference

method or laboratory.

n=20

n=40

3. Linearity (IFCC) The range of concentration or other quantity in the specimen

over which the method is applicable without modification (CLIS)

when analytical results are plotted against expected concentrations;

the degree to which the plot curve conforms to a straight line is a

measure of the system linearity.

4 data points each in duplicate as a

minimum requirement, but 5 data points are

preferred (over reportable range). Linearity

studies are expected on an initial method

work-up and further studies as defined by

the College guidelines (i.e.

troubleshooting).

4. Reference

range

validation

It is common convention to define the reference range or interval of a

laboratory test as the central 95% interval bounded by the 2.5 and

97.5 percentiles of the selected patient population. Validation of an

established reference range requires a minimum of 20 samples.

The minimum requirement is 20 data points

for confirmation of an established reference

range and 120 for the establishment of a

new reference range.

5. Accuracy Closeness of the agreement between the result of a measurement and

the accepted reference value (true value of the analyte)

3 data points using acceptable reference

material (i.e. CEQAL or CAP) 1 data point

may be acceptable for haematology

accuracy studies if related to sample

stability.

6. Sensitivity Measure of the ability of an analytical method to detect small

quantities of the measured component. When concern is performance

at a very low concentration it is useful to determine the detection

limit as influenced by imprecision.

Sensitive studies are only required for those

methods which have clinical relevance at

values close to “0” (i.e. TSH)

When

clinically

relevant

When clinically

relevant


7. Specimen

Stability

The conditions of handling and storage, which permits the

measurement and reporting of a clinically relevant result.

No data generally required. As per

manufacturer’s guidelines. If

manufacturer’s stability window is to be

extended a stability study is expected.

Storage and

transportation

dependent

Storage and

transportation

dependent

8. Interference The effect of any component of the sample on the accuracy of the

measurement of the desired analyte.

Document the manufacturer’s interference

information. The method should include a

disclaimer or a process for dealing with a

lipemic, icteric or hemolyzed sample.

Methods with

known

interferences

Methods with

known

interferences

9. Recovery A recovery procedure involves the addition of a known amount of

analyte to an aliquot of sample. Recovery is defined as the ratio of

the amount of the analyte recovered to amount added and is given as

percentage.

Recovery studies should only be necessary

for those methods or analytes where

organic extractions or equivalent are

required as part of the methodology (i.e.

Toxicology)

Method

specific/organic

extraction

Work-up requirements when an instrument is moved from site “A” to site “B”: (It is assumed that the instrument has been in recent use

with acceptable performance).

Work-up Element Minimum data requirements:

1. Imprecision studies, QC only As above

2. Patient correlation 10 data points, where feasible.


HEMATOLOGY


PRINCIPLES FOR HEMATOLOGY PRACTICE

Hematology incorporates leading edge technology to help decipher and treat troubling diseases.

As hematology has evolved, there are some generic principles that must be simply stated for

quality patient care.

Quality management practices are essential to ensuring quality care. Some of the core principles

include:

1) Hemoglobin, the single most common complex organic molecule (Hb) shall be determined

by spectrophotometric methodology.

2) WBC and platelet counts shall be tested by automated methodology as part of a CBC on

whole blood specimens. If necessary, WBC and platelet counts may be estimated on the

peripheral smear.

3) Manual PT (INR)/APTT testing shall be discontinued. Please refer to CLSI H21-A4 for

reference on collection and storage.

4) Reticulocyte Count shall be performed by automated methodology. Manual counts may used

as a QC method for automated analyzers.

5) All differential leukocyte counts shall be reported in absolute values. Reporting percentages

is optional, and would be in addition to absolute values.


HEMATOLOGY FILMS/LABELLING OF SLIDES

Unequivocal patient identification is the first step to ensuring a quality hematology slide.

With no formal standards in place for labelling slides, the basic principles require:

Unique identification of the patient (at least two identifiers)

Written instructions for labelling

Labels shall be clear and legible

Date of collection

CLSI Document H-20-A states: “Label the samples uniquely.”

Positive Patient ID

_______________________________ _______________________________ Last Name First Name

PHN______________________________________________________________

Date of Birth

Unique ID #

Date of Slide___________________________________


MORPHOLOGY OF LYMPHOCYTES

Benign versus Malignant

A variety of diseases and disorders may produce changes from the normal in numbers and/or

morphology and functions of one or more of the leukocytes. The most important feature of

variant lymphocyte morphology is the recognition of its benign nature. The pertinent fact is that

these lymphocytes are normal cells that have been altered as the result of a normal response to

stimulus. When changes in WBC’s are produced by non-malignant disorders (e.g. infections),

the cells formerly called atypical, are now referred to as reactive lymphocytes. When changes

are suspicious of being produced by malignant disorders (leukemias, lymphomas,

gammopathies) and additional investigations may be required, the cells are often referred to as

atypical and/or abnormal.

In non-malignant disorders, the variant lymphocytes, reactive lymphocytes, atypical

lymphocytes, virocytes, stress lymphocytes, Downey cells, transformed lymphocytes,

transitional lymphocytes, and glandular fever cells, among others, are normal cells reacting to a

stimulus, whether it be viral or other. The designation of reactive lymphocytes is preferred.

In Chronic Lymphocytic Leukemia, the lymphocytes are somewhat larger than normal, have

nuclei with clumped or condensed chromatin, and may have prominent nucleoli. The cytoplasm

may be abundant, nongranular and moderately basophilic, or it may be relatively scant.

In Prolymphocytic Leukemia, the prolymphocyte is a relatively large mononuclear lymphoid cell

with an oval to round nucleus, coarse-appearing chromatin strands and one or two large vesicular

nucleoli with perinuclear condensations of chromatin. The cytoplasm is abundant and usually

granular and is basophilic with Romanowsky stains.

In Waldenström’s Macroglobulinemia, the abnormal B-lymphocytes involved are transitional

cells. They have the ability to differentiate into large plasmacytoid lymphocytes and plasma

cells. These malignant cells circulate in the peripheral blood only in the terminal stages.

In Lymphomas, peripheral blood involvement (i.e., abnormal circulating cells) is seen late in the

disease. Lymphoma cells can exhibit a variety of appearances and the cellular morphology is

variable and depends on the underlying type of lymphoma. These cells can exhibit variable size,

shape, nuclear, and cytoplasmic characteristics. Lymphoma cells are usually round to oval, and

can be irregular. Cell size ranges from 8 to 30 µm and the N-C ratio varies from 7:1 to 3:1. In

diffuse small lymphocytic lymphoma (the tissue equivalent of chronic lymphocytic leukemia),

the cells are generally small with round to oval nuclei, compact and coarse chromatin, and have a

scant amount of basophilic cytoplasm. They may be the same size as normal lymphocytes or

may be slightly larger. Occasionally, the nuclei exhibit an angulated appearance with slightly

more open chromatin. A small nuclear indentation may be present. Nucleoli are not seen.

Scattered prolymphocytes, which are larger cells with a centrally placed nucleus, a prominent

single nucleolus, and moderate basophilic cytoplasm, often are seen. In the small-cleaved cell

lymphomas, the cells are slightly larger than normal lymphocytes and have an angulated


appearance. The majority of nuclei have clefts, indentations, folds, convolutions, and may even

be lobulated. The chromatin is moderately coarse and one or more nucleoli may be prominent.

Their cytoplasm is scant to moderate and basophilic. The cells in small noncleaved lymphomas

(Burkitt’s lymphoma) appear similar to L3 lymphoblasts. These cells are generally moderate in

size (10 to 25 µm) and have a round to oval nucleus with moderately coarse chromatin, and one

or more prominent nucleoli. The cytoplasm is moderate, stains dark blue, and may contain

numerous small vacuoles. Large cell lymphomas and immunoblastic lymphomas may exhibit

some of the most blast-like and abnormal morphology. These cells are large (20 to 30 µm) and

have scant to moderate amounts of deeply basophilic cytoplasm. The nuclei are generally round

to oval, but may be angulated, folded, indented, or convoluted. Nucleoli are prominent and may

be single or multiple. Vacuoles can occasionally be seen in the cytoplasm. These cells can be

easily confused with blasts. T cell lymphomas can exhibit similar morphology to any of the

above types of lymphomas. The typical appearance is a moderate-size cell with a markedly

convoluted nucleus giving a cerebriform or grooved pattern. Their chromatin is moderately

coarse and nucleoli are not apparent. The cytoplasm is generally scant and blue.

In Hairy Cell Leukemia, the abnormal lymphocytes (Hairy Cells) have scant to abundant,

agranular, light grayish-blue cytoplasm. The plasma membrane appears irregular with hair-like

or ruffled projections, which are seen more easily with phase microscopy. These cells often have

a round or oval nucleus; sometimes, the nucleus appears folded or bilobed. The chromatin is

loose and lacy, and one or two nucleoli are commonly seen.

In Sézary Syndrome, the abnormal lymphocyte is larger than normal with scanty cytoplasm, and

the nucleus is large with clefting. Nuclear folding can be so extensive as to suggest an image of

the brain, and these nuclei are thus described as cerebriform. The nuclear chromatin is fine with

little condensation. There may or may not be visible nucleoli.

Note: Performance of manual differentials is required when abnormalities/unexpected results are

found in the WBC.

References:

McTaggart Bill, SAIT Hematology Updating Correspondence Course, 5th Edition, 1993. pp. 10.

Stiene-Martin, 1998, pp. 355-356, 484-485, 490, 507-508. College of American Pathologists, Surveys, Hematology Glossary,

2001


DIFFERENTIAL PERFORMANCE AND REFERRAL PRACTICE GUIDELINE

Blood Film Reference Range Perform a Differential or Scan

on First Occurrence or

Significant Change

Referral

Unexpected or Unexplained

WBC Count – adults 4.0 - 11.0 x 109/L

Lower referral range <1.5 x 109/L (all age groups) <1.0 x 10

9/L (all age groups)

Upper referral range >25.0 x 109/L >25.0 x 10

9/L

children (2-14 years) 5.0 – 15.0 x 109/L

children (90 days – 2

yrs)

5.0 – 20.0 x 109/L

newborn (0 – 90 days) 7.0 – 20.0 x 109/L

Absolute Neutrophils 1.5 – 7.5 x 109/L <1.5 x 10

9/L (all age groups) <1.0 x 10


Absolute

Granulocytes

1.5 – 7.5 x 109/L <1.5 x 10

9/L (all age groups) <1.0 x 10


Absolute Eosinophils 0.0 – 0.6 x 109/L >1.0 x 10

9/L (all age groups) >2.0 x 10


Absolute Basophils 0.0 – 0.2 x 109/L >0.3 x 10



Absolute Lymphs 1.1 – 4.4 x 109/L

adults >5.0 x 109/L >7.0 x 10

9/L

children (0-14 years) >7.0 x 109/L >10.0 x 10

9/L

Absolute Monocytes 0.2 – 0.8 x 109/L >1.0 x 10



Hemoglobin

Lower referral range

adult female 120 – 160 g/L <100 g/L

(Combined with MCV <70 fL)

<100 g/L


adult male 135 – 180 g/L <100 g/L


<100 g/L


Upper referral range

adult female 120 – 160 g/L >165 g/L >165 g/L

adult male 135 – 180 g/L >185 g/L >185 g/L

Pediatric ranges

children (1 mo – 14

yrs)

105 – 145 g/L <100 g/L


<100 g/L

(Combined with MCV <70fL)

newborn (0 – 1 month) 135 – 195 g/L <160 g/L or >210 g/L <135 g/L or >210 g/L

HCT 0.37 – 0.50 L/L None >0.65 L/L

MCV

Lower referral range

> 3 months (adult) 79.0 – 97.0 fL <70.0 fL or >100.0 fL

(Combined with HGB <100.0

g/L)

<70.0 fL


g/L)

0 – 3 months 98.0 – 114.0 fL <97.0 fL


g/L)

<90.0 fL


g/L)

MCHC 310 – 360 g/L

Upper referral range >360 g/L >365 g/L

MCH 27.0 – 32.0pg None none

RDW 11.5 – 14.5 % None none

RBC COUNT >6.5 x 1012

/L >6.5 x 1012

/L

adult female 3.2 – 5.4 x 1012

/L

adult male 4.6 – 6.2 x 1012

/L


Blood Film Reference Range Perform a Differential or Scan

on First Occurrence or

Significant Change

Referral

Unexpected or Unexplained

MPV 7.4 – 10.4 fL None

Lower referral range <6.0 fL

Upper referral range >14.0 fL

Platelet Count 150 – 400 x 109/L

Lower referral range <100 x 109/L <100 x 10

9/L

Upper referral range >600 x 109/L >600 x 10

9/L

WBC Morphology > 10% Reactive Lymphs

Pelger-Huet anomaly

Hypogranulated neutrophils

Hairy Cells

Blasts/Immature Cells

Hypersegmented neutrophils

NUCLEATED RBC’S >5 NRBC/100 WBC

RBC Morphology RBC inclusions: Pappenheimer,

Howell-Jolly or Heinz Body,

basophilic stippling

Presence of schistocytes,

echinocytes, bite cells, sickle

cells, rouleaux,

autoagglutination, significant

polychromasia, oval or round

macrocytes, target cells, tear

drops, spherocytes, elliptocytes,

acanthocytes, stomatocytes

Dimorphic picture

Parasites – Malaria

PLATELET

MORPHOLOGY

none

OTHER CRITERIA

Specified Instrument

Flags

When indicated

Ordered by Physician Physician request Physician request

Technologist

Discretion

Technologist initiated Technologist initiated – if

suspicious cells are present, refer

to a pathologist

The Color Atlas of Hematology, Hematology and Clinical Microscopy Resource Committee,

CAP; Glassy, Eric F. is recommended as a resource.

NOTE: This table is provided as a guideline only. Consult a larger centre for more specific

ranges.


RED BLOOD CELL MORPHOLOGY REPORTING GUIDELINE

Red Blood Cells/100x oif Abnormality to be

Reported

Implications for Diagnosis

(200 RBC field) x 10 fields

Any present

Schistocytes Thrombotic thrombocytopenic

purpura (TTP), RBC

fragmentation syndromes such

as hemolytic uremic syndrome,

DIC, microangiopathic

hemolysis, malignant

hypertension, eclampsia,

Cardiac valve hemolysis, some

renal vascular diseases

Echinocytes/Burr Cells Kidney Disease

Bite Cells Drug or chemical induced

oxidative damage, unstable

hemoglobins

Sickle Cells Sickle Cell Anemia,

Hemoglobin SC/SD Disease

Basophilic Stippling Lead Poisoning, Thalassemia,

Sideroblastic & Megaloblastic

Anemia, Sickle Cell Anemia

Howell Jolly Bodies Megaloblastic Anemia, Post-

splenectomy state

Dimorphic Hemorrhage, response to

treatment, Sideroplastic anemia,

post-transfusion

RBC Rouleaux (5cells

stacked)

Paraproteinemia, increased

fibrinogen, inflammatory

disorders

RBC Agglutination Autoimmune Hemolytic Anemia

(Cold Agglutinin Disease)

Parasites Identify specific forms

Nucleated RBC Sever hemolysis, part of

Leukoerythroblastic picture,

Bone marrow stress

>5 Polychromasia Response to treatment, blood

loss, Hemolysis

Macrocytes (oval) Megaloblastic state, Aplastic

Anemia, Myelodysplastic

Syndrome


Target Cells Liver Disease, Post-

splenectomy/hyposplenism,

Hemoglobinopathy,

Thalassemia

Tear Drops Myelofibrosis, Pernicious

Anemia

Spherocytes Hereditary Spherocytosis,

Autoimmune Hemolytic Anemia

Pappenheimer Bodies Sideroblastic Anemia, Chronic

Hemolysis, Liver Disease

>10 Macrocytes (round) Liver Disease, Alcoholism

Elliptocytes Hereditary Elliptocytosis

Acanthocytes/Spur Cells Post-splenectomy state, Liver

Disease, Abetalipoproteinemia

Stomatocytes Liver Disease

Microcytes (hypochromic

cells)

Iron Deficiency, Thalassemias,

Treated Polycythemia

Avoid using the terms anisocytosis and/or poikilocytosis since they convey no specific meaning.

The numeric value is meant for internal use to indicate a significant abnormality presence. No

numeric value is reported, just the abnormality.


DIFFERENTIAL QUALITY CONTROL

The following is a list of measures undertaken by each laboratory to ensure the quality of

differential results reported on patients.

Good Laboratory Practice:

1. Develop a protocol for determining if a manual differential is required based on instrument

capabilities. (i.e. Hematology Guidelines—Differential Reporting Guidelines)

2. Develop a list of abnormalities, which must be reviewed by the supervisor or pathologist

before results are reported. Refer to Blood Film Guideline.

3. Differentials must be repeated if each cell does not meet the limits set out in the table below.

If the repeat is still not within the established limits, a second technologist should repeat the

differential.

4. Whenever the tech1 has concerns with a differential the rest of the CBC can be released with

a notation “Differential to follow”. The requesting physician can be invited to review the

smear if they so choose. The smear should be evaluated as soon as possible.

5. Leukocyte abnormalities seen during a smear review require a manual differential completed

regardless of the protocol for when to perform a manual differential.

6. Perform the manual differential and compare it to the automated differential using the 95%

confidence limits table. Each cell should compare within the range set.

7. Differentials between technologists should also fall within the established limits (95%

confidence limits).

8. If the manual differential performed by two technologists agrees within the established

limits, but is not in agreement with the automated differential, then the manual differential

should be reported out instead of the automated differential.

9. It is good laboratory practice to circulate unknown QA slides quarterly. The results will be

compared with peers and should be within the 95% confidence limits as set by the following

table. Any problem areas will be covered between the technologist and the supervisor.

Procedure:

How to use the following 95% confidence limits table:

1. Look up each cell number you want to compare to in column “a”.

E.g. 25% neutrophils

1 Tech refers to the medical laboratory technologist or combined laboratory and x-ray technologist.


2. If a 100 cell differential was performed go to the column “n=100” to determine the

acceptable range. If a 200 cell differential was done refer to column “n=200”.

E.g. you counted 32 neutrophils in a 100-cell differential.

3. Determine acceptability of the differential by checking if the number you counted for a

certain cell falls within the stated range.

E.g. Stated range is 16-35%; you are within this range.

4. Repeat for each cell type.

5. Determine acceptability of each cell line by comparing automated to manual differential. For

the neutrophils/granulocytes, segmented neutrophils, band neutrophils and other neutrophil

precursors must be added together and for lymphocytes, reactive lymphocytes and

lymphocytes must be added together.

E.g. 77 segs and 15 bands = 92%

6. You must be within this range, or the differential must be repeated.

Example:

The automated or technologist differential indicated the following differential and the acceptable

range for each number was looked up in “n=100” column:

Neu: 70 acceptable range 60-79

Lymph: 15 “ “ 8-24

Mono: 7 “ “ 1-12

Eos: 3 “ “ 0-9

A 100 cell differential is completed by another technologist and based on the acceptable range

the results are as follows:

Neu: 52 not within limits

Lymph: 27 not within limits

Mono: 12 within limits

Eos: 7 within limits

Baso: 2 within limits

This manual differential would have to be repeated.


The 95% confidence limits for various percentages of leukocytes of a given type as determined

by differential counts on stained blood smears.

n n=100 n=200 n=500 n=1,000

0 0-4 0-2 0-1 0-1

1 0-6 0-4 0-3 0-2

2 0-8 0-6 0-4 1-4

3 0-9 1-7 1-5 2-5

4 1-10 1-8 2-7 2-6

5 1-12 2-10 3-8 3-7

6 2-13 3-11 4-9 4-8

7 2-14 3-12 4-10 5-9

8 3-16 4-13 5-11 6-10

9 4-17 5-14 6-12 7-11

10 4-18 6-16 7-13 8-13

15 8-24 10-21 11-19 12-18

20 12-30 14-27 16-24 17-23

25 16-35 19-32 21-30 22-28

30 21-40 23-37 26-35 27-33

35 25-46 28-43 30-40 32-39

40 30-51 33-48 35-45 36-44

45 35-56 37-53 40-50 41-49

50 39-61 42-58 45-55 46-54

55 44-65 47-63 50-60 51-59

60 49-70 52-67 55-65 56-64

65 54-75 57-72 60-70 61-68

70 60-79 63-77 65-74 67-73

75 65-84 68-81 70-79 72-78

80 70-88 73-86 76-84 77-83

85 76-92 79-90 81-89 82-88

90 82-96 84-94 87-93 87-92

91 83-96 86-95 88-94 89-93

92 84-97 87-96 89-95 90-94

93 86-98 88-97 90-96 91-95

94 87-98 89-97 91-96 92-96

95 88-99 90-98 92-97 93-97

96 90-99 92-99 93-98 94-98

97 91-100 93-98 95-99 95-98

98 92-100 94-100 96-100 96-99

99 94-100 96-100 97-100 98-100

100 96-100 98-100 99-100 99-100

References:

1. Abbott training seminar—the following table was provided.

2. Clinical Hematology Principles, Procedures, Correlations, Cheryl A Lotspeich-Steininger et al, 1992.

3. FHHR protocol


SMUDGE CELLS

Distinguished by their naked amorphous nuclear chromatin material, smudge cells were initially

described as white blood cells with broken-down nuclei in patients with chronic lymphocytic

leukemia. Subsequently, these nuclear shadows have most often been referred to as smudge

cells, but the term basket cells is used synonymously.

The mechanism is often associated primarily with traumatic disruption of cells during blood film

preparation. In the process, the cell membrane ruptures and when viewed under a microscope,

what remains looks like a smudge, hence the term, smudge cells.

To ensure reliability of results, it is important to understand the effects of variables associated

with smudge cell formation, particularly the blood film preparation. Thus, the angle and the

degree of incline of the slide spreader, the type of slide spreader (sharp or smooth), the

cleanliness of the slides, and the overall quality of the blood films cannot be overemphasized.

For minimal morphologic alterations, blood films should be made within three hours and not

more than twelve hours after collection.

It is recommended to include smudge cells in the differential as an absolute count, especially

when the smudge cell numbers are noticeably increased. This identifies a more appropriate

count because smudge cells are actually lymphocyte artifacts. It also avoids the need for

repeating or verifying abnormal counts by the time - consuming albumin – treated method.

Education is needed (for the ordering physicians especially) to eliminate the risk of

misinterpreting this smudge cell count as a new cell type.

Criteria for Reporting Smudge Cells

Absolute lymphocyte count should be greater than 5.0 x109/L.

Patient age should be more than 30 years*.

Smudge cells should be reported if greater than 10 per 100 leukocytes. Report smudge cells in

absolute numbers.

*Although CLL is not often diagnosed in patients under the age of 40, patients over 30 years of age should be

considered potentially at risk. CLL is rare in patients under 30 years of age.

*In children (18 & under), the smudge cells are not counted as part of the differential. However, the presence of

smudge cells may be noted on the report.

*Smudge cells are present in those candidates for which definitive diagnostic criteria are well established. Examples

include: CLL & Acute Leukemia.


FLOW CYTOMETRY FOR THE DIAGNOSIS OF LYMPHOMA/LEUKEMIA

Immunophenotypic analysis of hematological malignancies is crucial for accurate diagnosis and

classification of these complex malignancies. Flow cytometric data shall be interpreted in the

context of additional information and correlation with other clinical findings, obtained through

genetic studies, and through conventional morphologic and cytochemical methods. Flow

cytometry by itself does not provide enough information for diagnosis.

“Flow cytometric analysis has become an acceptable medical practice in the diagnosis and

characterization of hematologic neoplasia and its role in the management of patients with these

diseases is well recognized. Despite its extraordinary power, there is great concern regarding

the inconsistent practices and wide variation in styles among laboratories involved in the flow

cytometric analysis of leukemias and lymphomas. Of particular importance are the deficiencies

in standardization and validation of procedures used in the analysis, the manner by which the

information is generated and reported to pathologists or treating physicians, and the

appropriate utililzation of this technology in patient care.” (US-Cdn Consensus

Recommendations on the Immunophenotypic Analysis of Hematologic Neoplasia by Flow

Cytometry)

a) A MLT with Hematology experience, and appropriate Immunology background, and

training in Flow Cytometry is required for the performance of Flow Cytometry clinical

testing of Lymphoma and Leukemia.

b) A qualified Pathologist/Hematologist who has training in both Hematopathology and

Flow Cytometry must perform interpretation of Flow Cytometry results.

c) Flow cytometry in diagnostic purposes for Lymphoma/Leukemia shall only be performed

in pathologist – directed laboratories.


MALARIA

Malaria can be a life threatening infection and a rapid diagnosis is necessary to institute

appropriate management in a timely manner. The diagnostic information required for patient

management is best obtained by microscopy; however, a rapid test (immunologically-based) is a

very useful screening test. The Rapid Diagnostic Tests (RDT) results shall be confirmed by

microscopy.

Rapid Diagnostic Tests (RDTs):

1. Should be available in all acute care sites in Saskatchewan

2. A single RDT should be recommended for use across the province.

3. Standardized reporting terminology should be used.

4. The test should be handled as a STAT procedure.

5. Results should be phoned to the requesting physician.

6. All requests should automatically trigger microscopic examination.

7. Results in a patient suspected of falciparum infection should include prompt referral (of

the patient or transport of sample) to a centre where microscopy is available on an urgent

basis.

8. Reports must indicate that the RDT is a screening test, with confirmation by microscopy

to follow.

Microscopic Diagnosis:

1. The test should be available 24/7.

2. Upon receipt of specimens at the testing laboratory, TAT is 4-6 hours.

3. The testing should be based on the protracted (20 min.) examination of four thick and

four thin blood smears.

4. Positive slides must be confirmed by a laboratory physician and/or pathologist.

5. The information in the report should include the following; a) positive or negative, b)

speciation, c) quantification of parasitemia.

6. All positive results should indicate the requirement to report Malaria cases to Public

Health and a recommendation to consult with an Infectious Disease specialist as soon as

practically possible.

7. Results must be called to the physician by the testing laboratory

8. The laboratories must participate in an external/internal quality assurance program.


3.2% Na CITRATE ANTICOAGULANT RECOMMENDED VERSUS 3.8%

FOR COAGULATION STUDIES

The International Standards Committee on Thrombosis and Hemostasis, and the CLSI have

recommended guidelines to standardize whole blood collection to 3.2% sodium citrate

anticoagulant.

Several publications have indicated that the clotting times for PT and APTT are significantly

shortened with the 3.2% NaCitrate. For this reason, exchange between the two concentrations is

not acceptable. When selecting an anticoagulant for collection, it is important to note that 3.2%

NaCitrate is used for ISI assignments for thromboplastin. For these reasons, it is important for

laboratories to standardize the choice of anticoagulant for sample collection.

The anticoagulant used for coagulation assays should be 3.2% trisodium citrate. The proportion

of blood to anticoagulant volume is 9:1. Inadequate filling of the collection device will decrease

this ratio, and may lead to inaccurate results. The manufacturer recommendations should always

be followed.

If the hematocrit is very high and the usual relative volumes of blood and citrate solution are

mixed, a prolonged prothrombin time results from overcitration of the reduced proportion of

plasma in the blood sample. This means the final citrate concentration in the blood should be

adjusted in patients who have hematocrit (PCV) values above 0.55 (55%). Adjust the volume of

NaCitrate in the draw tube by applying the calculation outlined in the CLSI H21-A3. Note: To

maintain the vacuum in the NaCitrate collection tube, use a tuberculin syringe to draw out the

anticoagulant.

The anticoagulant volume may be calculated from the expression:

X = (100 – PCV) x draw volume of tube (mLs)

(595 – PCV)

X = the volume of anticoagulant required to prepare volume of anticoagulated blood

PCV = packed cell volume (Hct) in %

For example: To determine the volume required for 5 mLs anticoagulated blood, calculate x 5

mLs

References:

H21-A3 CLSI, BD Vacutainer Systems, Azko Nobel

Assessment of the influence of citrate concentration on the international Normalized Ratio (INR) determined with twelve

reagent-instrument combinations. Chantarangkul, Tripodi, Clerici, Negri, Mannucci

Effect of concentration of trisodium Citrate Anticoagulant on Calculation of the international Normalized Ratio and the

International Sensitivity Index of Thromboplastin, Duncan, Casey, Duncan, and Lloyd

The Prothrombin Time Test: Effect of Varying Citrate Concentration, Ingram, Hills.

Comparison of 3.2% vs. 3.8% Sodium Citrate Anitcoagulant Collection Tubes for Coumadin, Heparin, Abnormal and Normal

Specimens. Phillips, Sunnybrook ON


VITAMIN B12

& FOLATE

Background:

The normal proliferation of cells depends on adequate folate and vitamin B12. Folate is

necessary for efficient thymidylate synthesis and production of DNA. B12 is needed to

successfully incorporate circulating folic acid into developing RBCs retaining folate in the RBC.

MEASURING BOTH B12 AND FOLATE LEVELS IS NOT NECESSARY IN ALL PATIENTS.

Serum B12:

The clinical indications for ordering serum B12 include:

1) Evaluation of patients with MACROCYTIC (high MCV in the CBC) anemia and the clinical

information suggesting possible B12 deficiency;

2) Evaluation of patients with psychiatric and neurologic impairment (symptoms of subacute

combined degeneration of spinal cord).

Red Cell Folate:

Red cell folate is ordered as it is an indication of the folate status over a longer period of time

(several months) as opposed to serum folate that reflects levels over the last few days.

Population studies have shown that dietary supplements have increased average folate levels and

therefore folate deficiency is much rarer. The clinical indications for ordering red cell folate

include the evaluation of MACROCYTIC (high MCV in the CBC) anemia and the clinical

information suggesting possible folate deficiency.

Practice Tips:

1. Persons on B12/folate supplements or other multivitamins do not require testing.

2. Lab tests are used to confirm a specific diagnosis and not as a fishing expedition.

3. Marginal B12 deficiency in any elderly patient with dementia, peripheral neurological

symptoms or impaired immunity should be taken seriously.

4. B12 should not be done on patients on oral contraceptives.

Prepared by Dr. A. Saxena,

Department of Laboratory Medicine,

Saskatoon Health Region


Current Indications for B12 and Folate Investigation

Measuring both B12 and Folate levels is not necessary in all patients.

Notes:

*B12 tests should not be done if the person is on B12

*False low B12 in pregnant women and women on oral contraceptives

Serum folates are not a useful screening tool

Folic Acid Deficiency is rare due to folate fortification in food

RBC FOLATE IS MORE INDICATIVE OF TISSUE FOLATE LEVELS

Folate tests will not be done if the person is on folate

Normal

CBC

MCV ↑

No further testing YES

B12 and RBC

folate

Abnormal Normal

N

o

r

m

a

l

Neurological or Clinical

symptoms suggestive of B12

&/or Folate deficiency

Requires further

patient evaluation

No further

testing


BLEEDING TIME

Bleeding Time (BT) is defined as the time between the making of a small incision and the

moment the bleeding stops. It indicates how well platelets interact with blood vessel walls to

form blood clots (a test of platelet and vascular function).

Purpose:

Bleeding time is used most often to detect qualitative defects of platelets, e.g. Von Willebrand’s

disease (vWD). However, it is prolonged in many situations including vascular disorders, e.g.

Ehler-Danlos’ syndrome, pseudoxanthoma elasticum.

Issue:

The use of BT is declining and at many institutions this test has been eliminated. This is because

of three main reasons:

It is neither a sensitive nor a specific test.

A specific diagnosis of vWD can be made based on history and other tests.

More reliable information about platelet function can be obtained by other tests.

Notes:

Use of bleeding time is not recommended (not by itself). Current practice varies and if BT is

required, the recommended method is the template device. BT is generally not recommended for

children under the age of 5.

Reference Interval:

Results are reported to the nearest half-minute. The reference range varies and is usually 1.5-9.5

minutes. The test should be discontinued if the patient hasn’t stopped bleeding by 15-20 minutes.

References:

Andrew M, Paes B, Bowker J, et al, "Evaluation of an Automated Bleeding Time Device in the Newborn,"Am J Hematol, 1990,

35(4):275-7.

Basili S, Ferro D, Leo R, et al, "Bleeding Time Does Not Predict Gastrointestinal Bleeding in Patients With Cirrhosis,"J Hepatol,

1996, 24(5):574-80.

Brown BA, Hematology: Principles and Procedures, 6th ed, Philadelphia, PA: Lea and Febiger, 1993, 267-70.

de Rossi SS and Glick MG, "Bleeding Time: An Unreliable Predictor of Clinical Hemostasis,"J Oral Maxillofac Surg, 1996,

54(9):1119-20.

George JN, Shattil SJ: The Clinical Importance of Acquired Abnormalities of Platelet Function. NEJM 324:27-29, 1991.

Gewirtz AS, Miller ML, and Keys TF, "The Clinical Usefulness of the Preoperative Bleeding Time,"Arch Pathol Lab Med, 1996,

120(4):353-6.

Henry, J. B. Clinical Diagnosis and Management by Laboratory Methods. Philadelphia: W. B. Saunders Co., 1996.

Munro J, Booth A, and Nicholl J, "Routine Preoperative Testing: A Systematic Review of the Evidence,"Health Technol Assess,

1997, 1(12):1-62.

Lewis SM, Ban BJ, Bates I. Dacie and Lewis Practical Haematology. 2006. Churchill Livingstone Elsevier, Philadelphia (PA).

Lind SE: Prolonged Bleeding Time. Am J Med 77:305-312, 1984.

Peterson P, Hayes TE, Arkin CF, et al, "The Preoperative Bleeding Time Test Lacks Clinical Benefit,"Arch Surg, 1998,

133(2):134-9.

Rodgers RP and Levin J, "A Critical Reappraisal of the Bleeding Time, "Semin Thromb Hemost, 1990, 16(1):1-20.

Triplett DA. Laboratory Evaluation of Coagulation, 1982. American Society of Clinical Pathologists Press, Chicago (IL).


CROSSCHECK/VALIDATION GUIDELINE FOR THOSE FACILITIES WITH MULTIPLE CHEMISTRY/HEMATOLOGY INSTRUMENTS

PERFORMING THE SAME TEST PROCEDURE

Proficiency testing registration is mandatory for each analytical ‘test’.

Rotating proficiency testing result submission between analyzers is not a requirement, but is suggested where appropriate (e.g. Blood

Gases).

Where there are multiple analyzers performing the same test procedure in larger facilities, it may be more appropriate for proficiency

testing submission to be consistently submitted from the same analyzer for tracking purposes.

An internal cross-check/validation protocol is required to ensure that there is correlation between all analyzers providing the same test

result in the same facility.

If this protocol is not followed, then each analyzer must be registered in the external proficiency testing program as mandated by LQAP.

This procedure is recommended every six months.

Chemistry/Hematology High Volume Analyzers (e.g. Electrolytes/CBC)








recommendations




Minimum of 20 patient specimens/2 times per year or

equivalent

(i.e. 10 patient specimens/4 times per year or on-going data

collection as appropriate)


Chemistry/Hematology Low Volume Analyzers (e.g. Fibrinogen)








recommendations




Minimum of 5-10 patient specimens/2 times per year or

equivalent



PROCEDURE/METHOD STATISTICAL WORK-UP/VALIDATION STUDY GUIDELINES: CHEMISTRY/HEMATOLOGY

Work-up guidelines and definitions (change in method/instrument):

Work-up

Element

Definition: CLIS or International Federation of Clinical

Chemistry (IFCC)

Minimum Data Requirements (where

appropriate):

Applicability:

Qualitative Quantitative

1. Imprecision

within run

between run

The variation in analytical results demonstrated when a particular

specimen of aliquot is analyzed multiple times or on multiple days.

Imprecision is expressed quantitatively by a statistic such as standard

deviation or coefficient of variations.

Within run – use preferably a patient

sample or pool close to the decision levels

with a minimum of 10 data points.

Between run – 20 results from 20 separate

runs on 2 levels over a 10-day minimum

time period using appropriate QC material.

2. Patient

Correlation

The correlation coefficient is a means to look for a relationship, not

agreement, between pairs. Two methods may have a perfect

correlation throughout the measuring range but may not agree in

value (i.e. one may be double the value of the other).

40 data points are recommended with a

minimum of 20 having 50% of the data

points outside the reference intervals, if

possible. Correlations should involve

comparison with an acceptable reference

method or laboratory.

n=20

n=40

3. Linearity (IFCC) The range of concentration or other quantity in the specimen

over which the method is applicable without modification (CLIS)

when analytical results are plotted against expected concentrations;

the degree to which the plot curve conforms to a straight line is a

measure of the system linearity.

4 data points each in duplicate as a

minimum requirement, but 5 data points are

preferred (over reportable range). Linearity

studies are expected on an initial method

work-up and further studies as defined by

the College guidelines (i.e.

troubleshooting).

4. Reference

range

validation

It is common convention to define the reference range or interval of a

laboratory test as the central 95% interval bounded by the 2.5 and

97.5 percentiles of the selected patient population. Validation of an

established reference range requires a minimum of 20 samples.

The minimum requirement is 20 data points

for confirmation of an established reference

range and 120 for the establishment of a

new reference range.

5. Accuracy Closeness of the agreement between the result of a measurement and

the accepted reference value (true value of the analyte)

3 data points using acceptable reference

material (i.e. CEQAL or CAP) 1 data point

may be acceptable for haematology

accuracy studies if related to sample

stability.

6. Sensitivity Measure of the ability of an analytical method to detect small

quantities of the measured component. When concern is performance

at a very low concentration it is useful to determine the detection

limit as influenced by imprecision.

Sensitive studies are only required for those

methods which have clinical relevance at

values close to “0” (i.e. TSH)

When

clinically

relevant

When clinically

relevant

7. Specimen

Stability

The conditions of handling and storage, which permits the

measurement and reporting of a clinically relevant result.

No data generally required. As per

manufacturer’s guidelines. If

Storage and

Storage and


manufacturer’s stability window is to be

extended a stability study is expected.

transportation

dependent

transportation

dependent

8. Interference The effect of any component of the sample on the accuracy of the

measurement of the desired analyte.

Document the manufacturer’s interference

information. The method should include a

disclaimer or a process for dealing with a

lipemic, icteric or hemolyzed sample.

Methods with

known

interferences

Methods with

known

interferences

9. Recovery A recovery procedure involves the addition of a known amount of

analyte to an aliquot of sample. Recovery is defined as the ratio of

the amount of the analyte recovered to amount added and is given as

percentage.

Recovery studies should only be necessary

for those methods or analytes where

organic extractions or equivalent are

required as part of the methodology (i.e.

Toxicology)

Method

specific/organic

extraction

Work-up requirements when an instrument is moved from site “A” to site “B”: (It is assumed that the instrument has been in recent use with

acceptable performance).

Work-up Element Minimum data requirements:

1. Imprecision studies, QC only As above

2. Patient correlation 10 data points, where feasible.


PROTOCOL FOR VALIDATION OF LINEARITY ON AUTOMATED HEMATOLOGY ANALYZERS

Purpose:

To validate the entire range through which patient values can be reported or to verify the

manufacturer’s stated linearity from the analysis of undiluted or diluted specimens for all

measured parameters.

Specimen Selection:

For each parameter requiring linearity validation, choose a specimen with results at/near or

above the high end of the linear range published by the manufacturer of the instrument.

Ensure that there were no interfering substances noted during analysis of the specimen. This

value is equivalent to 100% while patient’s plasma or instrument diluent is equivalent to 0%.

For RBC and HGB use a polycythemic or concentrated sample.

A concentrated specimen may be prepared by centrifuging the specimen and removing plasma to

produce a value near or above the high end of the linear range published by the manufacturer.

Linearity of MCV, MCH and MCHC can also be performed by concentrating the specimen.

For RBC and HGB, do not exceed an HCT of 0.60-0.65.

For WBC use a leukocytosis sample diluted in patient’s own plasma or instrument diluent.

For PLT count use a thrombocythemic sample diluted in patient’s own plasma or instrument

diluent.

Ensure that sufficient specimen is collected for dilution preparations and instrument

aspiration.

Procedure:

1. Verify that instrument reagents are not expired and that sufficient volume of reagents is

loaded on instrument to cycle all the prepared dilutions.

2. Check that background counts, instrument precision and calibration of the instrument are

acceptable before starting procedure.

3. Determine the volume per dilution by calculating the volume required for aspiration on the

instrument. One specimen may not provide sufficient volume. If more than one tube is

drawn, pool the tubes into one aliquot.

4. Label five clean plastic tubes 80%, 60%, 40%, 20% and 0%.

5. Prepare the dilutions using the original specimen as the 100% as shown in table provided

below. Make sure the 100% sample remains well mixed throughout the dilution preparation

process.


Dilution (%) Specimen (Parts) Diluent (Parts)

100 10 0

80 8 2

60 6 4

40 4 6

20 2 8

0 0 10

6. Analyze each well-mixed dilution in triplicate on the instrument. Results with suspect

parameter flags should not be used.

7. Record all results for each parameter from all three runs on table provided.

8. Calculate Obtained Mean value for each parameter.

9. Calculate Expected value for each parameter by multiplying the Reference Mean (100%

Obtained Mean) by the multiplication factor.

10. Calculate the Difference between Obtained Mean and Expected Mean.

11. To graphically illustrate linearity, plot each parameter on linear graph paper with the

obtained mean value for the parameter on the X axis and the expected value for the

parameter on the Y axis. Draw a line through all the points on the graph. When the

obtained mean is plotted against the corresponding expected value, the plotted curve will

approximate a straight line for a linear method. Excel spreadsheet can also be used to

show linearity.

Note: Linearity is performed initially and when calibration fails to meet the laboratory’s

acceptable limits.

References:

1. Package insert from R&D Systems Inc. which sells a CBC-LINE Full Range Hematology Linearity Kit and CBC-LINE Low

Range Hematology Linearity Kit 1994

2. Shinton NK, England JM, Kennedy DA, “Guidelines for the evaluation of instruments used in Haematology laboratories” J

Clin Path 1982;35; 1095-1102

3. Package insert from STRECK Calibration Verification Assessment 2007

4. Quam EF, “Method Validation – The Linearity of Reportable Range Experiment ASCP


WBC Linearity WBC Results 0% 20% 40% 60% 80% 100%

(Reference)

Run #1

Run #2

Run #3

Obtained Mean

Reference Mean

X Multiplication Factor

X 0.0

X 0.2

X 0.4

X 0.6

X 0.8

X 1.0

Expected Value

Difference

Allowable Evaluation Limits ± 0.4 0.4 0.4 0.4 0.4 0.4

RBC Linearity RBC Results 0% 20% 40% 60% 80% 100%

(Reference)

Run #1

Run #2

Run #3

Obtained Mean

Reference Mean


X 0.0

X 0.2

X 0.4

X 0.6

X 0.8

X 1.0

Expected Value

Difference

Allowable Evaluation Limits ± 0.1 0.1 0.1 0.1 0.1 0.1

HGB Linearity HGB Results 0% 20% 40% 60% 80% 100%

(Reference)

Run #1 Run #2 Run #3 Obtained Mean Reference Mean


X 0.0

X 0.2

X 0.4

X 0.6

X 0.8

X 1.0

Expected Value Difference Allowable Evaluation Limits ± 3 3 3 3 3 3

PLT Linearity PLT Results 0% 20% 40% 60% 80% 100%

(Reference)

Run #1 Run #2 Run #3 Obtained Mean Reference Mean


X 0.0

X 0.2

X 0.4

X 0.6

X 0.8

X 1.0

Expected Value Difference Allowable Evaluation Limits ± 10 10 10 10 10 10


PROCEDURE FOR WBC ESTIMATE

WBC Estimation is to be used for cases in which the instrument flags invalid data or suspect

WBC populations and for which you cannot obtain a valid WBC in any mode.

1. Examine the specimen for presence of a clot, strands of fibrin, or gross hemolysis before you

proceed.

2. Next examine the histograms for likely causes of interference. Histogram review may help

you decide which version to use if it is necessary to issue a partial CBC report. (If the HGB

or PLT results are critical or STAT, these should not be held back.) Most interferences are

visible in the lower region of your first WBC histogram, e.g. resistant RBC, NRBC,

megathrombocytes, clumped platelets, fibrin, etc. In some cases, the analyzer will

incorrectly include these interferences in the lymphocytes. This results in both a falsely

elevated WBC and lymphocyte count. Rule of thumb: the lowest WBC from the analyzer is

usually the most correct.

3. Make and stain two blood smears.

4. Two techs shall do an estimate, each using a separate slide (if a second tech is not available,

one tech can do two estimates, one on each slide)

a. Examine the smears under 10x objective for even distribution of WBC before

proceeding. If there is a ridge or layer of WBC in the tails, the estimate will be invalid.

Re-make the slide.

b. Choose an area from the tails, where the RBC are evenly spread, or just beginning to

overlap (where 50% of the red cells are overlapping doubles or triplets). This should be

the same area where you would do RBC morphology or platelet estimation. Be aware

that if the RBC/PCV are low, you must avoid going in too deep. Then count in ten

consecutive fields.

c. Switch to hpf. Count the total number of WBC seen in 20 fields. Include disintegrated

cells. Do not include NRBC.

d. Divide your total by 20 to obtain the average number of WBC/hpf, to two decimal places.

Multiply the average by the conversion factor for the microscope.

Refer to conversion factor procedure for hpf.

Average your result with that of the other tech.

e. If your estimate is 0.40 or lower, just call it “less than 0.5”

f. If your estimate is over 0.40 calculate the estimate as a range of +/- 40% from this

number. Round off the results of 2.0 or lower to one decimal place; round off results

over 2.0 to the nearest whole number.


The examples below are using a microscope conversion factor of 2.5.

Example 1:

Avg. number of WBC/hpf on #1 slide = 1.55 x conversion factor of 2.5 = 3.875

Avg. number of WBC/hpf on #2 slide = 2.00 x conversion factor of 2.5 = 5.00

Average estimate from two slides (3.875+5.00) ÷2=4.4375

40% Range=4.4375x4.0=1.775

Estimate =4.4375 +/- 1.775, or 2.6625 to 6.2125. Round off to whole numbers.

WBC Estimate is 3 to 6 x 10^9/L

Example 2:

Estimate is calculated as 0.30.

Estimate is 0.40 or lower, so interpret range as “less than 0.5”

Example 3:

Estimate is calculated as 0.45, range of 0.27 to 0.72.

Round off as 0.3 to 0.7.

Example 4:

Estimate is calculated to be 2.5, range of 1.5 to 3.5.

Round off as 1.5 to 4.

5. If the estimate range agrees with the analyzer WBC, report the best results from the analyzer.

Example:

Analyzer count = 5.5

Estimate from films is 4.4 +/- 40%, or 3 to 6.

Report the analyzer WBC of 5.5.

6. If the WBC estimate does not agree with the analyzer count, report the estimate as a range.

Examples:

WBC estimate on film appears to be less than 0.5 x 10^9/L.

WBC estimate on film appears to be 1.5 to 4 x 10^9/L.

WBC estimate on film appears to be 3 to 6 x 10^9/L.

7. If it is absolutely necessary to report a differential with a WBC estimate, it must be a manual

differential in relative units (%).

References:

Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company,

1998


ESTABLISHING CONVERSION FACTOR FOR WBC ESTIMATION

Purpose:

To provide for instruction for establishing a conversion factor for the hematology microscope by

calculating the ratio of electronic count to the number of WBC on either hpf or per oil immersion

field.

Frequency:

The conversion factor must be established once for every microscope used in hematology or

when there are major repairs or changes in the analyzer for electronic counts or a new

microscope is used for estimates.

Procedure:

Note: Total leukocyte counts can be estimated roughly either by hpf or 100x oil immersion field.

Therefore 100x oil immersion field can be substituted in procedure that states hpf.

Step: Action:

1. Perform electronic WBC count on 30 consecutive fresh patient blood samples.

Alternatively if unable to perform 30 consecutive samples it is acceptable to perform

10 samples per day on 3 consecutive days.

2. Prepare and stain one peripheral blood film for each sample.

3. For each film, under hpf microscopy, find an area where 50% of the red cells are

overlapping doubles or triplets. Then count WBC in 10 consecutive fields.

4. Divide by 10 the total number of WBC found to obtain the average number per hpf.

5. Divide the electronic WBC count by the average number of WBC per hpf.

6. Add the numbers obtained in step 5 and divide by 30 (number of observations in this

analysis) hpf.

7. Round the number calculated in step 6 to the nearest whole number to obtain an

estimation factor.

Reference:


1998.


WBC ESTIMATION CONVERSION FACTOR LOG SHEET

Microscope Model: _______________________ ID: ______________________________

Column 1 Column 2 Column 3 Column 4

SPECIMEN ID

AUTOMATED WBC

COUNT

WBC ESTIMATION

DIVIDE

AUTOMATED WBC

COUNT BY WBC

ESTIMATION

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

Average Estimation

Factor

__________________

Calculation:

To determine Average, total the values in Column 4, divide by 30. Round off to nearest whole

number to obtain the estimated factor.


PROCEDURE FOR PLATELET ESTIMATES

1. Platelet estimation is to be used for cases in which instrument flags identify invalid platelet

data, platelet clumps and for verifying platelet counts below 100x109/L. Scan the slide for

clumps under hpf. If platelet clumps are present, they cause a false decrease in platelet count.

Edit out the platelet count by replacing it with the message “Platelets clumped, results

invalid.” Do not report the MPV.

NOTE: For this purpose, platelet clumping is defined as at least 5 clumps, with at least 5

platelets per clump. Rare or occasional smaller clumps should be ignored.

2. Switch to 100x objective. Total the number of platelets in at least 10 fields. Determine the

average number per field. Multiply by the conversion factor established for your microscope.

The estimate should agree with the analyzer count +/- 40%

Example:

Average of 6.3 cells per field x conversion factor of 11 = estimate of 69 x 10^9/L.

Analyzer count of 88. Multiply by 0.6 or 1.4 for +/-40% range of 53 to 123.

Platelet estimate agrees with analyzer.

3. If your estimate disagrees with the instrument platelet count by more than 40% in the

absence of clumps:

a. Have another technologist do an estimate, when possible, to confirm the discrepency.

b. If the patient is anemic or polycythemic (RBC below 4.0 or over 6.0), the estimate may be

inaccurate. A second blood film should be prepared, (see Indirect Platelet Count

Procedure) and performed by two technologists, this may require referral.

c. Examine the platelet histograms and the blood smear for the following sources of error:

i) RBC fragments: may be small enough to be counted as platelets.

ii) Megathrombocytes: if many of the platelets on the film appear abnormally large, i.e.

approximately the size of RBC, the analyzer may not be including them in the count.

If your estimate varies from the analyzer by more than 40%, add the message

“Platelet estimate appears higher on film; megathrombocytes may be excluded from

count.” Do not comment anything if occasional large platelets are present, and your

estimate agrees within 40%.

iii) Leukocyte cytoplasmic fragments, if present in large numbers, may falsely elevate the

instrument platelet count. They appear as particles similar in size to platelets, but with

a homogeneous, rather than granular, structure.

4. If you are reporting the platelet estimate instead of the analyzer count, append an

appropriate comment:


“Platelet estimate ≤20 x 10^9/L”

“Platelet estimate > 20 x 10^9/L”

“Platelet estimate > 50 x 10^9/L”

“Platelet estimate >100 x 10^9/L”

Note: You should not use this method if RBC is abnormal.

5. Send to Senior Technologist/Pathologist for review on first occurrence for each patient for

whom the instrument count is determined to be invalid due to RBC fragments, leukocyte

fragments, or megathrombocytes.

Reference:


1998


ESTABLISHING CONVERSION FACTOR FOR PLATELET ESTIMATION

Purpose:

To provide instructions for establishing a conversion factor for the hematology microscope by

calculating the ratio of electronic count to the number of platelets per oil immersion field.

Frequency:

The conversion factor must be established once for every microscope used in hematology or

when there are major repairs or changes in the analyzer for electronic counts or a new

microscope is used for estimates.

Procedure:

Step: Action:

1. Perform electronic platelet count on 30 consecutive fresh patient blood samples.

Alternatively if unable to perform 30 consecutive samples it is acceptable to perform 10

samples per day on 3 consecutive days.

2. Prepare and stain one peripheral blood film for each sample.

3. For each film, under oil immersion (100x) microscopy, find an area where 50% of the red

cells are overlapping in doubles or triplets. Then count platelets in 10 consecutive fields.

4. Divide by 10 the total number of platelets found to obtain the average number per oil

immersion field.

5. Divide the electronic platelet count by the average number of platelets per oil immersion

field.

6. Add the numbers obtained in step 5 and divide by 30 (number of observations in this

analysis) to obtain the average ratio of the platelet count to platelets per oil immersion

field.

7. Round the number calculated in step 6 to the nearest whole number to obtain an

estimation factor, the number of peripheral blood platelets represented by one platelet in

an oil immersion field.

Reference:

Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al; J.B. Lippincott Company,

1998


Platelet Estimation Conversion Factor Log Sheet

Microscope Model: __________________________ ID: __________________________

Column 1 Column 2 Column 3 Column 4

PATIENT ID

AUTOMATED

PLATELET COUNT

PLATELET

ESTIMATION

DIVIDE

AUTOMATED

PLATELET COUNT

BY PLATELET

ESTIMATION

1.

2.

3.

4.

5.

6.

7.

8.

9.

10

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

Average Estimation

Factor

__________________

Calculation:

To determine Average, total the values in Column 4, divide by 30. Round off to the nearest

whole number to obtain the estimated factor.


INDIRECT PLATELET COUNT

Purpose:

An indirect platelet count may be obtained by determining the ratio of platelets on a blood film

to RBC. The ratio is then used to calculate the number of platelets in comparison to the number

of RBC x 1012

/L.

This method is useful for situations in which an accurate platelet count cannot be obtained from

the CBC analyzer e.g. samples with marked megathrombocytes, RBC fragments, or leukocyte

cytoplasmic fragments. It is particularly more accurate than conventional estimation when the

RBC is abnormal.

Procedure:

1. Make two good quality blood smears. Air dry and stain as for differentials. The peripheral

smears must be well made, not touching edges of slide, with rounded end and no visible tails.

2. Obtain the RBC count from the automated CBC analyzer.

3. Using a 100x oil immersion objective, count the number of platelets per 500 RBC. Two

technologists should count one slide each and the platelets counts should check within 10

platelets of each other. If the counts do not check, a third technologist counts in the same

manner.

4. Add the two agreeable counts together and multiply this result by the RBC count to obtain

the indirect platelet count in 109/L. Next calculate +/- 10 to report the result as a range rather

than an exact number.

Example: RBC = 4.10

1st count = 11 platelets per 500 RBC

2nd

count = 15 platelets per 500 RBC

11+15 = 26

26 x 4.10 = 107

Add and subtract 10 to establish the reportable range.

The indirect platelet count is reported as 97 to 117 x 109/L.

5. Compare the indirect platelet count to the analyzer count. If the indirect count is within the

analyzer count +/- 30%, report the analyzer count.

6. If the indirect count is not within the analyzer count +/- 30%, report the indirect platelet

count.


Procedure Notes:

The Indirect Platelet Count is preferable to platelet estimation when an accurate platelet count

cannot be obtained on the CBC analyzer. It is particularly more accurate than conventional

estimation when the RBC is abnormal.

Limitations of the Procedure:

The indirect method is not recommended as the method of choice for enumerating platelets.

However, it is useful when an accurate platelet count cannot be obtained by the CBC analyzer.

References:

1. Raphael SS. Lynch’s Medical Laboratory Technology, Third Edition, Philadelphia PA: WB Saunders Company, 1976, page

1084.

2. Hematology Laboratory Safety Manual, Royal University Hospital, Saskatoon, SK 1998

3. Constantino BT. Leukocyte Cytoplasmic Fragmentation: Its Causes and Laboratory Evaluation, Canadian Journal or Medical

Laboratory Science -60, 1998, page 195


INTERNATIONAL SENSITIVITY INDEX (ISI) VERIFICATION

Purpose:

Verification of ISI is mandated for all laboratories using assigned instrument-specific ISI’s.

Verification must be performed in each laboratory at least annually, to ensure the accuracy of

the International Normalized Ratio (INR) result. Verification is done using a set of certified

plasmas.

NOTE: This guideline does not apply to point-of-care/rapid cartridge-based technology, at

this time.

Frequency:

Verification of ISI is required:

when a new instrument is put into place;

with any change in reagent type;

with any change in reagent lot number;

with any change in instrument;

following instrument repair, if QC is outside acceptable limits;

at a minimum of once per year.

Procedure:

1. Determine Geometric Mean Normal Prothrombin Time (MNPT) for current lot. [*contact

LQAP office for Geometric Mean Calculator]

2. Obtain a set of certified plasmas from the manufacturer. A minimum of three certified

plasmas with an INR range of 1.5 - 4.5 are recommended. The certified plasma must be

appropriate for the instrument in which they will be used.

3. Perform INR test on certified plasma in duplicate over three sessions.

4. Determine mean INR from the three sessions of testing on all certified plasmas.

a) If the mean INR is within ± 15% of assigned INR on all certified plasmas then the ISI

is valid and verification is complete.

b) If the mean INR is not within ± 15% of assigned INR – revalidate the MNPT.

i. If MNPT is valid, then a local system calibration must occur; go to step 5.

ii. If MNPT is not valid, then it must be re-established, go back to step 1.

5. Contact the manufacturer for further instructions (ie. calibration). Return to step 3.


VERIFYING OR ESTABLISHING A NORMAL REFERENCE RANGE FOR

ROUTINE COAGULATION TESTING

Purpose:

To provide instructions for establishing or verifying a Normal Reference Range for routine

coagulation testing.

1. Verifying a Normal Reference Range: To verify a normal reference range, 20

representative donor samples should be used.

2. Establishing a Normal Reference Range: For an assay that has never previously had a

reference range study, 120 donor samples should be used.

To provide instructions for lot number changes of reagent or quality control (QC) material.

Frequency:

Verify a Normal Reference Range:

With any change in reagent lot number

Following instrument repair if QC is outside acceptable limits

At a minimum of once per year

Establish a Normal Reference Range:

When a new instrument-reagent system is put into place

With any change in instrument

With any change in reagent type

Sample Information:

Specimens must be the same type and collected in the same container required for a given test

(usually 3.2% citrated plasma; see specific procedure).

Accumulate the appropriate number of normal donors that meet the following guidelines:

1. Healthy with no known pathological conditions (in-patients should be avoided – they are

hospitalized for a medical reason).

2. On no medications affecting coagulation, including anticoagulants/blood thinners.

3. Span the adult age range (unless a pediatric range is required).

4. Equally divided between males and females. If unable to maintain equal male/female

ratio, a larger center may be able to assist with donor specimens.

Donor samples can be collected in advance to speed up the evaluation process. The samples

should be double spun, aliquoted and stored for up to 14 days at -20 ºC or up to 6 months at -80

ºC. Spun plasma should have <10x109/L platelet counts.


Procedure for Verifying a Normal Reference Range

Prompt Additional Information

Run the applicable tests(s) as

per procedure manual.

Print all results.

Run applicable QC for each

assay.

Examine all results for outliers. Repeat outliers to rule

out an analyzer error.

When determining new reference ranges, it is acceptable

to exclude outlier values – particularly any values that

fall outside the current normal range - providing a

minimal number of data points are involved.

An assay with more than 5% unexplained outliers should

be discussed with the laboratory supervisor.

Calculate the geometric mean

and SD from the data (Refer to

the Geometric Mean

Calculation). Establish a

reference range based on the

mean ± 2 SD.

Compare reference range with previous range and submit

to the lab supervisor for review before implementing.

Procedure for Establishing a Normal Reference Range

Consultation with Laboratory Manager/Director (or a coagulation specialist) is recommended

when establishing a normal reference range.

Sequestering Reagents/Quality Control Materials:

1. All routine coagulation reagents (Prothrombin Time, APTT and Fibrinogen) and related

QC materials should be sequestered from the supplier in appropriate volumes for long

term use (usually up to 1 year).

2. When sequestering new lot items, be sure to ask for an expiry date longer than the time

sequestered for.

3. When sequestering a new lot of PT reagent, always check the ISI assigned to your

specific instrument. An ISI close to 1.0 is preferable and improves correlation between

instruments and peer laboratories.

4. Be sure to allow adequate time for shipping and preliminary testing prior to depleting

your current stock.

Lot Number Changes:

Note: It is not recommended to change reagent and QC lots for the same assay, at the same time.

1. Reagent Lot Number Changes


a. Establish a new mean and 2 SD normal range for the applicable assay as described in the

procedure above.

b. Compare the new reference range with the previous. Variation should be minimal. If

there is a significant fluctuation (> 5%), consult with supervisor to obtain new lot number

of reagent.

c. Establish new mean and SD for all QC levels associated with the assay using the new lot

number of reagent (see below - Quality Control Lot Number Changes).

Note: If possible, run the new lot of reagent in parallel with your current lot whenever QC is

run. When performing parallel testing with new lots of reagent, it is not recommended to

load any new patient samples on the analyzer – this will prevent possible release of

erroneous results.

2. Quality Control Lot Number Changes

a. Establish a new mean and 2 SD reference range for each level of control using a

minimum of 20 data points. Run the new control in parallel with the current control

whenever QC is required.

Note: Running all QC points consecutively is not recommended as this will not reflect

changes in the age of reagents or controls, and may result in a reference range that is too

narrow. Run QC on multiple days and multiple shifts.

b. Compare the SD and mean of your current QC lot to that of the new lot. Generally there

should not be any significant fluctuations. Ensure the CV is acceptable (5% or less).

NOTES:

Fibrinogen

If the reagent being changed is for the fibrinogen assay, a new standard calibration curve must be

performed before any preliminary testing proceeds. Compare old and new curves – there should

not be a significant change between the lot numbers.

APTT

If the laboratory performs APTTs on heparinized patients, a “Heparin Therapeutic Range” for

APTT must be done using the same unfractionated heparin used for patients.

Prothrombin Time

1. If the analyzer requires a “PT Calibration” when changing reagent lot number, this must be

performed before any other testing.

2. Before implementing the new lot number of reagent, ensure that the new ISI and Mean

Normal PT (MNPT) are entered in the appropriate areas of the analyzer and/or LIS

(depending on how the INR’s are calculated).

3. When doing the patient correlations (old vs. new lot of reagent) enter the new ISI and MNPT

into the analyzer/LIS to enable comparison of INR’s, instead of PT’s (Prothrombin Times

may not compare well if the ISI value has changed significantly).

Reference: Adapted from the Regina Qu’Appelle Health Region


TRANSFUSION

MEDICINE


RETENTION OF TRANSFUSION MEDICINE RECORDS

The most recently published standards of the Canadian Society for Transfusion Medicine are

referenced.

Each transfusion service shall have in place an established system for record-keeping that

is written and abides by the following:

DOCUMENTS

Issue Vouchers from CBS Indefinitely

Correspondence Related to Blood Components Indefinitely

Documents Related to Traceback or Lookback Indefinitely

Daily Records for issue of Blood Components/Products Indefinitely

Transfused Patient Data

• includes antibody investigation & resolution

• transfusion reaction investigation

Indefinitely

Non-transfused patient data 5 years

Method Revision Indefinitely

Blood Component & Product Final Disposition Records Indefinitely

Tissue & Bone Banking Donor & Inventory Records Indefinitely

Quality Control Records

•Include reagents, serological test controls, external proficiency

testing

5 years

Utilization Reports

•Blood component product inventory

5 years

Direct & Stores Inventory

•Purchase & acquisition data

5 years

Meeting & Related Documents 5 years

Computer Program Validation & Exception List 5 years

Workload Records/Reports 3 years

In-service Education Documentation 3 years

Test & Blood Product Request Forms 13 months

SPECIMENS

Donor Segments from transfused red cells 3 weeks

Clotted and/or EDTA specimens, serum/plasma from transfused

patients (pre-transfusion)

1 to 3 weeks

Cord Blood 2 weeks

All Other Patient Specimens 1 week

Kleihauer-Betke Slides 1 year


PROCEDURE/METHOD STATISTICAL VALIDATION/WORK-UP GUIDELINES:

TRANSFUSION MEDICINE

When initiating new methodology or implementing new testing, a validation process must be

developed.

Validation Process

Object of the validation

Purpose

Manufacturer’s instructions/data

References

Description

Standard Operating Procedure (SOP)

Sample quantities and types

“known” and “unknown” samples

Patient population

Test methods and conditions

Isolated testing

Parallel testing

System stress points

Testing personnel

Training plan

Training documents

Data collection/documentation required

Acceptance criteria

Reviewing personnel

Summary

Results

Performance characteristics/limitations

Functionality

Method

SOP

Training plan and documents

Safety

Sources of error

Conclusion


Test New Method Recommendations Change in Methodology Recommendations Acceptable level of

result accuracy

ABO typing Known samples:

-10 group ‘O’

-10 group ‘A’

-5 A1 positive

-5 A1 negative

-10 group ‘B’

-10 group ‘AB’

Unknown patient samples:

-20

Known samples:

-10 group ‘O’

-10 group ‘A’

-5 A1 positive

-5 A1 negative

-10 group ‘B’

-10 group ‘AB’


-20

100%

Rh(D) typing Known samples:

- 10 Rh(D) positive

- 10 Rh(D) negative


-20

Known samples:

-10 Rh(D) positive

-10 Rh(D) negative


-5

100%

Direct antiglobulin

test (DAT) Known samples (may be patient samples, cord blood

samples or simulated samples):

-5 DAT negative

-5 DAT positive


-5

Known samples(may be patient samples, cord

blood samples or simulated samples):

-5 DAT negative

-5 DAT positive


-5

90%

Antibody screen Known samples:

-10 clinically significant antibodies

-10 antibody negative samples


-20

Known samples:

-10 clinically significant antibodies

-10 antibody negative samples


-10

90%

All tests Tested under routine laboratory conditions

Testing shall include challenges to:

-All shifts

-Variety of personnel

-Acceptable method variations (e.g. variation in

incubation time)

Tested under routine laboratory conditions

Testing shall include challenges to:

-All shifts

-Variety of personnel

-Acceptable method variations (e.g. variation in

incubation time)

Unknown patient samples shall be verified by a comparable validated method.

Depending on laboratory test volumes, unknown patient samples may be obtained from another laboratory.
