Laboratory Quality Assurance Program
College of Physicians & Surgeons of Saskatchewan
Laboratory
Guidelines
General
Anatomic Pathology
Chemistry
Hematology
Transfusion Medicine
2014 Edition
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)
Laboratory Guidelines 2014 Edition
Page 1
Table of Contents
General
Retention Guideline
Recommended Reference Textbook List for Laboratories
4
7
Anatomic Pathology
Specimens Exempt from all Gross &/or Microscopic Pathology Laboratories
Surgical Pathology Reports
Removal of Tissue, Blocks or Slides from the Original Hospital Site
Performance of Non-Gynecological Cytology
Follow-up Reports for Gynecological Cytology
Follow-up Program for Cytology
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12
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15
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Chemistry
Estimated Glomerular Filtration Rate – eGFR
Cholesterol/Triglyceride/Lipid Testing
Urinalysis
Reporting Sperm in Urine
Quality Control
Diagnosis and Monitoring of Thyroid Disease
Presence of Small Amounts of Albumin
Crosscheck/Validation Guideline for Those Facilities with Multiple Chemistry Instruments
Procedure/Method Statistical Work-up/Validation Study Guidelines
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20
21
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26
27
28
Hematology
Principles for Hematology Practice
Hematology Films/Labelling of Slides
Morphology of Lymphocytes
Differential Performance and Referral Practice Guideline
Red Blood Cell Morphology Reporting Guideline
Differential Quality Control
Smudge Cells
Flow Cytometry for the Diagnosis of Lymphoma/Leukemia
Malaria
3.2% Na Citrate Anticoagulant Recommended vs. 3.8% for Coagulation Studies
Vitamin B12 & Folate
Bleeding Time
Crosscheck Validation Guideline for Facilities with Multiple Hematology Instruments
Procedure/Method Statistical Work-up/Validation Study Guidelines
Protocol for Validation of Linearity on Automated Hematology Analyzers
Procedure for WBC Estimate
Establishing Conversion Factor for WBC Estimation
Procedure for Platelet Estimates
Establishing Conversion Factor for Platelet Estimation
Indirect Platelet Count
International Sensitivity Index (ISI) Verification
Verifying or Establishing a Normal Reference Range for Routine Coagulation Testing
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35
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55
57
59
61
63
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66
Transfusion Medicine
Retention of Transfusion Medicine Records
Procedure/Method Statistical Validation/Work-up Guidelines – Trans. Med.
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Page 2
GENERAL
Laboratory Guidelines 2014 Edition
Page 3
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
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Record Retention
Storage Time
Hematology
Biochemistry
Microbiology
Cytopathology
Surgical
Pathology
Accession record
Worksheets
Instrument print-outs
Paper copy of patient reports
1 year
1 year
1 year
3 months
1 year
1 year
1 year
3 months
1 year
1 year
1 year
3 months
2 years
6 months
n/a
indefinite
2 years
6 months
n/a
indefinite
Quality control/PT documents
Maintenance records
2 years
2 years
2 years
2 years
2 years
2 years
1 year
2 years
2 years
Life of the instrument, plus 2
years
Life of the instrument, plus 2 years
2 years after the method has been discontinued
2 years after procedure has been discontinued
1 year
1 year
1 year
1 year
3 months
3 months
2 years
3 months
3 months
2 years
3 months
1 year
2 years
Service records
Method /instrument evaluation
Procedure Manual
Technologist ID & initials
log/computer
Telephone logs
Requisition
Laboratory Information Systems
Records
- Validation records, including
transmission of results and
calculations
- Database changes
- Hardware and software
modifications
- LIS downtime and corrective action
Biomedical Waste
Laboratory Guidelines 2014 Edition
3 months
5 years
2 years
3 months
5 years
2 years
Manifests must be retained for a minimum of 1 year
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Specimen Retention
Hematology
Chemistry
Microbiology
Cytopathology
Surgical
Pathology
Peripheral Blood
Smear
-7days
Whole blood,
serum &
plasma
- 48 hrs after
report has been
finalized
Swabs or
specimens
- 24 hrs after
report has been
finalized
Slides
Blocks & slides
- 20 yrs (adults)
- 50 yrs (children)
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
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
Positive Blood
Culture
- 5 days after
reporting
Gram Stain
- one week or
until final report
is sent
Ova & Parasite
slides
- one month
neg/unsatisfactory
- 5 years
Slides
suspicious/pos
- 20 years
Autopsy
- 20 yrs
Fine-needle
aspiration slides
- 20 years
Gross specimen
- min. 8 weeks
after issue of
report
Cytology
Consultation/
Requisition
- Indefinitely
Wet Autopsy
Tissue
- 8 weeks after
issue of report
Male fertility
slides
- 1 year
Bone Marrow
Slides/ Reports
- 20 yrs (adults)
- 50 yrs (children)
Cytology
paraffin blocks
- 20 years
Photographic
Transparencies –
indexed and kept
indefinitely
Urine
- 24 hrs after
report has been
finalized
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Page 6
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 4th Edition; Rodak, Fritsma, Doig
4) Clinical Hematology Atlas 4th Edition; Carr/Rodak
Chemistry
1) Tietz Fundamentals of Clinical Chemistry 6th Edition, W. B. Saunders Company; Burtis,
Ashwood, Bruns
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Page 7
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
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ANATOMIC
PATHOLOGY
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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
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
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
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Laboratory Guidelines 2014 Edition
GROSS &/OR
MICROSCOPIC
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(By Chemistry Dept.)
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SPECIMEN TYPE
DISCRETIONARY
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
Torn menicus
Varicose veins
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Laboratory Guidelines 2014 Edition
GROSS &/OR
MICROSCOPIC
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(under 10 yrs
old)
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(over 10 yrs)
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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
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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.
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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
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Page 14
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.
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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.
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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.
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CHEMISTRY
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Page 18
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,
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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
eGFR 30-59
eGFR 15-29
eGFR < 15
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Normal or slightly decreased kidney function (stages 1 or 2)
Moderately decreased kidney function (stage 3)
Seriously decreased kidney function (stage 4)
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
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CHOLESTEROL / TRIGLYCERIDE / LIPID TESTING
Cholesterol results are based on optimal performance of testing. Considerations to be included:
a)
b)
c)
d)
treatment/preparation of the patient
the emergent or non-emergent nature of the test
appropriate technical equipment
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 methodspecific 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.
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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.).
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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.
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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 nonthyroidal 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.
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Recommended Testing Algorithm
TSH
Increased
FT4 low
Primary
hypothyroidism
Normal
If clinical suspicion
is high for secondary
hypothyroidsm
order FT4 as initial
test
Decreased
FT4 high
hyperthyroidism
FT4 normal
FT3 if clinical
suspicion is high
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.
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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.
2.
HSURC guidelines. Nov 1992
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.
Laboratory Guidelines 2014 Edition
Page 25
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.
Laboratory Guidelines 2014 Edition
Page 26
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:
Patient correlation
Frequency/Data Points:
Requirement:


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)
Validation/Crosscheck
Element:
Patient correlation
Laboratory Guidelines 2014 Edition
Frequency/Data Points:
Requirement:


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 5-10 patient specimens/2 times per year or
equivalent
As necessary per recalibration/validation event
Page 27
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):
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.
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).
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.
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.
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).
The minimum requirement is 20 data points
for confirmation of an established reference
range and 120 for the establishment of a
new reference range.
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.
Applicability:
Qualitative
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. 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.
Closeness of the agreement between the result of a measurement and
the accepted reference value (true value of the analyte)
5. Accuracy
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.
Laboratory Guidelines 2014 Edition
Sensitive studies are only required for those
methods which have clinical relevance at
values close to “0” (i.e. TSH)
Quantitative

n=20
n=40






When
clinically
relevant

When clinically
relevant
Page 28
7. Specimen
Stability
The conditions of handling and storage, which permits the
measurement and reporting of a clinically relevant result.
8. Interference
The effect of any component of the sample on the accuracy of the
measurement of the desired analyte.
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.
No data generally required. As per
manufacturer’s guidelines. If
manufacturer’s stability window is to be
extended a stability study is expected.
Document the manufacturer’s interference
information. The method should include a
disclaimer or a process for dealing with a
lipemic, icteric or hemolyzed sample.
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)

Storage and
transportation
dependent

Methods with
known
interferences

Storage and
transportation
dependent

Methods with
known
interferences

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
1. Imprecision studies, QC only
2. Patient correlation
Laboratory Guidelines 2014 Edition
Minimum data requirements:
As above
10 data points, where feasible.
Page 29
HEMATOLOGY
Laboratory Guidelines 2014 Edition
Page 30
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.
Laboratory Guidelines 2014 Edition
Page 31
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___________________________________
Laboratory Guidelines 2014 Edition
Page 32
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
Laboratory Guidelines 2014 Edition
Page 33
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
Laboratory Guidelines 2014 Edition
Page 34
DIFFERENTIAL PERFORMANCE AND REFERRAL PRACTICE GUIDELINE
Blood Film
Reference Range
WBC Count – adults
Lower referral range
Upper referral range
children (2-14 years)
children (90 days – 2
yrs)
newborn (0 – 90 days)
Absolute Neutrophils
Absolute
Granulocytes
Absolute Eosinophils
Absolute Basophils
Absolute Lymphs
adults
children (0-14 years)
Absolute Monocytes
Hemoglobin
Lower referral range
adult female
4.0 - 11.0 x 109/L
adult male
Upper referral range
adult female
adult male
Pediatric ranges
children (1 mo – 14
yrs)
newborn (0 – 1 month)
HCT
MCV
Lower referral range
> 3 months (adult)
Perform a Differential or Scan
on First Occurrence or
Significant Change
Referral
Unexpected or Unexplained
<1.5 x 109/L (all age groups)
>25.0 x 109/L
<1.0 x 109/L (all age groups)
>25.0 x 109/L
<1.5 x 109/L (all age groups)
<1.5 x 109/L (all age groups)
<1.0 x 109/L (all age groups)
<1.0 x 109/L (all age groups)
>1.0 x 109/L (all age groups)
>0.3 x 109/L (all age groups)
>2.0 x 109/L (all age groups)
>0.5 x 109/L (all age groups)
>5.0 x 109/L
>7.0 x 109/L
>1.0 x 109/L (all age groups)
>7.0 x 109/L
>10.0 x 109/L
>1.5 x 109/L (all age groups)
135 – 180 g/L
<100 g/L
(Combined with MCV <70 fL)
<100 g/L
(Combined with MCV <70 fL)
<100 g/L
(Combined with MCV <70 fL)
<100 g/L
(Combined with MCV <70 fL)
120 – 160 g/L
135 – 180 g/L
>165 g/L
>185 g/L
>165 g/L
>185 g/L
105 – 145 g/L
<100 g/L
(Combined with MCV <70 fL)
<160 g/L or >210 g/L
None
<100 g/L
(Combined with MCV <70fL)
<135 g/L or >210 g/L
>0.65 L/L
5.0 – 15.0 x 109/L
5.0 – 20.0 x 109/L
7.0 – 20.0 x 109/L
1.5 – 7.5 x 109/L
1.5 – 7.5 x 109/L
0.0 – 0.6 x 109/L
0.0 – 0.2 x 109/L
1.1 – 4.4 x 109/L
0.2 – 0.8 x 109/L
120 – 160 g/L
135 – 195 g/L
0.37 – 0.50 L/L
79.0 – 97.0 fL
0 – 3 months
98.0 – 114.0 fL
MCHC
Upper referral range
MCH
RDW
RBC COUNT
adult female
adult male
310 – 360 g/L
27.0 – 32.0pg
11.5 – 14.5 %
<70.0 fL or >100.0 fL
<70.0 fL
(Combined with HGB <100.0 (Combined with HGB <100.0
g/L)
g/L)
<97.0 fL
<90.0 fL
(Combined with HGB <135.0 (Combined with HGB <135.0
g/L)
g/L)
>360 g/L
None
None
>6.5 x 1012/L
>365 g/L
none
none
>6.5 x 1012/L
3.2 – 5.4 x 1012/L
4.6 – 6.2 x 1012/L
Laboratory Guidelines 2014 Edition
Page 35
Blood Film
MPV
Lower referral range
Upper referral range
Platelet Count
Lower referral range
Upper referral range
WBC Morphology
Reference Range
7.4 – 10.4 fL
Perform a Differential or Scan
on First Occurrence or
Significant Change
None
Referral
Unexpected or Unexplained
<6.0 fL
>14.0 fL
150 – 400 x 109/L
<100 x 109/L
>600 x 109/L
NUCLEATED RBC’S
RBC Morphology
PLATELET
MORPHOLOGY
OTHER CRITERIA
Specified Instrument
Flags
Ordered by Physician
Technologist
Discretion
<100 x 109/L
>600 x 109/L
> 10% Reactive Lymphs
Pelger-Huet anomaly
Hypogranulated neutrophils
Hairy Cells
Blasts/Immature Cells
Hypersegmented neutrophils
>5 NRBC/100 WBC
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
none
When indicated
Physician request
Technologist initiated
Physician request
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.
Laboratory Guidelines 2014 Edition
Page 36
RED BLOOD CELL MORPHOLOGY REPORTING GUIDELINE
Red Blood Cells/100x oif
(200 RBC field) x 10 fields
Abnormality to be
Reported
Schistocytes
Any present
Echinocytes/Burr Cells
Bite Cells
Sickle Cells
Basophilic Stippling
Howell Jolly Bodies
Dimorphic
RBC Rouleaux (5cells
stacked)
RBC Agglutination
Parasites
Nucleated RBC
>5
Polychromasia
Macrocytes (oval)
Laboratory Guidelines 2014 Edition
Implications for Diagnosis
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
Kidney Disease
Drug or chemical induced
oxidative damage, unstable
hemoglobins
Sickle Cell Anemia,
Hemoglobin SC/SD Disease
Lead Poisoning, Thalassemia,
Sideroblastic & Megaloblastic
Anemia, Sickle Cell Anemia
Megaloblastic Anemia, Postsplenectomy state
Hemorrhage, response to
treatment, Sideroplastic anemia,
post-transfusion
Paraproteinemia, increased
fibrinogen, inflammatory
disorders
Autoimmune Hemolytic Anemia
(Cold Agglutinin Disease)
Identify specific forms
Sever hemolysis, part of
Leukoerythroblastic picture,
Bone marrow stress
Response to treatment, blood
loss, Hemolysis
Megaloblastic state, Aplastic
Anemia, Myelodysplastic
Syndrome
Page 37
Target Cells
Tear Drops
Spherocytes
Pappenheimer Bodies
>10
Macrocytes (round)
Elliptocytes
Acanthocytes/Spur Cells
Stomatocytes
Microcytes (hypochromic
cells)
Liver Disease, Postsplenectomy/hyposplenism,
Hemoglobinopathy,
Thalassemia
Myelofibrosis, Pernicious
Anemia
Hereditary Spherocytosis,
Autoimmune Hemolytic Anemia
Sideroblastic Anemia, Chronic
Hemolysis, Liver Disease
Liver Disease, Alcoholism
Hereditary Elliptocytosis
Post-splenectomy state, Liver
Disease, Abetalipoproteinemia
Liver Disease
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.
Laboratory Guidelines 2014 Edition
Page 38
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.
Laboratory Guidelines 2014 Edition
Page 39
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.
Laboratory Guidelines 2014 Edition
Page 40
The 95% confidence limits for various percentages of leukocytes of a given type as determined
by differential counts on stained blood smears.
n
0
1
2
3
4
5
6
7
8
9
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
91
92
93
94
95
96
97
98
99
100
n=100
0-4
0-6
0-8
0-9
1-10
1-12
2-13
2-14
3-16
4-17
4-18
8-24
12-30
16-35
21-40
25-46
30-51
35-56
39-61
44-65
49-70
54-75
60-79
65-84
70-88
76-92
82-96
83-96
84-97
86-98
87-98
88-99
90-99
91-100
92-100
94-100
96-100
n=200
0-2
0-4
0-6
1-7
1-8
2-10
3-11
3-12
4-13
5-14
6-16
10-21
14-27
19-32
23-37
28-43
33-48
37-53
42-58
47-63
52-67
57-72
63-77
68-81
73-86
79-90
84-94
86-95
87-96
88-97
89-97
90-98
92-99
93-98
94-100
96-100
98-100
n=500
0-1
0-3
0-4
1-5
2-7
3-8
4-9
4-10
5-11
6-12
7-13
11-19
16-24
21-30
26-35
30-40
35-45
40-50
45-55
50-60
55-65
60-70
65-74
70-79
76-84
81-89
87-93
88-94
89-95
90-96
91-96
92-97
93-98
95-99
96-100
97-100
99-100
n=1,000
0-1
0-2
1-4
2-5
2-6
3-7
4-8
5-9
6-10
7-11
8-13
12-18
17-23
22-28
27-33
32-39
36-44
41-49
46-54
51-59
56-64
61-68
67-73
72-78
77-83
82-88
87-92
89-93
90-94
91-95
92-96
93-97
94-98
95-98
96-99
98-100
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
Laboratory Guidelines 2014 Edition
Page 41
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.
Laboratory Guidelines 2014 Edition
Page 42
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.
Laboratory Guidelines 2014 Edition
Page 43
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.
Laboratory Guidelines 2014 Edition
Page 44
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
Laboratory Guidelines 2014 Edition
Page 45
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
Laboratory Guidelines 2014 Edition
Page 46
Current Indications for B12 and Folate Investigation
Measuring both B12 and Folate levels is not necessary in all patients.
CBC
MCV ↑
Normal
Neurological or Clinical
symptoms suggestive of B12
&/or Folate deficiency
No further testing
YES
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
B12 and RBC
folate
Abnormal
Requires further
patient evaluation
Laboratory Guidelines 2014 Edition
Normal
No further
testing
Page 47
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).
Laboratory Guidelines 2014 Edition
Page 48
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:
Patient correlation
Frequency/Data Points:
Requirement:


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)
Validation/Crosscheck
Element:
Patient correlation
Laboratory Guidelines 2014 Edition
Frequency/Data Points:
Requirement:


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 5-10 patient specimens/2 times per year or
equivalent
As necessary per recalibration/validation event
Page 49
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):
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.
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).
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.
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.
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).
The minimum requirement is 20 data points
for confirmation of an established reference
range and 120 for the establishment of a
new reference range.
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.
Applicability:
Qualitative
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. 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.
Closeness of the agreement between the result of a measurement and
the accepted reference value (true value of the analyte)
5. Accuracy
6. Sensitivity
7. Specimen
Stability
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.
The conditions of handling and storage, which permits the
measurement and reporting of a clinically relevant result.
Laboratory Guidelines 2014 Edition
Sensitive studies are only required for those
methods which have clinical relevance at
values close to “0” (i.e. TSH)
No data generally required. As per
manufacturer’s guidelines. If
Quantitative

n=20
n=40






When
clinically
relevant

Storage and

When clinically
relevant

Storage and
Page 50
8. Interference
The effect of any component of the sample on the accuracy of the
measurement of the desired analyte.
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.
manufacturer’s stability window is to be
extended a stability study is expected.
Document the manufacturer’s interference
information. The method should include a
disclaimer or a process for dealing with a
lipemic, icteric or hemolyzed sample.
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)
transportation
dependent

Methods with
known
interferences
transportation
dependent

Methods with
known
interferences

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
1. Imprecision studies, QC only
2. Patient correlation
Laboratory Guidelines 2014 Edition
Minimum data requirements:
As above
10 data points, where feasible.
Page 51
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.
Laboratory Guidelines 2014 Edition
Page 52
Dilution (%)
100
80
60
40
20
0
Specimen (Parts)
10
8
6
4
2
0
Diluent (Parts)
0
2
4
6
8
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
Laboratory Guidelines 2014 Edition
Page 53
WBC Linearity
WBC Results
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor
Expected Value
Difference
Allowable Evaluation Limits ±
0%
X 0.0
20%
X
0.2
40%
X
0.4
60%
X
0.6
80%
X 0.8
100%
(Reference)
X
1.0
0.4
0.4
0.4
0.4
0.4
0.4
0%
20%
40%
60%
80%
100%
(Reference)
RBC Linearity
RBC Results
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor
Expected Value
Difference
Allowable Evaluation Limits ±
X 0.0
X
0.2
X
0.4
X
0.6
X 0.8
X
1.0
0.1
0.1
0.1
0.1
0.1
0.1
0%
20%
40%
60%
80%
100%
(Reference)
HGB Linearity
HGB Results
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor
Expected Value
Difference
Allowable Evaluation Limits ±
X 0.0
X
0.2
X
0.4
X
0.6
X 0.8
X
1.0
3
3
3
3
3
3
0%
20%
40%
60%
80%
100%
(Reference)
PLT Linearity
PLT Results
Run #1
Run #2
Run #3
Obtained Mean
Reference Mean
X Multiplication Factor
Expected Value
Difference
Allowable Evaluation Limits ±
X 0.0
10
Laboratory Guidelines 2014 Edition
X
0.2
10
X
0.4
10
X
0.6
X 0.8
10
10
X
1.0
10
Page 54
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.
Laboratory Guidelines 2014 Edition
Page 55
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
Laboratory Guidelines 2014 Edition
Page 56
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:
1.
2.
3.
4.
5.
6.
7.
Action:
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.
Prepare and stain one peripheral blood film for each sample.
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.
Divide by 10 the total number of WBC found to obtain the average number per hpf.
Divide the electronic WBC count by the average number of WBC per hpf.
Add the numbers obtained in step 5 and divide by 30 (number of observations in this
analysis) hpf.
Round the number calculated in step 6 to the nearest whole number to obtain an
estimation factor.
Reference:
Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company,
1998.
Laboratory Guidelines 2014 Edition
Page 57
WBC ESTIMATION CONVERSION FACTOR LOG SHEET
Microscope Model: _______________________ ID: ______________________________
Column 1
Column 2
Column 3
Column 4
DIVIDE
SPECIMEN ID
AUTOMATED WBC WBC ESTIMATION AUTOMATED WBC
COUNT
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.
Laboratory Guidelines 2014 Edition
Page 58
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:
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“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:
Clinical Hematology Principles, Procedures, Correlation, 2nd Edition, E. Anne Stiene-Martin et. al: J.B. Lippincott Company,
1998
Laboratory Guidelines 2014 Edition
Page 60
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
Laboratory Guidelines 2014 Edition
Page 61
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.
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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.
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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
Laboratory Guidelines 2014 Edition
Page 64
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.
Laboratory Guidelines 2014 Edition
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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.
Laboratory Guidelines 2014 Edition
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Procedure for Verifying a Normal Reference Range
Prompt
Run the applicable tests(s) as
per procedure manual.
Print all results.
Run applicable QC for each
assay.
Additional Information
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 Compare reference range with previous range and submit
and SD from the data (Refer to to the lab supervisor for review before implementing.
the Geometric Mean
Calculation). Establish a
reference range based on the
mean ± 2 SD.
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
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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
Laboratory Guidelines 2014 Edition
Page 68
TRANSFUSION
MEDICINE
Laboratory Guidelines 2014 Edition
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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
Correspondence Related to Blood Components
Documents Related to Traceback or Lookback
Daily Records for issue of Blood Components/Products
Transfused Patient Data
• includes antibody investigation & resolution
• transfusion reaction investigation
Non-transfused patient data
Method Revision
Blood Component & Product Final Disposition Records
Tissue & Bone Banking Donor & Inventory Records
Quality Control Records
•Include reagents, serological test controls, external proficiency
testing
Utilization Reports
•Blood component product inventory
Direct & Stores Inventory
•Purchase & acquisition data
Meeting & Related Documents
Computer Program Validation & Exception List
Workload Records/Reports
In-service Education Documentation
Test & Blood Product Request Forms
Indefinitely
Indefinitely
Indefinitely
Indefinitely
Indefinitely
5 years
Indefinitely
Indefinitely
Indefinitely
5 years
5 years
5 years
5 years
5 years
3 years
3 years
13 months
SPECIMENS
Donor Segments from transfused red cells
3 weeks
Clotted and/or EDTA specimens, serum/plasma from transfused 1 to 3 weeks
patients (pre-transfusion)
Cord Blood
2 weeks
All Other Patient Specimens
1 week
Kleihauer-Betke Slides
1 year
Laboratory Guidelines 2014 Edition
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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
Laboratory Guidelines 2014 Edition
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Test
ABO typing
Rh(D) typing
Direct antiglobulin
test (DAT)
Antibody screen
All tests
New Method Recommendations
Change in Methodology Recommendations
 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 Rh(D) positive
- 10 Rh(D) negative
 Unknown patient samples:
-20
 Known samples (may be patient samples, cord blood
samples or simulated samples):
-5 DAT negative
-5 DAT positive
 Unknown patient samples:
-5
 Known samples:
-10 clinically significant antibodies
-10 antibody negative samples
 Unknown patient samples:
-20
 Tested under routine laboratory conditions
 Testing shall include challenges to:
-All shifts
-Variety of personnel
-Acceptable method variations (e.g. variation in
incubation time)
 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 Rh(D) positive
-10 Rh(D) negative
 Unknown patient samples:
-5
 Known samples(may be patient samples, cord
blood samples or simulated samples):
-5 DAT negative
-5 DAT positive
 Unknown patient samples:
-5
 Known samples:
-10 clinically significant antibodies
-10 antibody negative samples
 Unknown patient samples:
-10
 Tested under routine laboratory conditions
 Testing shall include challenges to:
-All shifts
-Variety of personnel
-Acceptable method variations (e.g. variation in
incubation time)
Acceptable level of
result accuracy
100%
100%
90%
90%
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.
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