Laboratory Diagnostics

Introduction

Xiaosong Qin

Dept. of Laboratory Medicine

the 2nd clinical college

Shengjing hospital of CMU
COVID-19
Transmission COVID-19 patient
 Proof of diagnosis

Laboratory Pathogenic evidence

Role of lab eyes of doctors
 Proof of diagnosis

Laboratory Pathogenic evidence

Can a negative result rule out a diagnosis?

 A case of repeated SARS-CoV-2 RNA(-)

female, 40y
7X RNA(-)
manicures worker

RNA(-) Fever:37.5℃ RNA(-) Ab(+) Ab(++) RNA(-)

Mar 16th Mar 17th Mar 18th Mar 27th Mar 30th Mar 31th

normal body anti-nCOV IgM 1.348 3.374

IgM 1.348
temperature IgG 1.441
anti-nCOV IgG 1.441 8.655

patchy ground-glass opacities CutOff : S/CO 1

WBC 2.22X109/L
 Conditions that need to be met
for a positive RNA test result？

timing
1. There is a certain amount of virus in the infected cells；

viral load

days
conditions to be met for a positive RNA test result

1. There is a certain amount of virus in the infected cells；

site
2. When collecting specimens, cells containing viruses must be collected;
specimen collection
pharyngeal swab collection

（6）
（7）
Posterior pharyngeal wall
Pharyngopalatine arch Posterior pharyngeal wall
 Contents

01 history

key points
02 specimen collection

03 Factors affecting the analysis of the results

difficult points
 HISTORY

400BC: Hippocrates

The more you look the more you see

1673: Leeuwenhook: MICROSCOPE
 1796: Edawar Jennar: smallpox vaccination

Edawar Jennar

1860’s: Robert Koch:

*infectious diseases caused by microorganisms
*specific pathogens caused specific disease
Robert Koch

1890’s: Pasteur:
isolate microorganisms and culture bacterial
successfully
Louis Pasteur
 1888: exotoxin produced by Diphtheria

1891: Diphtheria antitoxin ( Behring )

• Passive immunity
• Humoral immunity
–antiserum , antibody (Ab)
–antigen (Ag)
–serology
Ehrlich
lab
 Contents

01 history

key points
02 specimen collection

03 Factors affecting the analysis of the results

difficult points
 Specimen types

1 2 3

Blood Urine Feces

... ...
 1 Blood

Sample type

Whole blood = liquid part + blood cell

In vivo:liquid part of circulating blood

Plasma In vitro: after anticoagulation, the yellowish
liquid obtained by centrifuging the blood

Serum liquid part of whole blood after

coagulation in vitro
= plasma - fibrinogen - clotting factor
 1 Blood

Sample treatment EDTA-K

Anticoagulant Heparin
Citrate
Fluoride
Mixed well
Native correct volume

Storage Metabolism
Centrifuge
Transport
Avoid light, heat, contaminant

Asepsis Bacteria culture

1 Blood

Sample type

Most of clinical chemistry tests

Serum
Immunology tests

Partial chemistry tests

Plasma
Coagulate factor tests
proteins？
serum K+,plasma K+ RBC,PLT
Blood cell count
Whole blood Morphology
Erythrocyte sedimentation rate
 1 Blood

Sampling

Venous Reflect the state of the whole body

Application: Most of tests

Large volume

Puncture site: peripheral vein

In arm or wrist
 1 Blood

Sampling

Venous

Attention:

1.Factors lead to a concentration of blood specimen

➢ posture
➢Duration of venous compression
 posture

sitting upright
 long venous compression

>30sec
Intravascular fluid flow to interstitial spaces

Caused condensed blood and false high test result

1 Blood

Sampling

Venous Attention:

2.Factors lead to hemolysis

3.Factors lead to Chylemia

 Hemolysis Difficult Point

concept Erythrocyte rupture ,hemoglobin escapes

out of the cell

Causes:
➢Blood sampling technique:strong suction
by thin syringe needle
➢Specimen placement time is too long
 What are the effects of hemolysis?

1. affect the detection results of substances with large

differences in concentration inside and outside red blood
cells.：potassium，AST, etc.

2. Erythrocyte rupture, hemoglobin escapes out of the

cell and serum appeared as red color.Those tests
which detect the optical dendisy will be affected

K+
Ca2+
Mg+
ALP
 Chylemia

an increase in the amount of

concept
chylous particles in the
plasma, the serum
appearance is chyllike.

causes • lymphatic obstruction,

• abnormal lipid metabolism
• blood transfusion after the fat milk
these components mixed with the blood without absorption.
 Effects of Chylemia

1、 those test results tested by optical density will be

affeced： enzyme etc.;
2、 affect turbidimetric detection by formation of turbidity：
total protein, MAO, ALT ,AST, CK, etc.
 1 Blood

Sampling
Capillary local

Application: Blood smears

POCT

Puncture site: End of ring finger

Heel (neonates)

Attention: Away from infected, swollen sites

Wait until disinfector has dried.
Limitation: Small volume
Large variations
Tissue fluid diluting
 1 Blood

Sampling

Arterial
Application: Blood gases test:
pH, partial Pco2 ,
partial Po2 ,
O2 saturation
Puncture site: Femoral artery

Attention: Sealed promptly

2 Urine

Instruction

1. Morning urine

2. Random urine

3. 24-h urine

4.Clean midstream urine

2 Urine

Container

1. Avoid contaminant

2. Free of interfering substance

3. Aseptic container for culture

4.volume: 12ml

5. label
3 Feces

Container Clean, dry, moisture unabsorbed, covered

Infant Pampers
Adoption Mucus, blood, purulence

Timing Within 1 hour

Immediately for ameba test
Early morning for pinworm test (anus vicinity)

Fasting Meat, animal blood, chalybeate, Vit C

(for occult blood test)
3 Cerebrospinal fluid

Microbiology

Lumbar vertebra puncture

Volume :3~5ml
Aseptic container
Test immediately

Do not refrigerate

Chemistry Cell count

3 Effusion

Location Pleural effusion

Ascites
Pericardial effusion

Treatment Aseptic container for culture

Anticoagulant: Heparin : chemistry
EDTA: cell counts

Volume Routine: 3-5ml

Tumor cell, culture: 20~200ml
3 Sputum

Preparation Gargle

Timing Early morning

from Catheter by aspirator

Trachea dissection
 Contents

01 history

key points
02 specimen collection

03 Factors affecting the analysis of the results

difficult points
 How is a test data obtained?

Pre-analytical phase

Specimen analysis

Analytical assessment

Medical assessment
⚫70% of the laboratory errors occurred in preanalytical phase

sample sample
Preparation
collection transportation

• position • condition
• test order
• site •time
•Patient
preparation
•label

Preanalytical phase
 How to order a test ？
• Markers at different stages of disease course
• RNA？IgM? IgG？
 How to order a test ？
• Markers at different stages of disease course
• RNA？IgM? IgG？

• Screening ? Diagnostic? Monitoring？

• Sensitivity requirements?

• Specific requirements?

• methodological principle

• significance of clinical diagnosis,

• interference factor
Factors affecting lab test interpretation

Sensitivity
the ability of the test to detect
patients with some specific disease

Probability of positive diagnostic tests in actual patients

Specificity
how well test abnormality is
restricted to those persons who have the disease
in question
 golden standard
（+） （-） total
（+） a b a+b
diganositic （-） c d c+d
test
total a+c b+d n

Sensitivity Specificity

Se=a/（a+c） Sp=d/（b+d）
 Pre-analytical variables

Age
Race
Invariable Gender
Hereditary

Biological factors Pregnancy

Nutrition
Physical activity
Variable Fasting
Diurnal rhythm
Environmental

Hemoglobinemia
Intrinsic Bilirubinemia
Hyperlipoproteinemia
Interference factors Anticogulants
hemolysis
extrinsic Bacteria
Yeasts
 Factors affecting lab test interpretation

Patient Preparation

Be quiet Fatty acid↓

Lactic acid↑
Postprandial:
temporarily Fatty acid,
•Activity ALT, GLU, catecholamine ↑
durative
Proteinic
BUN, Na ↑ diet:K,Na,Ca,
•Coffeeuric,
•Diet urea,amonia urea,WBC,CK,
↑ Lactic acid,
crea normalLDH,AST,GLU ↑ HDL,MCV,
•Bed rest Urine Na, •Alcohol
GGT ↑
Ca,Cl,amonia,
•Smoking Serum ALP•Drug
↑
Catecholamine, addiction
cortical, WBC, MCV,
Hemoglobin ↑
 Factors affecting lab test interpretation

Medications
Drug induced injury to tissues or organs
Drug induced alterations in organ function
Drug competition
Drug interference analysis method
medicine: antibiotic, antidepressant
treatment: transfusion, iv drop, operation, dialysis
 Factors affecting lab test interpretation

Reference is set to cover 95% of all values

Ranges from the general population.
Five percent (5%) of results consequently fall
outside the normal range.

Population representative Various methods

The normal range may differ depending on the patient's
age, size, sex, or ethnic background.

Some normal persons may have abnormal laboratory test values.

Persons with disease may have normal test values
The relationship of observed values with reference range

Reference individual
Comprise
Reference population
Select
Reference sample group
Determine
Reference value
Observe
Observed value Reference distribution
Compared with Calculate
Reference limits
Define
Reference interval
 Critical values

These values may indicate the need for

prompt clinical intervention. Any sudden
changes may also be critical. Also called
action values or automatic call back values.
Values will vary according to the laboratory
performing the tests as well as patient age
and other factors.
 key points

✓ Pre-analytical variables
hemolysis
chylemia

✓ Influence of Different Sample Types on Test Results

✓ Reference Ranges

✓ Critical values

✓ Sensitivity

✓ Specificity
THANKS