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Automated Hematology Dr.P.Dahim Health Reference Laboratory. Principles of Automation Calibration Quality Control Flagging. Hematology Automation. Two General Principles Electronic resistance ( impedance) Light scattering. Electronic Resistance (Impedance).

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Automated hematology dr p dahim health reference laboratory

Automated Hematology

Dr.P.Dahim

Health Reference Laboratory


Automated hematology dr p dahim health reference laboratory

  • Principles of Automation

  • Calibration

  • Quality Control

  • Flagging


Hematology automation

Hematology Automation

  • Two General Principles

    • Electronic resistance ( impedance)

    • Light scattering


Electronic resistance impedance

Electronic Resistance (Impedance)

  • Utilizes non-conductive properties of blood cells

    • as blood cell passes through orifice of aperture it displaces its own volume

    • increased resistance between electrodes results in an electrical pulse

    • RBCs and Platelets counted together, separated by pulse heights

    • hydrodynamic focusing forces cells to pass single file through sensing zone


Cell counting

Cell counting


Light scattering

Light scattering

  • Cells counted as passed through focused beam of light( LASER)

  • Sum of diffraction(bending around corners), refraction (bending due to change in speed) and reflection (light rays turned back by obstruction).

  • Multi angle polarized scatter separation (M.A.P.S.S)

    • 0° : indicator of cell size

    • 10° : indicator of cell structure and complexity

    • 90° polarized: indicates nuclear lobularity

    • 90° depolarized: differentiate eosinophils


Principles of counting sizing

Principles of counting &sizing

  • Counting and sizing red cells ,WBCs , Plts by counting the number of pulses and height of pulses

  • Measuring Hb by modified HiCN method

  • Measuring MCV by computing the mean of height of pulses

  • Hct (PCV ) derived from MCV & RBC

  • MCH derived from Hb & RBC

  • MCHC derived from Hb , RBC & MCV


Automated hematology dr p dahim health reference laboratory

  • RDW: SD or CV of individual measurement of red cell volume

  • HDW :The degree of variation in red cell hemoglobinization,CV of measurements of hemoglobin concentration of individual cells

  • NRBC: included in the WBC counting


Wbc differential count

WBC Differential Count :

  • Impedance technology with current of various frequencies

  • Light scattering & light absorbance


Reticulocyte counting

Reticulocyte counting :

  • Flourescence – based methods


Histograms

Histograms

  • RBC, PLT, and WBC plotted on histogram

  • X-Axis

    • Cell size in femtoliters (fL)

  • Y-Axis

    • # of cells


Histogram

Histogram

WBC: Distribution with three individual peaks and valleys at specific regions representing the lymphocytes, monocytes, and granulocytes.

All curves normally start and end at baseline


Automated hematology dr p dahim health reference laboratory

مشخصات شناسنامه دستگاههای شمارشگر سلولی خودکار

1-تاريخ خريد

2- تاريخ نصب ، کاليبراسيون و نام فرد راه اندازی کننده

3- تاريخ شروع بکار

3-کارخانه و کشور سازنده

4- مدل وشماره سريال دستگاه

5- شرايط دستگاه هنگام خريد (نو ، مستعمل ، بازسازی شده )

نکته : علاوه بر شناسنامه ، موارد زير نيز برای هر سل کانتر می بايست بصورت مکتوب در آزمايشگاه موجود باشد .


Automated hematology dr p dahim health reference laboratory

6- نام وآدرس شرکت پشتيبان

7-نام کاربر يا کاربران دستگاه

8- مشخصات و نحوه آموزش کاربر دستگاه توسط شرکت پشتيبان

9-ميزان عدم دقت و عدم دقت دستگاه در هنگام نصب


Automated hematology dr p dahim health reference laboratory

ساير مستندات

- مشخصات محلولها و مواد مصرفی

- تاريخ آخرين سرويس دوره ای و خدمات پشتيبانی

- تاريخ آخرين کاليبراسيون

- تاريخ آخرين تعمير با ذکر قطعه تعويض شده

- تاريخ آخرين مشکل و راه حل آن


Automated hematology dr p dahim health reference laboratory

اصول کار

  • كار با دستگاه سل كانتر مي بايست بطور كامل مطابق كاتالوگ دستگاه يا آموزش كارشناسان شركت پشتيبان صورت گيرد .


Automated hematology dr p dahim health reference laboratory

اصول کار

  • درصورت تعويض کاربر دستگاه ، می بايست از آموزش وی در مورد چگونگی کار با دستگاه و نحوه نگهداری و اصول کنترل کيفی اطمينان حاصل نمود .


Automated hematology dr p dahim health reference laboratory

اصول کار

  • کليه موارد مربوط به نگهداری دستگاه ، از قبيل تاريخ انجام شستشوهای لازم ، تعمير ، سرويس ويا تعويض محلولها می بايست ثبت ونگهداری شوند.


Automated hematology dr p dahim health reference laboratory

اصول کار

هر روز شمارش زمينه يا Back ground دستگاه ارزيابي و در صورت امکان ثبت و نگهداری شود .بطور معمول شمارش زمينه هر دستگاه در کاتالوگ مربوطه آمده است ولی مقادير قابل قبول سازمان جهانی بهداشت به شرح زير می باشند :

  • RBC: <0/03 x 1012/l

  • WBC: <0/04 x 109 / l

  • Haemoglobin: <0/2g/dl

  • Platelets: <5 x 109 /l


Calibration

Calibration

  • Calibration provides the most accurate results possible.

  • For best performance, calibrate all the CBC parameters.

  • The WBC differential is calibrated at the factory. They do not require calibration in the laboratory.


When to calibrate

When to Calibrate

  • At installation

  • After the replacement of any component that involves dilution characteristics or the primary measurements (such as the apertures)

  • When advised to do so by your service representative


Calibration1

Calibration

Normal fresh blood samples

Calibrators


Accuracy

Accuracy

  • 100×ميانگين روش دستگاهي - ميانگين روش دستيCF=

  • ميانگين روش دستگاهی


Automated hematology dr p dahim health reference laboratory

پايداری کاليبراسيون

_

Sd =√ ∑(d2)- (∑d)2 /n / n-1

tn = d √n 

sd


Precision check

Precision check

  • Purpose: check reproducibility of instrument

  • Precision quick method

    • Run 5 replicates of one patient’s sample free of elevated bilirubin, lipemia, and hemoloysis

  • Calculate % CV or SD

  • Refer to the methodology in procedure manual for expected % CV or SD


Quality control

Quality Control

  • Purpose of QC

    • Assure proper functionality of instrumentation

    • Monitoring the Integrity of the Calibration


Quality control methods

Quality Control Methods

  • Assayed stabilized material (Commercial)

  • Previously analyzed patient samples

    • Easily obtained

    • Cost effective

    • Results and samples readily available


Quality control1

Quality control

Replicate tests (control samples)

Control charts

Duplicate & Check tests

Correlation system

Delta check

Daily means of MCV,MCH,MCHC

Comparison the results with reference methods


Replicate tests

Replicate tests

  • بررسي عدم دقت دستگاه


Control charts

Control charts

  • استفاده از خون كنترل

  • تفسير با استفاده از قوانين سازمان جهاني بهداشت يا وستگارد


Quality control2

Quality Control

OUT OF CONTROL!!!

Repeat the assay ( One time occurrence )

Check integrity of material

Troubleshoot

Verify instrumentation


Duplicate test

Duplicate test

درهرسري کاري 2-3 نمونه به صورت مضاعف((Duplicate آزمايش ميشوند تا با بررسي اختلاف خوانده ها از طريق محاسبات آماري از وجود خطاهاي تصادفي آگاه گرديد . افزايش اختلاف بين دو خوانده بيش از 2SD محاسبه شده ، احتمال وجود خطاي تصادفي را مطرح مي نمايد.

  • SD= √∑d2

  • 2n


Check test

Check test

  • در ابتدای سری کاری يا صبح3- 2نمونه آزمايش شده وهمان نمونه ها در انتهاي سري كاري يا بعد از ظهرمجددا آزمايش مي شوند. نتايج حاصل از اين دو آزمايش با استفاده از فرمول Duplicate test قابل مقايسه می باشند . اختلاف نتايج در محدوده 2SD قابل قبول می باشد . در صورت نگهداری نمونه ها در شرايط مناسب ، هرگونه تغيير در نتايج خارج از اين محدوده نشاندهنده اشکال درعملکرد دستگاه و يا معرفها مي باشد.

  • اين آزمايش براي بررسي هموگلوبين و گلبولهای قرمز مناسب بوده و به ميزان کمتر براي شمارش گلبولهاي قرمز و پلاکت کاربرد دارد ولي براي هماتوکريت بخصوص اگر فاصله زماني آزمايش نمونه ها بيش از 6 ساعت باشد کارايي ندارد.


Delta check

Delta check

Hb 2 g/l

PCV 0.05 L/L

MCV >6 fL

MCH > 5 pg

WBC Normal to abnormal

Platelets Reduced or increased by more than 50%


Daily means of mcv mch mchc

Daily means of MCV,MCH,MCHC

مراکز آزمايشگاهي با پذيرش بيمار زياد (حداقل روزی 100 بيمار

تفاوت مقادير بيش از 3٪ از ميانگين، نشان دهنده بروز خطا و لزوم بررسی کاليبراسيون دستگاه می باشد.


Comparison the results with reference methods

Comparison the results with reference methods

انجام محاسبات آماري براي بررسي هم خواني نتايج الزامي است .


Errors

ERRORS

INACCURACY


Reliability of electronic counters

Reliability of Electronic Counters

High Precision

Accuracy?

- Recirculation of cells

- Red cell agglutination

- Lipid droplets, Microorganisms,

- Extraneous particles

- Faulty maintenance

- Incorrectly calibration


Interfering substances

Interfering substances

Cold Agglutinins

Cryoglobulins

Lipemia

Platelet Clumps

RBC fragments

NRBCs

Clot


Wbc count interferences

WBC Count Interferences

Unusual RBC abnormalities that resist lysing

malarial parasites

giant platelets, platelet clumps, NRBCs

fragmented white cells, agglutinated white cells,

lyse-resistant red cells,

cryoglobulin, some extremely elevate proteins,

unlysed particles greater than 35 fL in size


Rbc count interferences

RBC Count Interferences

Very high WBC count,

high concentration of very large platelets,

Auto-agglutination


Automated hematology dr p dahim health reference laboratory

Hgb

Very high WBC count,

Severe lipemia,

certain unusual RBC abnormalities that resist lysing


Automated hematology dr p dahim health reference laboratory

MCV

Very high WBC count,

high concentration of very large platelets,

Auto-agglutination


Flagging

Flagging

  • WBC Suspect flags

    • Blasts

    • Imm Grans

    • Variant lymphs


More flagging

More Flagging

  • RBC Suspect flags

    • NRBCs

    • Macrocytic RBCs

    • Dimorphic RBC population

    • Micro RBCs/RBC fragments

    • RBC agglutination


Troubleshooting flagged results

Troubleshooting Flagged Results:

  • Refer to the Instrument Manual to troubleshoot codes, flags, and messages displayed with patient results


Automated hematology dr p dahim health reference laboratory

Thank you for your attention


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