FLOW CYTOMETRY ² CLINICAL APPLICATIONS

Everything you need to know about flow cytometry at Ziv Medical Center 2010

THE FLOW CYTOMETRY UNIT AT ZIV MEDICAL CENTER

Beckman Coulter Epics XL-MCL: 488nm laser, 4 FL channels, manual or automated carousel sample loading

WHAT IS FLOW CYTOMETRY?
Cyto = Cell metry = measure Flow = fluid stream ´Cytometry is a process in which physical and/or chemical characteristics of single cells « are measured. In flow cytometry, the measurements are made as the cells or particles pass through the measuring apparatus, a flow cytometer, in a fluid stream.µ ² Howard Shapiro, Practical Flow Cytometry, available online free at www.coulterflow.com .

ADVANTAGES OF FLOW CYTOMETRY
Flow cytometer can make measurements on tens of thousands of cells in minutes. Multiple parameters can be measured at the same time. Relatively simple sample preparation. Use of monoclonal antibodies allows for measuring specific immunophenotypes, such as different kinds of lymphocytes.

INSTRUMENT BASICS ² TEST AT THE END

chamber

TYPICAL FLOW CHAMBER ² NO TEST AFTERWARDS, YOU·LL NEVER SEE IT

FRONT SCATTER RELATES TO SIZE

SIDE SCATTER (90O) RELATES TO CELL COMPLEXITY

WITH THESE ALONE, LEUKOCYTE GROUPING CAN BE SEEN

Granulocytes

Monocytes Lymphocytes

FLUORESCENCE BASICS ² TEST ONLY FOR
THOSE WHO WANT EXTRA CREDIT

http://biology.berkeley.edu/crl/flow_images/fluorescence_detector.gif

‡Common Fluorophores excited by a 488nm laser: FITC, PE, PI, PE-TR, PE-Cy5 ‡Overlap of emission spectra can cause signal in a nonspecific channel. ‡This overlap is corrected electronically by ´compensationµ, or subtraction of the electronic signal caused by the wrong dye.

MONOCLONAL ANTIBODIES ² START PAYING ATTENTION SOON
Monoclonal antibodies allow specific antigens on the membrane or inside a cell to be ´taggedµ with a fluorescent dye. This yields a wealth of data that is clinically useful, such as the percent of lymphocytes which are B-cells, T-cells, or NK-cells. Abnormal percentages can indicate suspicious cells. Aberrance of antigen expression and light scatter properties can indicate abnormal or suspicious cells.

EXAMPLES OF LINEAGE SPECIFIC MARKERS ² EXTRA CREDIT ONLY
T-cells: CD3, CD5, CD7, CD2« B-cells: CD19, CD20 Monocytes: CD14 bright Granulocytes: combinations of CD13, CD33, CD15, CD16, CD11b Platelets: CD41

Fluorchrome (FITC)

Antibody (anti-CD45) cell Membrane Antigen (CD45)

LEUKOCYTES DIFFER IN CD45 EXPRESSION AND SIDE SCATTER

Granulocytes

Monocytes Lymphocytes

DATA DISPLAY ²
TECHNICAL STUFF YOU PROBABLY WON·T NEED TO KNOW, BUT YOU CAN SAY YOU HEARD ABOUT IT

The electronic signals are now processed by the computer. For each cell we have now gathered data which can include SSC (side scatter), FSC (front scatter), and four FL channels. Most common displays are the histogram and the dot plot. The histogram is a count of each cell which had data in a particular ´channelµ, or value. The dot plot is a plot of dots! Each dot represents a cell and the value of the parameter chose.

SAMPLE DATA DISPLAY
A Histogram of the PE channel data for a monoclonal Lambda sample.

A dot plot of the PE vs. FITC channels for the same sample.

SAMPLE CD45 PC5/CD19 FITC /CD5 PE

ABNORMAL SAMPLE 45-19-5

TACHLIS ²WHAT SAMPLES CAN YOU SEND? REQUIRED KNOWLEDGE TO PASS TEST
Peripheral Blood ² purple or green caps (EDTA or heparin, citrate also possible) Bone Marrow -- purple or green caps (EDTA or heparin) Lymph Nodes (via pathology) Body fluids (peritoneal, plural, etc..) -- purple or green caps (EDTA or heparin)

Please consult with Dr. Dali first to confirm that flow cytometry is recommended. CLOTTED SAMPLES CANNOT BE ANALYSED.

PNH - PAROXYSMAL NOCTURNAL
HEMOGLOBINURIA

Symptoms PNH is a complex disease with signs and symptoms that are nonspecific, unpredictable, and often similar to those of other diseases. While symptoms can vary widely in severity among patients with PNH, they share a common cause of chronic hemolsyis. Some more common symptoms include:6,8 Debilitating pain, especially in the stomach Difficulty swallowing Severe anemia Shortness of breath Dark-colored urine (hemoglobinuria) Disabling fatigue Erectile dysfunction

FLOW CYTOMETRY FOR HEMATOLOGICAL DISORDERS

Lymphomas
B-cell y T-cell y NK-cell
y

Leukemias
y

Acute

Lymphoid Myeloid Lymphoid Myeloid

y

Chronic

PNH - Paroxysmal nocturnal hemoglobinuria MDS ² Myelodysplastic Syndromes Hereditary Spherocytosis

WHAT ELSE DO YOU NEED TO KNOW?
TAKE NOTES NOW, THIS IS THE INTERESTING PART

Samples are accepted Sunday morning ² Thursday noon. Urgent samples can be processed on weekends with approval of Dr. Dali only. CBC samples from the same day must be submitted to the hematology laboratory separately. Requisition form must be filled out including: y Patient information sticker y Referring doctor y Reason for immunophenotyping request (susp LY, susp blasts, etc..) y Relevant medical history (known illnesses, drug treatments, immune system problems) y Type of sample (peripheral blood, bone marrow, fluid) y Date of sample

§ª º ±» ´¡œŸŸ º ª ›À ›Ÿ ¿¨º ¨
Flow Cytometry Lab ± §¡š «¡Õ º ª¡ø¡º ´ Ÿš FCL2010-0396-01 :²« øŸ Õ¨ ª § Àø¨ : ´ ±ª›»º ¨±² 200396 :º ¨¡œª ²« Õ¨ 31/05/2010 :º ø¡± ¬¡²´ š 31/05/2010: º ¿¡ ± ¬¡²´ š 31/05/2010 :øŸ¡ª ¬¡²´ Ÿ š

SAMPLE OF REPORT

š ² ¡›ŸŸ Ÿ :º Ÿ §³ Õ » ¨Ÿ « øº .´ 5144762-1 : .æ 26/19/1952 :º ª¡ ¬¡²´ š ´ ¡œŸŸ º š ² :ø Ÿ §² ¿¨º Õ ¨ ³ Ÿ œ æ:« ¡¨ :´ ¡» ± º » ¡ ø›š º ¨Ÿ¨¡ Õ ª³ ø :º ¡¡» º ´ ›¡« Õ

:Õ » ŸŸ §ÀŸ š ª › ´Ÿ ¡¿Ÿ¡Õ» ¨¡š æ ¨œŸ ¡¿›³ ª ø º » š :º ±¡ª› §Ÿ ø›Ÿ ¥¡«
CD19+dim/CD20+/CD22+/CD79b+/CD23+dim/CD38+/CD71+/CD10+dim/CD25+dim/CD43- /FMC7- /CD11c-/LAMDA+

§¡¿¡ Ÿ¨¡ º ³ 63% Õ : §¡ªŸ øº §¡š º æ ³ ´ Ÿ øš )1-5 º ¨² øŸ¿( 0.06 = KAPPA/LAMBDA « Ÿ Ÿ » ø¡ )1- 1.5 º ¨² øŸ¿( 0.84 = Ÿ Ÿ » CD3+ ¬Ÿ ¨ CD4:CD8 « ´ ø¡ : CD45+/SSC ¡Õ §¡š ´ Ÿ Ÿ¥Ÿ ´ ¡« š Lympho:11.8% Mono:2% Gran: 73.6% .CD71+/CD38+ ± º ¡ ›¡¿±š ³ §¡» ³ ¡ : ´ ŸÀ ¨« ²º : §¡š §¡» º Ÿ ›º ¨« ±ª›» « ¡±´ CD13 « ¡±´ CD4 « ¡±´ CD56 4.8% of CD19+ KAPPA ¡±´ ¡±´ ¡›Ÿ ¡ø ¡›Ÿ ¡ø « ¡±´ « ¡±´ ¡›Ÿ ¡ø ¡›Ÿ ¡ø ¡±´ ¡›Ÿ ¡ø ¡›Ÿ ¡ø CD19 CD3 CD7 CD20 CD10 FMC7 CD22 CD38 « ¡±´ CD5 « ¡±´ CD16 « ¡±´ CD8 9% S-phase, possible DNA aneuploidy observed 83% of CD19+ LAMBDA ¡›Ÿ ¡ø CD23 ¡›Ÿ ¡ø CD25 ¡›Ÿ ¡ø CD79b §¡š º ´ Ÿ ¥¡

.marginal zone lymphoma Ÿ FCL Ÿ³ ² Õ ¡š.º ¨Ÿ¨¡ š ³ š Õ : º ª›À ¨º º » ´ ¨¡´ ø ¨ ¨ ŸÀ _________________š Ÿ ¡» š

 

 

   

CD43 CD11c CD71

: º ±¡ª›º À ›¨ ´ ¨¡´ ø ______________ ³ šº ¡´ ›

1 ¬Ÿ ¨ 1 ª Ÿ ´ ¨À

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8661 ² 8151 ²

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